HK1068898B - A virus causing respiratory tract illness in susceptible mammals - Google Patents

Description

Viruses causing respiratory disease in susceptible mammals

the present invention relates to the field of virology.

Over the past decades, several mammalian disease etiologies, particularly respiratory disease (RTI) etiologies, and particularly human disease etiologies, have been identified⁷. The classical etiology of mammalian RTIs is respiratory syncytial virus belonging to the Pneumovirus genus (pneumvirus) found in humans (hRSV) and ruminants such as cattle or sheep (bRSV and/or omsv). In human RSV, two antigenic subgroups of hRSV were identified using differences in the positive and negative cross-neutralization assay, reactivity of the G protein in the immunoassay, and the nucleotide sequence of the G gene. Within said subgroups, the amino acid sequences show 94% (subgroup a) or 98% (subgroup B) identity, whereas only 53% amino acid sequence identity is found between subgroups. Additional variability was observed within subgroups based on monoclonal antibodies, RT-PCR assays and rnase protection assays. Viruses from both subgroups have a worldwide distribution and can occur in one season. Infection may occur in the presence of existing immunity, and antigenic variation is not strictly required for reinfection. See, e.g., Sullerder, W.M., Respiratory synthetic viral genetics and antimicrobial universitylogy Reviews, 2000.13 (1): p.1-15; collins, p.l, MiIntosh, k. and Chanock, r.m., Respiratory synthetic vision, fieldvirology, b.n.knepe, Howley, p.m. editions, 1996, philidelphia: lippencott-raven.1313-1351; johnson, P.R. et al, The G glycoprotein of human respiratory viruses of subgroups A and B: extended sequence diversity between anti-genetic related proteins, Proc Natl Acad Sci U SA, 1987, 84 (16): p.5625-9; collins, P.L., The molecular Biology of human Research Synthetic Virus (RSV) of The Genus Pneumovirus, described in The Paramyxoviruses, D.W.Kingsbury, 1991, Plenum Press: new York, p.103-153.

Another classical pneumovirus is the mouse Pneumovirus (PVM), which is generally found only in laboratory mice. However, a certain percentage of the diseases observed in mammals still cannot be attributed to known pathogens.

The present invention provides an isolated substantially mammalian negative-sense single-stranded RNA virus (MPV) belonging to the Pneumovirinae subfamily of the Paramyxoviridae (Paramyxoviridae) family (Pneumovirinae) identifiable as phylogenetically corresponding to Metapneumovirus. The virus was tested by determining its nucleic acid sequence and in a phylogenetic analysis, for example in which a maximum likelihood evolutionary tree was generated using 100 leads and 3 jumbles, and was found to correspond phylogenetically more closely to the basic avian virus isolate corresponding to the Avian Pneumovirus (APV), also known as turkey rhinotracheitis virus (TRTV), a pathogen of avian rhinotracheitis, which was identified as phylogenetically corresponding to Metapneumovirus, and which corresponds to the viral isolate deposited at paris CNCM as I-2614. For the phylogenetic analysis, it is most useful to obtain non-MPV nucleic acid sequences as outer group(s) for comparison, and a very useful outer group isolate may be obtained from avian pneumovirus serotype C (APV-C), as demonstrated, for example, in FIG. 5 herein.

While phylogenetic analysis provides a convenient way to identify a virus as an MPV, several other potentially more direct but somewhat more procedural methods for identifying the virus or viral proteins or nucleic acids derived from the virus are also provided herein. Empirically, MPV can be identified by the percent homology of the viral protein or nucleic acid to be identified in comparison of the sequence or deposit with the isolate, viral protein or nucleic acid identified herein. It is well known that virus species, especially RNA virus species, often constitute a quasispecies, wherein the virus taxa (cluster) shows heterogeneity between its members. Thus, it is expected that the percentage relationship between each isolate and one of the various isolates provided herein may vary slightly.

When one wishes to compare with deposited virus I-2614, the invention provides an isolated substantially mammalian negative-sense single-stranded RNA virus (MPV) belonging to the Pneumovirinae subfamily of the Paramyxoviridae family, identifiable as phylogenetically corresponding to Metapneumovirus, by determining the amino acid sequence of said virus, and determining that the percentage of amino acid homology, in terms of L, M, N, P or F protein, with respect to the viral isolate deposited as I-2614 in Paris CNCM is substantially higher than the percentage provided herein compared to APV-C; alternatively, and as such, there is provided an isolated substantially mammalian negative-sense single-stranded RNA virus (MPV) belonging to the sub-family pneumovirinae of the family paramyxoviridae, said virus being identifiable as phylogenetically corresponding to metapneumvirus by: determining the nucleic acid sequence of said virus and determining that the percentage of nucleic acid identity of said nucleic acid sequence with respect to the nucleic acid encoding the L, M, N, P or F protein with respect to the viral isolate deposited at CNCM Paris as I-2614 is substantially higher than the percentage identified herein compared to APV-C.

Further empirically, when the percentage homology of the isolate to be identified or of the viral protein or nucleic acid of said isolate falls within the range of said percentage homology for each of the two groups identified herein (with isolate 00-1 or 99-1 as the respective comparative isolate), one can consider said MPV to belong to one of the two serological groups of MPVs identified herein. However, when the percentage homology is small, or it is more desirable to distinguish the viral isolate from, for example, APV-C, it is recommended that the phylogenetic analysis identified herein be preferred.

Furthermore, when selecting other isolates in determining the percentage of homology, one should remember that the percentage may vary slightly.

As a result of the provision of such MPVs, the present invention provides diagnostic tools and methods as well as therapeutic tools and methods for the diagnosis and/or treatment of diseases, in particular respiratory diseases, in particular mammalian diseases, more in particular human diseases. However, since the substantially mammalian MPV is genetically related, albeit distantly, to the substantially avian APV, particularly to APV-C, the present invention also provides tools and methods for diagnosing and treating avian diseases. In virology, the best advice is: diagnosing and/or treating a particular viral infection with an agent that is most specific for the particular virus causing the infection. In this case, this means that the diagnosis and/or treatment of the MPV infection is preferably carried out with the most specific agent for MPV. However, this by no means excludes the possibility of using reagents of lower specificity but sufficient cross-reactivity for substitution, for example because they are more readily available and sufficient to perform the task at hand. Here, for example, a virological and/or serological diagnosis of MPV infection in mammals with APV-derived reagents, in particular APV-C-derived reagents, is provided, which in the detailed description herein, for example, shows that a sufficiently reliable serological diagnosis of MPV infection in mammals can be achieved by using an ELISA specifically designed for the detection of avian APV antibodies. A particularly useful assay for this purpose is an ELISA designed to detect APV antibodies (e.g., in serum or yolk), a commercially available form known as APV-AbSVANOVIRProduced by SVANOVA Biotech AB (Uppsal Science Park GlunteSE-75183 Uppsala Sweden). The opposite is also the case, where, for example, the mammal is subjected to an MPV-derived reagentVirological and/or serological diagnosis of APV infection in the present detailed description, it is for example shown that a sufficiently reliable serological diagnosis of APV infection in birds can be achieved by using an ELISA designed for the detection of MPV antibodies. By selecting appropriate antibodies with sufficient cross-reactivity, detection of various antigens can be achieved, taking into account the lock and key relationship between antigen and antibody. Of course, because of the reliance on such cross-reactivity, it is desirable to select such reagents (such as antigens or antibodies) under the direction of amino acid homology between the various (carbohydrate) proteins of the various viruses, so that the reagent associated with the most homologous protein will be most useful for use in assays that rely on such cross-reactivity.

For nucleic acid detection, it is even simpler not to design primers or probes based on heterologous nucleic acid sequences of various viruses and therefore not to detect differences between the substantially mammalian or avian Metapneumoviruses, as long as primers or probes are designed or selected based on those sequence segments that show high homology in the virus-specific nucleic acid sequences. In general, for nucleic acid sequences, a percentage of homology of 90% or more than 90% ensures sufficient cross-reactivity to be relied upon in diagnostic assays employing stringent hybridization conditions.

The invention provides, for example, a method of virologically diagnosing an MPV infection in an animal, particularly a mammal, more particularly a human, said method comprising determining the presence of a viral isolate or a component thereof in a sample of said animal by reacting said sample with an MPV-specific nucleic acid or antibody of the invention; the present invention also provides a method of serologically diagnosing MPV infection in a mammal, the method comprising determining the presence of an antibody specific for MPV or a component thereof in a sample of said mammal by reacting said sample with an MPV-specific proteinaceous molecule, or fragment or antigen thereof, of the invention. The present invention also provides a diagnostic kit for diagnosing MPV infection, said kit comprising an MPV, an MPV-specific nucleic acid, a proteinaceous molecule or fragment thereof, an antigen and/or an antibody of the invention, preferably means for detecting said MPV, MPV-specific nucleic acid, proteinaceous molecule or fragment thereof, antigen and/or antibody, said means for example comprising an art-used excitable group, such as a fluorophore or an enzyme detection system (examples of suitable diagnostic kit formats include IF, ELISA, neutralization assays, RT-PCR assays). To determine whether an unidentified viral component or a synthetic analogue thereof, such as a nucleic acid, proteinaceous molecule or fragment thereof, can be identified as MPV-specific, it is sufficient to analyze the nucleic acid sequence or amino acid sequence of said component (respectively), e.g. to analyze a stretch of preferably at least 10, more preferably at least 25, more preferably at least 40 nucleotides or amino acids of said nucleic acid or amino acid, by comparison with the known MPV sequence and sequence homology to the known non-MPV sequence APV-C, using for example the phylogenetic analysis provided herein. The component or synthetic analogue may be identified by the degree of relationship to the MPV or non-MPV sequence.

The invention also provides a method of virologically diagnosing an MPV infection in a mammal, said method comprising determining the presence of a viral isolate, or a component thereof, in a sample from said mammal by reacting said sample with a cross-reactive nucleic acid derived from an APV, preferably of serotype C, or a cross-reactive antibody reactive with said APV; the invention also provides a method of serologically diagnosing MPV infection in a mammal, the method comprising determining the presence of cross-reactive antibodies also directed against APV or a component thereof in a sample from the mammal by reacting the sample with a proteinaceous molecule derived from APV, or a fragment or antigen thereof. Furthermore, the invention provides the use of a diagnostic kit originally designed for the detection of APV or APV antibodies for the diagnosis of MPV infection, in particular for the detection of said MPV infection in humans.

The invention also provides a method of virologically diagnosing an avian APV infection, said method comprising determining the presence of a viral isolate or component thereof in a sample of said avian by reacting said sample with a cross-reactive nucleic acid derived from an MPV or a cross-reactive antibody reactive with said MPV; the invention also provides a method of serologically diagnosing an avian APV infection, the method comprising determining in a sample of the avian the presence of cross-reactive antibodies also directed against MPV or a component thereof by reacting the sample with a proteinaceous molecule derived from MPV, or a fragment or antigen thereof. Furthermore, the invention provides the use of a diagnostic kit originally designed for the detection of MPV or MPV antibodies for the diagnosis of APV infections, in particular for the detection of said APV infections in poultry such as chickens, ducks or turkeys.

As already mentioned, the cross-reactivity found can be applied analogously for therapy, in particular in the present case by using a less direct route of higher homology. For example, when a more homologous MPV vaccine is not available, an unsustainable vaccination, such as an emergency vaccination against MPV infection, may be performed with a vaccine preparation derived from an APV (preferably type C) isolate; conversely, vaccination against APV infection with MPV-derived vaccine preparations may be considered. Furthermore, the reverse gene technology makes it possible to generate chimeric APV-MPV virus constructs that are sufficiently different from the field isolate of each respective strain to be attenuated to the desired level to be useful as vaccines. Similar reverse gene technology would make it possible to generate chimeric paramyxovirus-metapneumovirus constructs for vaccine formulations as well, such as RSV-MPV or PI3-MPV constructs. Such constructs are particularly useful as combination vaccines against respiratory diseases.

Thus, the present invention provides a novel pathogen-an isolated substantially mammalian negative-sense single stranded RNA virus (also referred to herein as MPV) belonging to the Pneumovirinae subfamily of the Paramyxoviridae, but not identified as a typical pneumovirus, but rather as Metapneumovirus-and MPV-specific components or synthetic analogues thereof. So far, metapneumvirus-like mammalian viruses, i.e. metapneumvirus which can be isolated from a mammal, which is substantially mammalian as the natural host for said virus or which is pathogenic in said mammal, have not been found. Metapneumovirus, also known as avian pneumovirus, is widely recognized as being essentially limited to poultry as a natural host or as a pathogen of poultry disease. Recently, an APV isolate of duck (FR 2801607) was described, further demonstrating that APV infection is essentially restricted to birds as the natural host.

The present invention provides isolated mammalian pneumoviruses (also referred to herein as MPVs) comprising a different genetic order and amino acid sequence from the pneumovirus genus and which are closely related, and whose phylogenetic relevance is believed to be likely to belong to Metapneumovirus in the pneumovirinae sub-family of the paramyxoviridae family. Although metapneumovirus has not been isolated from birds to date, it is now shown that viruses that are significantly different but related can be identified in other animal species, such as mammals. Here, we show that MPV is repeatedly isolated from humans without such reports on APV. Furthermore, unlike APV, MPV replicates substantially no, or only very little, in chickens and turkeys, but readily in rhesus monkeys. No report was found about the replication of APV in mammals. In addition, while specific antisera raised against MPV neutralize MPV, antisera raised against APV a, B or C do not neutralize MPV to the same extent, and this lack of full cross-reactivity provides another indication that MPV is a different metapneumovirus. Furthermore, although APV and MPV have similar gene sequences, the G protein and SH protein of MPV are significantly different from those of known APV because they do not show significant sequence homology at the amino acid level or nucleic acid level. Based on one or both of these proteins, diagnostic assays (e.g., IF, ELISA, neutralization assays, RT-PCR assays) for identifying APV isolates and MPV isolates or antibodies against these different viruses can be advantageously developed. However, in addition, sequence information and/or antigenic information from the more relevant N, P, M, F and L proteins of MPV and sequence homology analysis of the corresponding proteins to APV can also be used to identify APV and MPV. For example, phylogenetic analysis of sequence information from MPV indicates that MPV and APV are two distinct classes of viruses. In particular, phylogenetic evolutionary trees indicate that APV and MPV are two distinct viral lineages. We have also shown that MPV circulates in the population for at least 50 years, as such interpropagation may occur at least 50 years ago, rather than daily events. Since MPV CPE is virtually indistinguishable from CPE caused by hRSV or hPIV-1 in tMK or other cell cultures, it is possible that MPV has been overlooked to date. tMK (third generation monkey kidney cells, MK cells third passage in cell culture) is preferred for use because of its lower cost compared to primary or second generation cultures. The CPE, as well as CPE caused by certain viruses of the classical paramyxoviridae family, is characterized by syncytia formation, after which the cells exhibit rapid internal destruction, and then the cells are detached from the monolayer. The cells typically (but not always) show CPE at days 10-14 after inoculation after three passages of the virus from the original material, slightly later than CPE caused by other viruses such as hRSV or hPIV-1.

In general, paramyxoviruses cause death of many animals and humans worldwide each year as a destructive factor of the disease. Paramyxoviridae, a family of single negative strand RNA viruses (Mononegavirales) (negative-sense single-stranded RNA viruses) consists of the sub-family Paramyxoviridae (Paramyxoviridae) and the sub-family Pneumovirinae (Pneumovirinae). The latter subfamily is currently classified taxonomically into Pneumovirus (Pneumovirus) and Metapneumovirus¹. Human respiratory syncytial virus (hRSV) -a typical species of the Pneumovirus genus, is the most important single cause of lower respiratory tract infections in infants and young children worldwide². Other members of the pneumovirus genus include bovine and ovine respiratory syncytial virus and mouse Pneumovirus (PVM).

Avian Pneumovirus (APV), also known as turkey rhinotracheitis virus (TRTV), is the etiological agent of upper respiratory tract infection of turkey avian rhinotracheitis³APV is the only member of the recently assigned Metapneumovirus, and as has been described, APV has not been associated with infection, or more specifically, mammalian disease, to date. The serological subgroup of APV can be identified based on the nucleotide sequence or amino acid sequence of the G glycoprotein and a neutralization assay using monoclonal antibodies that also recognize the G glycoprotein. Within A, B and D subgroups, G proteins showed 98.5-99.7% amino acid sequence identity within subgroups, whereas only 31.2-38% amino acid identity was observed between subgroups. See, e.g., Collins, M.S., Gough, R.E., and Alexander, D.J., antibiotic differentiation of arianAvian Pathology, 1993.22: p.469-479; cook, j.k.a., Jones, b.v., Ellis, m.m., antibiotic differentiation of strain of tubular rhesus virus using monoclonal antibodies, avianpathology, 1993.22: p.257-273; Bayon-Auboyer, M.H. et al, nucleic acids sequences of the F, L and G protein genes of two non-A/non-B Alianpneumoviruses (APV) novel a non-APV subgroup J Gen Virol, 2000.81(Pt 11): p.2723-33; seal, b.s., Matrix Protein gene nucleotide and amplified amino acid sequence detected that is at the first US averagenegavirulent is distinguinated from European strains, virus Res, 1998.58 (1-2): p.45-52; Bayon-Auboyer, M.H., et al, Comparison of F-, G-and N-based RT-PCR protocols with a comprehensive viral procedure for the detection and typing of a turnkey research virus, Arch Virus, 1999, 144 (6): p.1091-109; juhasz, K, and A.J.Easton, Extensive sequence development in the attribute (G) protein gene of average pneumocirus: evidence of two discontint subgroups Gen Virol, 1994.75(Pt 11): p.2873-80.

Another APV serotype is provided in WO00/20600, and an APV Colorado isolate is described in WO00/20600 and compared to known APV or TRT strains using an in vitro serum neutralization assay. First, the Colorado isolate was tested against a monospecific polyclonal antiserum recognizing the TRT isolate. The Colorado isolate was not neutralized by monospecific antisera against any of the TRT strains. However, it was neutralized by hyperimmune antiserum raised against subgroup a strains. This antiserum neutralized the homologous virus at a titer of 1: 400, while the Colorado isolate was neutralized at a titer of 1: 80. The Colorado isolate was then tested against the TRT monoclonal antibody using the methods described above. In all cases, the cross-neutralization titer was less than 10. Monospecific antiserum raised against the Colorado isolate was also tested against two subgroups of TRT strains. None of the tested TRT strains was neutralized by antisera against the Colorado isolate.

The APV Colorado strain does not protect SPF chickens against challenge with subgroup a or subgroup B strains of the TRT virus. These results suggest that the Colorado isolate may be the first example of another serotype of avian pneumovirus, as suggested by Bayon-Auboyer et al (J.Gen. Vir.81: 2723-2733 (2000)).

In a preferred embodiment, the present invention provides an isolated MPV corresponding taxonomically to (hitherto unknown mammalian) metapneumovirus, said MPV comprising a different gene order than a pneumovirus in the pneumovirinae sub-family paramyxoviridae. The classification of these two genera is based mainly on their gene conformation (gene constellations); metapneumovirus generally lacks non-structural proteins such as NS 1or NS2 (see also Randhawa et al, J.Vir.71: 9849. 9854(1997), and the gene order differs from that of pneumovirus (RSV: '3-NS 1-NS2-N-P-M-SH-G-F-M2-L-5', APV: '3-N-P-M-F-M2-SH-G-L-5')^4，5，6. The MPV provided by the invention or the virus isolate corresponding to the MPV in taxonomy is revealed to be paramyxovirus-like particles based on EM analysis. In accordance with classification, MPV is sensitive to chloroform treatment, either phylogenetically or taxonomically, to its corresponding viral isolate; are most suitable for culture on tMK cells or functionally equivalent cells thereof and are essentially tyrosine-dependent in most cell cultures. Furthermore, the typical CPE and lack of hemagglutination activity for the most classically used erythrocytes suggest that the viruses provided herein are related, although distant only to classical pneumoviruses such as RSV. Although most paramyxoviruses have hemagglutination activity, most pneumoviruses do not have such activity¹³. MPVs according to the invention also contain a second overlapping ORF (M2-2) in the nucleic acid fragment encoding the M2 protein, as is common with most other pneumoviruses in general, such as also described in Ahmadian et al, j.gen.vir.80: 2011-2016 (1999).

In order to find further viral isolates provided by the present invention, it is sufficient to test for the presence of a pneumovirinae virus in a sample, optionally obtained from a diseased animal or human, and to test for the presence of a gene encoding (functional) NS 1or NS2 in the virus thus obtained, or to essentially demonstrate a different genetic order than a pneumovirus, such as RSV, as described above. Furthermore, viral isolates that phylogenetically, and thus classically correspond to MPV, can be found by cross-hybridization experiments with nucleic acids derived from MPV isolates provided herein, or in classical cross-serological experiments that employ monoclonal antibodies specific for MPV isolates and/or antigens and/or immunogens specifically derived from MPV isolates.

When the newly isolated virus contains a sufficiently similar gene sequence and/or amino acid sequence to, or corresponds structurally to, our prototype MPV isolate and is shown to be closely related to Metapneumovirus of the pneumovirinae, the virus corresponds phylogenetically and thus taxonomically to MPV. The highest amino acid sequence homology between MPV and any other virus known so far of the same family (APV C subtype) and the structural relatedness determined at the individual protein level is 87% for the matrix protein, 88% for the nucleoprotein, 68% for the phosphoprotein, 81% for the fusion protein and 56-64% for the portion of the polymerase protein, which can be derived when comparing the sequence given in fig. 6 with the sequences of other viruses, in particular APV-C. The individual proteins or complete virus isolates, which have a higher homology with respect to these above-mentioned maxima, respectively, are considered to correspond phylogenetically and thus taxonomically to MPV and comprise nucleic acid sequences which correspond in structure to the sequences shown in FIG. 6. The invention provides a virus which phylogenetically corresponds to the deposited virus. It should be noted that, similar to other viruses, a degree of variation is found between the different isolated substantially mammalian negative-sense single stranded RNA virus isolates provided herein. In phylogenetic trees, we identified at least 2 genetic taxa (genetic clusters) of viral isolates based on comparative sequence analysis of L, M, N and portions of the F gene. These MPV genotypes represent MPV subtypes based on nucleotide and amino acid differences in the viral nucleic acid or amino acid sequence (viral sequence), and similarity to other pneumoviruses, such as RSV. Within each genetic taxonomic group of MPV isolates, the percent identity at the nucleotide level was found to be 94-100 for L, 91-100 for M, 90-100 for N and 93-100 for F, and at the amino acid level, the percent identity was found to be 91-100 for L, 98-100 for M, 96-100 for N and 98-100 for F. In fig. 18-28, another comparison can be found. For the entire group of isolated essentially mammalian negative-sense single-stranded RNA viruses identified to date (MPV isolates) provided herein, the lowest percent identity at the nucleotide level is 81 for L and M, 83 for N, and 82 for F. At the amino acid level, the percentage is 91 for L and N, 94 for M and 95 for F. The viral sequences of the MPV isolates provided herein or the isolated MPV F gene, for example, exhibit a homology to, for example, Seal et al, vir. res.66: 139147(2000) provides an APV-C fusion (F) gene having a nucleotide sequence or amino acid sequence with less than 81% nucleotide sequence identity or less than 82% amino acid sequence identity to the corresponding nucleotide sequence or amino acid sequence.

Furthermore, the viral sequences of the MPV isolates provided herein or the isolated MPV L gene, for example, exhibit a homology to, for example, Ranhawa et al, j.gen.vir.77: 3047 the corresponding nucleotide sequence or amino acid sequence of the APV-A polymerase gene provided by (1996) has less than 61% nucleotide sequence identity or less than 63% amino acid sequence identity.

The percentage of sequence differences for MPV strains worldwide is similar to other viruses, possibly slightly higher. Thus, by analyzing the partial nucleotide sequences in the N, M, F and L ORFs of the 9 viral isolates, two potential genetic taxa were identified. 90-100% nucleotide identity was observed within one taxonomic group, and 81-88% identity was observed between taxonomic groups. Sequence information obtained with more virus isolates confirmed the presence of both genotypes. Viral isolate ned/00/01, which is the prototype of the A taxonomic group, and viral isolate ned/99/01, which is the prototype of the B taxonomic group, have been used in cross-neutralization assays to test whether the genotypes are associated with different serotypes or subgroups. From these data, we conclude that substantially mammalian single-stranded RNA isolates exhibiting percent amino acid homology with isolates 1-2614 can be classified as isolated substantially mammalian negative-sense single-stranded RNA viruses provided herein for L greater than 64, for M greater than 87, for N greater than 88, for P greater than 68, for F greater than 81, for M2-1 greater than 84, or for M2-2 greater than 58. In particular, the lowest percent identity, generally at the nucleotide sequence level, with a prototype MPV isolate provided herein is a member of the MPV isolate group provided herein for those viral isolates having L and M of 81, N of 83, and/or F of 82. At the amino acid level, these percentages are 91 for L and N, 94 for M, and/or 95 for F. When the percentage of amino acid sequence homology of a given viral isolate is above 90 for L and N, above 93 for M, or above 94 for F, the viral isolate is similar to the group of MPV isolates shown in figure 5.A given viral isolate can be identified as belonging to one of the genotypic taxa shown in figure 5 when the percentage of amino acid sequence homology for L is greater than 94, for N is greater than 95, or for M and F is greater than 97. It should be noted that these percentage homologies from which the genetic taxa are determined are similar to the degree of homology found in the genetic taxa of the corresponding genes of RSV.

Briefly, the present invention provides an isolated substantially mammalian negative-sense single stranded RNA virus (MPV) belonging to the sub-family pneumovirinae of the family paramyxoviridae and tested by determining the nucleic acid sequence of a suitable fragment in the genome of said virus and in a phylogenetic evolutionary tree analysis, wherein a maximum likelihood tree is generated using 100 bootstrap programs and 3 jumbles and it is found that it corresponds phylogenetically more closely to a viral isolate deposited in paris CNCM with I-2614, which virus can be identified as phylogenetically corresponding to metapneovirus, than to a viral isolate corresponding to Avian Pneumovirus (APV), also known as turkey rhinotracheitis virus (TRTV), the etiology of avian rhinotracheitis.

Each suitable nucleic acid genomic fragment that can be used in such phylogenetic tree analysis is, for example, any of RAP-PCR fragments 1-10 disclosed in the detailed description herein that generate the various phylogenetic tree analyses disclosed in FIG. 4 or FIG. 5 herein. Phylogenetic tree analysis of the nucleoprotein (N), phosphoprotein (P), matrix protein (M) and fusion protein (F) genes of MPV revealed the highest degree of sequence homology with the avian pneumovirus-APV C serotype, which is mainly found in american birds.

In a preferred embodiment, the invention provides an isolated substantially mammalian negative-sense single-stranded RNA virus (MPV) belonging to the subfamily pneumovirinae of the family paramyxoviridae and which has been tested by determining the nucleic acid sequence of a suitable fragment of the viral genome and in a phylogenetic evolutionary tree analysis, wherein a maximum likelihood evolutionary tree is generated using 100 bootstrap programs and 3 jumbles and it is found that it corresponds phylogenetically more closely to a viral isolate deposited in paris CNCM with I-2614, which virus can be identified as phylogenetically corresponding to Metapneumovirus, as compared to a viral isolate corresponding to Avian Pneumovirus (APV), also known as turkey rhinotracheitis virus (TRTV), the etiology of avian rhinotracheitis, wherein the suitable fragment comprises open reading frames encoding the viral proteins of the virus.

Suitable open reading frames (0RF) include ORFs encoding the N protein. When the overall amino acid identity of the analyzed N protein to the N protein of isolate I-2614 is found to be at least 91%, preferably at least 95%, then the analyzed viral isolate comprises the preferred MPV isolate according to the present invention. As indicated, the first gene in the MPV genomic map encodes a 394 amino acid (aa) protein and shows extensive homology to the N proteins of other pneumoviruses. The length of the N ORF is the same as that of the N ORF of APV-C (Table 5), but smaller than that of the N ORFs of other paramyxoviruses (Barr et al, 1991). Analysis of the amino acid sequence revealed the highest homology (88%) with APV-C and only 7-11% homology with other paramyxoviruses (Table 6).

Bart et al (1991) identified 3 regions with similarity between viruses belonging to the order Mononegavirales: A. b and C (fig. 8). Although the similarity is highest within the virus family, these regions are highly conserved between virus families. In all three regions, MPV showed 97% amino acid sequence identity to APV-C, 89% amino acid sequence identity to APV-B, 92% amino acid sequence identity to APV-A, and 66-73% amino acid sequence identity to RSV and PVM. The region between aa residues 160 and 340 appears to be highly conserved in metapneumovirus, to a lesser extent in the Pneumovirinae subfamily (Miyahara et al, 1992; Li et al, 1996; Barr et al, 1991). This is consistent with MPV being a metapneumovirus, this particular region showing 99% similarity to APV C.

Another suitable Open Reading Frame (ORF) that can be used in phylogenetic analyses includes the ORF encoding the P protein. When the overall amino acid identity of the analyzed P protein to the P protein of isolate I-2614 is found to be at least 70%, preferably at least 85%, then the analyzed viral isolate comprises the preferred MPV isolate according to the present invention. The second ORF in the MPV genomic map encodes a 294 aa protein with 68% amino acid sequence homology to the P protein of APV-C and only 22-26% amino acid sequence homology to the P protein of RSV (Table 6). The P gene of MPV contains a substantial ORF and is similar in this respect to P from many other paramyxoviruses (for review see Lamb and Kolakofsky, 1996; Sedlmeier et al, 1998). In contrast to APV A and B and PVM, and similar to RSV and APV-C, the MPV P ORF lacks a cysteine residue. Ling (1995) suggested that a region with high similarity among all pneumoviruses (aa 185-241) plays a role in RNA synthesis or in maintaining the structural integrity of the nucleocapsid complex. This region of high similarity is also present in MPV (FIG. 9), and especially when conservative substitutions are considered, shows 100% similarity to APV-C, 93% similarity to APV-A and B, and about 81% similarity to RSV. The C-terminus of MPV P proteins is rich in glutamate residues, as has been described for APV (Ling et al, 1995).

Another suitable Open Reading Frame (ORF) that can be used for phylogenetic analysis includes the ORF encoding the M protein. When the overall amino acid identity of the analyzed M protein to the M protein of isolate I-2614 is found to be at least 94%, preferably at least 97%, then the analyzed viral isolate comprises the preferred MPV isolate according to the present invention. The third ORF in the MPV genome encodes a 254 aa protein, which is similar to the M ORF of other pneumoviruses. The size of MPVM ORF was identical to that of M ORF of other metapneumovirus (Table 5), and showed high amino acid sequence homology to APV matrix protein (76-87%), low amino acid sequence homology to RSV and PVM (37-38%), and homology of 10% or less to other paramyxoviruses (Table 6). Easton (1997) compared the sequences of matrix proteins of all pneumoviruses and found that residues 14-19 had a conserved hexapeptide, which is also conserved in MPV (FIG. 10). For RSV, PVM and APV, a small second ORF within or overlapping the M major ORF has been identified (52 aa and 51 aa in bRSV, 75 aa in RSV, 46 aa in PVM and 51 aa in APV) (Yu et al, 1992; Easton et al, 1997; Samal et al, 1991; Satake et al, 1984). We note that there are two small ORFs in the MPV M ORF. A small ORF of 54 aa residues starting at nucleotide 2281 was found within the M major ORF, and a small ORF of 33 aa residues starting at nucleotide 2893, which overlaps the M major ORF (data not shown). Similar to the second ORFs of RSV and APV, there is no significant homology between these second ORFs and the second ORFs of other pneumoviruses, and there is a lack of apparent start or stop signals. In addition, no evidence has been reported about the synthesis of proteins corresponding to these second ORFs of APV and RSV.

Another suitable Open Reading Frame (ORF) that may be used in the phylogenetic analysis includes the ORF encoding the F protein. When the overall amino acid identity of the analyzed F protein with the F protein of isolate I-2614 is found to be at least 95%, preferably at least 97%, then the analyzed viral isolate comprises the preferred MPV isolate according to the present invention. The F ORF of MPV is located adjacent to the M ORF, a feature of Metapneumovirus members. The MPV F gene encodes a 539 aa protein that is 2 aa residues longer than F of APV-C (Table 5). Amino acid sequence analysis showed 81% homology to APV-C, 67% homology to APV-A and B, 33-39% homology to pneumovirus F protein, and only 10-18% homology to other paramyxoviruses (Table 6). One of the conserved features observed in paramyxovirus F proteins and also in MPV is the distribution of cysteine residues (Morrison, 1988; Yu et al, 1991). metapneumovirus shares 12 cysteine residues in F1 (7 are conserved in all paramyxoviruses) and 2 cysteine residues in F2 (1 is conserved in all paramyxoviruses). None of the 3 potential N-linked glycosylation sites present in the MPV F ORF were shared with RSV, 2 such sites with APV (positions 66 and 389). The third unique potential N-linked glycosylation site of MPV is at position 206 (fig. 11). Although having low sequence homology with other paramyxoviruses, the F protein of MPV exhibits characteristics of a typical fusion protein consistent with those described for F proteins of other members of the paramyxoviridae family (Morrison, 1988). The F protein of a member of the family paramyxoviridae is synthesized as an inactive precursor (F0), cleaved by proteases of the host cell to yield the amino-terminal F2 subunit and the large carboxy-terminal F1 subunit. Proposed cleavage sites (Collins et al, 1996) are conserved among all members of the Paramyxoviridae family. The cleavage site of MPV contains the residue RQSR. Two arginine (R) residues were shared with APV and RSV, but glutamine (Q) and serine (S) residues were shared with other paramyxoviruses such as human parainfluenza virus type 1, sendai virus and measles virus (data not shown). The hydrophobic region at the amino terminus of F1 is thought to act as a membrane fusion domain and to show high sequence similarity in paramyxoviruses and measles, but a lesser degree in pneumoviruses (Morrison, 1988). These 26 residues (position 137-163, FIG. 11) are conserved between MPV and APV-C, which is consistent with the region being highly conserved in metapneumovirus (Naylor et al, 1998; Seal et al, 2000).

As observed for the F2 subunit of APV and other paramyxoviruses, MPV showed a deletion of 22 aa residues compared to RSV (position 107-. Furthermore, for RSV and APV, the signal peptide and the anchor domain were found to be conserved within subtypes, while exhibiting high variability between subtypes (Plows et al, 1995; Naylor et al, 1998). The signal peptide at the amino terminus of F2 of MPV (aa 10-35, FIG. 11) shows some sequence similarity to APV-C (18 out of 26 aa residues are similar), with less conservation than other APVs or RSV. Much greater variability was observed in the membrane anchoring domain at the carboxy terminus of F1, although some homology to APV-C was still observed.

Another suitable Open Reading Frame (ORF) that can be used for phylogenetic analysis includes the ORF encoding the M2 protein. When the overall amino acid identity of the analyzed M2 protein to the M2 protein of isolate I-2614 was found to be at least 85%, preferably at least 90%, then the analyzed viral isolate comprised the preferred MPV isolate according to the present invention. The M2 gene is subcategory specific for pneumoviruses, and two overlapping ORFs were observed in all pneumoviruses. The first major ORF represents the M2-1 protein, which enhances the processivity of the viral polymerase (Collins et al, 1995; Collins, 1996) and the readthrough of its intergenic region (Hardy et al, 1998; Fearns et al, 1999). The M2-1 gene of MPV was located adjacent to the F gene, encoding a 187 aa protein (Table 5), and showed the highest homology (84%) to M2-1 of APV-C (Table 6). Comparison of all the Pneumovirus M2-1 proteins showed the highest conservation in the amino-terminal half of the protein (Collins et al, 1990; Zamora et al, 1992; Ahmandian et al, 1999), consistent with the observation that MPV shows 100% conservation of similarity to APV-C in the first 80 aa residues of the protein (FIG. 12A). The MPV M2-1 protein contains 3 cysteine residues located within the first 30 aa residues, conserved in all pneumoviruses. This concentration of cysteine is common in zinc binding proteins (Ahmadian et al, 1991; Cuesta et al, 2000).

The position of the second ORF (M2-2) that overlaps with the Pneumovirus M2-1ORF is conserved but not conserved in sequence, which ORF is believed to be involved in the control of the switch between viral RNA replication and transcription (Collins et al, 1985; Elango et al, 1985; Baybutt et al, 1987; Collins et al, 1990; Ling et al, 1992; Zamora et al, 1992; Alansari et al, 1994; Ahmadian et al, 1999; Berminham et al, 1999). For MPV, the M2-2 ORF starts at nucleotide 512 in the M2-1ORF (FIG. 7), which is the same starting position in APV-C. The M2-2 ORFs of APV-C and MPV were the same in length, with 71 aa residues (Table 5). Sequence comparison of the M2-2 ORF between MPV and APV-C (FIG. 12B) revealed 56% amino acid sequence homology, while MPV had only 26-27% amino acid sequence homology with APV-A and B (Table 6).

Another suitable Open Reading Frame (ORF) that can be used in phylogenetic analyses includes the ORF encoding the L protein. When the overall amino acid identity of the analyzed L protein with the L protein of isolate I-2614 is found to be at least 91%, preferably at least 95%, then the analyzed viral isolate comprises the preferred MPV isolate according to the present invention. Like other negative-strand viruses, the last ORF of the MPV genome is the RNA-dependent RNA polymerase component of the replication transcription complex. The L gene of MPV encodes a 2005 aa protein, which is 1 residue longer than the protein of APV-A (Table 5). MPV has an L protein that is 64% homologous to APV-A, 42-44% homologous to RSV, and about 13% homologous to other paramyxoviruses (Table 6). Poch et al (1989; 1990) identified 6 conserved domains within the L protein of non-segmented negative-strand RNA viruses, in which domain III contains 4 core polymerase motifs thought to be essential for polymerase function. These motifs (A, B, C and D) are well conserved in the MPV L protein: in motifs A, B and C: MPV has 100% similarity to all pneumoviruses, MPV 100% similarity to APV and 92% similarity to RSV in panel D. For the complete domain III (aa 625-847 in the L ORF), MPV was 83% identical to APV, 67-68% identical to RSV and 26-30% identical to other paramyxoviruses (FIG. 15). In addition to the polymerase motif, the L protein of the pneumovirus contains a consensus ATP-binding motif K (X)₂₁GEGAGN(X)₂₀K (Stec, 1991). The MPV L ORF contains a motif similar to APV, in which the distance of the intermediate residues is one residue less: k (x)₂₂GEGAGN(X)₁₉K。

More preferred suitable open reading frames (0RF) that can be used for phylogenetic analysis include ORFs encoding SH proteins. When the overall amino acid identity of the SH protein analyzed with the SH protein of isolate I-2614 is found to be at least 30%, preferably at least 50%, more preferably at least 75%, then the viral isolate analyzed comprises the preferred MPV isolate according to the present invention. The gene, located adjacent to M2 of MPV, encodes a 183 aa protein (FIG. 7). Analysis of the nucleotide sequence and its derived amino acid sequence showed no discernible homology to other RNA virus genes or gene products. The SH ORF for MPV is the longest SH ORF known to date (table 5). The composition of the aa residues of the SH ORF is quite similar to that of APV, RSV and PVM, with a high percentage of threonine and serine (serine/threonine content of MPV, APV, RSV a, RSV B, bvsv and PVM is 22%, 18%, 19%, 20.0%, 21% and 28%, respectively). The SH ORF of MPV contains 10 cysteine residues, while APV SH contains 16 cysteine residues. All pneumoviruses have a similar number of potential N-glycosylation sites (MPV 2, APV 1, RSV 2, bvsv 3, PVM 4).

The hydrophobic profiles of the MPV SH protein and SH of APV and RSV revealed similar structural features (FIG. 13B). The SH ORFs of APV and MPV have a hydrophilic N-terminus (aa1-30), a central hydrophobic domain (aa 30-53) that can serve as a potential transmembrane domain, a second hydrophobic domain around residue 160, and a hydrophilic C-terminus. In contrast, the SH of RSV appears to lack the C-terminal half of the APV and MPV ORFs. In the SH proteins of all pneumoviruses, the hydrophobic domain is adjoined by a plurality of basic amino acids, which are also present in the SH ORF of MPV (aa 29 and 54).

Another more preferred suitable Open Reading Frame (ORF) that can be used in phylogenetic analyses includes the ORF encoding the G protein. When the overall amino acid identity of the analyzed G protein to the G protein of isolate I-2614 is found to be at least 30%, preferably at least 50%, more preferably at least 75%, then the analyzed viral isolate comprises the preferred MPV isolate according to the present invention. The G ORF of MPV is located adjacent to the SH gene and encodes a 236 amino acid protein. Immediately after this ORF a second small ORF was found, possibly encoding 68 aa residues (position 6973-7179), but lacking the start codon. The third major ORF of 194 aa residues in a different reading frame (fragment 4, FIG. 7) overlaps with both ORFs, but also lacks the initiation codon (nucleotides 6416-7000). This major ORF is followed in frame by a fourth ORF (nt 7001-7198), possibly encoding 65 aa residues, but lacking the start codon. Finally, one possible 97 aa residue ORF (but lacking the start codon) was found in the third reading frame (nt 6444-6737, FIG. 1). Unlike the first ORF, the other ORFs do not have an apparent gene start sequence or gene stop sequence (see below). Although the 236 aa-residue G ORF may represent at least a part of an MPV adsorption protein, it cannot be excluded: the additional coding sequences are expressed by certain RNA editing events as individual proteins or as part of the adsorbed protein. It should be noted that for both APV and RSV, no second ORF was identified after the primary G ORF, but both APV and RSV have a second ORF within the primary ORF of G. However, there is a lack of evidence for expression of these ORFs and there is no homology between predicted amino acid sequences of different viruses (Ling et al, 1992). The second ORF in MPV G did not show the features of the other G proteins and whether the additional ORFs were expressed or not was to be investigated further. BLAST analysis of all 4 ORFs showed no discernable homology at the nucleotide or amino acid sequence level with other known viral genes or gene products. This is consistent with the finding of low sequence homology to other G proteins such as the G proteins of hRSV A and B (53%) (Johnson et al, 1987) and to the G proteins of APV A and B (38%) (Juhasz et al, 1994). Whereas most of the MPVORF are similar in length and sequence to the ORF of APV, the G ORF of MPV is much smaller than that of APV (Table 5). The amino acid sequence showed a serine and threonine content of 34%, which is even higher than 32% of RSV and 24% of APV. The G ORF also contains 8.5% proline residues, which is greater than 8% for RSV and 7% for APV. The unusual abundance of proline residues in the G proteins of APV, RSV and MPV is also observed in glycoproteins of mucin origin, where this is a major consideration of the three-dimensional structure of proteins (Collins et al, 1983; Wertz et al, 1985; Jentoft, 1990). The number of potential N-linked glycosylation sites in MPV G is similar to that of other pneumoviruses: there were 5 MPVs, 7 hRSV, 5 bRSV and 3-5 APVs.

The predicted hydrophobicity profile of MPV G revealed similar characteristics to other pneumoviruses. The amino terminus contains a hydrophilic region followed by a short hydrophobic region (aa 33-53) and a predominantly hydrophilic carboxy terminus (FIG. 14B). This overall organization is consistent with anchoring type II transmembrane proteins, corresponding well to these regions in the G proteins of APV and RSV. The G ORF of MPV contains only 1 cysteine residue, in contrast to RSV and APV (5 and 20, respectively).

According to classical serological analyses, for example those obtained from: francki, r.i.b., Fauquet, c.m., Knudson, D.L and Brown, f., Classification and nomenclature of viruses. file report of the international Committee on taxomonom of viruses. arch Virol, 1991.Supplement 2: p.140-144, MPV isolates can also be identified as belonging to the serotypes provided herein, based on immunological differences (immunological differentiation) determined by quantitative neutralization with animal antisera (antisera from, for example, ferrets or guinea pigs, provided in the detailed description). Such a serotype either does not cross-react with other serotypes or exhibits a ratio of homologous to heterologous titres in both directions of greater than 16. If neutralization indicates a degree of cross-reactivity of the two viruses in either or both directions (ratio of homologous to heterologous titres of 8 or 16), serotype differentiation is presumed if there is a significant biophysical/biochemical difference in the DNA. If neutralization indicates that the two viruses cross-react to different extents in either or both directions (the ratio of the homologous to heterologous titres is less than 8), it is assumed that the serotypes of the isolate under study are identical. As noted, a useful prototype isolate is provided herein, such as isolate I-2614, also referred to herein as MPV isolate 00-1.

Viruses can be further classified into the isolated substantially mammalian negative-sense single-stranded RNA viruses provided herein based on homology of the G and/or SH proteins. In the case where there is 64-88% overall amino acid sequence identity between the N, P, M, F, M2 and L ORFs of a typical APV (isolated from birds) and MPV (isolated from humans), and where nucleotide sequence homology is also found between the non-coding regions of the APV and MPV genomes, it was found that there is substantially no discernable amino acid sequence homology between two of the ORFs of the human isolate (MPV) and any of the ORFs of other paramyxoviruses. The amino acid content, hydrophobicity profile and position of these ORFs in the viral genome indicate that they represent G and SH protein analogs. Sequence homology between APV and MPV, their similar genomic organization (3 '-N-P-M-F-M2-SH-G-L-5'), and phylogenetic analyses provide further evidence that the proposed MPV is classified as the first mammalian metapneumovirus. Thus, a new MPV isolate can be identified, for example, by: by virus isolation and characterization with tMK or other cells, by RT-PCR and/or sequence analysis followed by phylogenetic tree analysis, by serological techniques such as virus neutralization assays, indirect immunofluorescent assays, direct immunofluorescent assays, FACs analysis, or other immunological techniques. Preferably these techniques are directed to SH and/or G protein analogues.

For example, the present invention provides herein a method for identifying other isolates of an MPV as provided herein, said method comprising inoculating guinea pigs or ferrets which are substantially not infected with an MPV or which are free of specific pathogens (in the detailed description, the animals are inoculated intranasally, although other modes of inoculation such as intramuscular or intradermal inoculation and the use of other laboratory animals are also possible) with prototype isolate I-2614 or a related isolate. Serum was collected from the animals on day 0, 2 weeks and 3 weeks after inoculation. In the immunological detection of the other isolates, animals specifically seroconverted and sera obtained from the seroconverted animals were determined using a Virus Neutralization (VN) assay and an indirect IFA assay against the corresponding isolate I-2614.

As an example, the present invention provides characterization of a new member of the paramyxoviridae family-human metapneumovirus or metapneumovirus-like virus that may cause severe stage RTI in humans (the MPV is described herein, for example, as corresponding to APV taxonomically (MPV) due to its ultimate taxonomy to be discussed by the virus classification committee). Clinical signs of disease caused by MPV are substantially similar to those caused by hRSV, e.g., cough, myalgia, vomiting, fever, bronchitis or pneumonia, possible conjunctivitis or complications thereof. Hospitalization may be required as observed in hRSV-infected children, particularly very young children. As an example, MPVs deposited as I-2614 at the Institute of Pasteur Paris CNCM in Paris, Institute Pasteur, 2001, on 19.1.2001, or virus isolates phylogenetically corresponding thereto, are provided. Furthermore, the invention provides a virus comprising a nucleic acid or functional fragment corresponding phylogenetically to the nucleic acid sequence shown in fig. 6a, 6b, 6c or corresponding structurally thereto. In particular, the present invention provides a virus characterized in that, after testing in a phylogenetic evolutionary tree analysis in which maximum likelihood evolutionary trees were generated with 100 leads and 3 jumbles, it was found to phylogenetically correspond more closely to the viral isolate deposited in paris CNCM with I-2614 than to the viral isolate corresponding to Avian Pneumovirus (APV), also known as turkey rhinotracheitis virus (TRTV), the etiological agent of avian rhinotracheitis. It is particularly useful to use APV-C virus isolates, which are most related, although essentially non-mammalian viruses, as the foreign group in the phylogenetic tree analysis.

Based on several observations, we propose that a novel human virus is named human metapneumvirus or metapneumvirus-like virus (MPV). EM analysis revealed paramyxovirus-like particles. Consistent with the classification, MPV appears to be sensitive to chloroform treatment. MPV is best suited for culture on tMK cells and is tyrosine dependent. Clinical symptoms caused by MPV, as well as typical CPE and lack of hemagglutination activity suggest that this virus is most associated with hRSV. Although most paramyxoviruses have hemagglutinating activity, most pneumoviruses do not have such activity¹³。

As an example, the present invention provides paramyxoviruses that have not been previously identified from nasopharyngeal aspirate samples taken from 28 children with severe RTI. The clinical symptoms of these children were largely similar to those caused by hRSV. Of the patients 27 were children under 5 years of age, half of which were 1-12 months old. Another patient was 18 years old. All individuals suffer from upper respiratory illness with symptoms ranging from cough, myalgia, vomiting and fever to bronchitis and severe pneumonia. Most of these patients are hospitalized for 1-2 weeks.

Viral isolates from these patients had the morphology of paramyxovirus under negative contrast electron microscopy, but did not react with specific antisera against known human and animal paramyxoviruses. They are all closely related by an indirect immunofluorescence assay (IFA) assay using sera raised against 2 of the isolates. Sequence analysis of 9 of these isolates showed that the virus was slightly associated with APV. Based on virological data, sequence homology and genome organization, we propose that the virus is a member of Metapneumovirus. Serological investigations have shown that this virus is a relatively common pathogen, since sero-positivity in the netherlands approaches 100% of the population aged 5 years. In addition, the seropositivity rate was found to be equally high in sera collected from humans in 1958, indicating that this virus has circulated in the population for more than 40 years. The identification of this proposed novel member of Metapneumovirus also allows for the development of tools and methods for diagnostic assays or kits and vaccines, or serum or antibody compositions against viral respiratory infections, and methods for testing or screening antiviral agents useful in the treatment of MPV infections.

In this regard, the invention provides, among other things, isolated or recombinant nucleic acids or virus-specific functional fragments thereof obtainable from the viruses of the invention. In particular, the invention provides primers and/or probes suitable for identifying MPV nucleic acids.

Furthermore, the present invention provides a vector comprising a nucleic acid of the invention. First, a vector, such as a plasmid vector containing (part of) the MPV genome, a viral vector containing (part of) the MPV genome (such as, but not limited to, other paramyxoviruses, vaccinia viruses, retroviruses, baculoviruses), or an MPV containing (part of) the genome of other viruses or other pathogens is provided. In addition, various reverse genetics techniques are described for generating recombinant negative-strand viruses based on two clinical parameters. First, the production of such viruses relies on the replication of partial or full-length copies of the negative-sense viral rna (vrna) genome, or their complementary copies (cRNA). Such vRNA or cRNA may be isolated from infectious virus produced by in vitro transcription or produced in cells upon transfection with nucleic acids. Second, the production of recombinant minus-strand viruses is dependent on a functional polymerase complex. Typically, the polymerase complex of pneumovirus consists of N, P, L and possibly M2 protein, but is not necessarily limited thereto. The polymerase complex or components thereof may be isolated from viral particles, cells expressing one or more of the components, or produced by transfection with a specific expression vector.

When the vRNA, cRNA or a vector expressing these RNAs is replicated by the polymerase complex^{16，17，18，19，20，21，22}Infectious copies of MPV can be obtained. In order to generate small replicons (minireplicons) or reverse genetics systems for generating full-length copies containing most or the entire MPV genome, it is sufficient to use nucleic acid sequences available, for example, from APV (Randhawa et al, 1997) or from the 3 'and/or 5' end of the MPV itself.

In addition, the invention provides a host cell comprising a nucleic acid or vector of the invention. Plasmid or viral vectors containing the MPV polymerase components (presumably N, P, L and M2, but not necessarily limited thereto) are produced in prokaryotic cells for expression of the components in relevant cell types (bacteria, insect cells, eukaryotic cells). Plasmids or viral vectors containing full-length copies or partial copies of the MPV genome will be produced in prokaryotic cells for expression of viral nucleic acids in vitro or in vivo. The latter vectors may contain additional viral sequences for the production of chimeric viruses or chimeric viral proteins, may lack portions of the viral genome in order to produce replication-defective viruses, and may contain certain mutations, deletions or insertions in order to produce attenuated viruses.

Infectious copies (wild-type, attenuated, replication-defective or chimeric) of MPV may be produced by co-expressing the polymerase components according to the prior art described above.

In addition, eukaryotic cells that transiently or stably express one or more full-length or partial MPV proteins may be used. Such cells may be prepared by transfection (protein or nucleic acid vectors), infection (viral vectors) or transduction (viral vectors) and may be used for complementation of wild-type, attenuated, replication-defective or chimeric viruses as described above.

Chimeric viruses may be particularly useful for generating recombinant vaccines against two or more viruses^23，24，26. For example, it is envisaged that an MPV viral vector expressing one or more proteins of RSV or an RSV vector expressing one or more proteins of MPV will protect an individual vaccinated with such vectors against infection by both viruses. Similar approaches can be envisaged for PI3 or other paramyxoviruses. Attenuated and replication-defective viruses may be useful for the purpose of vaccination with live vaccines, as suggested for other viruses^25，26。

In a preferred embodiment, the invention provides a proteinaceous molecule or a metapneumvirus specific viral protein or a functional fragment thereof encoded by a nucleic acid of the invention. Useful proteinaceous molecules are for example derived from any gene or genomic fragment obtainable from the virus of the invention. Such molecules, or antigenic fragments thereof, provided herein are useful, for example, in diagnostic methods or kits and in pharmaceutical compositions, such as subunit vaccines. Particularly useful are F, SH and/or G proteins or antigenic fragments thereof for inclusion as antigens or subunit immunogens, although inactivated whole viruses may also be used. Particularly useful are also proteinaceous substances encoded by the identified recombinant nucleic acid fragments for use in phylogenetic analyses, although those within the preferred ORFs useful in phylogenetic analyses are of course preferred, particularly for eliciting MPV-specific antibodies, whether in vivo (e.g.for protection purposes or for providing diagnostic antibodies) or in vitro (e.g.by phage display techniques or another technique useful for producing synthetic antibodies).

Also provided herein are antibodies specifically reactive with an antigen comprising a proteinaceous molecule of the invention or an MPV-specific functional fragment thereof, said antibodies being either natural polyclonal or monoclonal antibodies or synthetic (e.g. a (phage) library-derived binding molecule) antibodies. Such antibodies are useful in methods for identifying a viral isolate as an MPV, comprising reacting the viral isolate or a component thereof with an antibody provided herein. This can be achieved, for example, by using purified or unpurified MPV or parts thereof (proteins, peptides), using ELISA, RIA, FACS or similar forms of antigen detection assays (Current protocols in Immunology). Alternatively, viral antigens can be identified using infected cells or cell cultures using classical immunofluorescence or immunohistochemical techniques.

Other methods for identifying a viral isolate as an MPV include reacting the viral isolate or a component thereof with a virus-specific nucleic acid of the invention, particularly where the mammalian virus includes a human virus.

Thus, the present invention provides a viral isolate that can be used in the methods of the invention to identify a mammalian virus that taxonomically corresponds to a negative-sense single-stranded RNA virus identifiable as being likely to belong to Metapneumovirus in the Pneumovirinae of the Paramyxoviridae family.

The method may be used in a method of virologically diagnosing MPV infection in a mammal, e.g.comprising determining the presence of a viral isolate or a component thereof in a sample of said mammal by reacting said sample with a nucleic acid or an antibody of the invention. Examples are further given in the detailed description, for example using PCR (or other amplification or hybridization techniques well known in the art) or using immunofluorescence detection (or other immunological techniques known in the art).

The invention also provides a method of serologically diagnosing MPV infection in a mammal, said method comprising determining the presence of antibodies specific for MPV or a component thereof in a sample of said mammal by reacting said sample with a proteinaceous molecule, or fragment or antigen thereof, according to the invention.

The methods and tools provided herein are particularly useful in diagnostic kits for diagnosing MPV infection by virological or serological diagnosis. Such kits or assays may, for example, comprise a virus, nucleic acid, proteinaceous molecule or fragment thereof, antigen and/or antibody of the invention. Also provided is the use of a virus, nucleic acid, proteinaceous molecule or fragment thereof, antigen and/or antibody of the invention in the manufacture of a pharmaceutical composition, for example for the treatment or prevention of MPV infection and/or for the treatment or prevention of a respiratory disease, particularly a human disease. Attenuation of the virus can be achieved by developing established methods for this purpose including, but not limited to, use of related viruses from other species, serial passage through experimental animals and/or tissue/cell cultures, site-directed mutagenesis of molecular clones, and exchange of genes or gene fragments between related viruses.

Pharmaceutical compositions comprising a virus, nucleic acid, proteinaceous molecule or fragment thereof, antigen and/or antibody of the invention may, for example, be used in a method of treating or preventing MPV infection and/or respiratory disease, the method comprising providing to an individual a pharmaceutical composition of the invention. This is most useful when the individual comprises a human, especially when the human is less than 5 years old, since such infants and young children are most likely to be infected by the human MPV provided herein. Generally, in the acute phase, the patient will suffer from other respiratory diseases and other disease predisposing upper respiratory symptoms. A variety of other lower respiratory tract diseases may also occur which are predisposed to serious disease.

The invention also provides a method of obtaining an antiviral agent useful in the treatment of a respiratory disease, said method comprising establishing a cell culture or experimental animal comprising a virus of the invention, treating said culture or treating said animal with a candidate antiviral agent, and determining the effect of said agent on said virus or its infection of said culture or animal. One example of such an antiviral agent includes the MPV neutralizing antibodies or functional components thereof provided herein, but antiviral agents of other properties are also available. The invention also provides the use of an antiviral agent of the invention in the manufacture of a pharmaceutical composition, in particular a pharmaceutical composition for the treatment of a respiratory disease, especially when the respiratory disease is caused by MPV infection; and to provide a pharmaceutical composition comprising the antiviral agent of the present invention, which is useful in a method for treating or preventing MPV infection or a respiratory disease, the method comprising providing such a pharmaceutical composition to an individual.

The present invention is further explained in the detailed description, but the present invention is not limited thereto.

Description of the drawings

FIG. 1A is Table 1: the percentage homology found between isolate 00-1 and the amino acid sequences of other members of the Pneumovirinae subfamily. The percentage (. times.100) of the amino acid sequences of the two RAP-PCR fragments (8 and 9/10) in N, P, M, F and L is given. The accession numbers used for the analysis are described in the materials and methods section.

FIG. 1B is Table 2; seropositivity of MPV determined using immunofluorescence and virus neutralization assays in populations classified according to age group.

FIG. 2: graphical representation of the location and size of fragments obtained on viral isolate 00-1 using RAP-PCR and RT-PCR in the APV genome. Fragments 1-10 were obtained using RAP-PCR. Fragment A primers for one of RAP-PCR fragments 1 and 2 and based on APV and RSV leader and trailer sequences⁶And (3) aligning the designed primers. Fragment B was obtained using primers designed in RAP-PCR fragments 1 and 2 and RAP-PCR fragment 3. Fragment C was obtained using primers designed in RAP-PCR fragment 3 and RAP-PCR fragments 4, 5,6 and 7.

For all phylogenetic evolutionary trees, (FIGS. 3-5)The DNA sequences were aligned using the ClustalW package and the maximum likelihood evolutionary Tree was generated using the DNA-ML package using 100 bootstrap programs and 3 jumble Phylip3.5¹⁵. Previously published sequences for generating phylogenetic trees are available from Genbank under accession numbers: for all ORFs: hRSV: NC 001781; bRSV: NC 001989; for the F ORF: PVM, D11128; APV-A, D00850; APV-B, Y14292; APV-C, AF 187152; for the N ORF: PVM, D10331; APV-A, U39295; APV-B, U39296; APV-C, AF 176590; for the M ORF: PMV, U66893; APV-A, X58639; APV-B, U37586; apv.c, AF 262571; for the P ORF: PVM, 09649; APV-A, U22110, APV-C, AF 176591. Phylogenetic analyses were performed on 9 different MPV virus isolates, using the APV C strain as the outer group.

Abbreviations used in the figures: hRSV: human RSV; bRSV: bovine RSV; PVM: mouse pneumovirus; APV-A, B and C: avian pneumovirus A, B and type C.

FIG. 3: comparison of N, P, M and F ORFs for members of the Pneumovirinae subfamily and viral isolate 00-1. The sequence alignment shows the amino acid sequences of the complete N, P, M and F and partial L proteins of viral isolate 00-1. The amino acids that differ between isolate 00-1 and other viruses are shown, with identical amino acids indicated by periods and gaps indicated by dashes. Numbering corresponds to amino acid positions in the protein. The accession numbers used for the analysis are described in the materials and methods section. APV-A, B or C: A. avian pneumovirus type B or C, bRSV or hRSV: bovine or human respiratory syncytial virus, PVM: mouse pneumovirus. L8: fragment 8 in L obtained with RAP-PCR, L9/10 consensus sequence of fragments 9 and 10 in L obtained with RAP-PCR. For P sequence alignment, APV-B sequence was not available from Genebank. For the L sequence alignment, only sequences for bRSV, hRSV, and APV-A are available.

FIG. 4: phylogenetic analysis of N, P, M and F ORFs from members of the genus Pneumovirinae (Pneumovirinae) and viral isolate 00-1. Phylogenetic analyses were performed with viral sequences from the following genes: f (panel A), N (panel B), M (panel C) and P (panel D). The phylogenetic evolutionary tree is based on maximum likelihood analysis using 100 bootstrap programs and 3 jumbles. A scale representing the number of nucleotide changes per evolutionary tree is shown.

FIG. 5: phylogenetic relationship of 9 primary MPV isolates and the portions of the F (Panels, N (Panels B), M (Panels C) and L (Panels D) ORFs) of the APV-C that are genetically most relevant.

FIG. 6A: nucleotide sequence and amino acid sequence information from the 3' end of the MPV isolate 00-1 genome. The following ORFs are given: n: the ORF of the nucleoprotein; p: 0RF of phosphoprotein; ORF of M-bimatrin; f: the ORF of the fusion protein; GE: ending the gene; GS: gene initiation.

Fig. 6B and C: nucleotide sequence and hydrogen base sequence information of the fragment in the polymerase gene (L) obtained from isolate VIII V, isolate 00-1. The localization of the fragment in L is based on protein homology to APV-C (accession number U65312). The translated fragment 8 (FIG. 6B) is located at amino acid numbers 8-243 and the consensus sequence of fragments 9 and 10 (FIG. 6C) is located at amino acid number 1358-1464 of the APV-C L ORF.

FIG. 7

Genomic map of MPV isolate 00-1. Under each 0RF, the nucleotide positions of the start and stop codons are indicated. The double line of L0 RF crossing indicates shortening indicating the L gene. The three reading frames below the map indicate the first G ORF (nt 6262-6972) and the possible second ORF that overlaps.

FIG. 8:

sequence alignment of the nucleoprotein of MPV with the predicted amino acid sequences of said proteins of other pneumoviruses. The conserved regions identified by Barr (1991) are indicated in boxes and labeled A, B and C. Conserved regions in pneumovirus (Li, 1996) are shown in grey shading. Gaps are indicated by dashes and periods indicate the position of the same amino acid residue as compared to MPV.

FIG. 9:

the phosphoprotein of MPV is compared to the amino acid sequences of said proteins of other pneumoviruses. Regions of high similarity are boxed (Ling, 1995), and glutamate-rich regions are shaded in grey. Gaps are indicated by dashes and periods indicate the position of the same amino acid residue as compared to MPV.

FIG. 10:

comparison of the deduced amino acid sequences of the matrix proteins of MPV with said proteins of other pneumoviruses. The conserved hexapeptide sequence (Easton, 1997) is shaded in grey. Gaps are indicated by dashes and periods indicate the position of the same amino acid residue as compared to MPV.

FIG. 11:

alignment of the fusion protein of MPV with the predicted amino acid sequences of said proteins of other pneumoviruses. Conserved cysteine residues are boxed, the N-linked glycosylation site is underlined, the cleavage site of F0 is double underlined, and the fusion peptide, signal peptide, and membrane anchoring domain are shaded in grey. Gaps are indicated by dashes and periods indicate the position of the same amino acid compared to MPV.

FIG. 12

Comparison of the amino acid sequence of the M2 ORF of MPV with that of other pneumoviruses. The sequence alignment of the M2-1ORF is shown in Panel A, with the conserved amino terminus (Collins, 1990; Zamora, 1999) shown in gray shading. The three conserved cysteine residues are printed in bold and indicated with a #. The sequence alignment of the M2-2 ORF is shown in Panel B. Gaps are indicated by dashes and periods indicate the position of the same amino acid compared to MPV.

FIG. 13

Amino acid sequence analysis of MPV SH ORF. (A) The amino acid sequence of the SH ORF of MPV, serine and threonine residues are shaded in grey, cysteine residues are in bold, and the hydrophobic region is double underlined. Potential N-linked glycosylation sites are indicated by single underlining. The numbers indicate the position of the basic amino acid adjacent to the hydrophobic domain. (B) Comparison of the hydrophobicity profiles of SH proteins of MPV, APV-A and hRSV-B. The method of Kyte and Doolittle (1982) and a 17 amino acid window were used. The arrows represent the strongly hydrophobic domains. The positions within the ORF are given on the X-axis.

FIG. 14

Amino acid sequence analysis of MPV G ORF. (A) The amino acid sequence of G0 RF of MPV, serine, threonine and proline residues are shaded in grey, cysteine residues are in bold, and the hydrophobic region is double underlined. Potential N-linked glycosylation sites are indicated by single underlining. (B) Comparison of hydrophobicity plots for G proteins of MPV, APV-A and hRSV-B. The method of Kyte and Doolittle (1982) and a 17 amino acid window were used. Arrows indicate the hydrophobic regions. The positions within the ORF are given on the X-axis.

FIG. 15 shows a schematic view of a

Comparison of the amino acid sequences of the conserved domains in the polymerase genes of MPV and other paramyxoviruses. Domain III is shown, with 4 conserved polymerase motifs (a, B, C, D) in domain III in frame (Poch 1998, 1999). Gaps are indicated by dashes and periods indicate the position of the same amino acid residue as compared to MPV. hPIV 3: human parainfluenza virus type 3; SV; sendai virus; hPIV-2: human parainfluenza virus type 2; NDV: newcastle disease virus; MV: measles virus; nipah: nipah virus.

FIG. 16:

phylogenetic analysis of M2-1 and L ORFs of MPV and selected paramyxoviruses. The M2-1ORF was aligned with the M2-1ORF of other members of the Pneumovirinae (Pneumovirinae) genus (A), and the L ORF was aligned with the L ORF of members of the Pneumovirinae (Pneumovirinae) genus and other paramyxoviruses selected in the FIG. 15 graphic illustration (B). Phylogenetic evolutionary trees are generated using maximum likelihood analysis using 100 bootstrap programs and 3 jumbles. A scale representing the number of nucleotide changes per evolutionary tree is shown. The numbers in the evolutionary tree represent the values of the bootstrap of the evolutionary tree based on the consensus sequence.

FIG. 17:

non-coding sequence of hMPV isolate 00-1. (A) The non-coding sequences between the ORFs and at the ends of the genome are shown in sense orientation. From left to right, the stop codon for the indicated 0RF, followed by the non-coding sequence, the gene start signal and start codon for the indicated subsequent ORF are shown. The numbers indicate the position of the first start codon and stop codon in the hMPV map. Sequences showing similarity to published gene termination signals are underlined and sequences showing similarity to UAAAAAU/A/C are represented by lines on the sequences. (B) Nucleotide sequence of the end of the hMPV genome. The genomic ends of hMPV were aligned to each other and to the genomic ends of APV. The underlined regions represent primer sequences used in RT-PCR assays based on the 3 'and 5' sequences of APV and RSV (Randhawa et al, 1997; Mink et al, 1991). The nucleotides in bold italics are part of the initiation signal for the N gene. Le: leader sequence, Tr: a tail sequence.

FIG. 18:

comparison of the two prototype hMPV isolates with APV-A and APV-C; DNA similarity matrices for nucleic acids encoding various viral proteins.

FIG. 19:

comparison of the two prototype hMPV isolates with APV-A and APV-C; protein similarity matrices for various viral proteins.

FIG. 20:

amino acid sequence alignment of nucleoproteins of two prototype hMPV isolates

FIG. 21:

amino acid sequence alignment of phosphoproteins of two prototype hMPV isolates

FIG. 22:

amino acid sequence alignment of two prototype hMPV isolate matrix proteins

FIG. 23:

amino acid sequence alignment of two prototype hMPV isolate fusion proteins

FIG. 24:

amino acid sequence alignment of two prototype hMPV isolate M2-1 proteins

FIG. 25:

amino acid sequence alignment of two prototype hMPV isolates M2-2 proteins

FIG. 26:

amino acid sequence alignment of short hydrophobic proteins of two prototype hMPV isolates

FIG. 27 is a schematic view showing:

amino acid sequence alignment of glycoprotein adsorbed by two prototype hMPV isolates

FIG. 28:

alignment of amino acid sequences of polymerase protein N-terminals of two prototype hMPV isolates

FIG. 29: results of RT-PCR analysis of throat and nasal swabs in 12 guinea pigs vaccinated with ned/00/01 and/or ned/99/01.

FIG. 30A: IgG responses against ned/00/01 and ned/99/01 in guinea pigs infected with ned/00/01 and reinfected with either ned/00/01(GP 4, 5 and 6) or ned/99/01(GP 1 and 3).

FIG. 30B: IgG responses against ned/00/01 and ned/99/01 in guinea pigs infected with ned/99/01 and reinfected with either ned/00/01(GP 8 and 9) or ned/99/01(GP 10,11, 12).

FIG. 31: the ned/00/01 and ned/99/01 ELISAs were specific for sera taken from guinea pigs infected with either ned/00/01 or ned/99/01.

FIG. 32: average IgG responses to ned/00/01 and ned/99/01 ELISAs of 3 congeners (00-1/00-1), 2 congeners (99-1/99-1), 2 xenogeneic (99-1/00-1) and 2 xenogeneic (00-1/99-1) infected guinea pigs.

FIG. 33: average percentage of APV inhibition in hMPV infected guinea pigs.

FIG. 34: ned/00/01 and ned/99/01 infect guinea pigs against the virus neutralizing titers of ned/00/01, ned/99/01 and APV-C.

FIG. 35: RT-PCR assay results on throat swabs of rhesus monkeys inoculated with ned/00/01 (twice).

Fig. 36A (top two):

2 rhesus monkeys (re-) infected with ned/00/01 responded to IgA, IgM and IgG to ned/00/01.

FIG. 36B (lower view)

2 rhesus monkeys infected with ned/00/01 responded to APV with IgG.

FIG. 37: comparison of IgG antibodies in human serum using hMPV ELISA and APV inhibition ELISA.

Detailed Description

Isolation and characterization of viruses

From 1980 to 2000, we found 28 unidentified viral isolates isolated from patients with severe respiratory disease. These 28 unidentified virus isolates grew slowly in tMK cells, grew poorly in VERO cells and a549 cells, and did not or rarely propagate in MDCK or chicken embryo fibroblasts. Most of these virus isolates induced CPE between day 10 and day 14 after 3 passages on tMK cells. The CPE is virtually indistinguishable from CPE caused by hRSV or hPIV in tMK or other cell cultures, and is characterized by syncytia formation after which the cells exhibit rapid internal destruction before the cells are separated from the monolayer. The cells typically (and sometimes subsequently) show CPE slightly later than CPE caused by other viruses, such as hRSV or hPIV, after 3 passages of the virus from the starting material, at days 10-14 post inoculation.

We used supernatants of infected tMK cells for EM analysis, which indicated the presence of 150-600 nm paramyxovirus-like virus particles with short envelope processes of 13-17 nm. Consistent with the biochemical properties of enveloped viruses, such as Paramyxoviridae, standard chloroform treatment or ether treatment⁸Resulting in a reduction of infectivity of more than 10 for tMK cells⁴TCID 50. Virus infected tMK cell culture supernatants showed no hemagglutination activity on turkey, chicken and guinea pig erythrocytes. During culture, virus replication on the cells tested appears to be tyrosine dependent. These comprehensive virological data indicate that the newly identified viruses are taxonomically classified as members of the paramyxoviridae family.

We isolated RNA from tMK cells infected with 15 of the unidentified viral isolates, for use in Paramyxovirinae (Paramyxovirinae)⁹hPIV-4, Sendai virus, simian virus type 5, Newcastle disease virus, measles virus, hRSV mumps virus, Nipah virus, Hendra virus, Tupaia virus and Mapuera virus, and reverse transcription and polymerase chain reaction (RT-PCR) analysis. RT-PCR analysis was performed at low stringency to detect potentially related viruses, and RNA isolated from the same virus stocks was used as a control. The available control reacted positively with the corresponding virus-specific primer, and the newly identified isolate did not react with any primer set, indicating that the virus was not closely related to the virus tested.

We used two of the virus-infected tMK cell culture supernatants to inoculate guinea pigs and ferrets intranasally. Sera were collected from these animals on day 0, 2 weeks and 3 weeks after inoculation. The animals showed no clinical symptoms, but all animals developed seroconversion using the Virus Neutralization (VN) assay and indirect IFA assay against the homologous virus. The serum is non-reactive in indirect IFA with any of the known paramyxoviruses described above and with PVM. Next, we screened hitherto unidentified viral isolates with pre-and post-infection sera from guinea pigs and ferrets by performing an indirect IFA assay with post-infection sera, of which 28 were clearly positive, suggesting that they are serologically closely related or identical.

RAP PC R

To obtain sequence information on the unknown virus isolate, we used what we called RAP-PCR¹⁰Random PCR amplification strategy of (1). For this purpose, tMK cells were infected with one of the viral isolates (isolate 00-1) and hPIV-1 used as a control. After both cultures showed similar levels of CPE, the virus in the culture supernatant was purified on a continuous 20-60% sucrose gradient. The virus-like particles in each gradient fraction were examined by EM and RNA was isolated from the fraction containing about 50% sucrose, in which nucleocapsids were observed. RAP-PCR was performed with equal amounts of RNA isolated from both virus fractions, after which the samples were run side by side on a 3% NuSieve agarose gel. Subsequently, 20 bands which are specific for the virus yet unidentified and which show differences were purified from the gel, cloned in plasmid pCR2.1(Invitrogen) and sequenced with vector-specific primers. When we used these sequences to perform homology searches with BLAST software (www.ncbi.nlm.nih.gov/BLAST /) on sequences in the Genbank database, 10 of the 20 fragments showed similarity to APV/TRTV sequences.

These 10 fragments are located in the genes encoding the nuclear protein (N; fragments 1 and 2), the matrix protein (M; fragment 3), the fusion protein (F; fragments 4, 5,6, 7) and the polymerase protein (L; fragments 8, 9, 10) (FIG. 2). We next followed our RAP PCR fragments and published leader and trailer sequences of the Pneumovirinae subfamily⁶PCR primers were designed to complete the sequence information of the 3' end of the viral genome. Three fragments were amplified, with fragment a spanning the 3' most end of the N Open Reading Frame (ORF), fragment B spanning the phosphoprotein (P) ORF, and fragment C immediately adjacent to the gap between the M and F ORFs (fig. 2). Sequence analysis of these three fragments revealed that the NS1 and NS2 ORFs at the most 3' end of the viral genome were absent, and the F ORF was located immediately adjacent to the M ORF. This genome organization is similar to that of metapneumovirus APV, which is also identical to that of SEQ ID NOColumn homology is consistent. N, P, M and the F ORF show an average of 30-33% homology to Pneumovirus members and 66-68% homology to Metapneumovirus members. No discernable homology was found for the SH and G ORFs with any of the above members of the genus. The amino acid homology of N found indicates that there is about 40% homology with hRSV, 88% homology with apv.c, and the closest genetic relationship with APV-C, as can be derived, for example, by comparing the amino acid sequence of figure 3 with the amino acid sequences of the corresponding N proteins of other viruses. The amino acid sequence of P shows about 25% homology to hRSV, about 66-68% homology to APV-C, M shows about 36-39% homology to hRSV, about 87-88% homology to APV-C, F shows about 40% homology to hRSV, about 81% homology to APV-C, M2-1 shows about 34.36% homology to pneumovirus, 84-86% homology to APV-C, M2-2 shows 15-17% homology to pneumovirus, 56% homology to APV-C, and the fragment obtained in L shows an average of 44% homology to pneumovirus, 64% homology to APV-C.

Phylogenetic development

Although BLAST searches using nucleotide sequences from the unidentified viral isolates showed major homology to members of the pneumovirinae subfamily, homology based on protein sequences showed some similarity to other paramyxoviruses (data not shown). As an indication of the correlation between the newly identified viral isolates and members of the pneumovirinae subfamily, phylogenetic evolutionary trees were constructed based on the N, P, M and F ORFs of these viruses. The newly identified viral isolates were most closely related to APV in all 4 phylogenetic trees (fig. 4). 4 serotypes of APV according to what has been described¹¹APV serotype C, metapneumovirus found primarily in birds in the united states, appears to be most similar to the newly identified virus. It should be noted, however, that only partial sequence information is available for the APV D serotype.

To determine the relationship of our various newly identified viral isolates, we constructed phylogenetic evolutionary trees based on sequence information from 8-9 isolates (8 for F, 9 for N, M and L). To this end, we used RT-PCR and primers designed to amplify short fragments of the N, M, F and L ORFs, followed by direct sequencing of the fragments. It was also found that 9 viral isolates previously found to be serologically related (see above) were genetically closely related. In fact, all 9 isolates were more closely related to each other than to APV. Although the sequence information for these phylogenetic trees is limited, it appears that the 9 isolates can be divided into two groups, isolates 94-1, 99-1 and 99-2 into one group, while the other 6 isolates (94-2; 93-1; 93-2; 93-3; 93-4; 00-1) are divided into another group (FIG. 5).

Seropositive rate

To investigate the seropositivity of this virus in human populations, we tested sera from people of different ages by indirect IFA with tMK cells infected with one of the unidentified viral isolates. This analysis revealed that 25% of children at 6-12 months had antibodies to the virus, and by the age of 5, nearly 100% of children were seropositive. Among a total of 56 serum samples tested by indirect IFA, the test was performed with VN assay. For 51 of the samples (91%), the results of the VN assay (titres > 8) were consistent with those obtained with indirect IFA (titres > 32). 4 samples found positive in IFA were negative by VN test (titre < 8), while one serum reacted negative in IFA (titre < 32) but positive in VN test (titre 16) (Table 2).

72 parts of serum collected from human (8-99 years old) in 1958^12，27IFA performed revealed a 100% seroprevalence indicating that the virus has been circulating in the population for more than 40 years. Additionally, multiple sera from these sera were used in the VN assay to confirm IFA data (table 2).

N, M, P and F gene analysis revealed that iMPV has a higher sequence homology (on average 63%) to the recently proposed genus Metapneumovirus than to the genus Pneumovirinae (Pneumovirinae) (on average 30%), thus demonstrating a similar and analogous genome organization to that of an APV/TRTV. In contrast to the genomic organization of RSVs ('3-NS 1-NS2-N-P-M-SH-G-F-M2-L-5'), metapneumviruses lack the NS1 and NS2genes, and the location of the genes differs between M and L ('3-N-P-M-F-M2-SH-G-L-5'). The lack of ORF between M and F genes in our viral isolates, as well as the lack of adjacent N NS1 and NS2 and the high amino acid sequence homology found with APV, is the first mammalian (especially human-derived) member that suggests that MPV isolated from humans should be classified as Metapneumovirus.

Phylogenetic analysis revealed that the 9 MPV isolates from which sequence information was obtained were closely related. Although the sequence information is limited, they are actually more closely related to each other than to any avian metapneumovirus. Of the 4 serotypes of APV that have been described, serotype C is most closely related to MPV based on the N, P, M and F genes. It should be noted, however, that only a partial sequence of the F gene of serotype D is available from Genbank, and for serotype B, only M, N and F sequences are available. Our MPV isolates formed 2 taxa in the phylogenetic evolutionary tree. For both hRSV and APV, different gene subtypes and serological subtypes have been described. It is not known at present whether the two genetic taxa of an MPV isolate represent serological subgroups that are also functionally distinct. Our serological survey indicates that MPV is a common human pathogen. This virus was repeatedly isolated from clinical samples obtained from critically ill RTI children, suggesting that the clinical and economic impact of MPV may be high. A new diagnostic assay based on viral detection and serology would provide a more detailed analysis of the incidence of such viral pathogens and the clinical and economic impact of such viral pathogens.

The slight difference between the IFA results and VN results (5 samples) may be due to detection of only IgG serum antibodies in the IFA, whereas the VN assay detects both antibody classes and antibody subclasses, or the difference may be due to a difference in sensitivity between the two assays. For IFA, a cutoff of 16 is used, while VN uses a cutoff of 8.

On the other hand, differences between IFA and VN assays may also indicate possible differences between different serotypes of this newly identified virus. Since MPV appears to be most closely related to APV, we speculate that the human virus may originate from birds. Analysis of serum samples taken from humans in 1958 showed that MPV has spread widely in the human population for more than 40 years, suggesting that putative infectious animal disease events must have occurred well before 1958.

Materials and methods

Sample collection

During the past decades, our laboratory collected nasopharyngeal aspirates from children with RTI, which were routinely tested for the presence of virus. All nasopharyngeal aspirates were tested by direct immunofluorescence assay (DIF) using fluorescently labeled antibodies against influenza A and B viruses, hRSV and human parainfluenza virus (hPIV) types 1-3. The nasopharyngeal aspirates have also been treated for rapid shell visual technology¹⁴Viral isolation was performed on various cell lines including VERO cells, third generation rhesus monkey kidney (tMK) cells, human lung endothelial (HEL) cells, and marbin dock kidney (MDCK) cells. Samples that showed cytopathic effect (CPE) after 2-3 passages and were negative in DIF were tested by indirect immunofluorescence assay (IFA) with virus-specific antibodies against the following viruses: influenza viruses A, B and type C, hRSV A and B, measles virus, mumps virus, human parainfluenza virus (hPIV) types 1-4, Sendai virus, monkey virus type 5, and Newcastle disease virus. Although pathogens can be identified in many cases, some samples are negative for all of these viruses tested.

Direct immunofluorescence assay (DIF)

According to the method already described^14，15By naso-pharyngeal suction from RTI patientsSamples were taken for DIF and virus separation. Samples were stored at-70 ℃. Briefly, nasopharyngeal aspirates were diluted with 5ml of Dulbecco MEM (BioWhittaker, Walkersville, Md.) and mixed thoroughly on a vortex mixer for 1 minute. The suspension was centrifuged at 840 Xg for 10 min. The pellet was spread on a multi-spot (multispot) slide (Nutacon, Leimuiden, The Netherlands) and virus isolation was performed with The supernatant. After drying, the cells were fixed in acetone for 1 minute at room temperature. After washing, slides were incubated for 15 minutes at 37 ℃ with commercially available FITC-labeled virus-specific antisera, such as virus-specific antisera against influenza A virus and B, hRSV and hPIV 1-3 (Dako, Glostrup, Denmark). After 3 washes with PBS and 1 wash in tap water, slides were contained in glycerol/PBS solution (Citifluor, UKC, Canterbury, UK) and covered. Slides were analyzed with an Axioscop fluorescence microscope (Carl Zeiss B.V, Weesp, the Netherlands).

Virus isolation

For virus isolation, tMK cells (PIVM, Bilthoven, The Netherlands) were cultured in 24-well plates with slides (Costar, Cambridge, UK) with The following medium supplemented with 10% fetal bovine serum (BioWhittaker, Vervier, Belgium). Prior to inoculation, the plates were washed with PBS, then Eagle's MEM (ICN, Costamesa, Calif.) containing Hank's salt was added, and half liter was supplemented with 0.26 grams of NaHCO in the medium₃0.025M Hepes (Biowhittaker), 2mM L-glutamine (Biowhittaker), 100 units of penicillin, 100. mu.g streptomycin (Biowhittaker), 0.5 g whey protein (Sigma-A1drich, Zwijndrecht, The Netherlands), 1.0 g D-glucose (Merck, Amsterdam, The Netherlands), 5.0 g peptone (Oxoid, Haarlem, The Netherlands) and 0.02% tyrosine (Life Technologies, Bethesda, MD). The supernatant of the nasopharyngeal aspirate samples was inoculated in triplicate in the plates at 0.2ml per well and then centrifuged at 840 Xg for 1 hour. After inoculation, the plates were incubated at 37 ℃ for a maximum of 14 days, the medium was changed once a week and the cultures were checked daily for CPE. After 14 days, cells were scraped from the second subculture and then incubated for another 14 days. For theFor the third passage, the procedure was repeated. The presence of the virus was confirmed by indirect IFA using the slide as described below.

Animal immunization

Two ferrets and two guinea pigs, which were housed in separate pressurized glove boxes, were screened for experimental intranasal infection to generate ferret and guinea pig specific antisera against the newly discovered viruses. After 2-3 weeks, all animals were bled by cardiac puncture and their sera were used as reference sera. Indirect IFA was used to test all previously described viruses in the sera as described below.

Detection of antigens by indirect IFA

We performed indirect IFA on slides containing infected tMK cells. After washing with PBS, the slides were incubated with virus-specific antisera for 30 minutes at 37 ℃. We used monoclonal antibodies against influenza virus A, B and type C, hPIV 1-3 and hRSV in DIF as described above. For hPIV type 4, mumps virus, measles virus, sendai virus, simian virus type 5, newcastle disease virus, polyclonal antibodies (RIVM) and ferret and guinea pig reference sera were used. After 3 washes with PBS and 1 wash with tap water, slides were stained with secondary antibody against the serum used in the first incubation. Secondary antibodies to the polyclonal antiserum were goat anti-ferret (KPL, Guilford, UK, 40 fold dilution), mouse anti-rabbit (Dako, Glostrup, Denmark, 20 fold dilution), rabbit anti-chicken (KPL, 20 fold dilution) and mouse anti-guinea pig (Dako, 20 fold dilution). Slides were processed as described for DIF.

Detection of human antibodies by indirect IFA

To detect virus-specific antibodies, infected tMK cells were fixed on coverslips with cold acetone, washed with PBS, and then stained with 1-16 dilutions of serum samples. Subsequently, the samples were stained with FITC-labeled rabbit anti-human antibody (Dako) diluted 80-fold in PBS. Slides were processed as described above.

Viral cultures of MPV

Supernatants of samples showing CPE after 2-3 passages in 24-well plates were seeded in a sub-confluent monolayer of tMK cells in the above medium. Cultures were checked daily for CPE and media was changed once a week. Since CPE varies from isolate to isolate, all cultures were tested using indirect IFA on days 12-14 using ferret antibodies against the new viral isolate. The positive cultures were freeze-thawed 3 times, after which the supernatant was clarified by low speed centrifugation, divided into aliquots and frozen at-70 ℃. According to the method already described¹⁶The 50% tissue culture infectious dose of virus in the culture supernatant (TCID50) was determined.

Virus neutralization assay

VN assays were performed using 2-fold serial dilutions of human and animal sera starting at 8-fold dilutions. The diluted sera were incubated with 100 TCID50 virus for 1 hour, then seeded into tMK cells grown in 96-well plates, after which the plates were centrifuged at 840 × g. Media was changed after 3 and 6 days and IFA was performed 8 days after inoculation with ferret antibodies against MPV. VN titres were defined as the lowest dilution of serum samples that resulted in negative IFA and inhibition of CPE in cell culture.

Virus characterization

According to the method already described^8，14Hemagglutination assays and chloroform sensitivity tests were performed. For EM analysis, virus was concentrated from infected cell culture supernatants at 17000 Xg at 4 ℃ in a microcentrifuge, after which the pellet was resuspended in PBS and examined with negative contrast EM. For RAP-PCR, the virus was concentrated from infected tMK cell supernatants by ultracentrifugation (150000 Xg for 2 hours, 4 ℃) on a 60% sucrose cushion (session). The 60% sucrose intermediate phase was then diluted with PBS and then plated on top of a 20-60% continuous sucrose gradient and centrifuged at 275000 Xg for 16 hours at 4 ℃. Electrophoresis on EM and polyacrylamide gels followed by silver staining to check for sucroseGradient presence of virus like particles in each fraction. It appears to contain a nucleocapsid fraction of about 50% sucrose for RNA isolation and RAP-PCR.

RNA isolation

RNA was isolated from The supernatant or sucrose gradient fraction of infected cell cultures using The High Pure RNA Isolation kit (High purity RNA Isolation kit) according to The manufacturer's instructions (Roche Diagnostics, Almee, The Netherlands).

RT-PCR

The virus-specific oligonucleotide sequences for RT-PCR on known paramyxoviruses are described in appendix 1. In a medium containing 50mM Tris.HCl pH 8.5, 50mN NaCl, 4mM MgCl₂2mM dithiothreitol, dNTP 200. mu.M each, 10 units of recombinant RNAsin (Promega, Leiden, The Netherlands), 10 units of AMV RT (Promega, Leiden, The Netherlands), 5 units of Amplitaq Gold DNA polymerase (PE Biosystems, Nieuwerkerk aan de Ijssel, The Netherlands) and 5. mu.l of RNA in 50. mu.l reactions, a one-step RT-PCR was carried out. The cycling conditions were 42 ℃ for 45 min and 95 ℃ for 7min once, repeated 40 times at 95 ℃ for 1min, 42 ℃ for 2min and 72 ℃ for 3min, and once at 72 ℃ for 10 min. RAP-PCR

RAP-PCR essentially as described¹⁰The process is carried out. The oligonucleotide sequences are described in appendix 2. For the RT reaction, the reaction was carried out in a reaction system containing 10 ng/. mu.l oligonucleotide, 10mM dithiothreitol, dNTPs each 500. mu.m, 25 mM Tris-HCl pH 8.3, 75 mM KCl and 3mM MgCl₂The 10. mu.l reaction contained 2. mu.l of RNA. The reaction mixture was incubated at 70 ℃ for 5 minutes and at 37 ℃ for 5 minutes, followed by the addition of 200 units of Superscript RT enzyme (Life Technologies). Incubation was continued at 37 ℃ for 55 minutes and stopped by incubation at 72 ℃ for 5 minutes. The RT mixture was diluted to give a mixture containing 8 ng/. mu.l oligonucleotide, dNTPs each 300. mu.m, 15mM Tris-HCl pH 8.3, 65 mM KCl, 3.0 mM MgCL₂And 5 units of Taq DNA polymerase (PE Biosystems) in 50. mu.l. The circulation condition is 94 ℃ for 5minOnce at 40 ℃ for 5min and once at 72 ℃ for 1min, then repeated 40 times at 94 ℃ for 1min, at 56 ℃ for 2min and at 72 ℃ for 1min, and then once at 72 ℃ for 5min. After RAP-PCR, 15. mu.l each of The RT-PCR products were electrophoresed side by side on 3% NuSieve agarose gel (FMC Bioproducts, Heerhugowaard, The Netherlands). Fragments which differentially showed specificity for MPV were purified from The Gel using The Qiaquick Gel extraction kit (Qiagen, Leusden, The Netherlands) and cloned into The pCR2.1 vector (Invitrogen, Groningen, The Netherlands) according to The manufacturer's instructions.

Sequence analysis

The RAP-PCR product cloned in the vector pCR2.1(Invitrogen) was sequenced with M13-specific oligonucleotides. The DNA fragment obtained by RT-PCR was purified from agarose Gel using The Qiaquick Gel Extraction Kit (Qiagen, Leusden, The Netherlands) and directly sequenced using The same oligonucleotides used for PCR. Sequence analysis was performed using a Dynamic ETtermistering kit (Dynamic ET stop sequencing kit) (Amersham pharmacia Biotech, Rososendaal, The Netherlands) and ABI 373 automated DNA sequencer (PE biosystems). All techniques were performed according to the manufacturer's instructions.

Generation of genomic fragments of MPV by RT-PCR

To generate PCR fragments spanning gaps A, B and C (FIG. 2) between RAP-PCR fragments, we applied the RT-PCR assay described above on RNA isolated from viral isolate 00-1. The following primers were used:

for fragment a: design TR1 located in the leader sequence: (5'-AAAGAATTCACGAGAAAAAAACGC-3') and N1 (5'-CTGTGGTCTCTAGTCCCACTTC-3') designed to be located at the 3' end of the RAP-PCR fragment in the obtained N.

For fragment B: design N2 located at the 5' end of the RAP-PCR fragment in the obtained N: (5'-CATGCAAGCTTATGGGGC-3') and M1 designed to locate at the 3' end of the RAP-PCR fragment in the obtained M: (5'-CAGAGTGGTTATTGTCAGGGT-3').

For fragment C: design M2 located at the 5' end of RAP-PCR fragment in the obtained M: (5'-GTAGAACTAGGAGCATATG-3') and F1 designed to locate at the 3' end of the RAP-PCR fragment in the obtained F: (5'-TCCCCAATGTAGATACTGCTTC-3').

The fragments were purified from the gel, cloned and sequenced as described above.

RT-PCR for the diagnosis of MPV

To amplify and sequence the N, M, F and LORF fractions of 9 strains of the MPV isolate, we used primers N3 (5'-GCACTCAAGAGATACCCTAG-3') and N4 (5'-AGACTTTCTGCTTTGCTGCCTG-3') to amplify a 151 nucleotide fragment, M3 (5'-CCCTGACAATAACCACTCTG-3') and M4 (5'-GCCAACTGATTTGGCTGAGCTC-3') to amplify a 252 nucleotide fragment, F7 (5'-TGCACTATCTCCTCTTGGGGCTTTG-3') and F8 (5'-TCAAAGCTGCTTGACACTGGCC-3') to amplify a 221 nucleotide fragment, and L6 (5'-CATGCCCACTATAAAAGGTCAG-3') and L7 (5'-CACCCCAGTCTTTCTTGAAA-3') to amplify a 173 nucleotide fragment. RT-PCR, gel purification and direct sequencing were performed as described above. In addition, the probes used were:

probes for M: 5'-TGC TTG TAC TTC CCA AAG-3'

Probe for N: 5'-TAT TTG AAC AAA AAG TGT-3'

Probes for L: 5'-TGGTGTGGGATATTAACAG-3'

Phylogenetic analysis

For all phylogenetic trees, the DNA sequence alignments were carried out with the C1 UtalW package, with the DNA-ML package using 100 bootstrap programs and 3 jumble Phylip3.5 programs¹⁵And generating a maximum likelihood evolutionary tree. Previously published sequences for generating phylogenetic evolutionary trees are available from Genbank under accession numbers: for all ORFs:hRSV: NC 001781; bRSV: NC 001989; for the F ORF: PVM, D11128; APV-A, D00850; APV-B, Y14292; APV-C, AF 187152; for the N ORF: PVM, D10331; APV-A, U39295; APV-B, U39296; APV-C, AF 176590; for the M ORF: PMV, U66893; APV-A, X58639; APV-B, U37586; APV-C, AF 262571; for the P ORF: PVM, 09649; APV-A, U22110, APV-C, AF 176591. Phylogenetic analysis was performed on the 9 different MPV virus isolates using APV C strains as the outer group.

Abbreviations used in the figures: hRSV: human RSV; bRSV: bovine RSV; PVM: mouse pneumovirus; APV-A, B and C: avian pneumovirus A, B and type C.

Examples of methods for identifying MPVs

Sample collection

For the discovery of viral isolates, nasopharyngeal aspirates, laryngeal and nasal swabs, bronchoalveolar lavages, preferably from mammals such as humans, carnivores (dogs, cats, mustellites, seals, etc.), horses, ruminants (cows, sheep, goats, etc.), pigs, rabbits, birds (poultry, ostriches, etc.) should be examined. From birds, cloacal swabs and feces can also be examined. Sera should be collected for immunological assays, such as ELISA and virus neutralization assays.

The collected virus samples were diluted with 5ml of Dulbecco MEM medium (BioWhittaker, Walkersville, Md.) and mixed well for 1 minute on a vortex mixer. The suspension was centrifuged at 840 Xg for 10 min. For immunofluorescence techniques, The pellet was smeared onto a multi-spot (multispot) slide (Nutacon, Leimuiden, The Netherlands) and virus isolation was performed with The supernatant.

Virus isolation

For virus isolation, tMK cells (PaVM, Bilthoven, The Netherlands) were cultured in 24-well plates with slides (Costar, Cambridge, UK) in The following medium supplemented with 10% fetal bovine serum (BioWhittaker, Vervier, Belgium). In thatPrior to inoculation, the plates were washed with PBS, followed by addition of Eagle's MEM (ICN, Costamesa, Calif.) containing Hank's salt, the medium being supplemented with 0.52 g/l NaHCO₃0.025M Hepes (Biowhittaker), 2 mML-glutamine (Biowhittaker), 200 units/l penicillin, 200. mu.g/l streptomycin (Biowhittaker), 1 g/l lactalbumin (Sigma-Aldrich, Zwijndrecht, The Netherlands), 2.0 g/l D-glucose (Merck, Amsterdam, The Netherlands), 10 g/l peptone (Oxoid, Haarlem, The Netherlands) and 0.02% tyrosine (Life Technologies, Bethesda, MD). The supernatant of the nasopharyngeal aspirate samples was inoculated in triplicate in the plates at 0.2ml per well and then centrifuged at 840 Xg for 1 hour. After inoculation, the plates were incubated at 37 ℃ for a maximum of 14 days, the medium was changed once a week and the cultures were checked daily for CPE. After 14 days, cells were scraped from the second subculture and then incubated for another 14 days. This procedure was repeated for the third passage. The presence of the virus was confirmed by indirect IFA using the slide as described below.

CPE was generally observed at days 8-14 after the third passage, depending on the isolate. The CPE is virtually indistinguishable from CPE caused by hRSV or hPIV in tMK or other cell cultures. However, hRSV induced CPE starting at approximately day 4. CPE is characterized by syncytia formation, after which the cells exhibit rapid internal destruction, and then the cells are separated from the monolayer. For some isolates, CPE was difficult to observe, and IFA was used to confirm the presence of the virus in these cultures.

Viral cultures of MPV

Supernatants of samples showing CPE after 2-3 passages in 24-well plates were seeded in a sub-confluent monolayer of tMK cells in the above medium. Cultures were checked daily for CPE and media was changed once a week. Since CPE varies with each isolate, all cultures were tested on days 12-14 using indirect IFA using ferret antibodies against the new viral isolate. Positive cultures were freeze-thawed 3 times, after which the supernatant was clarified by low-speed centrifugation, divided into aliquots andfrozen at-70 ℃. According to established techniques used in the art¹⁶The 50% tissue culture infectious dose of virus in the culture supernatant (TCID50) was determined.

Virus characterization

According to the described techniques, which are well established and used in the art¹⁴Hemagglutination assays and chloroform sensitivity tests were performed. For EM analysis, virus was concentrated from infected cell culture supernatants at 17000 Xg at 4 ℃ in a microcentrifuge, after which the pellet was resuspended in PBS and examined with negative contrast EM.

Detection of antigens by indirect IFA

The collected samples were processed as described above and the sample pellets were smeared on a multi-spot slide. After drying, the cells were fixed in acetone for 1 minute at room temperature.

Alternatively, viruses were cultured on tMK cells in a 24-well slide (24 well slide) with slides. The slides were washed with PBS and fixed in acetone for 1 minute at room temperature.

After washing with PBS, the slides were incubated for 30 minutes at 37 ℃ with polyclonal antibody diluted 1: 50 to 1: 100 in PBS. We obtained polyclonal antibodies from immunized ferrets and guinea pigs, but these antibodies could be produced in a variety of animals, and the working dilution of the polyclonal antibody for each immunization could be varied. After 3 washes with PBS and 1 wash with tap water, slides were incubated with FITC-labeled goat anti-ferret antibody (KPL, Guilford, UK, 40-fold dilution) for 30 min at 37 ℃. After 3 washes with PBS and 1 wash with tap water, slides were contained in glycerol/PBS solution (Citifluor, UKC, cantbury, UK) and covered. Slides were analyzed with an Axioscop fluorescence microscope (Carl Zeiss b.v., Weesp, the Netherlands).

Detection of antibodies in humans, mammals, ruminants or other animals by indirect IFA

To detect virus-specific antibodies, MPV-infected tMK cells were fixed on coverslips with acetone (as described above), washed with PBS, and then incubated with 1-16 dilution of serum samples for 30 min at 37 ℃. After washing 2 times with PBS and 1 time with tap water, the slides were incubated with FITC-labeled secondary antibodies (Dako) against the species used for 30 minutes at 37 ℃. Slides were processed as described above.

The antibody can be directly labeled with a fluorescent dye, which will result in a direct immunofluorescence assay. FITC may be replaced with any fluorescent dye.

Animal immunization

Two ferrets and two guinea pigs, which were housed in separate pressurized glove boxes, were screened for experimental intranasal infection to generate ferret and guinea pig specific antisera against the newly discovered viruses. After 2-3 weeks, the animals were bled by cardiac puncture and their sera were used as reference sera. The sera were tested for all previously described viruses with indirect IFA as described below. Other animal species are also suitable for use in generating specific antibody preparations, and other antigen preparations may be used.

Virus neutralization assay (VN assay)

VN assays were performed using 2-fold serial dilutions of human and animal sera starting at 8-fold dilutions. Diluted sera were incubated with 100 TCID50 virus for 1 hour, then seeded into tMK cells grown in 96-well plates, after which the plates were centrifuged at 840 × g. The same medium as described above was used. The medium was changed after 3 and 6 days, and after 8 days IFA was performed (see above). VN titres were defined as the lowest dilution of serum samples that resulted in negative IFA and inhibition of CPE in cell culture.

RNA isolation

RNA was isolated from The supernatant or sucrose gradient fraction of infected cell cultures using The High Pure RNA Isolation kit (High purity RNA Isolation kit) according to The manufacturer's instructions (Roche Diagnostics, Almere, The Netherlands). RNA may also be isolated according to other methods known in the art (Current Protocols in Molecular biology).

RT-PCR

In the presence of 50mM Tris.HCl pH 8.5, 50mM NaCl, 4mM MgCl₂2mM dithiothreitol, dNTP 200. mu.M each, 10 units of recombinant RNAsin (Promega, Leiden, The Netherlands), 10 units of AMV RT (Promega, Leiden, The Netherlands), 5 units of Amplitaq Gold DNA polymerase (PE Biosystems, Nieuwerkerk aan deijsssel, The Netherlands) and 5. mu.l of RNA in 50. mu.l reactions, a one-step RT-PCR was carried out. The cycling conditions were 42 ℃ for 45 min and 95 ℃ for 7min once, repeated 40 times at 95 ℃ for 1min, 42 ℃ for 2min and 72 ℃ for 3min, and once at 72 ℃ for 10 min.

Primers used for diagnostic PCR:

in the nucleoprotein: n3 (5'-GCACTCAAGAGATACCGTAG-3') and N4(5 ' -AGACTTTCTGCTTTGCTGCCTG-3 ones) to amplify a 151-nucleotide fragment.

In the matrix protein: m3 (5'-CCCTGACAATAACCACTCTG-3') and M4 (5'-GCCAACTGATTTGGCTGAGCTC-3') to amplify a 252 nucleotide fragment.

In the polymerase protein: l6 (5'-CATGCCCACTATAAAAGGTCAG-3') and L7 (5'-CACCCCAGTCTTTCTTGAAA-3') to amplify a 173 nucleotide fragment.

Other primers can be designed based on the sequence of the MPV, and different buffers and assay conditions can be used for specific purposes.

Sequence analysis

Sequence analysis was performed using a Dynamic ET terminator sequencing kit (Dynamic ET stop sequencing kit) (Amersham Pharmacia Biotech, Roosendaal, The Netherlands) and ABI 373 automated DNA sequencer (PE biosystems). All techniques were performed according to the manufacturer's instructions. The PCR fragment was directly sequenced with The same oligonucleotides used for PCR, or The fragment was purified from The Gel using The Qiaquick Gel Extraction Kit (Qiagen, Leusden, The Netherlands) and cloned into The pCR2.1 vector (Invitrogen, Groningen, The Netherlands) according to The manufacturer's instructions followed by sequencing with Ml3 specific oligonucleotides.

An oligonucleotide for analysis of the 3' end of the genome (lacking NS1/NS 2).

Primer TR1 (5'-AAAGAATTCACGAGAAAAAAACGC-3') was designed based on the trailer and leader sequences of hRSV and APV published by Randhawa (1997), and primer N1 (5'-CTGTGGTCTCTAGTCCCACTTC-3') was designed based on the sequence in the obtained N protein. RT-PCR assays and sequencing were performed as described above.

The RT-PCR gave a product of about 500 base pairs which was too small to contain the information of two ORFs, and translation of these sequences did not reveal 0 RF.

Detection of antibodies in humans, mammals, ruminants or other animals by ELISA

In the paramyxoviridae family, the N protein is the most abundant protein, and immune responses against this protein occur early in infection. For these reasons, it is preferred to use the N protein of recombinant origin to develop an ELISA assay for the detection of anti-MPV antibodies. Antigens suitable for antibody detection include any MPV protein that binds to any MPV-specific antibody of a patient that has been contacted or infected with MPV virus. Preferred antigens of the invention include those which elicit an immune response primarily in patients who have been exposed to MPV and are therefore generally most readily recognized by the patient's antibodies. Particularly preferred antigens include N, F and the G protein of MPV. Antigens for immunological techniques may be natural antigens or may be modified forms thereof. Well-known molecular biological techniques can be applied to alter the amino acid sequence of an MPV antigen to produce a modified form of the antigen that can be used in immunological techniques.

Methods for cloning genes, manipulating the genes for addition to and removal from expression vectors, and expressing proteins encoded by the genes in heterologous hosts are well known and these techniques can be used to provide the expression vectors, host cells, and for expressing the cloned genes encoding antigens in hosts to produce recombinant antigens for use in diagnostic assays. See, for example: molecular cloning, A laboratory manual and Current protocols in Molecular Biology. A wide variety of expression systems can be used to produce MPV antigens. For example, a wide variety of expression vectors suitable for the production of proteins in e.coli (e.coli), b.subtilis, yeast, insect cells and mammalian cells have been described, any of which may be used to produce MPV antigens suitable for the detection of anti-MPV antibodies in patients exposed to MPV virus.

An advantage of baculovirus expression systems is that they provide the necessary processing of the protein, and therefore baculovirus expression systems are preferred. The system utilizes the polyhedrin promoter to direct expression of MPV antigens (Matsuura et al, 1987, J.Gen.Virol.68: 1233-1250).

Antigens produced by recombinant baculoviruses can be used in a wide variety of immunological assays to detect anti-MPV antibodies in patients. It is well established that recombinant antigens can be used to replace native viruses in virtually any immunoassay to detect virus-specific antibodies. These assays include direct and indirect assays, sandwich assays, solid phase assays such as those using plates or microbeads, and liquid phase assays. Suitable assays include those with primary and secondary antibodies, as well as those using antibody binding reagents such as protein a. In addition, a wide variety of detection methods can be used in the present invention, including colorimetric, fluorescent, phosphorescent, chemiluminescent, luminescent, and radioactive methods.

Example 1: indirect anti-MPV IgG EIA using recombinant N protein

Indirect IgG EIA using recombinant N protein (produced in insect (Sf9) cells using recombinant baculovirus) as antigen can be performed. For antigen preparation, Sf9 cells were infected with the recombinant baculovirus and harvested 3-7 days post infection. The cell suspension was washed 2 times in PBS, pH7.2, adjusted to 5.0X 10⁶Cell density of cells/ml, then freeze-thaw 3 times. Large cell debris was pelleted by low speed centrifugation (500Xg, 15min.) and the supernatant was collected and stored at-70 ℃ until use. Uninfected cells were similarly treated for the negative control antigen.

The microtiter plates were coated with 100. mu.l of freeze-thaw lysate with dilutions ranging from 1: 50 to 1: 1000. Uninfected cell lysates were added to both duplicate wells and used as negative controls. After incubation overnight, plates were washed 2 times with PBS/0.05% Tween. Test sera were diluted 1: 50 to 1: 200 in ELISA buffer (PBS, supplemented with normal goat serum to 2%, and supplemented with 0.5% bovine serum albumin and 0.1% milk powder), and then each well was incubated at 37 ℃ for 1 hour.

The plate was washed 2 times with PBS/0.05% Tween. To each well was added horseradish peroxidase-labelled goat anti-human (or for other species) IgG diluted 1: 3000 to 1: 5000 in ELISA buffer and incubated for 1 hour at 37 ℃. The plates were then washed 2 times with PBS/0.05% Tween, 1 time with weekly tap water, incubated with the enzyme substrate TMB-e.g.3, 3', 5, 5' -tetramethylbenzidine from Sigma for 15 minutes at room temperature, and the reaction was stopped with 100. mu.l of 2M phosphoric acid. The colorimetric readings at 450nm were measured using an automatic microtiter plate reader.

Example 2: capture of anti-MPV IgM EIA Using recombinant nucleoproteins

The previous Erdman et al (1990) j.clin.microb.29: 1466-1471, performed with a capture IgM EIA using a recombinant nucleoprotein or any other recombinant protein as antigen.

To each well of the microtiter plate, an affinity purified anti-human IgM capture antibody (or an antibody against other species), such as that from Dako, is added at a concentration of 250ng in 0.1M carbonate buffer ph9.6 per well. After incubation overnight at room temperature, the plates were washed 2 times with PBS/0.05% Tween. Mu.l of test serum diluted 1: 200 to 1: 1000 in ELISA buffer was added to three replicate wells and incubated at 37 ℃ for 1 hour. The plate was then washed 2 times with PBS/0.05% Tween.

The freeze-thawed (recombinant virus infected) Sf21 cell lysate was diluted 1: 100 to 1: 500 in ELISA buffer, added to each well, and incubated at 37 ℃ for 2 hours. Uninfected cell lysates were used as negative controls and added in duplicate wells. The plate was then washed 3 times with PBS/0.05% Tween and incubated with 100. mu.l of the most appropriate dilution of polyclonal anti-MPV antibody in ELISA buffer for 1 hour at 37 ℃. After washing 2 times with PBS/0.05% Tween, the plates are incubated with horseradish peroxidase-labeled secondary antibodies (e.g., rabbit anti-ferrets) and the plates are incubated for 20 minutes at 37 ℃.

The plate is then washed 5 times with PBS/0.05% Tween, incubated with the enzyme substrate TMB-e.g.3, 3', 5, 5' -tetramethylbenzidine from "Sigma" for 15 minutes at room temperature, and the reaction is stopped with 100. mu.l of 2M phosphoric acid. The colorimetric readings at 450nm were measured using an automatic microtiter plate reader.

The sensitivity of captured IgM EIA using recombinant nucleoproteins (or other recombinant proteins) and using MPV whole virus was compared with acute and convalescent serum pairs from humans with clinical MPV virus infection. The specificity of the capture of EIA by recombinant nucleoprotein was determined by examining serum samples obtained from healthy persons and persons with other paramyxovirus infections.

EIM using recombinant MPV fusion proteins and glycoproteins produced by baculovirus expression Possibility of。

Glycoproteins G and F are two transmembrane envelope glycoproteins of MPV virions, the major neutralizing protective antigens. Expression of these glycoproteins in a vector viral system, such as a baculovirus system, provides a source of recombinant antigen for use in assays for detecting MPV-specific antibodies. Furthermore, they are used in combination with, for example, nucleoproteins, further improving the sensitivity of enzyme immunoassays in the detection of anti-MPV antibodies.

A wide variety of other immunological assays (Currenf Protocols in immunology) can be used as alternatives to the methods described herein.

For the discovery of viral isolates, nasopharyngeal aspirates, throats and nasal swabs, bronchoalveolar lavages and laryngeal swabs, preferably but not limited to, from humans, carnivores (dogs, cats, seals, etc.), horses, ruminants (cows, sheep, goats, etc.), pigs, rabbits, birds (poultry, ostriches, etc.) can be examined. From birds, cloaca and small intestinal swabs and feces can also be examined. For all samples, serology (antibody and antigen detection, etc.), virus isolation and nucleic acid detection techniques can be performed to detect viruses. Monoclonal antibodies can be produced by immunizing mice (or other animals) with purified MPV or portions thereof (proteins, peptides) and then subsequently using established hybridoma technology (current protocols in Immunology). Alternatively, phage display technology (Current protocols in Immunology) can be used for this purpose. Similarly, polyclonal antibodies (Currentprotocols in Immunology) can be obtained from infected humans or animals, or from immunized humans or animals.

Detection of the presence of NS1 and NS2 proteins can be performed by western blot analysis, IFA, immunoprecipitation techniques using a variety of antibody preparations. The presence of the NS1 and NS2genes or homologues thereof in viral isolates can be detected by PCR using primer sets designed based on the known NS1 and/or NS2genes and a variety of nucleic acid hybridization techniques.

To determine whether the NS1 and NS2genes are present at the 3 'end of the viral genome, PCR can be performed with primers specific for this 3' end of the genome. In our case, we used one primer specific for the 3' untranslated region of the viral genome and one primer in NORF. Other primers can be designed for the same purpose. Based on the length and/or nucleotide sequence of the PCR product, the absence of the NS1/NS 2gene was revealed. Primers specific for the NS1 and/or NS2gene can be used in combination with primers specific for other parts of the 3' end of the viral genome (e.g. the untranslated region or N, M or the F ORF) to achieve positive identification of the presence of the NS1 gene or NS2 gene. In addition to PCR, various techniques such as molecular cloning, nucleic acid hybridization can be used for the same purpose.

Example 3: different serotypes/subgroups of MPV

By analyzing the partial nucleotide sequences in the N, M, F and L ORFs of the 9 viral isolates, two potential genetic taxa were identified. Nucleotide identities of 90-100% were observed within the taxa, and identities of 81-88% were observed between the taxa. With the sequence information obtained for more viral isolates, the presence of two genotypes was confirmed. Viral isolate ned/00/01, which is the prototype of the A taxonomic group, and viral isolate ned/99/01, which is the prototype of the B taxonomic group, have been used in cross-neutralization assays to test whether the genotypes are associated with different serotypes or subgroups.

Results

Using RT-PCR assays, we identified another 30 virus isolates from nasopharyngeal aspirate samples using primers located in the polymerase gene. The sequence information on the portions of the matrix genes and polymerase genes of these new isolates, as well as the sequence information on the previous 9 isolates, was used to construct a phylogenetic tree (FIG. 16). Analysis of these evolutionary trees confirmed the existence of two genetic taxa, with viral isolate ned/00/00-1 being the prototype virus of group A and viral isolate ned/99/01 being the prototype virus of group B. Nucleotide sequence identity within a panel is greater than 92%, while the identity between the taxa is 81-85%.

Viral isolates, ned/00/01 and ned/99/01, have been used to breed ferrets for the production of virus-specific antisera. These antisera were used in a virus neutralization assay with the two viruses.

Table 3:

virus neutralization titer

For isolate 00-1, the titers differed by a factor of 32(64/2)

For isolate 99-1, the titers differed by a factor of 16(64/4)

In addition, 6 guinea pigs had been vaccinated with either of the viruses (ned/00/01 and ned/99/01). RT-PCR assays on nasopharyngeal aspirate samples showed that virus replicated from day 2 to day 10 post infection. On day 70 post-infection, the guinea pigs were challenged with either the same or a different virus, and in all 4 cases, viral replication was observed.

TABLE 4

	First infection	Viral replication	Second infection	Viral replication
					Guinea pig 1-3	00-1	2/3	99-1	1/2
Guinea pig 4-6	00-1	3/3	00-1	1/3
					Guinea pig 7-9	99-1	3/3	00-1	2/2
Guinea pig 10-12	99-1	3/3	99-1	1/3

Note that: for the second infection, guinea pigs 2 and 9 were no longer included.

Virus neutralization assays with antiserum after the first challenge showed essentially the same results as VN assays with ferrets (> 16-fold difference in VN titres).

The results described in this example demonstrate that there are two genotypes, which correspond to the two serotypes of MPV, and indicate the possibility of repeated infection by both xenoviruses and homoviruses.

Example 4: further sequence determination

This example describes the further analysis of MPV Open Reading Frame (ORF) sequences and intergenic sequences and partial sequences of the ends of the genome.

Sequence analysis of the nucleoprotein (N), phosphoprotein (P), matrix protein (M) and fusion protein (F) genes of MPV revealed the highest degree of sequence homology with the avian pneumovirus APV serotype C found primarily in birds in the united states. These analyses also revealed that the non-structural proteins NS1 and NS2 located at the 3' end of the viral genome were absent and that the fusion protein was located next to the matrix protein. Here, we propose the sequences of the22K (M2) protein, the Small Hydrophobic (SH) protein, the adsorption (G) protein and the polymerase (L) protein genes, as well as the intergenic region and the trailer sequence. Together with the previously described sequences, the sequences presented herein complete the genomic sequence of MPV, except for the last 12-15 nucleotides at the end of the genome, thus establishing the genomic organization of MPV. Side-by-side comparison of the sequences of the MPV genome with those of APV subtypes a, B and C, RSV subtypes a and B, PVM and other paramyxoviruses provides strong evidence that MPV should be classified in Metapneumovirus.

Results

Sequence strategy

MPV isolate 00-1(van den Hoogen et al, 2001) was propagated in third generation monkey kidney (tMK) cells and RNA isolated from the supernatant 3 weeks after inoculation was used as a template for RT-PCR analysis. Primers were designed based on available partial sequence information for MPV 00-1(van den Hoogen et al, 2001) as well as the leader and trailer sequences of APV and RSV (Randhawa et al, 1997; Mink et al, 1991). Fragments ranging in size from 500 bp to 4 Kb between the previously obtained products were initially generated by RT-PCR amplification and directly sequenced. Subsequently, the genomic sequence was confirmed by generating a series of overlapping RT-PCR fragments of size range 500-800 bp representing the complete MPV genome. For all PCR fragments, both strands were directly sequenced to minimize amplification and sequencing errors. Using the nucleotide and amino acid sequences, the homology to sequences in the Genbank database was searched using BLAST software (www.ncbi.n1m.nih.gov/BLAST). Protein names are assigned based on homology to known viral genes and their location in the genome. From this information, a genomic map of MPV was constructed (fig. 7). The MPV genome is 13378 nucleotides in length and has a organization similar to that of APV. In the following, we present a comparison between the ORF and non-coding sequences of MPV and those of other paramyxoviruses, and discuss the important similarities and differences.

Nucleoprotein (N) gene

As shown, the first gene in the MPV genomic map encodes a 394 amino acid (aa) protein and shows extensive homology to the N proteins of other pneumoviruses. The length of the N ORF is the same as that of the N ORF of APV-C (Table 5), but less than that of the other paramyxovirus N ORFs (Barr et al, 1991). Analysis of the amino acid sequence revealed the highest homology (88%) with APV-C, but only 7-11% with other paramyxoviruses (Table 6).

Barr et al (1991) identified 3 regions with similarity between viruses belonging to the order Mononegavirales: A. b and C (fig. 8). Although the similarity is highest within the virus family, these regions are highly conserved between virus families. In all 3 regions, MPV showed 97% amino acid sequence identity to APV-C, 89% amino acid sequence identity to APV-B, 92% amino acid sequence identity to APV-A, and 66-73% amino acid sequence identity to RSV and PVM. The region between amino acid residues 160 and 340 appears to be highly conserved among metapneumovirus and slightly less conserved among the Pneumovirinae subfamily (Miyahara et al, 1992; Li et al, 1996; Barr et al, 1991). This is consistent with MPV being a metapneumovirus showing 100% similarity to APV C.

Phosphoprotein (P) gene

The second ORF in the genomic map encodes a protein of 294 amino acids which has 68% amino acid sequence homology with the P protein of APV-C and only 22-26% amino acid sequence homology with the P protein of RSV (Table 6). The P gene of MPV contains a substantial ORF, similar in this respect to P from many other paramyxoviruses (for reviews see Lamb and Kolakofsky, 1996; Sedlmeier et al, 1998).

In contrast to APV A and B and PVM, and similar to RSV and APV-C, the MPVP ORF lacks cysteine residues. Ling (1995) suggested that a region of high similarity among all pneumoviruses (aa 185-241) plays a role in RNA synthesis or in maintaining the structural integrity of the nucleocapsid complex. This region of high similarity is also present in MPV (FIG. 9), and especially when conservative substitutions are considered, shows 100% similarity to APV-C, 93% similarity to APV-A and B, and about 81% similarity to RSV. As described for APV (Ling et al, 1995), the C-terminus of MPV P protein is rich in glutamic acid residues.

Matrix (M) protein gene

The third ORF of the NIPV genome encodes a 254 amino acid protein, similar to the M ORF of other pneumoviruses. The MPV M ORF was identical in size to MORFs of other met-printed neumovirus (Table 5), and showed high amino acid sequence homology to the matrix proteins of APV (78-87%), low homology to RSV and PVM matrix proteins (37-38%), and homology of 10% or less to other paramyxovirus matrix proteins (Table 6).

Easton (1997) compared the sequences of all pneumovirus matrix proteins and found a conserved heptapeptide at residues 14-19, which is also conserved in MPV (FIG. 10). For RSV, PVM and APV, a small second ORF within or overlapping the M major ORF has been identified (52 aa and 51 aa in bRSV, 75 aa in RSV, 46 aa in PVM and 51 aa in APV) (Yu et al, 1992; Easton et al, 1997; Samal et al, 1991; Satake et al, 1984). We note that there are two small ORFs in MPV MORF. A small ORF of 54 aa residues starting at nucleotide 2281 was found within the M major ORF (fragment 1, FIG. 7) and a small ORF of 33 aa residues starting at nucleotide 2893 overlapping the M major ORF (fragment 2, FIG. 7). Similar to the second ORFs of RSV and APV, there is no significant homology between these second ORFs and the second ORFs of other pneumoviruses, and there is a lack of apparent start or stop signals. In addition, no evidence has been reported about the synthesis of proteins corresponding to these second ORFs of APV and RSV.

Melphalan (F) gene

The F ORF of MPV is located adjacent to the M ORF, a feature of Metapneumovirus members. The F gene of MPV encodes a 539 aa protein that is 2 aa residues longer than F of APV-C (Table 5). Amino acid sequence analysis showed 81% homology to APV-C, 67% homology to APV-A and B, 33-39% homology to pneumovirus F protein, and only 10-18% homology to other paramyxoviruses (Table 6). One of the conserved features observed in paramyxovirus F proteins and also in MPV is the distribution of cysteine residues (Morrison, 1988; Yu et al, 1991). metapneumovirus shares 12 cysteine residues in F1 (7 are conserved in all paramyxoviruses) and 2 cysteine residues in F2 (1 is conserved in all paramyxoviruses). None of the 3 potential N-linked glycosylation sites present in the MPV F ORF were shared with RSV, 2 such sites with APV (positions 74 and 389). The third unique potential N-linked glycosylation site of MPV is at position 206 (fig. 11).

Although having low sequence homology with other paramyxoviruses, the F protein of MPV exhibits characteristics of a typical fusion protein consistent with those described for F proteins of other members of the paramyxoviridae family (Morrison, 1988). The F protein of a member of the family paramyxoviridae is synthesized as an inactive precursor (F0), cleaved by proteases of the host cell to yield the amino-terminal F2 subunit and the large carboxy-terminal F1 subunit. Proposed cleavage sites (Collins et al, 1996) are conserved among all members of the Paramyxoviridae family. The cleavage site of MPV contains the residue RQSR. Two arginine (R) residues were shared with APV and RSV, but glutamine (Q) and serine (S) residues were shared with other paramyxoviruses such as human parainfluenza virus type 1, sendai virus and measles virus (data not shown).

The hydrophobic region at the amino terminus of F1 is thought to act as a membrane fusion domain and to show high sequence similarity in paramyxoviruses and measles, but a lesser degree in pneumoviruses (Morrison, 1988). These 26 residues (position 137-163, FIG. 11) are conserved between MPV and APV-C, which is consistent with the region being highly conserved in metapneumovirus (Naylor et al, 1998; Seal et al, 2000).

As observed for the F2 subunit of APV and other paramyxoviruses, MPV showed a deletion of 22 aa residues compared to RSV (position 107-. Furthermore, for RSV and APV, the signal peptide and the anchor domain were found to be conserved within subtypes, while exhibiting high variability between subtypes (Plows et al, 1995; Naylor et al, 1998). The signal peptide at the amino terminus of F2 of MPV (aa 10-35, FIG. 11) shows some sequence similarity to APV-C (18 out of 26 aa residues are similar), with less conservation than other APVs or RSV. Much greater variability was observed in the membrane anchoring domain at the carboxy terminus of F1, although some homology to APV-C was still observed.

22K (M2) protein

The M2 gene is subcategory specific for pneumoviruses, and two overlapping ORFs were observed in all pneumoviruses. The first major ORF represents the M2-1 protein, which enhances the processivity of the viral polymerase (Collins et al, 1995; Collins, 1996) and the readthrough of its intergenic region (Hardy et al, 1998; Feams et al, 1999). The M2-1 gene of MPV was located adjacent to the F gene, encoding a 187 aa protein (Table 5), and showed the highest homology (84%) to M2-1 of APV-C (Table 6). Comparison of all the Pneumovirus M2-1 proteins showed the highest conservation in the amino-terminal half of the protein (Collins et al, 1990; Zamora et al, 1992; Ahmandian et al, 1999), consistent with the observation that MPV shows 100% conservation of similarity to APV-C in the first 80 aa residues of the protein (FIG. 12A). The MPVM2-1 protein contains 3 cysteine residues located within the first 30 aa residues, conserved in all pneumoviruses. This concentration of cysteine is common in zinc binding proteins (Ahmadian et al, 1991; Cuesta et al, 2000).

The position of the second ORF (M2-2) that overlaps with the Pneumovirus M2-1ORF is conserved but not conserved in sequence, which ORF is believed to be involved in the control of the switch between viral RNA replication and transcription (Collins et al, 1985; Elango et al, 1985; Baybutt et al, 1987; Collins et al, 1990; Ling et al, 1992; Zamora et al, 1992; Alansari et al, 1994; Ahmadian et al, 1999; Berminham et al, 1999). For MPV, the M2-2 ORF starts at nucleotide 512 in the M2-1ORF (FIG. 7), which is the same starting position in APV-C. M2-20RF of APV-C and MPV were the same length with 71 aa residues (Table 5). Sequence comparison of M2-20RF between MPV and APV-C (FIG. 12B) revealed 64% amino acid sequence homology, while MPV had only 44-48% amino acid sequence homology with APV-A and B (Table 6).

Small vegetable water-based protein (SH) ORF

The gene, located adjacent to M2 of MPV, probably encodes a 183 aa SH protein (FIGS. 1 and 7). There is no discernable sequence identity between this ORF and other RNA viral genes or gene products. This is not unexpected because sequence similarity between pneumovirus SH proteins is generally low. The putative SH ORF for hMPV is the longest SH 0RF known to date (table 1). The aa composition of the SH ORF is quite similar to that of APV, RSV and PVM, with a high percentage of threonine and serine residues (serine/threonine content of hMPV, APV, RSVA, RSV B, bRSV and PVM is 22%, 18%, 19%, 20.0%, 21% and 28%, respectively). The SH ORF of hMPV contains 10 cysteine residues, while the APV SH contains 16 cysteine residues. The SH ORF of hMPV contains 2 potential N-linked glycosylation sites (aa 76 and 121), whereas APV has 1 such site, RSV has 2 or 3, and PVM has 4.

The putative hydrophilic profiles of hMPV SH protein and SH for APV and RSV revealed similar features (fig. 7B). The SH 0RF of APV and hMPV has a hydrophilic N-terminus, a central hydrophobic domain (aa 30-53 for hMPV) that can serve as a potential transmembrane domain, a secondary hydrophobic domain (aa 155-170) and a hydrophilic C-terminus. In contrast, the SH of RSV appears to lack the C-terminal portion of the APV and hMPV ORFs. In the SH proteins of all pneumoviruses, the hydrophobic domain is adjoined by a plurality of basic amino acid residues, which are also present in the SH ORF of hMPV (aa 29 and 54).

Adsorption of glycoprotein (G) ORF

The putative G ORF of hMPV was located adjacent to the putative SH gene, encoding a 236 aa protein (nt 6262-6972, FIG. 1). Immediately after this ORF a second small ORF was found, possibly encoding 68 aa residues (nt 6973-7179), but lacking the start codon. The third possible ORF at an 194 aa residue in the second reading frame overlaps with both ORFs, but also lacks the initiation codon (nt 6416-7000). This ORF was followed in frame by a fourth possible 0RF (nt 7001-7198) of 65 aa residues, but lacks the start codon. Finally, one possible 97 aa residue ORF (but lacking the start codon) was found in the third reading frame (nt 6444-6737, FIG. 1). Unlike the first ORF, the other ORFs do not have an apparent gene start sequence or gene stop sequence (see below). Although the 236 aa G ORF may represent at least a part of the hMPV adsorption protein, it cannot be excluded that: the additional coding sequences are expressed by certain RNA editing events as individual proteins or as part of the adsorbed protein. It should be noted that for both APV and RSV, no second ORF was identified after the primary G ORF, but both APV and RSV have a second ORF within the primary ORF of G. However, there is a lack of evidence for expression of these ORFs, and there is no sequence identity between the predicted amino acid sequences of different viruses (Ling et al, 1992). The second ORF in hMPV G did not show the features of the other G proteins and whether the additional 0RF was expressed or not was investigated further.

BLAST analysis of all ORFs showed no discernable sequence identity to other known viral genes or gene products at the nucleotide or amino acid sequence level. This is consistent with the finding of low percentage sequence identity with other G proteins such as the G proteins of hRSV A and B (53%) (Johnson et al, 1987) and with the G proteins of APV A and B (38%) (Juhasz and Easton, 1994).

Whereas most of the hMPV ORFs were similar in length and sequence to the 0RF of APV, the 236 aa-residue putative G ORF of hMPV was much smaller than that of APV (Table 1). The amino acid sequence showed that the putative G ORF contained 8.5% proline residues with serine and threonine contents of 34 even above 32% of RSV and 24% of APV, which is above 8% of RSV and 7% of APV. The unusual abundance of proline residues in the G proteins of APV, RSV and hMPV is also observed in glycoproteins of mucin origin, where this is a major consideration of the three-dimensional structure of proteins (Collins and Wertz, 1983; Wertz et al, 1985; Jentoft, 1990). The G ORF of hMPV contains 5 potential N-linked glycosylation sites, while there are 7 hRSV, 5 bRSV and 3-5 APV.

The predicted hydrophilicity profile of hMPV G revealed similar features to other pneumoviruses.

The amino terminus contains a hydrophilic region followed by a short hydrophobic region (aa 33-53 for hMPV) and a predominantly hydrophilic carboxy terminus (FIG. 8B). This overall organization is consistent with anchoring type II transmembrane proteins, corresponding well to these regions in the G proteins of APV and RSV. The putative G ORF of hMPV contains only 1 cysteine residue, in contrast to RSV and APV (5 and 20, respectively). Of course, only 2 of the 4 second ORFs contained an additional cysteine residue within the G gene, these 4 possible ORFs showed 12-20% serine and threonine residues and 6-11% proline residues.

Polymerase gene (L)

Like other negative-strand viruses, the last ORF of the MPV genome is the RNA-dependent RNA polymerase component of the replication transcription complex. The L gene of MPV encodes a 2005 aa protein, which is 1 residue longer than the protein of APV-A (Table 5). MPV has an L protein that is 64% homologous to APV-A, 42-44% homologous to RSV, and about 13% homologous to other paramyxoviruses (Table 6). Poch et al (1989; 1990) identified a non-segmented negative strand R6 conserved domains within the L protein of NA virus, wherein domain III contains 4 core polymerase motifs thought to be essential for polymerase function. These motifs (A, B, C and D) are well conserved in MPVL proteins: in motifs A, B and C: MPV has 100% similarity to all pneumoviruses, in motif D MPV has 100% similarity to APV and 92% similarity to RSV. For the complete domain III (aa 627-903 in LORF), MPV shares 77% identity with APV, 61-62% identity with RSV, and 23-27% identity with other paramyxoviruses (FIG. 15). In addition to the polymerase motif, the L protein of the pneumovirus contains a consensus ATP-binding motif K (X)₂₁GEGAGN(X)₂₀K (Stec, 1991). The MPVL ORF contains a motif similar to APV, in which the distance between the intermediate residues is one residue less:

K(x)₂₂GEGAGN(X)₁₉K。

phylogenetic analysis

As an index of the relationship between MPV and members of the Pneumovirinae subfamily, phylogenetic evolutionary trees based on the N, P, M and F ORFs have been previously constructed (van den Hoogen et al, 2001) and revealed a close relationship between MPV and APV-C. Because of the low homology of the MPV SH and G genes to said genes of other paramyxoviruses, a reliable phylogenetic evolutionary tree could not be constructed for these genes. In addition, the different genomic organization between members of the pneumovirus genus and Metapneumovirus makes it impossible to generate phylogenetic trees based on the complete genomic sequence. Therefore, we constructed phylogenetic trees only for the M2 gene and the L gene, in addition to those previously published. These two evolutionary trees demonstrate that APV and MPV are closely related within the pneumovirinae (fig. 16).

MPV non-coding sequence

The gene junctions of the paramyxovirus genome contain short highly conserved nucleotide sequences at the start and end of each gene (gene start signal and gene stop signal) that may play a role in transcription initiation and termination (Curran et al, 1999). Comparison of intergenic sequences between all genes of MPV revealed a consensus sequence of gene start signals for N, P, M, F, M2 and G: GGGACAAGU (FIG. 17A), which is identical to the consensus gene initiation signal of metapneumvirus (Ling et al, 1992; Yu et al, 1992; Li et al, 1996; Bayon-Auboyer et al, 2000). The gene initiation signals of the SH gene and the L gene of MPV were found to be slightly different from this consensus sequence (SH: GGGAUAAAU, L: GAGACAAAU). For APV, the gene initiation signal for L was also found to be different from the consensus sequence: AGGACCAAT (APV-A) (Randhawa et al, 1996) and GGGACCAGT (APV-D) (Bayon-Auboyer et al, 2000).

In contrast to the similarity of the gene start sequences of MPV and APV, the consensus gene end sequence of APV, UAGUUAAUU (Randhawa et al, 1996), cannot be found in the MPV intergene sequences. Apart from the G-L intergenic region, the repeat sequence found in most genes is UAAAAA U/A/C, which may serve as a termination signal for the gene. However, since we sequenced viral RNA, not mRNA, no defined gene termination signal could be assigned, and further investigation was required. The intergenic regions of the pneumovirus differ in size and sequence (Curan et al, 1999; Blumberg et al, 1991; Collins et al, 1983). The intergenic region of MPV did not show homology to the intergenic regions of APV and RSV and ranged in size from 10 to 228 nucleotides (FIG. 17B). The intergenic region between the M ORF and the F ORF of MPV contains a portion of the second ORF starting from the main M ORF (see above). The intergenic region between SH and G contains 192 nucleotides and appears to have no coding potential based on the presence of many stop codons in all three reading frames. The intergenic region between G and L contains 241 nucleotides, possibly including additional ORFs (see above). Interestingly, the start of the L ORF is located in these second ORFs. Whereas the L gene of APV does not start in the preceding G ORF, the L ORF of RSV also starts within the preceding M2 gene. At the 3 'and 5' ends of the paramyxovirus genome, short extra-gene regions are called leader and trailer, the first 12 nucleotides of the leader and the last 12 nucleotides of the trailer being approximately complementary, probably because they contain the essential elements of the viral promoter, respectively (Curran et al, 1999; Blumberg et al, 1991; Mink et al, 1986). The 3' leader sequences of both MPV and APV were 41 nucleotides in length, and some homology (18 out of 26 nucleotides) was observed in the region between nucleotides 16 and 41 of both viruses (FIG. 17B). As described above, the first 15 nucleotides of the MPV genomic map are based on the APV genome-based primer sequences. The length of the MPV 5' trailer sequence (188 nucleotides) is similar to the size of the RSV 5' trailer sequence (155 nucleotides), which is much longer than the 5' trailer sequence of APV (40 nucleotides). Sequence alignment of the 40 terminal nucleotides of the MPV and APV tail sequences revealed that 21 of the 32 nucleotides were homologous, except for the 12 most terminal nucleotides of the primer sequence representing the APV-based genomic sequence. Our sequence analysis revealed that NS1 and NS2genes were absent at the 3' end of the genome and that the genomic organization was similar to that of metapneumvirus (3 ' N-P-M-F-M2-SH-G-L-5 '). The high sequence homology between MPV and APV genes was found, further emphasizing the close relationship between these two viruses. For the N, P, M, F, M2-1 and M2-2 genes of MPV, an overall amino acid homology of 79% to APV-C was found. In fact, for these genes, APV-C and MPV show sequence homology in the same range as found between subgroups of other genera, e.g., RSV-A and B or APV-A and B. This close relationship of APV-C and MPV was also observed in phylogenetic analyses which revealed that MPV and APV-C were always in the same branch (branch), separate from the branch containing APV-A and B. The same genomic organization, sequence homology and phylogenetic analyses all supported the classification of MPV as the first member within Metapneumovirus to be isolated from mammals. It should be noted that the sequence variation in the N, M, F and L genes found between different viral isolates of MPV suggests that different genotypes may be present (van den Hoogen et al, 2001). The close relationship between MPV and APV-C is not reflected in the host range, since APV infects birds in contrast to MPV (van den Hoogen et al, 2001). This host-wide difference is probably due to the highly divergent differences between the SH and G proteins of the two viruses. The SH and G proteins of MPV do not show significant amino acid sequence homology to the SH and G proteins of any other virus. Although the amino acid content and hydrophobicity profiles support the identification of these ORFs as SH and G, experimental data is required to evaluate their function. Such an analysis would also provide clues to the role of the additional overlapping ORFs described in these SH and G genes. In addition, sequence analysis of SH and G genes of APV-C may allow a more thorough understanding of the functions of SH and G proteins of MPV and their relationship to said proteins of APV-C. The non-coding region of MPV was found to be quite similar to that of APV. The 3 'leader and 5' trailer sequences of APV and MPV show high homology. Although the length of the intergenic regions of APV and MPV are not always the same, it was found that the consensus gene start signals for the majority of the ORFs were identical. In contrast, the gene termination signal of APV is not found in the MPV genome. Although we did find a repeat sequence (U AAAAA U/A/C) in most intergenic regions, sequence analysis of viral mRNA was required to formally delineate those gene termination sequences. It should be noted that by using the modified Rapid Amplification of CDNA Ends (RACE) method, sequence information was obtained on the 3 '-most 15 nucleotides and 5' -most 12 nucleotides. This technique has been demonstrated to be successful with related viruses by others (Randhawa, J.S. et al, research of synthetic microorganisms such as the present invention of the NS1 and NS2genes from avian influenza virus.J.virol, 71, 9849. 9854 (1997); Mink, M.A. et al, nucleic acids of the 3 'leader a Ild 5' trailer region of human respiratory viral RNA. virology 185,615-24 (1991)). To determine the sequence of the 3' vRNA leader sequence, a homologous polymer a tail was added to the purified vRNA using poly-a polymerase, followed by PCR amplification of the leader sequence with poly-T primers and primers in the N gene. To determine the sequence of the 5' vRNA trailer sequence, a cDNA copy of the trailer sequence was prepared using reverse transcriptase and primers in the L gene, and the cDNA was then tagged with the cognate polymer dG tail using terminal transferase. Subsequently, the tail sequence region was amplified using a poly-C primer and a primer in the L gene. As an alternative strategy, vRNA is ligated to itself, or to a synthetic linker, after which the leader and trailer regions are amplified using primers in the L and N genes and linker specific primers. For the 5' tail sequence, direct dideoxynucleotide sequencing of purified vRNA is also feasible (Randhawa, 1997). Using these methods, we can analyze the actual sequence of the ends of the hMPV genome. The sequence information provided herein is important for generating diagnostic tests, vaccines and antiviral drugs against MPV and MPV infection.

Materials and methods

Sequence analysis

Viral isolate 00-1 was propagated to high titers (about 10,000 TCID50/ml) on third generation monkey kidney cells according to the method previously described (van den Hoogen et al, 2001). Viral RNA was isolated from The supernatant of infected cells using The high-purity RNA isolation Kit (high-purity RNA isolation Kit) according to The manufacturer's instructions (Roch Diagnostics, Almee, The Netherlands). In addition to the published sequences relating to the leader and trailer sequences of APV star SVs (Randhawa et al, 1997; Mink et al, 1991), primers were designed based on previously published sequences (van Hoogen et al, 2001), which were available upon request. Using a single tube assay in a single tube containing 50mM Tris pH 8.5, 50mM NaCl, 4.5 mM MgCl₂RT-PCR assays were performed with viral RNA in a total volume of 50. mu.l of viral RNA in 2mM DTT, 1. mu.M forward primer, 1. mu.M reverse primer, 0.6 mM dNTP, 20 units of RNAsin (Promega, Leiden, The Netherlands), 10U AMV reverse transcriptase (Promega, Leiden, The Netherlands), and 5 units of Taq polymerase (PE applied biosystems, Nieuwerkerk aan de IJssel, The Netherlands). Reverse transcription was performed at 42 ℃ for 30 minutes and then inactivated at 95 ℃ for 8 minutes. The cDNA was amplified as follows: 40 cycles of 95 ℃ for 1min, 42 ℃ for 2min, 72 ℃ for 3min, and finally 10min extension at 72 ℃. After examination on a 1% agarose Gel, Qiaquick Gel Extraction was usedKit (gel extraction Kit) (Qiagen, Leusden, The Netherlands), RT-PCR products were purified from The gel and then directly sequenced using a Dynamic ET terminator sequencing Kit (Dynamic ET terminator sequencing Kit) (Amersham Pharmacia Biotech, Roosendal, The Netherlands) and ABI 373 automated DNA sequencer (PE Applied Biosystem, Nieuwerkerk aan de Ussel, The Netherlands) according to The manufacturer's instructions.

For use in BioEdit vetson 5.0.6

(http:// jwbrown. mbio. ncsu. edu/Bioedit// Bioedit. htm 1; Hall, 1999) the clusta1 software package was available for sequence alignment.

Phylogenetic analysis

To construct the phylogenetic tree, alignments of DNA sequences were performed with the ClustalW package and the maximum likelihood tree was generated with the DNA-ML package using 100 bootstrap programs and 3 jumble Phylip3.5 programs. Bootstrap values for the consensus evolutionary tree created with the sense software package (felsense, 1989) were calculated.

MPV genomic sequences are available from Genbank under accession numbers: AF 371337. All other sequences used herein are available from Genbank under accession numbers: AB046218 herpes virus, all ORFs), NC-001796 (human parainfluenza virus type3, all ORFs), NC-001552 (Sendai virus, all ORFs), X57559 (human parainfluenza virus type 2, all ORFs), NC-002617 (Newcastle disease virus, all ORFs), NC-002728(Nipah virus, all ORFs), NC-001989(bRSV, all ORFs), M11486(hRSV A, all ORFs except L), NC-001803(hRSV, L ORF), NC-781001 (hRSV B, all ORFs), D10331(PVIM, N ORF), U09649(PVM, P ORF), U66893(PVM, M ORF), U66893(PVM, SH ORF), D11130(PVM, G ORF), D11128 (F' ORF). PViM 2 ORFs were taken from Ahmandian (1999), AF176590(APV-C, NORF), U39295(APV-A, N ORF), U39296(APV-B, N ORF), AF262571(APV-C, M ORF), U37586(APV-B, M ORF), X58639(APV-A, MORF), AF176591(APV-C, P ORF), AF325443(APV-B, P ORF), U22110(APV-A, P ORF), AF187152(APV-C, F ORF), Y14292(APV-B, FORF), D00850(APV-A, F ORF), AF 592(APV-C, M2 ORF), AF35650(APV-B, M2 ORF), X63408(APV-A, M2), U65V-A, LOV-6585 (APV-A, SH 40185).

Table 5: the length of the ORF of MPV and other paramyxoviruses.

Footnotes:

1. the length of the amino acid residues.

2. Failure to obtain a sequence

3. And (3) the other: human parainfluenza virus types 2 and 3, sendai virus, measles virus, nipah virus, phonine distemper virus, and newcastle disease virus.

Absence of ORF in the viral genome

Table 6: amino acid sequence identity between the ORF of MPV and the ORFs of other paramyxoviruses¹

1.No sequence homology was found with the known G and SH proteins and was therefore excluded.

2. No sequence was obtained.

3. See footnote 3 of the list in table 5.

ORF is absent from the viral genome.

Reference to the literature

Current Protocols in Molecular Biology, volume 1-3 (1994-1998). Ausubel, f.m., Brent, r., Kinston, r.e., Moore, d.d., Seidman, j.g., Smith, j.a., and strhl, k. eds., John Wiley and sons, inc., USA.

Current Protocols in immunology, Volnme 1-3.Coligan, J.E., Kruisbeam, A.M., Margulies, D.H., Shevach, E.M., and Strobe, eds, W., eds, John Wiley and sons, Inc., USA.

Sambrook et al, Molecular cloning, a Laboratory manua1, second ed., vol.1-3 (Cold Spring Harbor Laboratory, 1989).

Fields, virology.1996. Vol.l.23 rd.edition, Fields, B.N., Knipe, D.M. and Howley, p.M. eds., Lippi1lcott-Raven, Philadelpia, USA.

1.Pringle，C.R.Virus taxonomy at the Xith international congess ofvirology，Sydney，Australia 1999.Arch.Virol，144/2，2065-2070(1999)。

Domachowsk, j.b. and Rosenberg, h.f. respiratory synthetic viral infection: immune response, immunopathogenesis, and treatment, Clin.Microbio.Rev.12(2), 298 (1999). For an overview.

3, Gitaud, p., Bennejean, g., Guittet, m. and Toquin, d.turkeyhirogastris in France: predimine involvement on a ciriostic virus (turkey rhinotracheitis, France: preliminary study on ciriostic virus). Vef.Rec.119,606-607 (1986).

Ling, R., Easton, A.J. and Pringle, C.R.sequence analysis of the22K, SH and G genes of turkey rhinotracheitis virus and the intergenic regions of the genes a gene order from the tha of other pneumocoviruses, J.Gen.Virol.73, 1709-1715 (1992).

Yu, Q.S., Davis, P.J., Li, J.and Cavanagh, D.cloning and sequencing of the matrix protein (M) gene of turkey rhinotracheitis virus substrate protein (M) gene from gene of respiratory syncytial virus, Virology 186, 426-434 (1992).

Randhawa, J.S., Marriott, A.C., Pringle, C.R., and Easton, A.J.Rescue of synthetic miniature mutants of the sensitivity of the NS1 and the NS2genes from avian pneumovirus (rescue of synthetic minireplicons established that avian pneumoviruses lack NS1 and NS2 genes). J.Virol.71, 9849-.

Evans, A.S.: viral Infections of human. epidemiology and control.3th edn. (Evans, A.S eds.) 22-28(Plenum Publishing Corporation, New York, 1989).

Osterhaus, A.D.M.E., Yang, H, Spijkers, H.E.M., Groen, J., Teppema, J.S., and van Steenis, G.the isolation and partial characterization 0f a highlypat 1log 1lic herpes virus from the Harbor Seal (Phoca Vitulina) (isolation and partial characterization of highly pathogenic herpes viruses from the Harbor Seal (Phoca Vitulina)). Of Virol.86,239-251 (1985).

Chua et al, Nipah virus: a inventory essential depolyparamyxovirus (Nipah virus: a newly emerged lethal paramyxovirus) Science 288, 1432-.

Welsh, J., Chada, K., Dalal, S.S., Cheng, R., Ralph, D, and McClelland, M.arbitrarly printed PCR fingerprinting of RNA (arbitrary primer PCR fingerprinting of RNA). NAR.20, 4965-4970 (1992).

Bayon-Auboyer, M., Arnauld, C., Toquin, D, and Eterradossi, N.nucleotide sequences of the F, L and G protein genes of two non-A/non-B Avian Pneumoviruses (APV) whose nucleotide sequences F, L and G protein genes reveal a new APV subgroup, J.of Gen.Virol.81, 2723-2733 (2000).

Mulder, J. and Masurel, N.Pre-epidemic antibody against 1957strain of an antigenic antifluenza in serum of an alcoholic dosage in The Netherlands (epidemic antibodies against The 1957 Asian influenza strain in The serum of elderly living in The Netherlands). Lancet april19,810 and 814 (1958).

Pringle, C.R., The Paramyxovfruses.1th edn. (D.W.Kingsbury) 1-39(Plenum Press, New York, 1991).

Rothbarth, p.h., Groen, j., Bohnen, a.m., Groot, de r, and osteerhaus, a.d.m.e.infiluenza virus seriology-a comparative study, j.of viro.methods 78, 163-plus 169 (1999).

Braildenburg, A.H., Groen, J., Van Steense1-Moll, H.A., Claas, EIJ.C., Rothbarth, P.H., Neijens, H.J., and Osterhaus, A.D.M.E.Respirarynyrykinetic drugs in antigens units of six months of: limited diagnostic response on infection (respiratory syncytial virus specific serum antibodies in 6 month infants: limited serum response upon infection.) J Med. Virol.52, 97-104 (1997).

Lennette, D.A. et al, Diagnostic, procedures for viral, rickettsial and chlamydial, infections.7th edn. (Lennette, E.H., Lennette, D.pill and Lennette, E.T. eds.) 3-25; 37-138; 431-463; 481-; 539-563(American public health association, Washington, 1995).

15.Felsenstein，J.Department of Genetics，Universtity of Washington.Http：//evolution.genetics.washington.edu/phylip.html

Schnell et al EMBO J13, 4195-

Collins, P.L, Hill, M.G., Camargo, E., Grosfe1d, H., Chanock, R.M. and Murphy, B.R.Production of infectious human respiratory disease from the 5 'promoter expression frame of the M2 mRNn gene expression and expression a capacity for vaccine development [ production of infectious human respiratory syncytial virus from cloned cDNA confirms the important role of the transcriptional elongation factor from the M2mRNA 5' proximal open reading frame in gene expression and provides the capacity for vaccine development ] PNAS 92, 11563 (1995).

Hoffinan, E., Neunlan, G., Kawakao, Y., Hobom, G., and Webster, R.G.A DNA transfection system for generation of influenza virus (DNA transfection system for influenza virus production from 8 plasmids). PNAS 97, 6108-A6113 (2000).

Bridge, A., E11iot, R.M. research of a segmented negative-strand viral entry from cloned complete DNAs, PNAS 93, 15400-.

Palese, P., Zheng, h., Engemardt, o.g., P1eschka, s. and Garcia-Sastre, a.negative-strand RNA viruses; genetic engineering and applications PNAS 93, 11354-11358 (1996).

Peeters, b.p., de leew, o.s., Koch, g., and gilkens, a.l. research of new cast disease virus from bound cDNA: evidence of the cleavage ability of the fusion protein a. major or a tertiary for virus (rescue of Newcastle disease virus from cloned cDNA: evidence that the cleavage ability of the fusion protein is a major determinant of virulence) J.Virol' 73,5001-5009 (1999).

Durbin, a.p., Hall, s.l, Siew, j.w., Whitehead, s.s., Collins, P.L and Murphy, b.r.recovery of infectious human parainfluenza virus type3 from cDNA (recovery of infectious human parainfluenza virus type3 from cDNA). virology235,323-332 (1997).

Tao, T.T., Durbin, A.P., Whitehead, S.S., Davoodi, F.E., Collins, P.L and Murphy, B.R.Recovery of a full viable polymeric human parainfluenza virus (PIV) type3 in which hemagglutinin-neuraminidase and fusion glycoprotein have been replaced by hemagglutinin-neuraminidase and fusion glycoprotein active replaced by a fully chimeric human parainfluenza virus (PIV) type3 recovery J.Virol.72, 2955 (1998).

Durbin, A.P., Skodopoulos, M.H., McAuliffe, J.M., Riggs, J.M., Surman, S.R., Collins, P.L., and Murphy, B.R. human parainfluenza virus 3(PIV3) expresses the hemagglutinin protein of measles virus a potential method for immunization against measles virus and PIV3 in early infarnation (PIV3) provides a potential method for immunization against measles virus and PIV3 in infants J.Virol.74, J.683.1 (PIV 683 2000).

Skoadopoulos, M.H., Durbin, A.P., Tatem, J.M., Wu, S.L., Paschalis, M., Tao, T, Collins, P.L., and Murp1ly, B.R.Three amino acid dependencies in the L protein of the human parainfluenza virus type3 cp45live affected Vaccine composition to regulatory site-sensitive activity phenotypes (three amino acid substitutions in the L protein of human parainfluenza virus type3 cp45 attenuated live Vaccine candidate result in its temperature sensitive and attenuated phenotype). J.virol.72, 2 (1998).

Teng, n., Whitehead, s.s., Bermingham, a., st.claire, m., Elkins, w.r., Murphy, b.r., and Collins, p.l.j.virol.74, 9317-.

Masurcl, N.relation between The parent Hong Kbng viruses and The former human A2 isolates and The A/EQU12 viruses in human serum collected before 1957 collected under The collected before The collected after 1957 collected under The collected before May 3,907- "1959".

Other references used in example 4.

AHMADAN, G., CHAMBERS, P., and EASTON, A.J. (1999) Detection and characterization of protein encoded by the second of the second 0RF of the M2 gene of pneumoviruses J Gen Virol 80, 2011-6.

ALANSARI, H. and POTGIETER, L.N. (1994). Molecular cloning and analysis of the phosphoprotein, nucleomapped protein, the matrixprotein and 22K (M2) protein of the viral respiratory syncytial virus (Molecular cloning and sequence analysis of the phosphoprotein, nucleocapsid protein, matrix protein and 22K (M2) protein). J Gen, 75, 3597-.

BARR, J., CHAMBERS, P., PRINGLE, C.R., and EASTON, A.J. (1991) Sequence of the major nuclear associated protein gene of microorganism: sequence compositions supplement structural homology between nucleocapsid proteins of pneumoviruses, paramyxoviruses, rhabdovimses and filoviruses (sequence of major nucleocapsid proteins of murine pneumoviruses: sequence comparison suggests structural homology between nucleocapsid proteins of pneumoviruses, paramyxoviruses, rhabdoviruses and filoviruses). J Gen Virol 72,677-85.

BAYBUTT, H.N. and PRINGLE, C.R. (1987) Molecular cloning and sequencing of the F and 22K membrane protein genes of the RSS-2 strain of respiratory syncytial virus J Gen Virol68, 2789-96.

BAYON-AUTOYER, M.H., ARNAULD, C., TOQUIN, D, and ETERRADOSSI, N. (2000) Nucleotide sequences of the F, L and Gprotein genes of two non-A/non-B Avian Pneumoviruses (APV) F, L of two non-A/non-B Avian Pneumoviruses (APV) and the Nucleotide sequence of the G protein gene reveal a new APV subgroup, J Gen Virol81, 2723-33.

BERMINGHAM, A. and COLLINS, P.L. (1999) The M2-2 protein of human respiratory syncytial virus a regulatory factor involved in The balance between RNA replication and transcription Proc NatlAcad Sci U S A96, 11259-64.

BLUIVlBERG, b.m., CHAN, j. and UDEM, s.a. (1991), Ftraction of paramyxovirus 3 'and 5' end sequences: in therory and practice (function of paramyxovirus 3 'and 5' end sequences: theory and practice) is described In "the Paramyxoviruses" (D.Kingsbury), pp.235-247.Plerlnm, New York.

COLLINS, P.L and WERTZ, G.W. (1983). cDNA cloning and mapping 1 mapping of the encoded poly-adenylated RNAs by the genome of human respiratory syncytial virus. Proc Notl Acadsi U S A80.3208-12.

COLLINS, P.L and WERTZ, G.W. (1985) The envelope-associated22K protein of human respiratory synthetic virus: nucleotide sequence of the mRNA and a related polytript (envelope-associated 22K protein of human respiratory syncytial virus: nucleotide sequence of mRNA and related multiple transcripts) J Virol 54, 65-71.

COLLINS, P.L., DICKENS, L.E., BUCKLER-WI-IITE, A., OLMSTED, R.A., SPRIGGS, M.K., CAMARGO, E.and COELINGH, K.V.W. (1986) Nucleotide sequences for the gene junctions of human respiratory syncytial virus specific reactive defects of interactive stmctural gene orders (Nucleotide sequences of human respiratory syncytial virus gene junctions reveal unique properties of intergenic structure and gene order). Proc Natl Acad Sci USA 83, 4594-98.

COLLINS, P.L., HILL, M.G., and JOHNSON, P.R. (1990) The twOopen reading frames of The22K mRNA of human respiratory synthetic viruses: sequence composition of antigenic subgroups A and B and expression invitro (two open reading frames of the22K mRNA of human respiratory syncytial virus: sequence comparison and in vitro expression of antigenic subgroups A and B). J Gen Virol, 71, 3015-20.

COLLINS, p.l., HILL, m.g., camrgo, e., grosfed, h., chanck, r.m., and MURPHY, b.r. (1995) Production of infectious output responsive synthesis 1 from a cloned cDNA conjugate for the transformation of expression factor from the 5 'promoter region of the M2mRNA in expression of the M2 gene expression and the protein a capacity expression vector Production of infectious human respiratory syncytial virus from a cloned cDNA confirms the important role of the transcription elongation factor derived from the 5' proximal open reading frame of M2mRNA in gene expression and provides the capability of vaccine development, procisc scientific 92, a 7-a 1157.

COLLINS, P.L., MCINTOSH, K.AND CHANOCK, R.M. (1996) "Respiratory synthetic video," by: fields virology (b.n. knepe, Howley, p.m. eds.) Lippencott-Raven, philiadelphia.

COOK, J.K. (2000). Avian rhinotracheitis. Rev SciTech 19,602-13.

CLIESTA, I., GENG, X., ASENJO, A. and VILLANUEVA, N. (2000). Stmctural phosphoprotein M2-1 of the human respiratory syncytial virus RNA binding protein (the structural phosphoprotein M2-1 of human respiratory syncytial virus is an RNA-binding protein). J.Gen.Virol 74, 9858-67.

CURRAN, j. and KOLAKOFSKY, D. (1999) Replication of paramyxoviruses, adv. virus res.50, 403-422.

EASTON, A.J. and CHAMBERS, P. (1997). Nucleotide sequence of viruses encoding the matrix and minor 1l hydrophic proteins of pneumoconivirus of microorganisms (Nucleotide sequences of genes encoding matrix and hydrophobic proteins of mice). Virus Res 48, 27-33.

ELANGO, n., SATAKE, m. and VENKATESAN, s. (1985) tuRNAsequence of three respiratory syncytial virus genes encoding two non-structural proteins and one 22K structural protein jjirol 55,101-10.

FEARNS, R. and COLLINS, P.L (1999). Role of the M2-1transcription initiation protein of respiratory syncytial virus infection transcription (Role of M2-1transcription anti-terminator protein of respiratory syncytial virus in continuous transcription). J Virol 73, 5852-64.

FELSENSTEIN，J.(1989).”PHYL'-Phylogeny Inference Package(Version 3.2.Cladistics 5).”。

GIRAUD, p., BENNEJEAN, g., GUITTET, m., and TOQUIN, d. (1986) turnkey rhizotheracheitis in France: preliminary infectious agents on opportunistic viruses (turkey rhinotracheitis, France: preliminary study on the ciliostatic virus) Vet Rec 119,606-7.

Hall, T.A, (1999). a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT (Bio-editor: a user-friendly program for biological sequence alignment editing and analysis for Windows 95/98/NT.) Nucl.

HARDY, R.W. and WERTZ, G.W. (1998) The product of therapeutic synthetic virus M2 gene ORF1 genes readfrom Viral junction Viral transcription (The product of respiratory syncytial virus M2 gene ORF1 enhances The readability of The junctions between genes during Viral transcription). J Virol 72,520-6.

HORVATH, c.m. and LAMB, r.a. (1992) Studies on the fusion peptide of a paramyxvirus fusion protein: rolls of conserved residues cell fusion (investigation of fusion peptides of paramyxovirus fusion glycoproteins: role of conserved residues in cell fusion). J Virol 66, 2443-55.

JENTOFT, N. (1990). Why area proteins O-glycosylated? (why is the protein O-glycosylated.

JOINSON, P.R., JR., OLMSTED, R.A., PRINCE' G.A., MURPHIT, B.R., ALLING, D.W., WALSH, E.E., and COLL1NS, P.L. (1987) an adaptor related beta carbohydrate copolymers of human respiratory viruses subgroups A and B; evaluation of the associations of F and G glycoproteins to immunity (antigenic correlation between glycoproteins from subgroups A and B of human respiratory syncytial viruses: evaluation of the immune contribution of the F and G glycoproteins). J Virol 61, 3163-6.

JUHASZ, K. and EASTON, A.J. (1994). Extensive sequence variation of the attachment (G) protein gene of average plasmid: evidence for twodrosistinct subgroups groups (extensive sequence variation in the attachment (G) protein gene of avian pneumovirus: evidence for two different subgroups). J Gen Virol 75, 2873-80

Kyte, J. and Doolittle, R.F. (1982). A Simple Method for Displaying the hydrophic Character of a Protein (a Simple Method for demonstrating the hydrophobic properties of proteins). J mol.biol.157, 105-142.

LAMB, r.a. and KOLAKOFSKY, D. (1996). "Paramyxoviridae; the viruses and the virus replicahon (Paramyxoviridae: viruses and replication thereof) "are described in: fieldsvirology (b.n. knepe, Howley, p.m. eds.) Lippencott-Raven, Phi1 adelphia.

LI, J., LING, R., RANDHAWA, J.S., SHAW, K., DAVIS, P.J., JUHASZ, K., PRINGLE, C.R., EASTON, A.J., and CAVANAGH, D. (1996) Sequence of the nucleo-associated protein gene of avian pneumoviruses of the subgroups A and B.) Virus Res41, 185-91.

LING, R., EASTON, A.J. and PRINGLE, C.R. (1992) sequence analysis of the22K, SH and G genes of turnkey rhinotracheitis virus and the endogenous region of genes a gene order from a gene of other pneumoviruses J Gen Virol, 73, 1709-15.

LING, R., DAVIS, P.J., YU, Q., W00D, C.M., PRINGLE, C.R., CAVANAGH, D. and EASTON, A.J. (1995), Sequence and in vitro expression of the phosphoprotein gene of avian pneumovirus, Virus Res 36,247-57.

MARRIOT, a.c., SMITH, j.m., and EASTON, a. (2001) Fidelity of the use of leader and trailer sequence usage by the respiratory tract synthetic viruses and the trailer viral replication complex j.v. 75, 6265-72.

MINK, M.A., STEC, D.S. and COLLINS, P.L. (1991). Nucleotides sequences of me 3 'leader and 5' trailer regions of human respiratory syncytial viral genomic RNA (nucleotide sequence of the 3 'leader and 5' trailer regions of human respiratory syncytial viral genomic RNA.) Virology 185,615-24.

MIYAHARA, K., KITADA, S., YoshiMOT0, M., MATSUMURA, H., KAWAN0, M., KOMADA, H., TSURUDOME, M., KUSAGAWA, S., NISHIO, M. and ITO, Y. (1992) Molecular evaluation of human parainfluenza viruses, nucleic acid sequences analysis of human parainfluenza virus 1 viruses NP and M protein genes and transformation of human parainfluenza virus 1 genes for the purpose of the human parainfluenza viruses, Arrowol 124,255-3968 is constructed.

MORRISON, t.g. (1988), Structure, function, and intracellular processing of paramyxovims membrane proteins Virus Res 10,113-35.

NAYLOR, C.J., BRITTON, P. and CAVANAGH, D. (1998) Theeoplastic proteins but not the transmembrane domains of the fusion proteins of the human respiratory syncytial viruses A and B avians pulmonary artery conserved to a similar extent as the extracellular domains, but not the transmembrane domains, of the fusion proteins of avian respiratory viruses of the subtypes A and B. J Gen Virol 79, 1393-8.

PLOWS, D.J. and PRINGLE, C.R. (1995) Variation in the fusion glycoprotein gene of human respiratory syncytial Virus subgroup A. Virus Genes 11, 37-45.

POCH, O, BLUMBERG, b.m., bouguerret, L. and TORDO, N. (1990) Sequence composition of fine polymers (L proteins) of unsaturated reactive-strand RNA viruses: the theoretical assignment of functional domains by the 5 polymerases (L proteins) of non-segmented negative-strand RNA viruses).

POCH, o., savaget, i., delaree, m, and TORDO, n. (1989) Identification of 4 conserved motifs in the coding elements of RNA-dependent polymerases.

Randhwa, j.s., MARRIOTT, a.c., PRINGLE, c.r., and EASTON, a.j. (1997) research of synthetic miniature peptides of the present invention of the NS1 and NS2genes from pneumvirus (Rescue of synthetic minireplicons established that avian pneumoviruses lack NS1 and NS2 genes) J Virol 71, 9849-54.

Randdawa, j.s., WILSON, s.d., TOLLEY, k.p., CAVANAGH, d., PR1NGLE, c.r., and EASTON, a.j. (1996) Nucleotide sequence of the gene encoding the viral polymerase of avian pneumovirus J Gen Virol 77, 3047-51.

SAMAL, S.K. and ZAMORA, M. (1991). Nucleotide sequence analysis of the matrix protein of bovine respiratory syncytial virus and the dicistronic sequence dicistronic mRNA of matrix and sma1l hydrophic protein 1l of two citric mRNA of bovine respiratory syncytial virus, 1l hydrophic protein 1l from the same of human respiratory syncytial virus, GenJ Virus 72, 1715-20.

SATAKE, M. and VENKATESAN, S. (1984). Nucleotide sequence of gene encoding respiratory syncytial virus matrix protein J Virol 50, 92-9.

SEAL, b.s., SELLERS, h.s., and MEINERSMANN, R.J (2000) Fusion protein predicted amino acid sequence of the first US avermectin isolate and 1ack of heterologous amplification other US isolates (Fusion protein of the first US avian pneumovirus isolate predicts the amino acid sequence and lack of heterogeneity in other US isolates) Virus Res 66,139-47.

SEDLMEIER, R. and NEUBERT, W.J. (1998) The regenerative complex of paramyxoviruses: stmcture and function (replication complex of paramyxovirus: structure and function). Adv Virus Res 50,101-39.

STEC, D.S., HILL, M.G., 3RD and COLLINS, P.L. (1991) sequencing analysis of the polymerase L gene of human respiratory syncytial virus and amplified phenyl analysis of non-segmented negative strand viruses (sequence analysis of polymerase L gene of human respiratory syncytial virus and predicted phylogeny of non-segmented negative strand viruses). Virology 183,273-87

VAN DEN HOOGEN, b.g., DE JONG, j.c., GROEN, j.j., KUIKEN, t, DE GR00T, r., FOUCI-IIER, r.a., and ostrich, a.d. (2001) Anewly dj schoverred human pneumocovus isolated from young children with respiratory disease Nat et al 7(6), 719-24.

VIRUS TAXONOMY(2000).Seventh report of the internationalCommittee on Taxonomy of Viruses。

WERTZ, G.W., COLLINS, P.L., HUANG, Y., GRUBER, C., LEVINE, S. and BALL, L.A. (1985). Nucleotide sequence of the G protein of human respiratory syncytial virus: 1s an unused type of viral membrane protein, Proc Natl Acad Sci USA 82, 4075-9.

YU, q., DAVIS, p.j., BARRETT, t., BINNS, m.m., bour, snfll, m.e., and CAVANAGH, d. (1991). reduced amino acid sequence of the fusion protein of viral rhinotracheitis virus genome, with human respiratory syndrome virus, a pneumvirus, a tha of paramyxovirus and morbidiviruses (the Deduced amino acid sequence of the fusion glycoprotein of turkey rhinotracheitis virus has higher identity to human respiratory syncytial virus-a pneumovirus than to paramyxovirus and measles virus). J view 72, 75-81.

YU, Q., DAVIS, P.J., LI, J, and CAVANAGH, D. (1992) Cloning and sequencing of the matrix protein (M) gene of turkey rhinotracheitis virus matrix protein (Cloning and sequencing of the genes reveals a different genetic order from that of respiratory syncytial virus). Virology 186,426-34.

ZAMORA, M.and SAMAL, S.K. (1992). Sequence analysis of M2mRNA of a bovine respiratory synthetic virus extracted from an F-M2 diacetstric mRNA deletion structure with a that of human respiratory syncytial virus (Sequence analysis of M2mRNA of bovine respiratory syncytial virus from F-M2 bicistronic mRNA suggests structural homology to human respiratory syncytial virus). J Gen Virus 73,737-41.

Primers for RT-PCR detection of known paramyxoviruses. Primers for hPEV-1 to 4, mumps virus, measles virus, Tupaja virus, Mapuera virus and Hendra virus have been developed internally and are based on alignments of available sequences. Primers for newcastle disease virus were taken from Sea1, j., j, etc.; micro b., 2624. 2630, 1995. Primers for Nipah virus and general paramyxovirus PCR were taken from: chua, k.b. etc.; science, 28826 may2000

Viral primers are located on proteins

HPIV-1 fwd 5’-TGTTGTCGAGACTATCCAA-3’ HN

Rev 5’-TGTTG(T/A)ACCAGTTGCAGTCT-3’

HPIV-2 Fwd 5’-TGCTGCTTCTATTGAGAAACGCC-3’ N

Rev 5’-GGTGAC/T TC(T/C)AATAGGGCCA-3'

HPIV-3 Fwd 5’-CTCGAGGTTGTCAGGATATAG-3’ HN

Rev 5’-CTTTGGGAGTTGAACACAGTT-3'

HPIV-4 Fwd 5’-TTC(A/G)GTTTTAGCTGCTTACG-3’N

Rev 5’-AGGCAAATCTCTGGATAATGC-3'

Fwd 5'-TCGTAACGTCTCGTGACC-3' SH

Rev 5’-GGAGATCTTTCTAGAGTGAG-3’

NDV Fwd 5’-CCTTGGTGAiTCTATCCGIAG.3， F

Rev 5’-CTGCCACTGCTAGTTGiGATAATCC.3’

Tupaia Fwd 5’-GGGCTTCTAAGCGACCCAGATCTTG-3’ N

Rev 5’-GAATTTCCTTATGGACAAGCTCTGTGC-3’

Mapuera Fwd 5’-GGAGCAGGAACTCCAAGACCTGGAG-3’ N

Rev： 5’-GCTCAACCTCATCACATACTAACCC-3’

Hendra Fwa 5’-GAGATGGGCGGGCAAGTGCGGCAACAG-3’ N

Rev 5’-GCCTTTGCAATCAGGATCCAAATTTGGG-3’

Niyah Fwd 5’-CTGCTGCAGTTCAGGAAACATCAG-3’ N

Rev 5’-ACCGGATGTGCTUACAGAACTG-3’

HRSV Fwd 5’-TTTGTTATAGGCATATCATTG-3’ F

Rev 5’-TTAACCAGCAAAGTGTTA-3’

Fangchu Fwd 5'-TTAGGGCAAGAGATGGTAAGG-3' N

Rev 5’-TTATAACAATGATGGAGGG-3’

The general family paramyxoviridae:

positive direction 5'-CATTAAAAAGGGCACAGACGC-3' P

Reverse direction 5'-TGGACATTCTCCGCAGT-3'

Primers for RAP-PCR

ZF1：5'-CCCACCACCAGAGAGAAA-3’

ZF4：5'-ACCACCAGAGAGAAACCC-3’

ZF7：5'-ACCAGAGAGAAACCCACC-3’

ZF10：5’-AGAGAGAAACCCACCACC-3’

ZF13：5'-GAGAAACCCACCACCAGA-3’

ZF16：5'-AAACCCACCACCAGAGAG-3’

CS1：5'-GGAGGCAAGCGAAGGCAA-3’

CS4：5'-GGCAAGCGAACGCAAGGA-3’

CS7：5'-AAGCGAACGCAAGGAGGC-3’

CS10：5'-CGAACGCAAGGAGGCAAG-3’

CS18：5'-ACGCAAGGAGGCAAGCGA-3’

CS16：5’-CAAGGAGGCAAGCGAACG-3’

The 20 fragments were successfully purified and sequenced:

10 fragments of sequence homology found in APV

Fragment 1 ZF 7, 335 bp N gene

Fragment 2 ZF 10, 235 bp N gene

Fragment 3 ZF 10, 800 bp M gene

Fragment 4 CS 1, 1250 bp F gene

Fragment 5 CS 10, 400 bp F gene

Fragment 6 CS 13, 1450 bp F gene

Fragment 7 CS 13, 750 bp F gene

Fragment 8 ZF 4, 780 bp L Gene (protein level)

Fragment 9 ZF 10, 330 bp L Gene (protein level)

Fragment 10 ZF 10, 250 bp L Gene (protein level)

Primers for amplifying nucleic acids from prototype isolate RAP-PCR.

Example 5

Further exploration of the two subtypes of hMPV

Based on phylogenetic analysis of different isolates of hMPV obtained to date, two genotypes were identified, viral isolate 00-1 being the prototype of genotype A, and isolate 99-1 being the prototype of genotype B.

We hypothesize that the genotypes are associated with subtypes and that reinfection with viruses from both subgroups occurs with prior immunization, and that antigenic variation may not be strictly necessary to allow reinfection.

Furthermore, it appears that hMPV is closely related to an avian pneumovirus, which is mainly found in poultry. The nucleotide sequences of these two viruses show a high percentage of homology, with the exception of the SH and G proteins. Here we show that the viruses cross-react in assays based primarily on nucleoprotein and matrix protein, but that they react differently in assays based on adsorbed protein. The difference in virus neutralization titers further provides evidence that the two genotypes of hMPV are two different serotypes of one virus, while APV is a different virus.

Cross-reactivity between the two serotypes and cross-reactivity between APV and hMPV Method of producing a composite material

Protocol for detection of hMPV with IgG, IgA, and IgM antibodies:

peothbarth, p.h. et al, 1999; influnzavirus serigraphy-a comparative study. J.of vir. Memods 78(1999)163-169), indirect IgG EIA to hMPV was performed in microtiter plates.

Briefly, concentrated hMPV was solubilized by treatment with 1% Triton X-100, which was coated into microtiter plates at room temperature for 16 hours in PBS after determination of optimal working dilution by checkerboard titration (checkerbard titration). Subsequently, a volume of 100 μ l in EIA buffer at 1: 100 diluted human serum samples were incubated at 37 ℃ for 1 hour. Binding of human IgG was detected by addition of goat anti-human IgG peroxidase conjugate (Biosource, USA). TMB was added as a substrate, the plate was developed, and OD at 450nm was measured. The results are expressed as the ratio of S (i.e., signal)/N (i.e., negative) of OD. Sera were considered IgG positive if the S/N ratio exceeded the negative control plus three times the standard.

hMPV antibodies of the IgM and IgA classes in serum were detected by capturing EIA (Rothbarth, P.H et al, 1999; Influenza virus sero1gy-a comparative study. j. vir. methods78 (1999)163-169) essentially as described previously. For detection of IgA and IgM, commercially available microtiter plates coated with monoclonal antibodies specific to anti-human IgM or IgA were used. After incubation at 37 ℃ for 1 hour, sera were incubated at 1: 100 dilutions and the optimal working dilution of hMPV (100. mu.l) were added to each well. Incubate at 37 ℃ for 1 hour. After washing, polyclonal anti-hMPV, labeled with peroxidase, was added and the plates were incubated at 37 ℃ for 1 hour. The plate was developed by adding TMB as a substrate, and OD at 450nm was measured, and the result was expressed as a ratio of S (i.e., signal)/N (i.e., negative) of 0D. Sera were considered IgG positive if the S/N ratio exceeded the negative control plus three times the standard.

APV antibodies were detected using an APV inhibition assay. Protocols for APV inhibition assays include APV-Ab SVANOVIRAn enzyme immunoassay produced by SVANOVA Biotech AB (Uppsal Science Park Glunten SE-75183 Uppsala Swedeh). The results are expressed as the ratio of S (i.e., signal)/N (i.e., negative) of OD. Sera were considered IgG positive if the S/N ratio exceeded the negative control plus three times the standard.

1. Guinea pig

A.(Re-) infection of guinea pigs with two hMPV subtypes

6 guinea pigs (guineapig) of each subtype (intratracheal, nasal and ocular) have been inoculated with viral isolates ned/00/01 (subtype A) and ned/99/01 (subtype B).

Infection of 6 GP with hMPV 00-1(10e6, 5 TCD50)

6 GP was infected with hMPV 99-1(10e4, 1 TCD50)

54 days after primary infection, the guinea pigs were vaccinated with the same subtype and a different subtype (10e4TCID50/m 1):

2 guinea pigs: first infection 00-1; infection 99-1 (xenogeneic)

3 guinea pigs: first infection 00-1; infection for the second time 00-1 (same race)

2 guinea pigs: first infection 99-1; infection for the second time 00-1 (xenogeneic)

3 guinea pigs: first infection 99-1; a second infection 99-1 (allogenic),

throat and nasal swabs were collected either 12 days (first infection) or 8 days (second infection) after infection and tested for the presence of the virus as determined by RT-PCR.

Results of RT-PCR assay:FIG. 29

The results are summarized as follows: guinea pigs vaccinated with the virus isolate ned/oo/01 showed upper respiratory tract infections 1-10 days post infection. Guinea pigs vaccinated with the virus isolate ned/99/01 showed upper respiratory tract infections on days 1-5 post infection. It appears that the severity of infection with ned/99/01 is lower than with ne para 00/01. The second vaccination of the guinea pigs with the xenovirus caused reinfection in 3 out of 4 guinea pigs, whereas the second vaccination with the allovirus caused reinfection in 2 out of 6 guinea pigs. Note that there are no or few clinical symptoms in those animals that were reinfected, and no clinical symptoms were observed in those animals that were protected against the reinfection, demonstrating that the protective effect of the first infection is evident even with wild-type virus, suggesting that it is possible to use xenogenic (and certainly allogeneic) isolates as vaccines, and even isolates that may be in non-attenuated form.

Both subtypes of hMPV are able to infect guinea pigs, although it appears that infection with subtype B (ned/99/01) is less severe (the time of viral presence in the nose and throat is short) than with subtype a (ned/oo/01). This may be due to the higher dose given by subtype a, or the lower virulence of subtype B.

Although the presence of prior immunity does not completely protect against re-infection by both alloviruses and xenoviruses, the infection appears to be less pronounced as it is noted that the virus is present for a shorter period of time and not all animals become virus positive.

B. Serology of guinea pigs infected with two hMPV subtypes

On days 0, 52, 70, 80, 90, 110, 126 and 160, sera were collected from guinea pigs at a rate of 1: 100, tested in a whole virus ELISA for the ned/00/01 and ned/99/01 antigens.

FIGS. 30A and B: IgG response in each guinea pig to ned/00/01 and ned/99/01

FIG. 31: the specificity of the ned/00/01 and ned/99/01 ELISAs. Only data from the same re-infected guinea pigs were used.

FIG. 32: average IgG responses against the ned/00/01 and ned/99/01 ELISAs of 3 congeners (00-1/00-1), 2 congeners (99-1/99-1), 2 xenogeneic (99-1/00-1) and 2 xenogeneic (00-1/99-1) infected guinea pigs.

Summarizing the results;

only minor differences were observed in the response to the two different EUSAs. The whole virus EUSA against 00-1 or 99-1 cannot be used to distinguish the two subtypes.

C. The Dai meat has toxic effect on the response of the Xinhao and APV antigens to the hviVLuben

Sera collected from the infected guinea pigs have been tested using an APV inhibition ELISA.

FIG. 33(ii) a Average APV inhibition in H-footed V infected guinea pigsAnd (4) percent.

The results are summarized as follows:

guinea pig sera raised against hMPV reacted in the same way in the APV inhibition assay as they did in the hMPV IgG ELISA.

Sera generated against ned/99/01 showed a lower percent inhibition in the APV inhibition ELISA than sera generated against ned/00/01. Titers of the guinea pigs infected with ned/99/01 may be lower (as seen in the hMPV ELISA), or the cross-reaction of ne mountain 99/01 with APV was lower than that of ned/00/01. However, APV-Ab inhibition ELISA can be used to detect guinea pig hMPV antibodies.

D. Virus neutralization assays with guinea pig sera raised against hMPV.

Sera collected at day 0, 52, 70 and 80 post-infection were used in the virus (cross) neutralization assay with ned/00/01, ned/99/01 and APV-C. The initial dilution was 1-10, 100 TCD50 virus per well. After neutralization, the virus was attached to tMK cells, centrifuged at 3500 RPM for 15 minutes, and the medium was changed.

The APV test was grown for 4 days, while the cardiac rhythm V test was grown for 7 days. Cells were fixed with 80% acetone and positive A was performed with FITC-labeled monkey anti-hMPV. Wells that stained negative were considered to be neutralizing titers. For each virus, 10-10g serial dilutions of the virus stock (transcription) and 2-fold serial dilutions of the working solution (transcription) were included.

FIG. 34: viral neutralization titers against ned/00/01, ned/99/01 and APV-C of ned/00/01 and ned/99/01 infected guinea pigs

2. Rhesus monkey

A. (re) infection of rhesus monkeys with two hMPV subtypes f

2 rhesus monkeys (endotracheal, nasal and ocular) were inoculated with each subtype of virus isolates ned/00/01 (subtype A) and ned/99/01 (subtype B) (1 ℃ 5 TCD 50). The rhesus monkeys were vaccinated a second time with ned/00/01 6 months after the first infection. After infection, 14 days (first infection) or 8 days (second infection) laryngeal wipes were collected and tested for the presence of the virus by RT-PCR assay.

FIG. 35 is a schematic view of a: RT-PCR assay results on throat swabs of rhesus monkeys inoculated with ned/00/01 (twice).

The results are summarized as follows:

rhesus monkeys inoculated with the viral isolate ned/00/01 showed upper respiratory tract infections 1-10 days post infection. Clinical symptoms include rhinitis purulenta. The rhesus monkeys were inoculated with the same virus a second time and confirmed by PCR to cause reinfection, however no clinical symptoms were observed.

B. Serology of sera collected from hMPV infected rhesus monkeys.

Sera were collected from rhesus monkeys receiving ned/00/01 during the 6 month period after the first infection (reinfection occurred on day 240 for monkey 3 and on day 239 for monkey 6).

Serum was used to test for the presence of IgG antibodies to either ned/00/01 or APV, as well as IgA and IgM antibodies to ned/00/01.

As a result:FIG. 36A

IgA, IgM and IgG responses to ned/11/01 in 2 rhesus monkeys (re) infected with ned/0/0 l.

FIG. 36B

IgG response to APV in 2 rhesus monkeys infected with ned/00/01.

The results are summarized as follows:

2 rhesus monkeys have been successfully infected with ned/00/01 and reinfected with the same virus in the presence of anti-ned/00/01 antibody. IgA and IgM antibody responses showed an increase in IgM antibodies after the first infection, and no increase in IgM antibodies after reinfection. IgA antibodies were detectable only after reinfection, indicating the immediacy of the immune response after the first infection. Sera from rhesus monkeys raised against h-footed V showed a similar response to the flank four V IgG EUSA when tested in the APV inhibition ELISA.

Test porcelain/Xiao' an Rong

The sensitivity of detection of rhesus monkey hMPV antibodies by APV inhibition ELISA is similar to that using hMPV EUSA, therefore, APV inhibition ELISA is suitable for testing human samples for the presence of hMPV antibodies.

C. Virus (Cross-) neutralization assay using sera from the hMPV-infected rhesus castanea

The results are summarized as follows: sera collected from day 0 to day 229 after the first infection showed only low virus neutralization titers against ned/00/01 (0-80), and sera collected after the second infection showed high neutralization titers against ned/00/01: 1280. Sera collected only after the second infection showed neutralizing titers against ned/99/01 (80-640), with none neutralizing APV C virus.

In the virus (cross) neutralization assay, there was no cross-reaction between APV-C and hMPV; whereas there was a cross-reaction between ned/00/01 and ned/99/01 after a boost of the antibody response.

3. Human being

Sera from patients less than 6 months to greater than 20 years of age, have been previously tested for ned/00/01 in both IFA assays and virus neutralization assays (see table 1 of the patent).

Here we have tested many of these sera for the presence of IgG, Igm and IgA antibodies using ELISA for ned/00/01, and we tested the samples using APV inhibition ELISA.

And (6) obtaining the result.FIG. 37Detection of IgG antibody in human serum by using bMPV ELISA and APV inhibition ELISAIn comparison, there is a strong correlation between the IgG hMPV assay and the APV-Ab assay, such that the APV-Ab assay is substantially capable of detecting anti-hMPV IgG antibodies in humans.

4. Poultry raising

96 chickens were tested for the presence of IgG antibodies to APV using an APV inhibition ELISA as well as the ned/00/01 ELISA.

The results are summarized as follows: antibodies to APV were detected by both hMPV ELISA and APV inhibition ELISA (data not shown).

Summary of the results

We found two genotypes of bMPV, ned/00/01 being the prototype of subgroup A, and ned/99/01 being the prototype of subgroup B.

"two subtypes can be determined according to the immunological distinction (stictionalness) determined by quantitative neutralization assays with animal antisera according to classical serological analyses (e.g. known Francki, R.I.B., Fauquet, C.M., Knudson, D.L and Brown, F., Classification and nomenclature of viruses. Fifth report of the International Committee on Taxolomy of viruses. Arch Virol, 1991.Supplement 2: p.140-144). Two different serotypes either do not cross-react with each other or show a ratio of homological to xenogenic titers of > 16 in both directions. If neutralization indicates a degree of cross-reactivity between the two viruses in either or both directions (ratio of homologous to heterologous titers of 8 or 16), a serological distinction is presumed if there is a significant biophysical/biochemical difference in the DNA. If neutralization indicates a different degree of cross-reactivity between the two viruses in either or both directions (ratio of homologous to heterologous titres less than 8), it is assumed that the serotypes of the isolate under study are identical. "

For RSV, reinfection (allogenic as well as xenogenic) is known to occur in the presence of prior immunity. Infection of guinea pigs and rhesus monkeys with hMPV of the same serotype and of different serotypes indicates that this is also the case for hMPV. In addition, IgA and IgM ELISAs against hMPV revealed that IgA antibody responses occurred only after reinfection. Sera raised against hMPV or APV reacted in the same way in APV ELISA and hMPV ELISA. Based on the comparison of nucleotide sequences, the virus was known to show about 80% amino acid sequence homology to N, P, M and the F gene. In ELISA, N and M proteins are the main antigens that elicit a response. Virus neutralization assays (known to react against surface glycoproteins G, SH and F) indicated differences between the two different sera. Although APV and hMPV cross-react in ELISA, phylogenetic analysis of the nucleotide sequences of hMPV and APV, differences in virus neutralization titers of sera generated against the two different viruses, and differences in host usage again suggest that APV-C and hMPV are two different viruses. Based on the results, we speculate that hMPV infection in mammals may be the result of an infectious disease event in animals from birds to mammals. However, the virus has been adapted in such a way (i.e. G-protein and SH-protein) that it appears possible to revert (from mammalian to avian) infectious disease events in animals, taking into account the presence of APV in birds.

Appendix

Background information of pneumoviruses

The Paramyxoviridae (Paramyxoviridae) family has two subfamilies: paramyxovirinae (Paramyxovirinae) and Pneumovirinae (Pneumovirinae). The pneumovirinae consists of two genera: pneumovirus (Pneumovirus) and Metapneumovirus. Pneumovirus belongs to human, bovine, ovine and caprine respiratory syncytial virus and mouse Pneumonia Virus (PVM). Metapneumovirus has avian pneumovirus (APV, also known as TRTV).

The classification of genera in the pneumovirinae is based on classical viral characteristics, gene order and gene conformation. Pneumovirus viruses are unique in the Paramyxoviridae family because of the two non-structural proteins at the 3' end of the genome (3 ' -NS1-NS2-N-P-M-SH-G-F-M2-L-5 '). In contrast, Metapneumovirus lacks NS1 and NS2genes, and the genetic organization differs between the M coding region and the L coding region: 3 '-N-P-M-F-M2-SH-G-L-5'.

All members of the sub-family paramyxoviridae have hemagglutinating activity, but this function is not a limiting feature of the sub-family pneumovirinae, and is absent in RSV and APV, but is present in PMV. Neuraminidase activity is found in members of the genera Paramyxovirus (Paramyxovirus) and mumps virus (rubulavirus) (paramyxoviridae), but not in Morbillivirus (paramyxoviridae) and pneumovirus and Metapneumovirus (pneumovirinae).

The second identifying feature of the pneumovirinae is the limited use of RSV for the appearance of variable ORFs within mRNA. In contrast, several members of the subfamily Paramyxovirinae, such as Sendai virus and measles virus, can use a variable ORF encoding a phosphoprotein (P) in the mRNA to direct the synthesis of new proteins.

The G protein of Pneumovirinae has no sequence correlation or structural similarity with the HN protein or H protein of Paramyxoviridae, and is only about half of its chain length. In addition, both the N and P proteins are smaller than their counterparts of the subfamily paramyxoviridae and lack clear sequence homology. Most non-segmented negative-strand RNA viruses have a matrix (M) protein. Members of the Pneumovirinae subfamily are exceptional in that they have two such proteins-M and M2. The M protein is smaller than its paramyxoviridae counterpart and has no sequence association with paramyxoviridae.

Members of the Pneumovirinae subfamily exhibit typical cytopathic effects when grown in cell culture; they induce the characteristic syncytia formation of cells (Col [ ms, 1996).

Pneumovirinae, Pneumovirus

hRSV is a representative species of the pneumovirus genus and is the cause of a major and widespread lower respiratory tract disease during infancy and childhood (Selwyn, 1990). In addition, hRSV is increasingly recognized as an important pathogen in other patient populations, including immunocompromised individuals and the elderly. RSV is also an important cause of community-acquired pneumonia in adults of all ages (Eh shund, 1991, Falsey, 2000; Dowell, 1996). Two major antigenic types (A and B) of RSV have been identified based on reactivity with monoclonal and polyclonal antibodies and nucleic acid sequence analysis (Anderson, 1985; Johnson, 1987; Sullender, 2000). In particular, the two subtypes are distinguished by the G protein. RSV-A and B have only 53% amino acid sequence homology in G, while other proteins show higher homology between the subtypes (Table 1) (Collins, 1996).

The use of monoclonal and polyclonal antibodies to detect RSV infection in immunofluorescence techniques (DIF, IFA), virus neutralization assays, and ELISA or RT-PCR assays has been described (Rothbarth, 1988; Van Milaan, 1994; Coggins, 1998). Closely related to hRSV are bovine rsv (rsv), ovine rsv (rsv), and caprine rsv (rsv), with bRSV being the most widely studied. Ruminant RSV is classified in the Pneumovirus genus of the Pneumovirinae subfamily based on sequence homology to hRSV (Collins, 1996). The diagnosis and subtyping of RSV infection in ruminants is based on the combined use of serology, antigen detection, virus isolation and RT-PCR assays (Uttentha1, 1996; Valarocher, 1999; Oberst, 1993; Vilcek, 1994).

Several analyses of the molecular organization of bRSV have been performed with human and bovine antisera, monoclonal antibodies, and cDNA probes. These analyses revealed that the protein composition of hRSV and bRSV are very similar, and the genome organization of bRSV is similar to that of hRSV. For both bRSV and hRSV, the G and F proteins represent the major neutralizing protective antigens. The G protein is highly variable (53% and 28%, respectively) between subtypes of hRSV as well as between hRSV and bRSV (Prozzi, 1997; Lerch, 1990). There are comparable structural features and antigenic correlations of the F proteins of hRSV and bssv strains. The F protein of bRSV shows 80-81% homology to hRSV, while the two hRSV subtypes have 90% homology in F (Walravens, k.1990).

Studies based on the use of hTSV and monoclonal antibodies specific for bvsv have suggested the existence of different antigenic subtypes of bvsv. A. Subtypes B and AB are distinguished by the pattern of response of monoclonal antibodies specific for the G protein (F' urze, 1994; Prozzi, 1997; Elvander, 1998). The epidemiology of bRSV is very similar to that of hRSV. Spontaneous infections in calves are usually associated with severe respiratory signs, whereas experimental infections generally lead to mild disease with slight pathological changes (EIvander, 1996).

RSV has also been isolated from naturally infected sheep (oRSV) (LeaMaster, 1983) and goats (cRSV) (Lehmkuhl, 1980). These two strains share 96% of the nucleotide sequence with bovine RSV and are cross-reactive antigenically. Therefore, these viruses are also classified in the genus pneumovirus.

One particular member of the genus pneumovirus of the subfamily pneumovirinae is the mouse Pneumovirus (PVM).

PVM is a common pathogen in experimental animal populations, particularly those including athymic mice. Naturally acquired muramily infections are considered asymptomatic, with passage of the virus through the mouse lungs, causing significant signs of illness, from upper respiratory infection to lethal pneumonia (Richter, 1988; Weir, 1988).

Limited serological cross-reactivity between nucleocapsid protein (N) and phosphoprotein (P) has been described for PVM and hRSV, but none of the external proteins showed cross-reactivity and the viruses could be distinguished from each other in virus neutralization assays (Chambers, 1990 a; Gimenez, 1984; Ling, 1989 a). The glycoproteins of PVM appear to be different from those of other paramyxoviruses, and similar to those of RSV, with respect to their glycosylation pattern. However, they differ in processing. Unlike RSV, and similar to other paramyxoviruses, PVM has hemagglutination activity with murine erythrocytes and the G protein appears to be responsible for this because monoclonal antibodies directed against this protein inhibit hemagglutination (Ling, 1989 b).

The genome of PVM resembles that of hRSV, includes two nonstructural proteins at its 3' end, and has a similar genomic organization (Chambers, 1990 a; Chambers, 1990 b). The nucleotide sequence of the PVM NS1/NS 2gene has no detectable homology to the nucleotide sequence of the gene of hRSV (ChaInbers, 1991). Certain proteins of PVM show strong homology to hRSV (N; 60%, F: 38-40%), whereas G is significantly different (31% longer amino acid sequence) (Barr, 1991; Barr, 1994; Chambers, 1992). It was reported that the PVMP gene, but not the P gene of RSV or APV, encodes a second ORF, representing a unique PVM protein (Collins, 1996). New PVM isolates were identified by virus isolation, hemagglutination assays, virus neutralization assays and various immunofluorescence techniques.

The table in the appendix: amino acid homology between different viruses within the genus pneumovirus of the subfamily pneumovirinae.

Gene	hRSV	bRSV	oRSV versus hRSV	bRSV versus hRSV	bRSV vs oRSV	PVM vs hRSV
NS1	87			68-69	89	*
							NS2	92			83-84	87	*
N	96		93			60
							P	-		81
M	-		89
							F	89			80-81		38-40
G	53	88-100	21-29	38_41	60-62	*
							M2	92		94			41
SH	76		45-50		56
							L	-

No detectable sequence homology

Metapneumovirus

Avian Pneumovirus (APV) has been identified as the etiological agent of turkey rhinotracheitis (McDougall, 1986; Collins, 1988), and is therefore commonly referred to as turkey rhinotracheitis virus (TRTV). The disease is an upper respiratory infection in turkeys, resulting in high morbidity and variable but often high mortality. In the hens, the virus can also induce a significant drop in egg production. The virus can also infect chickens, but in this species the role of the virus as the main pathogen is certainly not well understood, although it is usually associated with Swollen Head Syndrome (SHS) in breeding hens (Cook, 2000). The virions are polymorphic, although predominantly spherical, in size from 70-600 nm, and the nucleocapsid containing the non-segmented negative-sense RNA genome of rouleau dry moat outside a city wall exhibits helical symmetry (Collins, 1986; Giraud, 1986). This morphology resembles members of the paramyxoviridae family. Analysis of the proteins and RNAs encoded by APV suggests that, of the two subfamilies of this family (Paramyxovirinae and Pneumovirinae), APV most closely resembles the Pneumovirinae (Collins, 1988; Ling, 1988; Cavanagh, 1988).

APV has no non-structural proteins (NS1 and NS2) and the gene sequence (3 '-N-P-M-F-M2-SH-G-L-5') is different from that of mammalian pneumoviruses such as RSV. Thus, APV was recently classified as a representative species of the new genus Metapneumovirus (Pringle, 1999). Differences in neutralization patterns, ELISA and reactivity with monoclonal antibodies have revealed the existence of different APV antigen types. Nucleotide sequencing of the G gene allowed the identification of two virus subtypes (A and B) which share only 38% amino acid homology (Collins, 1993; Juhasz, 1994). An APV isolated from Colorado, USA (Cook, 1999) showed poor cross-neutralization with subtype A and B viruses and was assigned a new subtype-C based on sequence information (Sea1, 1998; Sea 12000). Two non-a/non-B APVs were isolated in france, indicating that they differ antigenically from the A, B and C subtypes. These viruses are further classified into a new subtype, D, based on the amino acid sequences of F, L and the G gene (Bayon-Auboyer, 2000).

Diagnosis of APV infection can be achieved by isolation of the virus in chicken or turkey Tracheal Organ Cultures (TOC) or in Vero cell cultures. Cytopathic effects (CPE) were generally observed after one or two additional passages. This CPE is characterized by a discrete, localized cellular stunning zone that leads to syncytia formation (Buys, 1989). Various serological assays have been developed, including smart' and virus neutralization assays. Detection of antibodies against APV by ELISA is the most commonly used method (O' Loan, 1989; Gulati, 2000). More recently, polymerase chain reaction (PC (5) has been used to diagnose APV infections, swabs taken from the esophagus can be used as starting material (Bayon-Auboyer, 1999; Shin, 2000).

Alansari, H and Potgieter, l.n.d.1994.nucleotide and predictive amino acid sequence analysis of the over respiratory synthetic non-stmctual lC and lB genes and the small hydrophobic protein gene (nucleotide sequence and predicted amino acid sequence analysis of the ovine respiratory syncytial virus non-structural lC and lB genes and small hydrophobic protein genes) j.gen.virol.75: 401-404.

Alansari, h, Duncan r.b., Baker, J.C and Potgieter, l.n.1999.analysis of mammalian respiratory syncytial virus iso1ates by RNAseprotection of the G glycoprotein transcripts (analysis of ruminant respiratory syncytial virus isolates by rnase protection of G glycoprotein transcripts.) j.vet.diagn.invest.11; 215.20

Anderson, L.J, hirerholzer, j.c., Tsou, c., Hendry, r.m., Fernic, b.f., Stone, Y and mcinosh, k.1985. diagnostic chromatography of respiratory syncytial virus strains with monoclonal antibodies (antigenic characterization of respiratory syncytial virus strains with monoclonal antibodies), j.inf.dis.151; 626-633.

Bar, J., Chambers, Pringle, C.R., Easton, A.J.1991.sequence of the major nuclear associated protein gene of pUronia virus of micron: sequence compositions proteins structural homology between nucleocapsid proteins of pneumoviruses, paramyxoviruses, rhabdoviruses and filoviruses (sequence of major nucleocapsid protein genes of murine pneumoviruses: sequence comparison suggests structural homology between nucleocapsid proteins of pneumoviruses, paramyxoviruses, rhabdoviruses and filoviruses). j.gen.virol.72; 677-685.

Bar, J., Chambers, P., Harriott, P., Pringle, C.R and Easton, A.J.1994.sequence of the phosphoprotein gene of pneumoconia virus of microorganism: expression of multiple protein 1ls from two overlapping radiating frames (sequence of phosphoprotein genes of mouse pneumovirus: multiple proteins are expressed from two overlapping reading frames.) J.Virol.68: 5330-5334.

Bayon-Auboyer, M.H., Jestri I1, V., T0q.uiI1, D., Cherbone 1, M and Eterratosi, N.1999.Comparison of F-, G-and N.based RT-PCR protocol protective viral procedures for the detection and typing of turkey rhinotracheitis virus comparing F, G and N based RT-PCR protocols to conventional virology procedures. 1091-1109.

Bayon-autoyer, m.h., Amauld, c., Toquin, d, and etrarossi, n.2000.nucleotide sequences of the F, L and G protein genes of two non-a/non-B Avian Pneumoviruses (APV) temporal a non APV subgroup (nucleotide sequences of F, L and G protein genes of two non-a/non-B Avian Pneumoviruses (APV)) reveal a new APV subgroup j.gen.virol.81: 2723-2733.

Buy, s.b., Du Preez, j.h and Els, h.j.1989.the isolation and attenuation of a virus harboring rhinotracheitis in South african turkey, on der steport.j.ve.res.56: 87.98.

cavanagh, D and Barrett, T.1988. Pneumovrus-like charctehstics, mRNA and proteins of turkey rhinotracheitis virus pneumovirus-like characteristics of mRNA and proteins, Virus Res.11: 241-256.

Chalnbers, p., Prinle, c.r and Easton, a.j.1990a. molecular cloning of pneumoconia virus of mice j.virol.64: 1869-1872.

Chambers, P.P., Matthews, D.A, Pringle, C.R and Easton, A.J.1990b.the nucleotide sequences of endogenous genes of pnuemunonja viruses of the microbial metabolism of the p1 physiological order of the gene in the 1 genes (the nucleotide sequence of the intergenic regions between 9 genes of the murine pneumovirus establishes the physical order of these genes in the viral genome.) Virus Res.18: 263-270.

Chambers, p., prism, c.r and Easton, a.j.1991.genes 1 and 2 of pneumoconia viruses of human coding proteins white salt viruses, with the 1C and 1B proteins of human respiratory syncytial viruses (genes 1 and 2 of mouse pneumoviruses encode proteins with low homology to the 1C and 1B proteins of human respiratory syncytial viruses), j.gen.vir.72: 2545-2549.

Chambers, p.prism CR, Easton aj.1992.sequence analysis of the gene encoding the fusion protein of the murine pneumovirus, conserved second structural elements that are possible in paramyxovirus fusion glycoproteins are suggested by sequence analysis of the gene encoding the fusion glycoprotein, j.gen.view.73; 1717-1724.

Coggins, w.b., Lefkowitz, E.J and sulllender, w.m.1998.genetic variability among, group a and, group B respiratory syntychia 1 viruses in achilden's host (genetic variability in group a and group B vacuolar syncytial viruses in pediatric hospitals), j.chn.microbiol.36; 3552-3557.

Collins, m.s and Gough, r.e., Lister, s.a., chemical, N and Eddy, r.1986. furter characterization of a virus associated with turkey rhinotracheitis. vet.rec.119; 606.

collins, m.s and Gough, r.e.1988. characteristics of viral infection with viral tissue rhinotracheitis (characterization of viruses associated with turkey rhinotracheitis). 909-916.

Collins, m.s., Gough, r.e., and Alexander, d.j.1993.antigenic differentiation of Avian pneumovirus isolates using polyclonal antisera and mouse monoclonal antibodies. Avian pneumovirus isolate antigenic identification with polyclonal antisera and mouse monoclonal antibodies. Avian pathway 22: 469-479.

Collins, P.L, McIntosh, K., Chanock, R.M.1996.respiratory syncytial virus. P.1313-1351, in: fields, d.m. knepe and p.m. howley (eds.). Fields virology, 3rd ed., vol.1 Lippi1lcott-Raven, Philadelphia, Pa, USA.

Cook, j.k.a., Huggins, m.b., Orbell, S.J and Senne, d.a.1999 a prelimetric antigenic characterization of an Avian pneumovirus isolated from commercial turkeys, Colorado, USA (preliminary antigenic characterization of Avian pneumovirus isolated from commercial turkeys, Colorado), Avian pathway.28: 607-617.

Cook, j.k.a.2000.avian rhinotracheitis, rev.sci.tech.off int.epiz.19: 602-613.

Dowell, s.f., Anderson, l.j., Gary, h.e., Erdman, d.d., Plouffe, j.f., File, t.m., Marston, B.J and Breiman, r.f.1996.respiratory syndrome virus an infectious cause of respiratory-acquired lower respiratory infection in hospitalized adults j.infection.dis.174: 456-462.

Elvender, m.1996. driver respiratory disease in dairy cow disease systemic respiratory disease caused by infection with bovine respiratory syncytial virus. vet.rec.138: 101-105.

Elvender, m., Vilcek, s., Baule, c., ottenthal, a., Ba1 lag-porany, a and Belak, s.1998.genetic and antigenic analysis of the G attachment protein of bovine respiratory syncytial virus strain. j.gen.virol.79: 2939-2946.

Englund, j.a., Anderson, l.j. and Rhame, f.s.1991.nosocomial transmission of respiratory syncytial virus in immunocompromised addts (hospital rebroadcasting of respiratory syncytial virus in immunocompromised adults). j.clin.microbiol.29: 115-119.

Falsey, A.R and Walsh, e.e.2000.respiratory syncytial viral infection in adults, clinn.micro b.rev.13: 371-84.

Furze, j., Wertz, g., Lereh, R and Taylor, g.1994.antigenic specificity of the attachment protein of bone respiratory syncytial Virus antigen heterogeneity j.gen.virol.75: 363-370.

Gimenez, H.B., Cash, P and Melvin, W.T.1984.monoclonal antibodies to human respiratory syncytial Virus and their response in comparison of different Virus isolates. J.Gen.V. 01.65 two 963. jar 971.

Gulti, b.r., Cameron, k.t., Sea1, B.S, Goya1, s.m., Halvorson, D.A and njega, m.k.2000.development of a high throughput sensitive and specific enzyme-linked immunological sorbent assay for detecting avian pneumovirus antibodies development of a high sensitivity specific adsorption assay based on recombinant enzyme-linked immunosorbent assay of avian pneumovirus antibodies j.clin.microbiol.38: 4010-4.

Johnson, P.R., Spriggs M.K., olmested, R.A and Collins, P.L 1987.The G glycoprotein of human respiratory synthetic video subgroups A and B: external sequence based on the antigen related proteins (G glycoprotein of the human respiratory syncytial virus subgroups a and B: extensive sequence differences between antigenically related proteins) proc. natl.acad. sci. usa 84: 5625-5629.

Juhasz, K and Easton, A.J.1994. extension sequence variation in the attribute (G) protein gene of average pUnovices: evidence for two distinguishments groups (extensive sequence variation in the avian pneumovirus attachment (G) protein gene: evidence for two different subgroups), j.gen.virol.75: 2873-2880.

LeaMaster, b.r., Evermann, j.f., Mueller, m.k., Prierr, M. K and Scldie, J.V.1983. serological students on natural curative systemic viral and Haemophilus infection in sheet (serological study of naturally occurring respiratory syncytial virus and sleep Haemophilus infection in sheep.) America1l Association of viral Laboratory Diagnostics 26: 265-276.

Lehmkuhl, h.d., Smith, m.h., Cutlip, r.c.1980. morphogeneses and stmcture of goat respiratory syncytial virus) arch.vir.65: 269-76.

Lereh, r.a., Anderson, K and Wertz, g.w.1990.nucleotide sequence analysis and expression from a recombinant vector present adsorbed protein G of a bovine respiratory syncytial virus is a different from that of a human respiratory syncytial virus, j.view.64: 5559-5569.

Ling, R and Pringle, C.R.1988.Turkey rhinotracheitis virus: in vivo and in vitro polypeptide synthesis (turkey rhinotracheitis virus: in vivo and in vitro polypeptide synthesis) j.gen.virol 69: 917-923.

Ling, R and Ptingle, c.r.1989a. polypeptides of pneumoconia virus of micro.i.immunological cross-reactions-reactivs and post-translational modifications (polypeptides of mouse pneumovirus i.immune cross-reactions and post-translational modifications) j.gen.virol.70: 1427-1440.

Ling, R and print, c.r.1989b.polypeptides of pneumoconia viruses of ii. characterization of the glycoprotens (polypeptides of mouse pneumoviruses ii. characterization of glycoproteins) j.gen.virol.70: 1441-1452.

McD0ugall, J.S and Cook, j.k.a. 1986, Turkey rhinotracheitis; preliminary intagoniations (turkey rhinotracheitis: preliminary study), vet.Rec.118: 206-207.

Oberst, r.d., m.p.hays, k.j.hennessy, l.c.stine, j.f.evermann, and Kelling, c.l.1993.characteristics of the sequential differentiation in reverse transcription polymerase chain reaction products of ovane, bovine and human respiratory syncytial viruses (characteristic differences of reverse transcription polymerase chain reaction products of ovine, bovine and human respiratory syncytial viruses), j.vet.diagn.investig.5: 322-328.

O' Loan, c.j., alan, g., Baxter-Jones, C and McNulty, m.s.1989. animal promoted ELISA and serum neutralization test for the detection of turkey rhinotracheitis virus antibodies j.v. metal.25: 271-282.

Paccaud, M.F and jacquer, c., 1970.a respiratory syncytial virus of bovine origin. arch.ges. virusforsch.30: 327-342.

Pringle，C.R.1999 Virus taxonomy at the Xith intemational congressof virology，Sydney，Australia 1999.Arch.Virol.144/2：2065-2070.

Prozzi, D.D., Walravens, K., Langedijk, J.P.M, Daus, F.s., Kramps, J.A and Letesson, J.J.1997.antigenic and molecular analysis of the variabilityof bovine respiratory syncytial virus G glycoprotein antigenic analysis and molecular analysis), J.Gen.Virol.78: 359-366.

Randhawa, j.s., Marriott, a.c., prline, c.r., and a.j.easton 1997. research of synthetic miniseps estimpulse the sensitivity of the NS1 and NS2genes from avian pneumoviruses pneumviruses (Rescue of synthetic minireplicons established that avian pneumoviruses lack NS1 and NS2 genes). J.Virol.71: 9849-9854.

Richter, c.b., thigh, j.e., Richter, C.S and Mackenzie, j.m.1988. total pneumoconia, with terminal email in nuclear microorganism used byypneumonia vrius of mice (fatal pneumonia with end-to-end wasting in nude mice caused by mouse pneumovirus), lab.anim.sci.38: 255-261.

Rothbarth, p.h., Habova, J.J and mass 1, n.1988. Rapid diagnosis of Infection caused by respiratory syncytial virus, Infection 16; 252.

sea1, B.S.1998.matrix protein gene nucleotide and predicted amino acid sequence modified mutant that is at the first US average plasmid vector isolate for European strains (nucleotide sequence and predicted amino acid sequence of matrix protein gene demonstrate that the first US avian pneumoVirus isolate is different from European strain). Virus Res.58, 45-52.

Sea1, b.s., Sellers, h.s., meinserman, r.j.2000.fusion protein predicted amino acid sequence of the first US avians pneumocirus and lack of heterogeneity in the fusion protein of the first US pneumovirus isolate and other US isolates, Virus res.66: 139-147.

Selwyn, B.J.1990.the epidemic of acid respiratory traction in your child: complexity definitions from secondary respiratory infections of young children epidemiology: comparison results from several developing countries, rev. S870-S888.

Shin, h.j., Rajashekara, G: jirjis, f.f., Shaw, d.p., Goyat, s.m., Halvorson, D.A and Nagaraja, K.V. Specific detection of Avinpneumovirus (APV) US isolates RT-PCR (Specific detection of Avian Pneumovirus (APV) US isolate by RT-PCR). arch.virol.145: 1239-1246.

Sullender, W.M.2000.respiratory synthetic viral acquired diversity 1lic diversity (genetic and antigenic diversity of respiratory syncytial virus). Clin.Microb.Rev.13: 1-15.

Trudel, m., nano, f., Sinnard, c., Belanger, f., Alain, r, Seguin, C and luciser, g.1989. company of caprine, human and bone strains of respiratory syncytial virus. arch.v. 107: 141-149.

Ottenthal, a., Jensen, n.p.b. and Blom, j.y.1996.viral aetiology of enzootic pnettmo1lia in Danish dairies, diagnostic to o1s and diagnosis (viral etiology, diagnostic tools and epidemiology of enzootic pneumonia in Danish herds.) vet.rec.139, 114-117.

Valarcher, J., Bourhy, h., Gelfi, J and Schelcher, f.1999.evaluation of a novel reverse transcription-PCR assay on the nuclear protein gene diagnosis, of spontaneous and experimental bovine respiratory syncytial virus infection (evaluation of nested reverse transcription-PCR assays based on the nuclear protein gene for diagnosis of spontaneous and experimental bovine respiratory syncytial virus infection). j.clin.micro.37: 1858-1862

Van Milaan, a.j., Sprenger, j.j., Rothbarth, p.h., Brandenburg, a.h., Masurel, N and claus, e.c.1994.detection of respiratory syncytial RNA-polymerase chain reaction and differentiation of subgroups with oligonucleotide probes j.med.virol.44: 80-87.

Vilcek, S, Elvander, M., Ballagi-Portanny, A. and Belak, S.1994.development of nested PCR assay of bovine respiratory syncytial viruses in clinical samples J.Clin.Microb.32: 2225-2231.

Wakavens, K., Kettmann, R., Collard, A., Coppe, P and Bumy, A.1990.sequence composition between the fusion proteins of human and bovine respiratory syncytial viruses.) J.Gen.Virol.71: 3009-.

Weir, e.c., Brownstein, d.g., Smith, a.l and Johnson, e.a.1988.respiratory disease and fashion in aqueous microorganism infected with pneumoconiosis of mice (respiratory disease and wasting disease in athymic mice infected with murine pneumovirus), lab.anim.sci.34: 35-37.

Claims (30)

1. An isolated negative-sense single stranded RNA virus mammalian MPV, said virus belonging to the sub-family Pneumovirinae (Pneumovirinae) of the family Paramyxoviridae (Paramyxoviridae) and identified phylogenetically as corresponding to the genus Metapneumovirus, wherein said virus is MPV isolate 00-1 or 99-1 comprising the sequence shown in figures 20-28.

2. An isolated negative-sense single stranded RNA mammalian metapneumvirus of claim 1, wherein the genome of said virus comprises the nucleotide sequence of HMPV depicted in figure 6A, figure 6B, or figure 6C.

3. The virus of claim 1or 2, wherein the virus is an attenuated virus.

4.An immunogenic composition, wherein the immunogenic composition comprises the virus of claim 1or 2.

5. The immunogenic composition of claim 4, wherein said virus is an attenuated virus.

6. An isolated nucleic acid, wherein said nucleic acid encodes a protein comprising the amino acid sequence of the N protein of MPV isolate 00-1 or 99-1 as set forth in figure 20.

7. Use of the nucleic acid of claim 6 for the preparation of a kit for detecting mammalian metapneumovirus in a sample.

8. The use of claim 7, wherein the mammalian MPV is a human MPV.

9.A vector comprising the nucleic acid of claim 6.

10. A host cell comprising the nucleic acid of claim 6.

11. An antibody, wherein the antibody specifically binds to a viral protein expressed in the host cell of claim 10.

12. Use of an antibody according to claim 11 for the manufacture of a kit for the detection of mammalian metapneumovirus in a sample.

13. A viral isolate identifiable as a mammalian negative-sense single-stranded RNA virus of the subfamily pneumovirinae of the family paramyxoviridae using the nucleic acid of claim 6 or the antibody of 11 and phylogenetically identified as corresponding to the genus metapneumvirus.

14. Use of the nucleic acid of claim 6 in the preparation of a kit for virologically diagnosing MPV infection in a mammal.

15. A pharmaceutical composition, wherein the pharmaceutical composition comprises (i) the virus of claim 1or 2, and (ii) a pharmaceutically acceptable carrier.

16. A pharmaceutical composition, wherein the pharmaceutical composition comprises a nucleic acid encoding the genome or genomic component thereof of the virus of claim 1or 2; wherein the nucleic acid also encodes sequences of other viruses.

17. A pharmaceutical composition, wherein the pharmaceutical composition comprises a nucleic acid encoding the genome or genomic component thereof of the virus of claim 1or 2; wherein the genome may lack the viral genome components that produce the replication-defective virus and may contain mutations, deletions or insertions that produce an attenuated virus.

18. A pharmaceutical composition, wherein the pharmaceutical composition comprises (i) a subunit of the virus of claim 1or 2; and (ii) a pharmaceutically acceptable carrier.

19. An isolated or recombinant nucleic acid or MPV-specific functional fragment thereof obtainable from the virus of claim 1or 2.

20. An isolated or recombinant proteinaceous molecule encoded by the nucleic acid of claim 19, or an MPV-specific functional fragment thereof.

21. An antigen comprising the proteinaceous molecule of claim 20, or an MPV-specific functional fragment thereof.

22. A diagnostic kit for diagnosing MPV infection, the kit comprising the virus of claim 1or 2, the nucleic acid of claim 19, the proteinaceous molecule or fragment thereof of claim 20, the antigen of claim 21 or the antibody of claim 11.

23. Use of a pharmaceutical composition according to any one of claims 15, 16, 17 or 18 in the manufacture of a medicament for the treatment or prevention of MPV infection.

24. Use of a pharmaceutical composition according to any one of claims 15, 16, 17 or 18 in the manufacture of a medicament for the treatment or prevention of a respiratory disease.

25. Use of an antibody or fragment thereof for the preparation of a kit for detecting mammalian metapneumovirus in a sample, wherein the antibody or fragment thereof specifically recognizes a protein of the virus of claim 1or 2.

26. Use of a first set of one or more nucleic acids that hybridize under stringent conditions to a second set of one or more nucleic acids encoding a protein comprising the amino acid sequence of the N protein of MPV isolates 00-1 and 99-1 shown in figure 20, or a fragment thereof, in the preparation of a kit for detecting mammalian metapneumvirus in a sample.

27. The use of claim 26, wherein the nucleic acids of the first set are at least 18, at least 20, at least 23, or at least 25 nucleotides in length.

28. A chimeric parainfluenza virus type3 comprising a mammalian MPV nucleotide sequence, wherein the mammalian MPV nucleotide sequence is encoded by the genome of the virus of claim 1or 2.

29. Use of a primer in the preparation of a kit for detecting mammalian metapneumovirus in a sample, wherein the primer is selected from the group consisting of primers N3, N4, M3, M4, F7, F8, L6 and L7.

30. Use of a probe for the manufacture of a kit for detecting mammalian metapneumvirus in a sample, wherein the probe has a nucleic acid sequence selected from the group consisting of:

(i)TGC TTG TAC TTC CCA AAG；

(ii) TAT TTG AAC AAA AAG TGT, respectively; and

(iii)TGG TGT GGG ATA TTA ACA G。