WO1996003507A1

WO1996003507A1 - Method of isolating attenuated virus and vaccine thereof

Info

Publication number: WO1996003507A1
Application number: PCT/GB1995/001749
Authority: WO
Inventors: Daniel Todd; M. Stewart Mcnulty
Original assignee: Mallinckrodt Veterinary, Inc.
Priority date: 1994-07-23
Filing date: 1995-07-24
Publication date: 1996-02-08
Also published as: GB9414888D0; ZA956150B; AU2989095A

Abstract

The present invention provides a method of separating a virus isolate, in particular an attenuated virus isolate, form a mixed pool of viruses. In the method the viral genetic information is cloned into a vector, thus creating a vector library of the viral pool. The verctors are used to transfect host cells which are then grown up and separated into individual clones. The virus isolate required can be reconstituted from the relevant transformed clone. Using this technique with respect to Chicken Anaemia Virus an attenuated virus was isolate. The invention provides this isolate, the genetic sequence of this isolated and a vaccine against Chicken Anaemia Virus.

Description

METHOD OF ISOLATING

ATTENUATED VIRUS AND VACCINE THEREOF This invention relates to a method of isolating

attenuated virus strains, an attenuated strain of Chicken Anaemia Virus (CAV) and to the nucleotide sequence encoding therefor. Chicken Anaemia Virus (hereinafter designated CAV) was first isolated in Japan (Yuasa et al, Avian Dis 23 : 366-385 (1979)). CAV was noted to cause anaemia, asplasia of the bone marrow and atrophy of the lymphoid organs in susceptible chicks. Serological evidence indicates that CAV infections are common throughout the world (Yuasa et al, Nat Inst of Animal Health Quarterly (Yatabe) 23 : 78-81 (1983); McNulty et al, Avian

Pathology 18 : 215-220 (1989)). CAV is thus recognised as an economically important avian pathogen (McNulty, Avian Pathology 20 : 187-203 (1991)). Vertical transmission of the virus through the eggs of infected breeder flocks can result in increased

mortality in 10-14 day-old chicks associated with anaemia, haemorrhages and lymphoid depletion (McNulty, Avian Pathol 20 : 187-203 (1991)). Experimentally, similar clinical signs are observed in 14 day-old birds following intramuscular inoculation of day-old chicks. Birds infected after 2 weeks post-hatching fail to develop clinical signs. However, field studies have shown that sub-clinical disease in commercial broiler chicks resulting from infection with horizontally acquired virus, when maternal antibody has disappeared, adversely affects growth and profitability (McNulty, Avian Dis 35 : 263-268, (1991)). CAV is now known to be a non-enveloped icosahedral virus of 23.5nm in diameter which contains a circular single-stranded 2.3kb DNA genome (see Todd et al, J Gen Virol 71 : 819-823 (1990)). The replicative form (RF) of CAV has been shown to consist of both closed and open circular double-stranded DNA (see Meehan et al, Arch Virol 124 : 301-319 (1992)). Irrespective of the country of origin, naturally occurring CAV isolates belong to the same serotype group and produce similar pathological effects

following intramuscular inoculation of day-old chicks (see Connor et al, Aust Vet J 6. : 199-201 (1991);

McNulty et al, Avian Path 19 : 67-93 (1990) and McNulty et al, Avian Dis 33 : 691-694 (1989)). There is thus an established need for an effective vaccine to combat CAV infection, but no suitable vaccine has yet been produced. It was reported by von Bulow et al ( in J Vet Med B33 : 568-573 (1986)) that repeated passage of CAV in cell culture results in lowering of the overall

pathogenicity (ie, the ability of CAV to cause anaemia in experimentally-infected chicks) of the virus

suspension. However, at no stage was pathogenicity reduced below 27.3%, which remains an unacceptably high level of disease incidence for a candidate vaccine. In this paper von Bulow describes the use by Vielitz and Landgraf (Europaische Konferenz der World Poultry

Science Association, Paris, 8: 24-28, (1986)) of a "partially"-attenuated CAV isolate, which had been passaged 12 times in MSB1 cells in von Bulow's

laboratory, as a "vaccine" for the infection of broiler breeders so as to avoid clinical disease in young chicks. This isolate still produced anaemia in

approximately 90% of infected chicks but had a lower (4.2 logs) infectious titre in chicks, as judged by the ability to cause anaemia, than in cell culture.

Further, the results of von Bulow were contradicted by later work performed by Yuasa (see Natl Inst Anim

Health Quarterly 23 : 13-20 (1983)) and Goryo et al, (see Avian Pathol 16 : 149-163 (1987)) who found no evidence for attenuation after 19 and 40 cell culture passages, respectively. More recently, CAV has been studied using recombinant DNA technology and the sequence information of two isolates has been reported. Claessens et al, in J Gen Virol 72 : 2003-2006 (1991) reports the sequence of CAV isolate 26P4, and Noteborn et al, in J Virology 65(6) : 3131-3139 (1991) and Meehan et al, in Arch Virol 124 : 301-319 (1992) both report the sequence of the Cux-1 isolate of CAV. From this research, putative open reading frames (ORFs) have been identified which could code for the viral proteins, including the viral caps id protein. The viral capsid protein is of particular interest with regard to vaccine production since a successful vaccine must induce formation of antibodies which interact with the proteins exposed naturally in the wild-type virus. Identification of possible ORFs has led to speculation regarding the application of genetic engineering techniques to produce an effective vaccine by genetic manipulation of the genome. This general concept is described in EP-A-0, 483,911 and WO-A-92/04446 which are patent applications covering the work done by Claessens et al (supra) and Noteborn et al (supra), respectively. However, to date the description of an actual

manipulated or attenuated virus suitable for a CAV vaccine has not been disclosed and there has been no publication of an attenuated CAV isolate which could form the basis of a vaccine. Practical problems of manipulating CAV include

difficulties in growing the virus in cell culture. The number of suitable cell lines in which CAV will grow adequately are limited. Generally the virus is grown in a Marek's disease virus transformed chicken

lymphoblastoid cell line (MDCC-MSB1) (see Goryo et al, (1987) supra) or Avian Leukaemia Virus (ALV)

transformed lymphoblastoid cell lines from lymphoid leukosis (1104-X5 cells described by Yuasa (1983) supra). CAV, in common with certain other viruses, does not grow in monolayer culture. Thus, CAV isolates cannot be separated from a mixed viral pool by the technique of "plaque purification" where viral plaques, each comprised of a single virus isolate, are produced. CAV may be grown satisfactorily in suspension culture, but infects only actively dividing cells. Thus, at any one time CAV will have infected only about 30% of the total cells in the suspension culture. One commonly used technique to separate a single virus isolate from a mixed viral pool which is in suspension form, is to dilute the pool in a number of serial dilutions until statistically satisfied that each end-dilution contains only one virus isolate. However, this technique is biased towards selection of fast-dividing viral

isolates and is unlikely to allow separation of an attenuated virus strain, which is of interest here. Other viruses, in addition to CAV, may be of interest with regard to selection of attenuated isolates and the techniques disclosed herein are also applicable

thereto, (especially for viruses with a relatively small genome, such as RNA viruses) and are thus covered by the present invention. In one aspect, the present invention thus provides a method of separating a viral isolate having certain required characteristics from a mixed viral pool, said method comprising: a) isolating viral genetic material from said mixed viral pool; b) ligating said viral genetic material into a

suitable vector, optionally after copying into a more stable form and/or after treatment, for example with restriction enzyme(s); c) transforming a host cell with said vector; d) isolating transformed host cell clones and

selecting for clones having viral genetic

information leading to the required viral

characteristics; and e) producing virus from a clone so selected. The term "mixed viral pool" used herein refers to a mixture of genetically distinct viruses of the same or similar type. Thus, this term includes a mixture of different virus types (eg CAV with non-CAV virus (es)) as well as genetically, and optionally phenotypically, distinct CAV isolates. The term "viral genetic material" as used herein includes genetic material isolated directly from the virus in its natural form, DNA or RNA equivalent forms thereof and single-stranded or double-stranded forms thereof. As an example (and for the avoidance of doubt) this term includes the RF (double stranded) DNA of CAV. Also included are cDNA copies of an RNA virus. Preferably sufficient viral genetic material is

isolated and ligated into the cloning vector to allow production of viral particles following transfection of cultured cells. Desirably the process produces self- replicating viruses and therefore the coding regions needed for replication and production of viral

particles should be present. Conveniently,

substantially all of the viral genetic material is isolated from the mixed viral pool and ligated into the vector. However, deletion of portions of the viral genome which does not affect it's ability to produce virus particles may be tolerated. Rather than attempting to separate the virus directly from the mixed viral pool, the method of the invention involves the creation of a vector library of the viral genetic material which is used to transform a host for the vector. The transformed host cells containing the vector with viral genetic material can then be

separated by conventional techniques such as by plating out. Optionally the vector library is expanded by growth of the host cell population before or after separation of the transformed host cells into separate clones. After separation, the viral genetic material is excised from the vector(s) for each separate host cell clone. Thus, the separation stage occurs in relation to the transformed host cell, rather than the virus itself. The mixed viral pool may be generated by any convenient means or obtained from any convenient source. Thus, for example, a suitable pool may be obtained from an infected animal or cell culture. Preferably, the mixed viral pool starting material has a relatively high degree of genetic variation. Such a pool may be obtained, for example, by repeated passage of the virus in cell culture. This methodology of repeated viral passage is believed to increase viral genetic diversity. Generally numerous passages are required to achieve the variation required, for example 50-200 passages. It is believed that viral variation increases with the number of passages completed and preferably over 120 passages are performed. The mixed viral pool may be treated to promote

attenuated strains, for example by passage through particular host cells. Alternatively, the viral pool may be exposed to mutation inducing agents or to UV light. Optionally, the pool may contain defective viruses which are able to persist due to the presence of a co-transfected "helper" virus which fulfils the deficiency of the first virus. A method of isolating viral isolates may be of

particular utility in obtaining an attenuated variation of the wild-type virus. In a further aspect, the present invention provides a method of producing an attenuated virus isolate, said method comprising: a) production of a mixed viral pool; and b) separating a viral isolate which is attenuated as compared to the wild-type, using the methodology as described above. Preferably the step of producing the mixed viral pool comprises multiple passaging of the virus. For CAV multiple passaging has two effects. Firstly, it has been found that multiple passaging of CAV in MSB1 cells causes selection of isolates which are better adapted for growth in MSB1 cells and it appears that growth in lymphoblastoid cells selects out a virus population of CAV which is generally less able to cause anaemia in experimentally-infected chicks. Secondly, we have found that such multiple passage of CAV generates genetic heterogenicity and this phenomenon has not been observed previously for CAV. With the introduction of genetic diversity, at least some of the virus in the mixture could be expected to possess at least a degree of attenuation. In a yet further aspect, the present invention provides a viral isolate, preferably an attenuated viral isolate obtainable by use of the methodologies described above. Desirably a viral isolate obtained by such means may be substantially attenuated as compared to the wild-type. This attenuated virus may therefore substantially lack the ability to cause symptoms of disease, whilst retaining its immunogenicity (its ability to invoke an immune response against it and production of antibodies specific thereto). Cloning of the viral genome may involve a preliminary step of converting a single stranded DNA to a double stranded form. Likewise, where the viral DNA is initially circular, it may be necessary to linearise the molecule. The cloning technique described may also be applicable to RNA viruses, where the cloning stage would normally be preceded by preparation of a DNA copy of the RNA genome. Restriction endonucleases may be used to cut the viral genome so that insertion into a suitable cloning vector can be achieved. Mention may be made of BamHI, EcoRI, Haelll, Hpall, Pstl, Xbal or Nrul, although selection of other suitable restriction enzymes would be within the ability of the skilled practioner. Alternatively, the restriction sites produced may be modified into blunt ends. This may be achieved by converting the single stranded portion termini to double-stranded form by the use of a DNA polymerase, by digesting single-stranded portions of DNA, or by blunt end ligation using T4 DNA ligase. Alternatively the technique of homopolymer tailing may be used. Both of these latter techniques involve modification of the sequence to produce the required restriction site and desirably should be used only in a part of the viral genome which will not affect production of the desired viral particles. It is possible to create restriction sites in either the vector or viral DNA by selective mutation of the nucleotide sequence or by ligating thereto an

additional sequence containing the required restriction enzyme site. Once the viral genome is in a form compatible with the cloning vector it may be ligated thereto, optionally containing portions of DNA fusion protein sequences, such as β- galactosidase or marker sequences. Linker sequences may be ligated between the viral DNA and the vector, if convenient. Vectors suitable for cloning the viral genome are known in the art, but include pBR322, pUC, pGEM (for example pGEM-1) vectors, bacteriophages (such as λgt-WES-λB, Charon 28, M13) or viral vectors (such as SV40, adenovirus, pβG1, vaccina, polyoma virus) or

derivatives or modifications thereof. RNA vectors may be used for carrying DNA viral genetic material but a preliminary stage of converting the viral genome into a compatible form may be required. A similar conversion step may be necessary where the viral genetic material is available in RNA form and is to be cloned into a DNA vector. The construction of a cloning vector including a portion of foreign DNA (such as a viral genome) is well-known in the art and is summarised in Maniatis et al in "Molecular Cloning, A Laboratory Manual: Cold Spring Harbor Laboratory, 1982" and by Old et al in "Principles of Gene Manipulation" (3rd edition,

Blackwell). Generally the viral DNA will be cut with one or more restriction enzymes so that the ends produced will be complementary to the ends of the cut vector. The vector and viral DNA will thus ligate together. Vectors containing viral genetic information which are formed according to the methods described above form a further aspect of the present invention. Preferably the viral genetic information contained within the vector encodes for a virus which is attenuated as compared to the wild-type. Once the viral DNA has been inserted into a suitable vector, the complete vector may be used to transform a suitable host cell. The host cell selected would be dependent upon the vector used but mention may be made of E coli and yeast cells as well as cells from higher organisms. A host cell transformed with a vector produced as described above forms a further aspect of the present invention. After expansion and separation of the clones, the viral genetic material from each clone may be excised from the vector and used to transfect cells in which the virus may normally be cultivated. Optionally, the excised viral genetic material may be reannealed before transfection. It may be necessary for the cloned isolate to be homologously recombined. Genetically identical virus is produced from a clone since all the virus from that particular clone originates from the viral sequence in the single vector which transformed the primary host cell from which the clone was

originally derived. Use of the present invention has led to the production of a particular novel attenuated strain of Chicken Anaemia virus hereinafter designated "Isolate 10". A clone of E coli bacterium, which has been transformed with the transcription vector pGEM-1 containing the 2.3 kb Isolate 10 CAV replicative form DNA has been

deposited at the National Collections of Type Cultures (NCTC) under No. 12869 on 7 July 1994 and forms a further aspect of the present invention. The sequence of Isolate 10 is reported in SEQ ID No 1 and the variations in this sequence as compared to the original Cux-1 isolate have been analysed. Thus, the present invention further comprises a

nucleotide sequence as presented in SEQ ID No 1, or a fragment or functional equivalent thereof. Such a nucleotide sequence may be incorporated into a vector and used to transfect a suitable host cell. Both of these aspects form a further part of this invention. Comparison of the genome sequence of Isolate 10 with the genome sequence of the original Cux-1 isolate used in the passage experiments yielded 17 variations (see Meehan et al Arch Virol 121 : 301-319 (1992) or M81223 for the sequence data of the unpassaged virus used to produce Isolate 10). The positions of these variations in the genome of CAV is schematically illustrated in Figure 4. The genome sequence of Isolate 10 has also been compared to other published sequence data of virulent CAV isolates (namely Soine et al, 1994 L14767; Kato et al, 1994 D31965; Claessens et al, 1991 D10068; and Noteborn et al, 1991 M55918) and to Isolates 3, 4, 14 and 16 (see Examples 1 and 2). Any one or any combination of these nucleotides may cause the attentuation observed. In particular altered nucleotides Nos 3, 4, 9, 11, 13, 16 and 17 (see Figure 4) at positions 510, 628, 1301, 1639, 1797, 1866 and 2302 respectively or combinations thereof may

contribute to the attenuation observed in Isolate 10. Altered nucleotides Nos 3, 4, 9 and 11 or combinations thereof are in particular believed to be responsible for the attenuation observed. The present invention therefor provides a mutated form of CAV having one or more of the 17 variations in SEQ ID No 1. Further the present invention comprises a polypeptide and vaccine derived from said mutated form of CAV. One or more of these variations located in the sequence give rise to the attenuation found. A stable variant of Isolate 10 may be produced using known genetic

engineering methodologies such as point mutations, deletions or insertions of particular sections of the genome and such a stably transmittable variant is also within the scope of this invention. The present invention also provides a vaccine which comprises an attenuated virus produced by the methods described above. In particular, Isolate 10 or a

derivative thereof is suitable for use in a vaccine to prevent the disease caused by Chicken Anaemia Virus. The vaccine may be formulated in any convenient manner and would normally include pharmaceutically acceptable inert carriers or excipients. It may be useful for the vaccine to be prepared in liquid or tablet form and simply be added to the drinking water or feed of the animals or birds to be inoculated. Alternatively, the vaccine may be prepared for parenteral administration (such as by injection) or for enteral administration. Topical application, for example by applying a skin patch containing the vaccine, is also possible. In a further aspect the present invention also provides a method of diagnosis which comprises the use of a virus isolated according to the method described above. SEQ ID No 1 is the sequence of Isolate 10 as deposited at NCTC under No 12869. Figure 1A shows PAGE of Hpall analysis of CAV DNA at different numbers of passage. Figure 1B shows a restriction enzyme analysis of 1507bp CAV DNA. Figure 2 shows PAGE of Hae III analysis of PGEM-1 plasmids containing CAV DNA passaged 173 times. Figure 3 shows PAGE of Hpa II analysis of pGEM-1 plasmids containing CAV DNA passaged 173 times. Figure 4 shows the genome organisation of high passage CAV DNA (Isolate 10) showing the position of each of the 17 mutations relative to the original Cux-1 isolate used. Figure Legends Figure 1(A) Restriction endonuclease (Hpa II) of PCR- products amplified from DNAs specified by CAV isolates at different passage number. Following PAGE, DNA bands were silver stained. Lane a, passage number 1; lane b, passage number 33; lane c, passage number 67; lane d, passage number 100; lane e, passage number 133.

Fragments of 0X174 RF DNA digested with Hae III were included for size reference purposes (lane f). The 271- and 281-bp 0X174 RF fragments, which are subject to anomalous electrophoretic behaviour are left

unnumbered. Restriction fragments that are indicated by letters at the left of lane a correspond to those shown in Figure 1B. An arrow indicates band c,

modified by the 21 bp insertion. Figure 1(B) Restriction map of the 1507 bp CAV DNA amplified by PCR. The positions of the Hpa II restriction sites and the fragment sizes (in base pairs) were predicted from the sequence of CAV (Cux-1 isolate) RF DNA ( EMBL accession number M81233). Figure 2 Restriction endonuclease (Hae III) analysis of recombinant pGem-1 plasmids containing RF inserts specified by high passage (number 173) CAV. Following PAGE, DNA bands were silver stained. Lane b, plasmid number 1;

lane c, plasmid number 2;

lane d, plasmid number 3;

lane e, plasmid number 10;

lane f, plasmid number 11;

lane g, plasmid number 14;

lane i, plasmid number 5;

lane j, plasmid number 6;

lane k, plasmid number 12;

lane l, plasmid number 13;

lane m, plasmid number 15;

lane n, plasmid number 16. Recombinant pGem-1 plasmids containing similarly cloned RF inserts specified by low passage CAV in each of the 2 possible orientations are included (lanes a and h). Letters located to the left of a band indicate

differences in the banding patterns. Figure 3 Restriction endonuclease (Hpa II) analysis of recombinant pGem-1 plasmids containing RF inserts specified by high passage (number 173) CAV. Following PAGE, DNA bands were silver stained. Lane a, plasmid number 1;

lane b, plasmid number 2;

lane c, plasmid number 3; lane d, plasmid number 10;

lane e, plasmid number 11;

lane g, plasmid number 5;

lane h, plasmid number 6;

lane i, plasmid number 12

lane j, plasmid number 13;

lane k, plasmid number 15;

lane l, plasmid number 16. Recombinant pGem-1 plasmids containing a similarly cloned RF insert specified by low passage CAV is located in lane f. Letters located to the left of bands indicate differences in the banding patterns. The present invention will now be further described by reference to the following, non-limiting, Examples.

EXAMPLE 1 Cells and virus growth The Cux-1 isolate of CAV and MDCC-MSB1 cells were obtained from V. von Bulow (Free University, Berlin, Germany). CAV 87/11/52 was

isolated in this laboratory from clinical material obtained from a UK poultry organisation (see McNulty et al, Avian Pathol 19 : 67-93, (1990)). In the repeated passage experiment each passage involved the addition of virus-infected cells to fresh cells or growth medium at intervals of 2-3 days (McNulty et al, Avian Pathol, 17: 315-324 (1988)). Experimental infection Day-old specific pathogen free (SPF) chicks hatched from eggs from a CAV-free source (Lohmann Tierzucht, Cuxhaven, Germany), were inoculated intramuscularly (0.1 ml) with selected CAV isolates (10⁵ to 10⁷ 50% tissue culture infections doses per ml). At 14 days post-hatching the birds were bled and packed cell volumes (PCVs) estimated. At post-mortem on day 14 or day 15 post-hatching the birds were examined for thymus atrophy and paleness of bone marrow, as

previously described (McNulty et al, Avian Dis, 33:

691-694 (1989)). In the experiment comprising the repeated passage of CAV in chicks, each passage involved the intramuscular inoculation of day-old chicks with homogenates of thymus and bone marrow that were recovered from chicks at 6 days postinfection. Cloning of CAV Replicative Form DNA Replicative form (RF) DNA specified by the CAV isolate that had been passaged 173 times in tissue culture was isolated from infected MDCC-MSB1 cells using the Hirt extraction method and agarose gel electroelution as described (see Todd et al, J Clin Microbiol, 29: 933-939 (1991)). RF DNA, which had been digested with the restriction endonuclease EcoRI, was ligated into the multiple cloning site of the transcription vector pGem-1

(Promega, Dublin, Ireland). Clones of E coli (ED8767) transformed with recombinant plasmids containing CAV RF DNAs were isolated. Similar recombinant plasmids containing RF specified by low passage CAV (Cux-1 isolate) were produced as previously described (see Meehan et al, Arch Virol, 124: 301-319 (1992)). Transfection MDCC-MSB1 cells were transfected with CAV DNAs using the DEAE Dextran method of Sompayrac and Danna (PNAS USA 78: 7575-7578 (1981)) as described by Todd et al, supra 1991. Cloned CAV RF DNAs were prepared for transfection by digesting plasmid DNAs (1μg) which had been recovered from 3 ml bacterial cultures using the alkaline lysis method (Sambrook et al in "Molecular Cloning: a Laboratory Manual", Cold Spring Harbor Laboratory, 1989). Transfected cells were examined by indirect immunofluorescence (IIF) after 4 or 5 passages and viruses isolated after 7-10 passages when cell death was apparent (McNulty et al, 1988, supra). Restriction endonuclease analysis PCR-amplified DNAs and recombinant pGem-1 CAV RF plasmid DNAs were

digested with the restriction endonucleases Hpa II or Hae III using conditions stipulated by the

manufacturers (Promega, Dublin, Ireland). Restriction fragments were separated by electrophoresis in gels of polyacrylamide (PAGE) (Todd et al, J Clin Microbiol, 30: 1661-1666 (1992)). Fragments of 0X174 replicative form DNA resulting from digestion with Hae III were used as molecular weight markers. Gels were stained with silver using the kit purchased from Bio-Rad (Milton Keynes, UK). Polymerase chain reaction amplification The

complementary oligonucleotide primers

5'-TCGCACTATCGAATTCCGAGTG-3' and

5'-GGCTGAAGGATCCCTCATTC-3' which encompass EcoRI and BamHI sites respectively and which are specified by the sequence of the CAV (Cux-1 isolate) RF DNA, were used to enzymatically amplify CAV-specific fragments of approximately 1.5kb. For use in the polymerase chain reaction (PCR) DNA was extracted from CAV-infected MDCC-MSB1 cells using phenol : chloroform: isoamylalcohol as described (Todd et al, 1991, supra). Amplification was carried out in 50μl volumes using buffer components and Taq polymerase obtained from Promega (Dublin,

Ireland) and deoxyribonucleotides (final concentration 200mM) obtained from Pharmacia (Milton Keynes, UK).

Following an initial 2 minute denaturation step at 94°C, 30 thermal cycles with each comprising 1 minute at 94°C, 2 minutes at 47°C and 3 minutes at 72°C, were carried out. A final extension period of 7 minutes at 72°C preceded storage of the PCR products at 4°C. DNA sequencing The 1.5 kb PCR product that was

amplified from DNA previously extracted from CAV

(passage 133 Cux-1)-infected MDCC-MSB1 cells as

described earlier, was digested with EcoRI and Pstl and subcloned into the mp derivatives of M13 (Gibco-BRL). The nucleotide sequence was determined by the dideoxy termination method (Sanger et al, PNAS USA, 74: 5463- 5467 (1977)), using a commercial deaza T7 DNA

polymerase kit (Promega, Dublin, Ireland) in accordance with the manufacturer's instructions. The sequence data were analysed using the computer program DNASIS (Pharmacia, Milton Keynes, UK). Dot blot hybridization Samples of thymus DNA that were extracted using phenol :chloroform: isoamylalcohol

(25:24:1) following treatment with SDS (1%) and

proteinase K (1 mg/ml ) were investigated for the presence of CAV DNA using dot blot hybridization as described (Todd et al, 1991, supra). Briefly this involved dotting DNA samples onto Hybond-N nylon membranes (Amersham, Aylesbury, UK) using a 96-well manifold apparatus (Gibco-BRL) and hybridizing with a ³²P-labelled cloned CAV-specific DNA probe. Samples of DNA from CAV-infected and uninfected MDCC-MSB1 cells were used as controls.

RESULTS Effects of cell culture passage on virus growth in cell culture and pathogenicity. The Cux-1 isolate of CAV grew to a higher titre in MDCC-MSB1 cells following multiple passages in cell culture. Thus, whereas virus pools that had received less than 20 passages,

possessed infectivity titres of 10^5.5 to 10^6.0 50% infectious tissue culture doses per ml, the infectivity titres of virus pools that were passaged 121, 139 and 171 times were 10^6.5, 10^6.5, 10^7.0 50% infectious tissue culture doses per ml respectively. Possibly as a consequence of its higher infectivity titre, CAV that had been passaged 171 times appeared to infect a greater proportion of MDCC-MSB1 cells as determined by IIF. Inoculation of day-old chicks with high- and low- passage virus, indicated that the high-passage virus was less pathogenic, in terms of its ability to cause anaemia, than low-passage virus. The reduction in pathogenicity was not apparent when virus at passage numbers 10 and 20 were tested. With virus that had been passaged 50, 70, 82 or 121 times, however, the proportion of anaemic birds were 6 of 9 (67%), 6 of 9 (67%), 2 of 10 (20%), and 3 of 11 (27%) respectively. Under conditions in which 12 of 12 (100%) chicks became anaemic after infection with low (less than 20)-passage virus (10^5.75 50% infectious tissue culture doses per ml), 3 of 9 (33%) chicks developed anaemia following infection with the virus that had been passaged 139 times (10^6.5 50% infectious tissue culture doses per ml). Further propagation to passage number 172 failed to eliminate the pathogenicity of this virus, 2 of 11 (18%) chicks becoming anaemic following inoculation with this virus pool. Cloning of Replicative Form DNA specified by high- passage CAV. Selection of individual virus isolates from the mixed population constituting the high passage virus pool, was accomplished by the cloning of RF DNAs specified by high (number 173) -passage CAV and

subsequent transfection of MDCC-MSB1 cells with cloned RF inserts. Of 25 cloned plasmid DNAs processed in this way 22 produced infectious virus, giving rise to full

cytopathic effect between 6 and 10 passages post transfection. RF clones 3, 5 and 17 remained negative for infectious virus following a repeated transfection attempt. Although virus pools derived from most cloned inserts possessed infectivity titres of 10^6.0 to 10^6.5 50% infectious tissue culture doses per ml, some cloned virus isolates (numbers 4 and 16) produced higher titres of 10^7.0 and one (number 12) as low as 10^5.0 50% infectious tissue culture doses per ml. Genetic diversity of tissue culture passaged CAV. Restriction endonuclease analysis of PCR-amplified DNAs showed that virus that had received different numbers of cell culture passages were genetically different (Fig 1). The most significant change involved the insertion of a small DNA fragment which became

established in the virus DNA population between passage number 20 and passage number 30 (data not shown). This insertion, which decreased the electrophoretic mobility of the Hpa II restriction fragment C (Fig 1) was shown by sequencing to constitute a 21 bp fragment (data not shown). Analysis revealed that the insert contained a 19 bp sequence which was identical to that of 4 repeat sequences, tandemly-arranged within the putative regulatory region of CAV RF DNA (Meehan et al, Arch Virol, 124:301-319 (1992)). The existence of 2

separate virus populations which differed with regard to the possession of the Hpa II restriction site by which fragments A and F were separated, provided a further example of the genetic changes that had

occurred within the virus pool as a result of passage in cell culture. Thus, whereas virus DNA derived from first passage virus contained this restriction site, the majority of virus DNAs from virus passage number 33 did not. Analysis of DNA from virus at passage number 133 showed that both populations were present in substantial proportions. Further evidence for the genetic diversity that existed within the pool of high-passage CAV was provided by a limited study in which 12 recombinant plasmid DNAs containing RF inserts specified by high-passage CAV were compared by restriction endonuclease analysis. Preliminary investigation showed that plasmid numbers 1, 2, 3, 10, 11 and 14 were in the same orientation relative to the multiple cloning site of pGem-1 and that plasmid numbers 5, 6, 12, 13, 15 and 16 were in the opposite orientation. When the profile differences resulting from orientation were taken into

consideration, 3 of the 12 plasmids, numbers 3, 10 and 16 could be differentiated after treatment with the Hae III restriction endonuclease (Fig 2). Thus, with DNAs from plasmid numbers 3 and 10, additional restriction sites have resulted in the disappearance of one of the 2 bands at Z and at Y respectively and the appearance of bands at Z' and Y'. With DNA from plasmid number 16, the loss of a restriction site resulted in the appearance of band X' and the disappearance of X. The profile of plasmid number 16 also differed from its counterparts including low passage CAV RF control (Fig 2 h) in having no band W and an additional band V.

Smaller differences in the electrophoretic mobilities of bands such as U in plasmid numbers 6, 15 and 16, and band T in plasmid number 13 may be due to changes in secondary structure resulting from sequence mutations. Restriction endonuclease analysis using Hpa II showed that 3 different profiles could be differentiated in the 6 plasmids with one of the orientations (numbers 1, 2, 3, 10, 11 and 14) and that 5 of the 6 plasmids in the alternative orientation could be differentiated (Fig 3). In one of the plasmids, number 13, the restriction site, by which fragment S was generated from fragment S', was absent. The other plasmid profiles were different with regard to the

electrophoretic mobilities of fragments S, R, Q and P which are in the 250 to 300 bp range. Such differences could not, at this stage, be definitively attributed to the acquisition of additional restriction sites. It did appear, however, that band R was absent from the profiles of all plasmids except number 5, suggesting that this genetic change had become established in most of the high-passage virus population. Detailed examination of the Hae III and Hpa II profiles

indicated that all 12 cloned RF DNAs contained the insertion sequence. Pathogenicity of cloned CAV isolates. CAV isolates that were derived by transfection with recombinant plasmid DNAs (cloned CAV isolates) were assessed for their ability to produce anaemia in 14 day-old birds. Gross pathological changes, paleness in the bone marrow and atrophy of the thymus were also recorded at post- mortem (Table 1). Two separate experiments were performed, A and B, each involving the use of control chicks that were inoculated with a pool of low-passage CAV. The cloned virus isolates varied substantially in their pathogenicities. For example, cloned isolate numbers 14 and 15 which were tested in separate

experiments, appeared to be as pathogenic as the low- passage CAV isolate. On the other hand and most significantly, in the two experiments performed cloned isolate number 10 produced anaemia in only 1 of 20 birds and no gross pathological changes. It should also be noted that the single anaemic chick resulting from the inoculation with this isolate possessed a PCV of 26, one unit less than the cut-off value of 27. The detection of CAV DNA by dot blot hybridization in 10 of 10 thymuses recovered from 15 day-old chicks that were inoculated with cloned isolate number 10 confirmed that this virus had replicated in the birds (data not shown). For a more thorough evaluation of the pathogenicity of cloned isolate number 10, an additional experiment using a larger number of birds was performed. The results obtained with the uninfected control groups provided a base level with which the results obtained using the virus-inoculated groups were compared (Table 2). Inoculation of chicks with the cloned virus isolate generated insignificant levels of anaemia. In contrast to previous results, however, a substantial proportion (23%) of birds that were inoculated with this isolate were judged to have pale bone marrows. Although only 20 of 51 (39%) chicks that were

inoculated with the pool of low-passage CAV developed anaemia, the proportion of birds that had pale bone marrows (80%) and atrophied thymuses (27%) were

considerably greater than their counterparts that were inoculated with cloned isolate number 10. Pathogenicity of cloned isolate number 10 following passage in birds. When criteria such as development of anaemia, paleness of bone marrow and thymus atrophy were considered it was apparent that, following 10 passages in chicks, cloned isolate number 10 had partially recovered the pathogenicity possessed by the parent Cux-1 virus strain (Table 3). In the same experiment, under conditions in which low-passage pools of the Cux-1 isolate and the UK 87/11/52 isolate produced anaemia in 5 of 10 chicks and 6 of 10 chicks respectively, cloned isolate number 10 produced no anaemia. In an additional small-scale experiment, the low-passage UK isolate 87/11/52 virus and the bird- passaged, cloned isolate number 10 produced anaemia in 4 of 8 (50%) and 6 of 8 (75%) chicks respectively.

DISCUSSION This example describes the generation, using repeated cell culture passage and recombinant DNA cloning technology, of an attenuated CAV isolate. We have observed a progressive reduction in the

pathogenicity of the Cux-1 isolate of CAV following large numbers (from 50 to 173) of passages in MDCC-MSB1 cells. These results support the view that adaption for growth in this lymphoblastoid cell line selects a virus population that is less able to cause anaemia in 14 day-old chicks. Research in our laboratory has shown that, in vivo, CAV replicates in precursor T- cells in the thymus and mature T-lymphocytes in the spleen and that MDCC-MSB1 cells, which support in vitro replication, possess the characteristics of mature T helper lymphocytes (Adair et al, Avian Dis 37 : 943-950 (1993)). In addition, evidence was presented that CAV also infects a population of bone marrow cells that are not T-lymphocytes and this population may well be a precursor cell of erythroblastoid origin (Adair et al, Avian Dis (in press)). Histopathological

investigations of CAV-infected chicks support this view (Smyth et al, Avian Dis, 37 : 34-338 (1993)). It is possible that virus which has been adapted to growth in the mature lymphoblastoid MDCC-MSB1 cells is less well able to replicate in precursor, erythroblastoid cells and as such is less effective in causing anaemia. We found no pathological evidence that the repeatedly- passaged virus causes more severe depletion of thymus or spleen as might be expected of a virus that has been adapted for growth in lymphoblastoid cells . On the contrary, our results with one such virus, cloned isolate number 10, has demonstrated a reduced level of thymus atrophy following infection. At the molecular level, one of the most significant changes to have occurred in the virus as a consequence of high numbers of cell culture passages was the insertion of a 21 bp sequence, a genetic change that became established between passage 20 and 30.

Subsequent sequencing indicated that the insertion was almost identical to 4 other repeated sequences that are located in the non-coding region of the virus genome, and with which transcription regulatory activity has been putatively associated (Meehan et al, Arch Virol, 124: 301-319 (1992) and Noteborn et al, (J Virol, 65 : 3131-3139 (1991)). The presence of a similar fifth repeat in the nucleotide sequence of Cux-1 DNA as reported elsewhere (Noteborn et al, J Virol, 65: 3131- 3139 (1991)) together with results described here, support the view that this insertion is not uncommon, and thus its introduction probably contributes to improved growth in MDCC-MSB1 cells. The findings of Noteburn et al, (J Virol, 65: 3131-3139 (1991)) indicate that the CAV isolate containing a fifth repeat can be fully pathogenic. It is therefore unlikely that this insertion contributes greatly to the reduced pathogenicity of high-passage CAV. Whereas the

insertion constituted a genetic change shared by most viruses in the pool of high-passage CAV, restriction analysis of recombinant plasmids containing high- passage CAV RF inserts provided evidence that this virus population was genetically diverse. In the absence of an effective plaque assay procedure for CAV, recombinant DNA cloning and transfection methodologies, both of which are established for CAV by this invention (Todd et al, J Clin Microbiol 29: 933- 939 (1991)) provided the means whereby individual, genetically homogeneous, virus populations could be selected from the mixture present in the pool of high- passage CAV. The repeated failure of 3 of the 25 cloned DNA inserts tested, to produce infectious virus following transfection probably indicates that these RFs have been specified by defective genomes, that were maintained in the virus pool through the utilisation of complementary gene functions from non-defective

genomes. The presence of defective CAVs in the pool of high-passage CAV is not unexpected since defective viruses, particularly those possessing RNA genomes, can commonly occur following repeated tissue culture passage using high multiplicities of infection

(Steinhauer et al, Ann Rev Microbiol, 41 : 409-434

(1987)). The results of pathogenicity experiments carried out with pools of low-passage CAV over the 2 year period encompassed by this investigation are worthy of

comment. While earlier experiments routinely resulted in very high proportions of birds with anaemia, thymus atrophy and muscle haemorrhages (McNulty et al, Avian Dis, 33: 691-694 (1989)), in the experiments performed later in this study, the degree of pathogenicity of the same or similar virus pools was apparently reduced.

Such findings not only apply to pools of low-passage Cux-1 but also to pools of the low-passage UK 87/11/52 isolate, which was used as an alternative "pathogenic" control virus in some of our later experiments. We found no evidence that the SPF chicks, that are used in such experiments, contained CAV antibody that might have conferred protection against infection. A

possible explanation for our findings concerns the natural resistance to CAV that particular chicken genetic lines may possess. The eggs, from which our experimental chicks were hatched, were obtained as a mixture from a number of genetically different supply flocks. Variation in the resistance to CAV among the supply flocks may explain the differences in

pathogenicity that we have observed in different experiments using the same virus inocula. If the susceptibility of the SPF chicks to CAV disease had indeed changed, caution must be exercised regarding the extent of the reduction in pathogenicity that has been described for cloned isolate number 10. Thus, though our results indicate that this isolate is much less pathogenic than low-passage virus pools, the

possibility cannot be dismissed that it may still have a substantial pathogenic effect in birds that, for genetic reasons, are more susceptible to CAV infection. The stability of the attenuation possessed by cloned isolate number 10 was assessed by evaluating its pathogenicity after passage in chicks. With each passage involving the inoculation of homogenates prepared with bone marrow and thymus material that was recovered at 6 days post-infection, virus populations that grew more quickly in precursor cells were possibly selected. EXAMPLE 2 Evaluation of Isolate 10 The sequence of SEQ ID No 1 has 17 nucleotides

different to the original Cux-1 isolate used (see

Meehan et al supra and 1992, M81223). Figure 4 schematically illustrates the genome of

Chicken Anaemia Virus, and shows the open reading frames for the 3 major structural proteins VP1, VP2 and VP3. Also marked are the positions of the 17

variations between the genome of Isolate 10 compared to the original Cux-1 isolate prior to passage. The 17 altered nucleotides are located at the following positions.

Of the above alterations, Nos 1, 2 and 17 appear in a non-transcribed portion of the genome. Alterations Nos 13 and 16 cause no alteration in the amino acid

sequence of the expressed polypeptide. The remaining alterations have been compared to Isolate 3 (a defective isolate), Isolate 4 (partially

attenuated isolate), Isolate 14 (non-attenuated

isolate) and Isolate 16 (non-attenuated isolate), and to the CAV sequence data of Noteborn (supra and 1991, M55918), Claessens (supra and 1991, D10068), Kato

(1994, D31965) and Soine (1994, L14767). It is believed that any one of the abovementioned alterations, (but especially Nos 3, 4, 9, 11, 13, 16 or 17) or a combination thereof are responsible for the attenuation observed. In particular, alteration Nos 3, 4, 9 and 11

(especially 4 and 11) or combinations of these

alterations with any of the other alterations are likely candidates for causing the attenuation observed.

SEQUENCE LISTING

(1) GENERAL INFORMATION:

(i) APPLICANT:

(A) NAME: Mallinckrodt Veterinary, Inc

(B) STREET: 421 East Hawley Street

(C) CITY: Mundelem

(D) STATE: Illinois

(E) COUNTRY: United States of America

(F) POSTAL CODE (ZIP): 60060

(ii) TITLE OF INVENTION: Method of Isolating Attenuated Virus and

Vaccine thereof

(in) NUMBER OF SEQUENCES: 1

(iv) COMPUTER READABLE FORM:

(A) MEDIUM TYPE: Floppy disk

(B) COMPUTER: IBM PC compatible

(C) OPERATING SYSTEM: PC-DOS/MS-DOS

(D) SOFTWARE: Patentln Release ≠1.0, Version #1.30 (EPO)

(vi) PRIOR APPLICATION DATA:

(A) APPLICATION NUMBER: GB 9141888.9

(B) FILING DATE: 23-JUL-1994

(2) INFORMATION FOR SEQ ID NO: 1:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 2319 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

( D ) TOPOLOGY : circular

(ii) MOLECULE TYPE: DNA (genomic)

(ill) HYPOTHETICAL: NO

(iv) ANTI-SENSE: NO

(vi) ORIGINAL SOURCE:

(A) ORGANISM: Chicken Anaemia Virus

(B) STRAIN: Cux-1

(vii) IMMEDIATE SOURCE:

(B) CLONE: Isolate 10

(ix) FEATURE:

(A) NAME/KEY: variation

(B) LOCATION: (325, "G") (ιx) FEATURE:

(A) NAME/KEY: variation

(B) LOCATION: (341, "T" )

(ix) FEATURE:

(A) NAME/KEY: variation

(B) LOCATION: (510, "T" )

(ix) FEATURE:

(A) NAME/KEY: variation

(B) LOCATION: (628, "C")

(ix) FEATURE:

(A) NAME/KEY: variation

(B) LOCATION: (721, "T" )

(ix) FEATURE :

(A) NAME/KEY: variation

(B) LOCATION: (864, "C" )

(ix) FEATURE:

(A) NAME/KEY: variation

(B) LOCATION: (965, "G")

(ix) FEATURE:

(A) NAME/KEY: variation

(B) LOCATION: (1252, "C")

(ix) FEATURE :

(A) NAME/KEY: variation

(B) LOCATION: (1301, "T" )

(ix) FEATURE :

(A) NAME/KEY: variation

(B) LOCATION: (1311, "G" )

(ix) FEATURE :

(A) NAME/KEY: variation

(B) LOCATION: (1639, "C")

(ix) FEATURE:

(A) NAME/KEY: variation

(B) LOCATION: (1673, "C")

(ix) FEATURE:

(A) NAME/KEY: variation

(B) LOCATION: (1797, "T" )

(ix) FEATURE :

(A) NAME/KEY: variation

(B) LOCATION: (1840, "G" )

(ix) FEATURE:

(A) NAME/KEY: variation (B) LOCATION: ( 1341, "C" )

(ix) FEATURE :

(A) NAME/KEY: variation

(B) LOCATION: (1866, "G" )

(ix) FEATURE :

(A) NAME/KEY: variation

(B) LOCATION: (2302, "A")

Claims

CLAIMS 1. A method of separated a virus isolate from a mixed viral pool, said method comprising: a) isolating viral genetic material from said mixed viral pool; b) ligating said viral genetic material into a suitable vector; c) transforming a host cell with said vector; d) isolating transformed host cell clones and selecting for clones having viral genetic information having the required

characteristics; and e) producing virus from a clone so selected.

2. A method as claimed in Claim 1 wherein said viral genetic material is converted to a more stable form prior to ligation into said vector.

3. A method as claimed in either one of Claims 1 and 2 wherein said viral genetic material is processed by restriction enzymes prior to ligation into said vector.

4. A method as claimed in any of Claims 1 to 3

wherein the mixed virus pool is generated by repeated passage of the virus, by exposure to UV light or mutation inducing agents or by a

comibation of such techniques.

5. A method as claimed in any one of Claims 1 to 4 for isolating an attenuated virus clone.

6. A method as claimed in any one of Claims 1 to 5 wherein said virus is Chicken Anaemia Virus.

7. An attenuated form of Chicken Anaemia Virus

produced by the method of any one of Claims 1 to 6.

8. Chicken Anaemia Virus Isolate 10 deposited at NCTC under No 12869.

9. A polynucleotide having the sequence of SEQ ID No 1 or a fragment or functional equivalent thereof.

10. A polynucleotide having a sequence corresponding to at least part of the genome of a CAV isolate, said sequence having one or more of the variations located at nucleotides Nos 325, 341, 570, 628, 721, 864, 965, 1252, 1301, 1311, 1639, 1673, 1797, 1840, 1841, 1866 and 2302 of SEQ ID No 1.

11. A polynucleotide as claimed in Claim 10 comprising one or more of the variations located at

nucleotides Nos 510, 628, 1301 and 1639 of SEQ ID No 1.

12. A vector comprising a polynucleotide sequence as claimed in any one of Claims 9 to 11.

13. A host cell transformed with a vector as claimed in Claim 12.

14. A vaccine comprising an attenuated virus produced by the method of any one of Claims 1 to 6.

15. A vaccine comprising a polypeptide expressed from a coding sequence comprising a polynucleotide as claimed in any one of Claims 9 to 11.

16. A vaccine according to Claim 12 wherein said

attenuated virus is attenuated Chicken Anaemia Virus.

17. A vaccine according to Claims either one of 15 and 16 wherein said virus is Chicken Anaemia Virus Isolate 10 deposited at NCTC under No 12869.

18. A method, attenuated form of Chicken Anaemia

Virus, polynucleotide or vaccine substantially as hereinbefore defined with reference to the

Examples.