WO2019162532A1

WO2019162532A1 - Improved pneumovirus reverse genetics

Info

Publication number: WO2019162532A1
Application number: PCT/EP2019/054775
Authority: WO
Inventors: Martin LUDLOW; Albertus Dominicus Marcellinus Erasmus Osterhaus
Original assignee: Stiftung Tierärztliche Hochschule Hannover (Tiho)
Priority date: 2018-02-26
Filing date: 2019-02-26
Publication date: 2019-08-29

Abstract

The invention provides a reverse genetics system combining bacterial artificial chromosome (BAC) recombination-mediated mutagenesis with reverse genetics for a negative strand RNA virus classified in the family Pneumoviridae, such as a Orthopneumovirus and/or Metapneumovirus, that is also suitable for rescuing pneumoviruses having a nucleotide G gene duplication. In one particular embodiment, a respiratory syncytial virus (RSV) reverse genetics rescue system for strains encoding a nucleotide duplication in the G gene that are notoriously unstable in bacterial expression systems is herewith established. An important advantage of this system is that RSV antigenomic cDNA is stabilized in the vector, allowing reliable rescue of recombinant RSVs with sequences identical to clinical isolates. Herewith, the invention provides a method and means to rapidly mechanistically investigate point mutations, e.g. to be used in a human challenge study, or in an antiviral drug screen, performed with a clinically up-to-date Pneumovirus isolate.

Description

Title: Improved Pneumovirus reverse genetics.

The field of the invention

The invention relates to viruses within the family Pneumoviridae, which comprises varies species that cause respiratory disease, especially in young, old and immunosuppressed humans and young animals. The invention provides a n improved reverse genetic system for the production of recombinant viruses of the family Pneumoviridae that allows reverse genetic rescue of otherwise difficult to access clinical isolates.

Background

The family Pneumoviridae comprises enveloped negative-sense non-segmented RNA viruses. This taxon was formerly a subfamily within the Paramyxoviridae but was reclassified in 2016 as a family with two genera, Orthopneumovirus and Metapneumovirus (Rima et al., Journal of General Virology 2017;98:2912-2913). Pneumoviruses infect a range of mammalian species, while some members of the Metapneumovirus genus may also infect birds. Some viruses are specific and pathogenic for humans, such as huma n respiratory syncytial virus and human metapneumovirus. Transmission is thought to be primarily by aerosol droplets and contact.

Human respiratory syncytial virus (HRSV) and human metapneumovirus (HMPV) are globally ubiquitous respiratory pathogens of the Pneumoviridae family. Both viruses comprise two genetic groups, A and B, distinguishable genetically and serologically, which co-circulate with fluctuating frequencies. The two HRSV genetic groups are referred to as subgroups; these comprise genotypes distinguished on the basis of antibody cross reactivity or phylogeny. Each of the two HMPV genetic groups are referred to somewhat paradoxically as genotypes, and each genotype comprises two sub-genotypes (Al, A2, B1 and B2). HMPV genotypes are distinguishable serologically and sub-genotypes are discerned

phylogenetically. Fluctuating circulation frequencies of HRSV subtypes and HMPV genotypes give rise to the observation of switching of the predominantly circulating subtype (HRSV) or genotype (HMPV) between respiratory seasons. HMPV was discovered in 2001 and so longitudinal epidemiologic studies are infrequent, though for HRSV a theme of cyclicity whereby subtype A predominates for a number of seasons then subtype B predominates (usually for a shorter duration) are reported. HRSV-A is considered the major subtype in terms of both frequency and associated morbidity. Similarly, HMPV-A strains are generally detected at a higher frequency than HMPV-B strains and clinical differences are reported between HMPV genotypes. Repeat HRSV infections occur throughout life with decreasing morbidity, and increasingly evidence suggests the same is also true for HMPV.

The HRSV and HMPV virions both express two highly immunogenic surface proteins against which adaptive immune responses are directed. The fusion (F) protein mediates fusion of viral and cell membranes and is highly conserved. Anti-HRSV antibody directed against F protein is cross-reactive for strains of both subtypes, and studies on HMPV using human sera and animal models have indicated similar cross-reactive antibody reactivity patterns. It is conceivable that the conformational changes arising on activation of the fusion protein serve to expose the conserved functional (and immunogenic) regions which are otherwise, in the native state, sheltered from immunologic recognition.

The attachment (G) glycoprotein of pneumoviruses conversely portrays several immune evasive traits. Specificity of antibody raised against the G protein extends to, and possibly beyond, the genotype level (HRSV) or sub-genotype level (HMPV). In both viruses the G protein is extensively glycosylated with both N- and O-linked sugars and a high proportion of proline residues thought to reduce ordered secondary structure of the protein.

The genus Orthopneumovirus includes human, rodent, bovine and ovine viruses. Members of the human Orthopneumovirus species are divided into the co-circulating subgroups A and B. Viruses closely related to murine Orthopneumovirus have been isolated from dogs and pigs. The gene organization of Orthopneumovirus differs from that of Metapneumoviruses in the order of the envelope genes; the Orthopneumovirus also possess two genes (NS1 and NS2) upstream of the N gene that encode proteins that inhibit the synthesis and action of the host type 1 and type 3 interferon responses and inhibit apoptosis. Human respiratory syncytial virus (RSV) is an enveloped, non-segmented negative-strand RNA virus classified in the genus Orthopneumovirus. In the past 30 years, we have seen a tremendous increase in understanding of the molecular biology of RSV (as reviewed in Curr Top Microbiol Immunol. 2013; 372: 3-38). This is based on a succession of advances involving molecular cloning, reverse genetics, and detailed studies of protein function and structure. However, much remains to be learned. RSV disease is complex and variable, and the host and viral factors that determine tropism and disease are poorly understood. RSV is a major cause of lower respiratory tract infection (LRTI) in young children and the elderly. RSV infections are the leading cause of severe lower respiratory tract infections in children worldwide, being responsible for up to 80% of the cases of acute bronchiolitis and frequent subsequent hospital admission in industrialized countries. It has been estimated that RSV causes 33.8 million acute lower respiratory tract infections globally each year, resulting in 66,000 to 199,000 deaths among children under 5 years of age, 99% of which occur in developing countries. Although certain populations, such as children with chronic lung disease, congenital heart disease, prematurity, or immunodeficiency, are at increased risk for severe RSV disease, most infants hospitalized for RSV infection are previously healthy and have no known risk factors. RSV disease is associated with acute airway obstruction (AO), long-term airway hyper-responsiveness (AHR), and chronic lung inflammation. In addition to young children, immunocompromised individuals and the elderly are at increased risk for severe RSV disease and hospitalization.

Notable features of RSV are: Replication and budding in vitro are inefficient, infectivity is unstable, particles grown in vitro are mostly large filaments. RSV encodes additional proteins that are either unique to the Orthopneumovirus genus (NS1 and NS2) or found only in a subset of viruses in Paramyxoviridae (SH, M2-1, and M2-2). Two genes, NS1 and NS2, are dedicated to expressing proteins that interfere with the host type I interferon system, among other functions. Pneumoviruses possess an M2 gene that encodes two proteins, M2- 1 and M2-2, from overlapping open reading frames. The highly conserved M2-1 protein enhances virus RNA synthesis through its action as a processivity factor. Expression of the second ORF M2-2 protein utilizes a unique mechanism of re-initiation of translation of the mRNA. The M2-2 protein is involved in the switch of virus RNA synthesis from transcription to replication. The M2 and L genes overlap, and L mRNA is expressed by a backtracking mechanism. The small hydrophobic (SH) protein forms a pentameric ion channel, but its function is unclear. The F protein precursor is activated by cleavage at two furin recognition sites. The F protein activates TLR-4 signaling pathways, but this is inhibited by the G protein. Viral attachment appears to involve both the F and G proteins, but F fuses independently of G. The G protein is heavily glycosylated, nonglobular, and highly variable. The G protein bears a CX3C fractalkine-like motif that may modify the cellular immune response. The G protein is expressed in membrane-bound and secreted forms; the latter interferes with antibody-mediated neutralization and interacts directly with antigen presenting cells to modify their function. The HRSV field has been hampered by the reliance on lab-adapted strains which grow very well in tissue culture, but which contain a number of mutations and are not representative of currently circulating strains. Comparatively few HRSV reverse genetics systems are available and those systems that are based on the lab adapted A2 or Long strains. The major problem in reverse genetic engineering Pneumoviruses is plasmid stability, the G protein region is very susceptible to deletion, and no recombinant wild-type HMPV or HRSV A strain has been published which is representative of currently circulating strains.

Several diagnostic methods have been developed since respiratory syncytial virus (RSV) was defined as an important respiratory human pathogen. As RSV is a thermolabile virus, specimens destined for inoculation into cell culture require special transport, handling, and storage. During the 1980s and early 1990s, viral isolation in tissue culture was widely used in the diagnosis of RSV infections. Although this method is considered the gold standard for laboratory diagnosis of RSV, results are not available quickly enough to be the basis for initiating antiviral therapy or infection control measures. Primary isolation of RSV in conventional cell culture generally takes three to seven days but can range from two to ten days. For faster diagnosis, the isolation of RSV in cell culture has been replaced by antigen detection-based assays and new sensitive molecular techniques such as reverse

transcriptase-polymerase chain reaction (RT-PCR). RSV is a highly thermolabile virus requiring stringent adherence to transport and storage guidelines, and immediate inoculation of specimens in permissive cell lines for optimal recovery of the virus. When this is not possible, specimens need be stored carefully. RSV does not tolerate slow freezing and thawing. Approximately 50% of RSV infectivity is lost when samples are submitted to a single freeze-thaw cycle, with a complete loss of viability when they are frozen slowly at - 20^C and then thawed. The residual viral titer can be maintained for several years when the clinical samples with RSV are stored at 70^C. Although detection of viral replication in cell culture previously was the "gold standard" for RSV detection, molecular methods are now preferred by many clinicians because of the increased sensitivity and more rapid reporting of results.

The 1950s and 1960s were remarkable for the successful development of other inactivated vaccines, such as the Salk polio vaccine, and such success induced both public and scientific appreciation of and support for vaccines in general. The development of formalin- inactivated RSV vaccine candidates began during the mid-1960s; however, the history of these early RSV vaccine trials has haunted researchers. At present, no RSV vaccine candidate is ready for wide-scale clinical testing. Vaccine development has been hindered by difficulties associated with the development of animal models, the immunologic immaturity of the neonatal target population, the potential confounding of immunogenicity in the presence of maternal antibodies, and the existence of 2 antigenic subgroups of RSV.

Nonetheless, new approaches to the creation of an RSV vaccine have been developed in the laboratory, and several candidates have undergone clinical trials in humans. During the past decade, RSV protein vaccine candidates, recombinant RSV subunit vaccine candidates, fusion protein vaccine candidates for maternal immunization, and live attenuated vaccine candidates have all been evaluated in humans. To date, only live attenuated vaccine candidates have been tested in young infants— the group at highest risk for severe RSV disease.

Human metapneumovirus (hMPV), discovered in 2001, most commonly causes upper and lower respiratory tract infections in young children, but is also a concern for elderly subjects and immune-compromised patients. hMPV is the major etiological agent responsible for about 5% to 10% of hospitalizations of children suffering from acute respiratory tract infections. hMPV infection can cause severe bronchiolitis and pneumonia in children, and its symptoms are indistinguishable from those caused by human respiratory syncytial virus. Initial infection with hMPV usually occurs during early childhood, but re-infections are common throughout life. Due to the slow growth of the virus in cell culture, molecular methods (such as reverse transcriptase PCR (RT-PCR)) are the preferred diagnostic modality for detecting hMPV. A few vaccine candidates have been shown to be effective in preventing clinical disease, but none are yet commercially available.

The hMPV genome is comprised of negative-sense single-stranded RNA and contains eight genes that code for nine proteins. The order of the genes in the genome (from 3' to 5' end) is N-P-M-F-M2-SH-G-L. The proteins are: the nucleoprotein (N protein), the

phosphoprotein (P protein), the matrix protein (M protein), the fusion glycoprotein (F protein), the putative transcription factor (M2-1 protein), the RNA synthesis regulatory factor (the M2-2 protein), the small hydrophobic glycoprotein (SH protein), the attachment glycoprotein (G protein), and the viral polymerase (L protein).— The RNA core is surrounded by M protein and covered by a lipid envelope. This envelope contains the three surface glycoproteins (F, SH, and G), in the form of spikes of approximately 13-17 nm. The core nucleic acids are associated with the P, N, L, M2-1, and M2-2 proteins and form a nucleocapsid 17 nm in diameter. With the help of the G and F proteins, hMPV attaches and fuses to heparan sulphate receptors on the cell surface. After the fusion process, the viral nucleocapsid enters the cytoplasm of the host cell and undergoes replication. The newly synthesized viral genome assembles with the viral P, N, L, and M2 proteins, and moves towards the host cell membrane. The virion now buds out of the cell, with the F, SH, and G proteins exposed on the outer side of the membrane. The P protein acts as a co-factor to stabilize the L protein, allowing the formation of the virus ribonucleoprotein (RNP) complex during virus replication. The M protein plays a crucial role in virus assembly and budding by interacting with the RNP complex. The N protein encapsidates the viral genome and protects it from nuclease activity. In addition to regulating viral transcription and replication, the M2-2 protein plays a major role in virulence by decreasing the host's innate immunity.

Currently, the treatments available for hMPV infection are primarily supportive. But a few reports have raised the possibility of using ribavirin, immunoglobulin, fusion inhibitors, and small interfering ribonucleic acids for the treatment and control of hMPV infection. Several vaccine candidates against hMPV have undergone testing in rodent models and non-human primate models. Although they have shown some promising results, none has yet been tested in human volunteers. There may be problems - a heat inactivated viral vaccine against hMPV enhanced lung disease when tested in mice.

Despite these important steps forward, ongoing clinical development of Pneumovirus vaccine candidates remains extremely limited. Despite over 50 years of research, to date no safe and efficacious RSV vaccine has been licensed. RSV is notable for a historic vaccine failure in the 1960s involving a formalin-inactivated vaccine that primed for enhanced disease in RSV naive recipients. Unfortunately, the formalin-inactivated, alum-precipitated RSV vaccine candidate not only failed to protect young seronegative infants against RSV disease during the following RSV season, but the vaccine recipients actually experienced enhanced disease after wild-type RSV infection and had increased rates of pneumonia. Up to date, there has not been universal agreement with respect to the disease processes that led to enhanced disease in these vaccine recipients, further hampering RSV vaccine development. These findings, and the death in 1967 of 2 vaccine recipients who were naturally infected with RSV after receiving the inactivated vaccine, effectively slowed RSV vaccine development for decades. Development of subunit or other protein-based vaccines for pediatric use is again hampered by the possibility of enhanced disease and the difficulty of reliably demonstrating its absence in preclinical studies. Many experimental vaccination strategies failed to induce balanced T-helper (Th) responses and were associated with adverse effects such as hypersensitivity and immunopathology upon challenge. Live vaccine candidates have been shown to be free of this complication, on the other hand,

development of live attenuated RSV vaccines that are deemed more likely to induce balanced host responses appears difficult to tune, commonly these live vaccines are either over- or under-attenuated, and some of the vaccine candidates were genetically unstable which resulted in the loss of the attenuated phenotype.

Bovine respiratory syncytial virus, a major cause of respiratory disease in calves, is closely related to human RSV, a leading cause of respiratory disease in infants. Recently, promising human RSV-vaccine candidates have been engineered that stabilize the metastable fusion (F) glycoprotein in its prefusion state; however, the absence of a relevant animal model for human RSV has complicated assessment of these vaccine candidates. In cows, such a vaccine stabilizing the metastable fusion (F) glycoprotein in its prefusion state has been shown to induce protective immunity from bovine-RSV in vaccinated calves. However, in 20% of vaccinated cases, calves were not protected from clinical signs of disease, lung inflammation and macroscopic lung lesions, again indi cating that a subunit or killed-vaccine approach to protect against RSV may still result in enhanced disease in vaccinated subjects after wild-type RSV infection.

RSV has an epidemic seasonality similar to influenza viruses, with increased numbers of cases during the winter in temperate climates and during the monsoon season in tropical and sub-tropical climates. RSV can be classified into two antigenic groups (A and B), each containing several distinct subgroups based on antigenic and genomic sequence differences, especially in the G glycoprotein. Studies suggest group A viruses cause more severe disease and transmit more readily than group B viruses in infants. These two groups tend to alternate in prevalence between RSV seasons and also show evidence of multiple co circulating intra-group viral genotypes, or clades, during any given season, resulting in a diverse set of circulating viruses that can adapt to herd immunity. It is unclear if this represents a gradual evolution of viral genomes or stochastic differences in infection rates by co-circulating strains. RSV sequencing studies have largely focused on sequencing only complete or partial G gene sequences because the C-terminal, second hypervariable portion of G is sufficient and required for distinguishing the two RSV groups and the various genotypes within each group. In 1999, a G gene variant was identified in RSV-B that contained a 60-nucleotide (20 amino acid) duplication in the C-terminal third of G, within the second hypervariable mucin-like domain. This genotype has now spread globally. In 2010, a similar G gene variant was identified in RSV-A from several locations around the globe that contained a 72-nucleotide (24 amino acid) duplication in the second mucin-like domain.

Eshagi et al (PLoS ONE 7(3): e32807.) investigated the genetic variability of HRSV circulating in Ontario during 2010-2011 winter season by sequencing and phylogenetic analysis of the G glycoprotein gene. Among the 201 consecutive HRSV isolates studied, HRSV-A (55.7%) was more commonly observed than HRSV-B (42.3%). On phylogenetic analysis of the second hypervariable region of the 112 HRSV-A strains, 110 (98.2%) clustered within or adjacent to the NA1 genotype; two isolates were GA5 genotype. Eleven (10%) NAl-related isolates clustered together phylogenetically as a novel HRSV-A genotype, named ONI, containing a 72-nucleotide duplication in the C-terminal region of the attachment (G) glycoprotein. The predicted polypeptide is lengthened by 24 amino acids and includes a 23-amino acid duplication. Using RNA secondary structural software, a possible mechanism of duplication occurrence was derived. The 23-amino acid ONI G gene duplication results in a repeat of 7 potential O-glycosylation sites including three O-linked sugar acceptors at residues 270, 275, and 283. Using Phylogenetic Analysis by Maximum Likelihood analysis, a total of 19 positively selected sites were observed among NA1 isolates studied; six were found to be codons which reverted to the previous state observed in the prototype RSV-A2 strain. This tendency of codon regression in the G-ectodomain gene may infer a decreased avidity of antibody to the current circulating strains. Eshagi et al (ibid) and others in the field of HRSV concluded that further work is needed to document and further understand the emergence, virulence, pathogenicity and transmissibility of the HRSV genotype with a nucleotide G gene duplication. However, existing HRSV reverse genetics systems (J Virol. 2010 Aug; 84(15); 7770-7781 J Virol. 2015 Mar 1; 89(5): 2849-2856; Virology. 2012 Dec 5; 434(1): 129-136, [also described in US 20160030549 and US 20120264217]); do not allow for studying G gene duplications and in particular strive to avoid genetic reversion towards undesirable wild-type viral sequences. In particular, necessarily rapid mechanistic investigation of point mutations in RSV, in particularly performed with up-to-date clinical isolates has until now not been possible. For HPMV, the field has even been less well developed.

The invention

The invention provides a reverse genetics system combining bacterial artificial chromosome (BAC) recombination-mediated mutagenesis with reverse genetics for a negative strand RNA virus classified in the family Pneumoviridae, such as an Orthopneumovirus and/or

Metapneumovirus, that is also suitable for rescuing pneumoviruses having a duplication of nucleotides in the G gene. In one particularly preferred embodiment, a respiratory syncytial virus (RSV) reverse genetics rescue system for strains encoding a nucleotide duplication in the G gene that are notoriously unstable in bacterial expression systems is herewith provided. In a preferred embodiment, such strains (herein also called G-duplicate strains) are selected from those as presented in figure 31 herein. I n another preferred embodiment, the invention provides a bacterial artificial chromosome (BAC), comprising both an ori2 (oriS) origin of replication allowing for a single copy state in bacteria as well as an oriV medium-copy origin of replication, allowing for a multiple copy state in bacteria. An important advantage of this system is that virus antigenomic cDNA is stabilized in the vector, allowing reliable rescue of recombinant virus with sequences identical to clinical isolates. Herewith, the invention provides a method and means to rapidly mechanistically investigate point mutations, e.g. to be used in a human challenge study, or in an antiviral drug or antibody screen, performed with a clinically up-to-date Pneumovirus isolate. In a first aspect, the invention provides a nucleic acid comprising a bacterial artificial chromosome (BAC-) nucleic acid, said BAC-nucleic acid prefera bly at least pa rtly obtai na ble from a vector preparation as provided for example in US20040038401, or as at least partly obtainable as pSMART^®BAC v2.0 from Lucigen Corporation ( at 2905 Parmenter St, Middleton, Wl 53562 USA), from which vector preparation preferably one Notl site is removed (e.g. by using point mutagenesis) said nucleic acid preferably comprising a Notl restriction site and a Kasl restriction site, said BAC-nucleic acid also comprising a nucleic acid encoding an antigenome of an enveloped, non-segmented negative-strand RNA virus classified in the genus Pneumovirus, said virus preferably having a duplication in the gene encoding the G-protein, said nucleic acid also comprising a nucleic acid encoding a hammerhead ribozyme, preferably preceding the leader sequence of the nucleic acid encoding the antigenome, said nucleic acid also comprising a nucleic acid encoding a hepatitis delta virus ribozyme, preferably following the trailer sequence of the antigenome nucleic acid , said nucleic acid preferably also comprising a nucleic acid preceding the antigenome nucleic acid and encoding a T7 promotor, preferably followed by 'GGG' nucleic acid, said nucleic acid preferably also comprising a nucleic acid encoding at least one T7 terminator, preferably at least two T7 terminators, preferably following the nucleic acid encoding the hepatitis delta virus ribozyme. In a preferred embodiment, said nucleic acid facilitates wild-type virus strain reverse genetics. In another preferred embodiment, said antigenome comprises a fully consecutive antigenome, herein also identified as a consecutive viral antigenome sequence not interspersed or interjected with artificially introduced restriction sites separating nucleic acid stretches that encode viral proteins.

In a preferred embodiment said virus is a respiratory syncytial virus, preferably selected from the group of human or ruminant respiratory syncytial viruses. In one embodiment said virus is a respiratory syncytial virus, selected from the group of human respiratory syncytial viruses (HRSV). In one embodiment, said virus is HRSV-A. In another embodiment, said virus is HRSV-B. In yet another embodiment, said virus is bovine respiratory syncytial virus (BRSV). In yet another embodiment, said virus is ovine respiratory syncytial virus (ORSV). The invention provides a nucleic acid comprising a bacterial artificial chromosome additionally provided with DNA dependent RNA-polymerase promotor/terminator nucleic acid sequences, said nucleic acid also comprising a nucleic acid encoding a hammerhead ribozyme, said nucleic acid also comprising a nucleic acid encoding an antigenome of an enveloped, non-segmented negative-strand RNA virus classified in the family

Pneumoviridae, said nucleic acid also comprising a nucleic acid encoding a hepatitis delta virus ribozyme.

The invention provides a nucleic acid comprising a bacterial artificial chromosome additionally provided with DNA dependent RNA-polymerase promotor/terminator nucleic acid sequences, said nucleic acid also comprising a nucleic acid encoding a hammerhead ribozyme, said nucleic acid also comprising a nucleic acid encoding an antigenome of an enveloped, non-segmented negative-strand RNA virus classified in the family

Pneumoviridae, said nucleic acid also comprising a nucleic acid encoding a hepatitis delta virus ribozyme wherein said virus is an Orthopneumovirus.

Pneumoviridae, said nucleic acid also comprising a nucleic acid encoding a hepatitis delta virus ribozyme wherein said virus is selected from the group of human or ruminant respiratory syncytial viruses.

The invention provides a nucleic acid comprising a bacterial artificial chromosome additionally provided with DNA dependent RNA-polymerase promotor/terminator nucleic acid sequences, said nucleic acid also comprising a nucleic acid encoding a hammerhead ribozyme, said nucleic acid also comprising a nucleic acid encoding an antigenome of an enveloped, non-segmented negative-strand RNA virus classified in the family Pneumoviridae, said nucleic acid also comprising a nucleic acid encoding a hepatitis delta virus ribozyme wherein said virus is a human respiratory syncytial virus (HRSV).

Pneumoviridae, said nucleic acid also comprising a nucleic acid encoding a hepatitis delta virus ribozyme wherein said virus is HRSV-A.

The invention provides a nucleic acid comprising a bacterial artificial chromosome additionally provided with DNA dependent RNA-polymerase promotor/terminator nucleic acid sequences, said nucleic acid also comprising a nucleic acid encoding a hammerhead ribozyme, said nucleic acid also comprising a nucleic acid encoding an antigenome of an enveloped, non-segmented negative-strand RNA virus classified in the family Pneumoviridae, said nucleic acid also comprising a nucleic acid encoding a hepatitis delta virus ribozyme wherein said virus is HRSV-A and has a duplication in the gene encoding the G-protein. In a preferred embodiment, said HRSV-A virus is selected from the group of G-duplicate strains comprising a nucleic acid as shown in figure 31 NZL/UHRSV76/2011, KX765960 (SEQ ID No 29); Lebanon/14UF289/2015, MG793382 (SEQ ID No 30); TOp-13-203, KY654517 (SEQ ID No 31); TEv-12-138, KY654512 (SEQ ID No 32); USA/TH_10195/2012, KU950680 (SEQ ID No 33); USA/LA2_90/2012, KM042383 (SEQ ID No 34); USA/TH_10305P/2013 , KX894797 (SEQ ID No 35); USA/LA2_13/2012, KJ672465 (SEQ ID No 36); USA/LA2_100/2013, KJ672457 (SEQ ID No 37); HRSVA 0594 SEQ ID No 38).

Pneumoviridae, said nucleic acid also comprising a nucleic acid encoding a hepatitis delta virus ribozyme wherein said virus is HRSV-A and wherein said virus has a nucleic acid sequence homology of at least 80%, preferably at least 90%, more preferably at least 95%, most preferably of at least 99% with the nucleic acid of SEQ ID NO: 1. The invention also provides HRSV-A-0594 with SEQ ID NO: 1.

Pneumoviridae, said nucleic acid also comprising a nucleic acid encoding a hepatitis delta virus ribozyme wherein said virus is HRSV-B.

The invention provides a nucleic acid comprising a bacterial artificial chromosome additionally provided with DNA dependent RNA-polymerase promotor/terminator nucleic acid sequences, said nucleic acid also comprising a nucleic acid encoding a hammerhead ribozyme, said nucleic acid also comprising a nucleic acid encoding an antigenome of an enveloped, non-segmented negative-strand RNA virus classified in the family Pneumoviridae, said nucleic acid also comprising a nucleic acid encoding a hepatitis delta virus ribozyme wherein said virus is HRSV-B wherein said virus has a duplication in the gene encoding the G- protein. In a preferred embodiment, said HRSV-B virus is selected from the group of G- duplicate strains comprising a nucleic acid as shown in figure 31 NZL/UHRSV20/2014, KX765973 (SEQ ID No 43); USA/LA2_16/2012, KM042393 (SEQ ID No 44); 1856, KR350475 (SEQ ID No 45); TB5_CA-15-0741_1, LC384998 (SEQ ID No 46); NZL/UHRSV51/2013, KX765959 (SEQ ID No 47); WI/629-Q0190/10, JN032120 (SEQ ID No 48); PER/FPP01374/2012, KJ627262 (SEQ ID No 49); NZL/UHRSV67/2012, KX765976 (SEQ ID No 50); JOR/D3772/2013, KX655693 (SEQ ID No 51); RSVB 9671 (SEQ ID No 52).

Pneumoviridae, said nucleic acid also comprising a nucleic acid encoding a hepatitis delta virus ribozyme wherein said virus is HRSV-B wherein said virus has a nucleic acid sequence homology of at least 80%, preferably at least 90%, more preferably at least 95%, most preferably of at least 99% with the nucleic acid of SEQ ID NO: 13. The invention also provides HRSV-B-9671 with SEQ. ID NO: 13.

Pneumoviridae, said nucleic acid also comprising a nucleic acid encoding a hepatitis delta virus ribozyme wherein said virus is bovine respiratory syncytial virus (BRSV).

Pneumoviridae, said nucleic acid also comprising a nucleic acid encoding a hepatitis delta virus ribozyme wherein said virus is ovine respiratory syncytial virus (ORSV).

Pneumoviridae, said nucleic acid also comprising a nucleic acid encoding a hepatitis delta virus ribozyme wherein said virus is a Metapneumovirus.

Pneumoviridae, said nucleic acid also comprising a nucleic acid encoding a hepatitis delta virus ribozyme wherein said virus is human metapneumovirus (HMPV).

Pneumoviridae, said nucleic acid also comprising a nucleic acid encoding a hepatitis delta virus ribozyme wherein said virus is avian metapneumovirus.

Pneumoviridae, said nucleic acid also comprising a nucleic acid encoding a hepatitis delta virus ribozyme wherein said virus is human metapneumovirus (HMPV) and wherein said virus has a duplication in the gene encoding the G-protein.

Pneumoviridae, said nucleic acid also comprising a nucleic acid encoding a hepatitis delta virus ribozyme wherein said virus is avian metapneumovirus (AMPV) and wherein said virus has a duplication in the gene encoding the G-protein.

It is herein preferred that the nucleic acid comprising a bacterial artificial chromosome (BAC-) nucleic acid according to the invention is at least pa rtly obtai na ble from a vector preparation as provided for example in US20040038401, or as at least partly obtainable as pSMART^®BAC v2.0 from Lucigen Corporation; from which vector preparation preferably one Notl site is removed to facilitate RSV antigenome insertion.

The invention also provides a prokaryote and a eukaryote cell comprising a nucleic acid comprising a bacterial artificial chromosome additionally provided with DNA dependent RNA-polymerase promotor/terminator nucleic acid sequences, said nucleic acid also comprising a nucleic acid encoding a hammerhead ribozyme, said nucleic acid also comprising a nucleic acid encoding an antigenome of an enveloped, non-segmented negative-strand RNA virus classified in the family Pneumoviridae, said nucleic acid also comprising a nucleic acid encoding a hepatitis delta virus ribozyme. The invention also provides a prokaryote and a eukaryote cell comprising a nucleic acid comprising a bacterial artificial chromosome additionally provided with DNA dependent RNA-polymerase promotor/terminator nucleic acid sequences, said nucleic acid also comprising a nucleic acid encoding a hammerhead ribozyme, said nucleic acid also comprising a nucleic acid encoding an antigenome of an enveloped, non-segmented negative-strand RNA virus classified in the family Pneumoviridae, said nucleic acid also comprising a nucleic acid encoding a hepatitis delta virus ribozyme wherein said virus is an Orthopneumovirus.

The invention also provides a prokaryote and a eukaryote cell comprising a nucleic acid comprising a bacterial artificial chromosome additionally provided with DNA dependent RNA-polymerase promotor/terminator nucleic acid sequences, said nucleic acid also comprising a nucleic acid encoding a hammerhead ribozyme, said nucleic acid also comprising a nucleic acid encoding an antigenome of an enveloped, non-segmented negative-strand RNA virus classified in the family Pneumoviridae, said nucleic acid also comprising a nucleic acid encoding a hepatitis delta virus ribozyme wherein said virus is selected from the group of human or ruminant respiratory syncytial viruses.

The invention also provides a prokaryote and a eukaryote cell comprising a nucleic acid comprising a bacterial artificial chromosome additionally provided with DNA dependent RNA-polymerase promotor/terminator nucleic acid sequences, said nucleic acid also comprising a nucleic acid encoding a hammerhead ribozyme, said nucleic acid also comprising a nucleic acid encoding an antigenome of an enveloped, non-segmented negative-strand RNA virus classified in the family Pneumoviridae, said nucleic acid also comprising a nucleic acid encoding a hepatitis delta virus ribozyme wherein said virus is a human respiratory syncytial virus (HRSV)

The invention also provides a prokaryote and a eukaryote cell comprising a nucleic acid comprising a bacterial artificial chromosome additionally provided with DNA dependent RNA-polymerase promotor/terminator nucleic acid sequences, said nucleic acid also comprising a nucleic acid encoding a hammerhead ribozyme, said nucleic acid also comprising a nucleic acid encoding an antigenome of an enveloped, non-segmented negative-strand RNA virus classified in the family Pneumoviridae, said nucleic acid also comprising a nucleic acid encoding a hepatitis delta virus ribozyme wherein said virus is

HRSV-A. The invention also provides a prokaryote and a eukaryote cell comprising a nucleic acid comprising a bacterial artificial chromosome additionally provided with DNA dependent RNA-polymerase promotor/terminator nucleic acid sequences, said nucleic acid also comprising a nucleic acid encoding a hammerhead ribozyme, said nucleic acid also comprising a nucleic acid encoding an antigenome of an enveloped, non-segmented negative-strand RNA virus classified in the family Pneumoviridae, said nucleic acid also comprising a nucleic acid encoding a hepatitis delta virus ribozyme wherein said virus is HRSV-A and has a duplication in the gene encoding the G-protein.

The invention also provides a prokaryote and a eukaryote cell comprising a nucleic acid comprising a bacterial artificial chromosome additionally provided with DNA dependent RNA-polymerase promotor/terminator nucleic acid sequences, said nucleic acid also comprising a nucleic acid encoding a hammerhead ribozyme, said nucleic acid also comprising a nucleic acid encoding an antigenome of an enveloped, non-segmented negative-strand RNA virus classified in the family Pneumoviridae, said nucleic acid also comprising a nucleic acid encoding a hepatitis delta virus ribozyme wherein said virus is HRSV-A and wherein said virus has a nucleic acid sequence homology of at least 80%, preferably at least 90%, more preferably at least 95%, most preferably of at least 99% with the nucleic acid of SEQ ID NO: 1. The invention also provides a eukaryote cell comprising HRSV-A-0594 with SEQ ID NO: 1.

The invention also provides a prokaryote and a eukaryote cell comprising a nucleic acid comprising a bacterial artificial chromosome additionally provided with DNA dependent RNA-polymerase promotor/terminator nucleic acid sequences, said nucleic acid also comprising a nucleic acid encoding a hammerhead ribozyme, said nucleic acid also comprising a nucleic acid encoding an antigenome of an enveloped, non-segmented negative-strand RNA virus classified in the family Pneumoviridae, said nucleic acid also comprising a nucleic acid encoding a hepatitis delta virus ribozyme wherein said virus is HRSV-B.

The invention also provides a prokaryote and a eukaryote cell comprising a nucleic acid comprising a bacterial artificial chromosome additionally provided with DNA dependent RNA-polymerase promotor/terminator nucleic acid sequences, said nucleic acid also comprising a nucleic acid encoding a hammerhead ribozyme, said nucleic acid also comprising a nucleic acid encoding an antigenome of an enveloped, non-segmented negative-strand RNA virus classified in the family Pneumoviridae, said nucleic acid also comprising a nucleic acid encoding a hepatitis delta virus ribozyme wherein said virus is HRSV-B wherein said virus has a duplication in the gene encoding the G-protein.

The invention also provides a prokaryote and a eukaryote cell comprising a nucleic acid comprising a bacterial artificial chromosome additionally provided with DNA dependent RNA-polymerase promotor/terminator nucleic acid sequences, said nucleic acid also comprising a nucleic acid encoding a hammerhead ribozyme, said nucleic acid also comprising a nucleic acid encoding an antigenome of an enveloped, non-segmented negative-strand RNA virus classified in the family Pneumoviridae, said nucleic acid also comprising a nucleic acid encoding a hepatitis delta virus ribozyme wherein said virus is HRSV-B wherein said virus has a nucleic acid sequence homology of at least 80%, preferably at least 90%, more preferably at least 95%, most preferably of at least 99% with the nucleic acid of SEQ ID NO: 13.

The invention also provides a eukaryote cell comprising HRSV-B-9671 with SEQ. ID NO: 13 The invention also provides a prokaryote and a eukaryote cell comprising a nucleic acid comprising a bacterial artificial chromosome additionally provided with DNA dependent RNA-polymerase promotor/terminator nucleic acid sequences, said nucleic acid also comprising a nucleic acid encoding a hammerhead ribozyme, said nucleic acid also comprising a nucleic acid encoding an antigenome of an enveloped, non-segmented negative-strand RNA virus classified in the family Pneumoviridae, said nucleic acid also comprising a nucleic acid encoding a hepatitis delta virus ribozyme wherein said virus is bovine respiratory syncytial virus (BRSV).

The invention also provides a prokaryote and a eukaryote cell comprising a nucleic acid comprising a bacterial artificial chromosome additionally provided with DNA dependent RNA-polymerase promotor/terminator nucleic acid sequences, said nucleic acid also comprising a nucleic acid encoding a hammerhead ribozyme, said nucleic acid also comprising a nucleic acid encoding an antigenome of an enveloped, non-segmented negative-strand RNA virus classified in the family Pneumoviridae, said nucleic acid also comprising a nucleic acid encoding a hepatitis delta virus ribozyme wherein said virus is ovine respiratory syncytial virus (ORSV).

The invention also provides a prokaryote and a eukaryote cell com prising a nucleic acid comprising a bacterial artificial chromosome additionally provided with DNA dependent RNA-polymerase promotor/terminator nucleic acid sequences, said nucleic acid also comprising a nucleic acid encoding a hammerhead ribozyme, said nucleic acid also comprising a nucleic acid encoding an antigenome of an enveloped, non-segmented negative-strand RNA virus classified in the family Pneumoviridae, said nucleic acid also comprising a nucleic acid encoding a hepatitis delta virus ribozyme wherein said virus is a Metapneumovirus.

The invention also provides a prokaryote and a eukaryote cell comprising a nucleic acid comprising a bacterial artificial chromosome additionally provided with DNA dependent RNA-polymerase promotor/terminator nucleic acid sequences, said nucleic acid also comprising a nucleic acid encoding a hammerhead ribozyme, said nucleic acid also comprising a nucleic acid encoding an antigenome of an enveloped, non-segmented negative-strand RNA virus classified in the family Pneumoviridae, said nucleic acid also comprising a nucleic acid encoding a hepatitis delta virus ribozyme wherein said virus is human metapneumovirus (HMPV).

The invention also provides a prokaryote and a eukaryote cell comprising a nucleic acid comprising a bacterial artificial chromosome additionally provided with DNA dependent RNA-polymerase promotor/terminator nucleic acid sequences, said nucleic acid also comprising a nucleic acid encoding a hammerhead ribozyme, said nucleic acid also comprising a nucleic acid encoding an antigenome of an enveloped, non-segmented negative-strand RNA virus classified in the family Pneumoviridae, said nucleic acid also comprising a nucleic acid encoding a hepatitis delta virus ribozyme wherein said virus is avian metapneumovirus.

The invention also provides a prokaryote and a eukaryote cell comprising a nucleic acid comprising a bacterial artificial chromosome additionally provided with DNA dependent RNA-polymerase promotor/terminator nucleic acid sequences, said nucleic acid also comprising a nucleic acid encoding a hammerhead ribozyme, said nucleic acid also comprising a nucleic acid encoding an antigenome of an enveloped, non-segmented negative-strand RNA virus classified in the family Pneumoviridae, said nucleic acid also comprising a nucleic acid encoding a hepatitis delta virus ribozyme wherein said virus is human metapneumovirus (HMPV) and wherein said virus has a duplication in the gene encoding the G-protein. The invention also provides a prokaryote and a eukaryote cell comprising a nucleic acid comprising a bacterial artificial chromosome additionally provided with DNA dependent RNA-polymerase promotor/terminator nucleic acid sequences, said nucleic acid also comprising a nucleic acid encoding a hammerhead ribozyme, said nucleic acid also comprising a nucleic acid encoding an antigenome of an enveloped, non-segmented negative-strand RNA virus classified in the family Pneumoviridae, said nucleic acid also comprising a nucleic acid encoding a hepatitis delta virus ribozyme wherein said virus is avian metapneumovirus (AMPV) and wherein said virus has a duplication in the gene encoding the G-protein.

It is herein provided that a prokaryote cell comprising or provided with a BAC-nucleic acid having a nucleic acid antigenome of a Pneumovirus according to the invention, is preferably additionally provided with a nucleic acid encoding a TrfA replication protein. A preferred prokaryote cell herein is E. coli, more preferred is BAC-Optimized Replicator v2.0 Electrocompetent Cells. It is herein also provided that a eukaryote cell, comprising or provided with a BAC-nucleic acid having a nucleic acid antigenome of a Pneumovirus according to the invention, is preferably additionally provided with a nucleic acid encoding a DNA dependent RNA polymerase, preferably a T7 DNA dependent RNA polymerase, more preferably, said cell is additionally provided with at least one helper nucleic acid (herein also identified as helper plasmid) encoding at least one protein selected from the group of N, P, M2-1 and L proteins from a virus classified in the family Pneumoviridae, more preferably, said eukaryote cell is provided with helper nucleic acid or helper plasmids encoding proteins N, P, M2-1 and L. It is preferred that said proteins stem from the same genus or species of Pneumovirus as the antigenome. A preferred eukaryote cell herein is a human epithelial cell, more preferred is HEp-2 (ATCC^® CCL-23^™).

The invention also provides a virus obtainable by rescue from a eukaryote cell comprising a nucleic acid according to the invention comprising a bacterial artificial chromosome additionally provided with DNA dependent RNA-polymerase promotor/terminator nucleic acid sequences, said nucleic acid also comprising a nucleic acid encoding a hammerhead ribozyme, said nucleic acid also comprising a nucleic acid encoding an antigenome of an enveloped, non-segmented negative-strand RNA virus classified in the family Pneumoviridae, said nucleic acid also comprising a nucleic acid encoding a hepatitis delta virus ribozyme.

The invention also provides use of a virus according to the invention as provided herein in a method of assessment of binding of an antibody to a Pneumovirus. Distinct and specific mutations (for example K272Q. and S275F in antigenic site A, a highly conserved region on the RSV F protein) are inserted individually into the F glycoprotein gene of full-length antigenomic clones of HRSV A and B strains as provided herein, Recombinant wild-type and mutant viruses are rescued following transfection of HEp-2 cells with full-length

antigenomic HRSV A or B clones together with helper plasmids expressing HRSV N, P, M2-1 and L proteins as provided herein. HEp-2 cells for example also provided with an additional plasmid expressing codon optimized T7 DNA dependent RNA polymerase. Stocks of rescued viruses are prepared by passage on HEp-2 cells and virus stocks are completely sequenced to ensure retention of the introduced mutation in the F glycoprotein. Resulting rescued viruses with or without said mutations are tested for their reactivity with Palivizumab.

Similarly, the invention also provides use of a virus according to the invention as provided herein in a method of assessment of efficacy of a vaccine directed against a Pneumovirus, wherein for example immunoreactivity against said rescued viruses having said mutations in the F glycoprotein is tested in vaccine challenge models. The invention also provides use of a virus according to the invention as provided herein in a method of assessment of efficacy of an antiviral agent directed against a Pneumovirus. For this purpose, additional rHRSV-A- 0594 and rHRSV-B-9671 is generated which contain for example a single point mutation (Y1631H) in the polymerase gene which has been shown to confer resistance to a number of polymerase inhibitors. This is achieved using the BAC mutagenesis protocols as provided herein which enable single mutations to be rapidly introduced into BAC-nucleic acid vectors of the invention, a technique which is not possible with standard low copy or high copy plasmid vectors. Stocks of rescued viruses are prepared by passage on HEp-2 cells and virus stocks are completely sequenced to ensure retention of the introduced mutation in the polymerase gene. The invention also provides use of a virus according to the invention as provided herein in a method of assessment of binding of Pneumovirus entry in a eukaryote cell, and the invention also provides use of a virus according to the invention as provided herein in a method of assessment of a T-helper cell response to a Pneumovirus. For such phenotypic HRSV analysis to study binding, Hep-2 cells are inoculated with rHRSV-A-0594 or rHRSV-B-9671 strains expressing renilla luciferase with and without the listed point mutations and subsequently incubated in presence of a range of Synagis or HRSV

polymerase inhibitor concentrations, until the optimal virus signal is achieved in control cultures without inhibitor. Herewith, the invention provides Pneumovirus, with methods and means, to rapidly mechanistically investigate point mutations, e.g. to be used in a human challenge study, or in an antiviral drug screen, performed with a clinically up-to-date Pneumovirus isolate.

Figure legends

Figure 1A. Schematic representation of recombinant human respiratory syncytial (HRSV) strains. The transcription units and the open reading frames (ORFs) of the non-structural protein 1 and 2, nucleocapsid (N), the phospho- (P), the matrix (M), small hydrophobic (SH), glycoprotein (G), fusion (F), M2, and large (L) protein. rRSVA⁰⁵⁹⁴ (A), rRSVA°⁵⁹⁴EGFP (B), rRSVB⁹⁶⁷¹ (C), rRSVB⁹⁶⁷¹EGFP (D). The additional transcription unit (ATU) encoding the ORF of EGFP or a different fluorescent or luminescent protein is inserted between the P and M genes.

Figure IB. Overview of HRSV reverse genetics systems. Schematic representation of (A) pSM- RSVA-0594 and (B) pSM-RSVB-9671 full-length cDNA clones in which pSMART-BAC (Lucigen) is used as the vector backbone. At the 5' end flanking sequences (yellow) include a T7 promoter sequence (T7) and a hammerhead ribozyme (HH). The virus antigenome is flanked at the 3' end by a hepatitis delta ribozyme (d) and two T7 terminator sequences (TF). (C) Verification of stability of pSMART BAC clones. Restriction endonuclease digest profile of Spel cut pSMART BAC clones containing the full-length antigenome of RSV-A0594 or RSV-B9671.

Figure 2A. Overview of HRSV reverse genetics systems. Schematic representation of (A) pSMRSV^{A 0594} and (B) pSMRSV^{6 9671} full-length cDNA clones in which pSMART-BAC (Lucigen) is used as the vector backbone. At the 5' end flanking sequences (yellow) include a T7 promoter sequence (T7) and a hammerhead ribozyme (HH). The virus antigenome is flanked at the 3' end by a hepatitis delta ribozyme (d) and two T7 terminator sequences (TF). (C) Non-recombinant RSV^{A 0594} produces large syncytia upon infection of HEp-2 cells, (D), Non recombinant RSV^{b 9671} infection of HEp-2 cells results in less cytopathic effects with cell rounding observed most frequently. (E-F) Rescue of rRSV^{B 9671}EGFP in HEp-2 cells with infected cells and limited cell-to-cell fusion observed in a fluorescence overlay on a phase contrast image (E) and by fluorescent microscopy (F).

Figure 2B. (A-D) Schematic representation of recombinant human respiratory syncytial (RSV) strains. The transcription units and the open reading frames (ORFs) of the non-structural protein 1 and 2, nucleocapsid (N), the phospho- (P), the matrix (M), small hydrophobic (SH), glycoprotein (G), fusion (F), M2, and large (L) protein. rRSV-A⁰⁵⁹⁴ (A), rRSV-A⁰⁵⁹⁴EGFP (B), rRSV- B⁹⁶⁷¹ (C), rRSV-B⁹⁶⁷¹EGFP (D). The additional transcription unit (ATU) encoding the ORF of EGFP or a different fluorescent or luminescent protein is inserted between the P and M genes. (E) Rescue of recombinant RSVs. rRSV^{A 0594} and rRSV-A⁰⁵⁹⁴EGFP produces large syncytia upon infection of HEp-2 cells, rRSV-B⁹⁶⁷¹EGFP infection of HEp-2 cells results in less cytopathic effects with cell rounding observed most frequently.

Figure 3. Schematic representation of the vector backbone pSMART-BAC (Lucigen,

http://www.lucigen.com/pSMART-BAC/)

Figure 4. Schematic representation of pSMART-BAC with HRSV-A-0594.

Features:

1-44: HRSV leader sequence

99-518: HRSV NS1 open reading frame

628-1002: HRSV NS2 open reading frame

1140-2315: HRSV Nucleocapsid open reading frame

2347-3072: HRSV Phosphoprotein open reading frame

3255-4025: HRSV Matrix open reading frame

4295-4489: HRSV SH open reading frame

4681-5646: HRSV G open reading frame

5726-7450: HRSV F open reading frame

7669-8256: HRSV M2-1 open reading frame 8228-8494: HRSV M2-2 open reading frame

8561-15058: HRSV L open reading frame

15125-15277: HRSV Trailer

15278-15361: Hepatitis delta ribozyme sequence

15432-15478: T7 terminator sequence

15574-15620: T7 terminator sequence

23213-23229: T7 Promoter

23230-23281: Hammerhead ribozyme

Figure 5. Schematic representation of pSMART-BAC with HRSV-B-9671. Features:

1-44: HRSV leader sequence

99-518: HRSV NS1 open reading frame

626-1000: HRSV NS2 open reading frame

1139-2314: HRSV Nucleocapsid open reading frame

2347-3072: HRSV Phosphoprotein open reading frame

3262-4032: HRSV Matrix open reading frame

4301-4498: HRSV SH open reading frame

4688-5641: HRSV G open reading frame

5718-7442: HRSV F open reading frame

7669-8256: HRSV M2-1 open reading frame

8222-8494: HRSV M2-2 open reading frame

8560-15060: HRSV L open reading frame

15133-15278: HRSV Trailer

15279-15362: Hepatitis delta ribozyme sequence

15433-15479: T7 terminator sequence

15575-15621: T7 terminator sequence

23214-23230: T7 Promoter

23231-23282:

Figure 6. HRSV-A-0594 nucleotide sequence (SEQ. ID NO: 1) Figure 7. HRSV-A-0594 NS1 protein sequence (SEQ ID NO: 2)

Figure 8. HRSV-A-0594 NS2 protein sequence (SEQ. ID No: 3)

Figure 9. HRSV-A-0594 N protein sequence (SEQ. ID No: 4)

Figure 10. HRSV-A-0594 P protein sequence (SEQ ID No: 5)

Figure 11. HRSV-A-0594 M protein sequence (SEQ ID No: 6)

Figure 12. HRSV-A-0594 SH protein (SEQ ID No: 7)

Figure 13. HRSV-A-0594 G protein (SEQ ID No: 8)

Figure 14. HRSV-A-0594 F protein (SEQ ID No: 9)

Figure 15. HRSV-A-0594 M2-1 protein (SEQ ID No: 10)

Figure 16. HRSV-A-0594 M2-2 protein (SEQ ID No: 11)

Figure 17. HRSV-A-0594 L protein (SEQ ID No: 12)

Figure 18. HRSV-B-9671 nucleotide sequence (SEQ ID NO: 13)

Figure 19. HRSV-B-9671 NS1 protein (SEQ ID NO: 14)

Figure 20. HRSV-B-9671 NS2 protein (SEQ ID No: 15)

Figure 21. HRSV-B-9671 N protein (SEQ ID No: 16)

Figure 22. HRSV-B-9671 P protein (SEQ ID No: 17) Figure 23. HRSV-B-9671 M protein (SEQ ID No: 18)

Figure 24. HRSV-B-9671 SH protein (SEQ ID No: 19)

Figure 25. HRSV-B-9671 G protein (SEQ ID No: 20)

Figure 26. HRSV-B-9671 F protein (SEQ ID No: 21)

Figure 27. HRSV-B-9671 M2-1 protein (SEQ ID No: 22)

Figure 28. HRSV-B-9671 M2-2 protein (SEQ ID No: 23)

Figure 29. HRSV-B-9671 L protein (SEQ ID No: 24)

Figure 30. Schematic representation of recombinant human metapneumovirus (HMPV) strains. The transcription units and the open reading frames (ORFs) of the nucleocapsid (N), the phospho- (P), the matrix (M), fusion (F), M2, small hydrophobic (SH), glycoprotein (G) and large (L) protein. rHMPVA (A), rHMPV^AEGFP (B), rHMPV^B (C), rHMPV^BEGFP (D). The additional transcription unit (ATU) encoding the ORF of EGFP or a different fluorescent or luminescent protein is inserted between the P and M genes.

Figure 31. Alignment of RSV whole genome sequences demonstrating the presence of a duplicated 72 bp region in the G gene of G-duplicate strains of RSV-A(A) and a 60 bp region in G-duplicate strains of RSV-B (B).

Sequences were aligned using BioEdit with RSV-A⁰⁵⁹⁴ and RSV-B⁹⁶⁷¹ sequences highlighted in the top row in each panel.

(A) RSV-A nucleic acid sequences from bottom to top:

G-single strains A2, KT992094 (SEQ ID No 25); Long, KF713490 (SEQ ID No 26); 19, FJ614813 (SEQ ID No 27); Memphis-37, KM360090 (SEQ ID No 28); G-duplicate strains NZL/UHRSV76/2011, KX765960 (SEQ ID No 29);

Lebanon/14LJF289/2015, MG793382 (SEQ ID No 30); TOp-13-203, KY654517 (SEQ ID No 31); TEv-12-138, KY654512 (SEQ ID No 32); USA/TH_10195/2012, KU950680 (SEQ ID No 33); USA/LA2_90/2012, KM042383 (SEQ ID No 34); USA/TH_10305P/2013 , KX894797 (SEQ ID No 35); USA/LA2_13/2012, KJ672465 (SEQ ID No 36); USA/LA2_100/2013, KJ672457 (SEQ ID No 37); HRSVA 0594 SEQ ID No 38).

(B) RSV-B nucleic acid sequences from bottom to top:

G-single strains USA/84I-250A-01/1984, KJ723485 (SEQ ID No 40); 9320, AY353550 (SEQ ID No 41); Bl, AF013254 (SEQ ID No 42);

G-duplicate strains NZL/UHRSV20/2014, KX765973 (SEQ ID No 43);

USA/LA2_16/2012, KM042393 (SEQ ID No 44); 1856, KR350475 (SEQ ID No 45); TB5_CA-15-0741_1, LC384998 (SEQ ID No 46); NZL/UHRSV51/2013, KX765959 (SEQ ID No 47); WI/629-Q0190/10, JN032120 (SEQ ID No 48); PER/FPP01374/2012, KJ627262 (SEQ ID No 49); NZL/UHRSV67/2012, KX765976 (SEQ ID No 50); JOR/D3772/2013, KX655693 (SEQ ID No 51); RSVB 9671 (SEQ ID No 52).

Detailed description

RSV is the type species of Genus Orthopneumovirus, Family Pneumoviridae,

Order Mononegavirales. Human RSV exists as two antigenic subgroups (HRSV-A and HRSV- B) that exhibit genome-wide sequence divergence. The other members of this genus are bovine RSV (BRSV), ovine RSV(ORSV), and pneumonia virus of mice (PVM). More

pneumoviruses remain to be identified: recent wide-ranging fieldwork provided sequence evidence of RSV-like viruses in African bats and possibly humans. The RSV virion consists of a nucleocapsid packaged in a lipid envelope derived from the host cell plasma membrane. Virions produced in cell culture consist of spherical particles of 100-350 nm in diameter and long filaments that usually predominate and are 60-200 nm in diameter and up to 10 pm in length. In vitro, 95 % of progeny virus remains associated with the cell surface as particles that seemingly have failed to fully bud. In preparing virus stocks, infected cells typically are subjected to freeze-thawing, sonication, or vortexing to release attached virus, although this reduces infectivity and increases cellular contamination. RSV readily loses infectivity during handling and freeze-thawing due to particle instability and aggregation, although this can be partly overcome by excipients such as sucrose. There is indirect evidence that the surface glycoproteins, especially F, are factors in the instability. The long filamentous shape of the particle likely also confers fragility.

The RSV envelope contains three viral transmembrane surface glycoproteins: the large glycoprotein G, the fusion protein F, and the small hydrophobic SH protein. The non- glycosylated matrix M protein is present on the inner face of the envelope. The viral glycoproteins form separate homo-oligomers that appear as short (11-16 nm) surface spikes. RSV lacks neuraminidase or hemagglutinin activity and the F is known to be heavily sialylated, presumably because of the lack of a neuraminidase. There are four

nucleocapsid/polymerase proteins: the nucleoprotein N, the phosphoprotein P, the transcription processivity factor M2-1, and the large polymerase subunit L.

The RSV genome is a single-stranded non-segmented negative-sense RNA of 15,191-15,226 nt for six sequenced strains (subgroup A strain A2, 15,222 nt, GenBank accession

number M74568, is the reference strain). RNA replication involves a complementary copy of the genome called the antigenome. The genome and antigenome lack 5' caps or 3' polyA tails. The first 24-26 nt at the 3' ends of the genome and antigenome have 88 % sequence identity, representing conserved promoter elements. The genome and antigenome are bound separately for their entire length by the N protein to form stable nucleocapsids.

These are the templates for RNA synthesis and remain intact throughout the replicative cycle and in the virion. In addition, encapsidation protects the RNA from degradation and shields it from recognition by host cell pattern recognition receptors that initiate innate immune responses.

The genome has 10 genes in the order 3' NS1-NS2-N-P-M-SH-G-F-M2-L. Each gene encodes a corresponding mRNA. The mRNAs have methylated 5' caps and 3' polyA tails. Each mRNA encodes a single major protein except for M2, which has two separate ORFs that overlap slightly and encode the M2-1 and M2-2 proteins. The downstream M2-2 ORF is accessed by ribosomes that exit the M2-1 ORF and reinitiate, a process that is influenced by upstream structure in the M2 mRNA. The 3' end of the genome consists of a 44-nt extragenic leader region that precedes the NS1 gene. The 5' end of the genome consists of a 155-nt extragenic trailer region that follows the L gene. Each gene begins with a highly conserved 9-nt gene- start (GS) signal and terminates with a moderately conserved 12-14-nt gene-end (GE) signal that ends with 4-7 U residues (genome-sense) that encode the polyA tail by polymerase stuttering. The first nine genes are separated by intergenic regions that vary in length from 1 to 58 nt for the strains sequenced to date. These lack any conserved motifs, are poorly conserved between strains, and appear to be unimportant spacers, except that at some gene junctions the first nucleotide of the intergenic region is important for mRNA termination. A tolerance for intergenic variability is illustrated by the finding that incrementally increasing the length of an intergenic region in recombinant RSV up to 160 nt had little effect on gene expression or viral replication in vitro; however, this was moderately attenuating in mice, indicating that excessive length is restrictive The last two genes, M2 and L, overlap by 68 nt: specifically, the L GS signal is located 68 nt upstream of the end of the M2 gene. The same overlap occurs in BRSV, and gene overlaps occur for some genes in some members of Rhabdoviridae and Filoviridae.

RSV encodes 11 separate proteins, and thus is more complex than most members of the closely related family Paramyxoviridae, which typically have 6-7 mRNAs encoding 7-9 separate proteins. The N, P, M, F, and L proteins of RSV have clear orthologs

throughout Paramyxoviridae, and their relative genome order is conserved. Amino acid sequence relatedness between RSV and paramyxoviruses is low and is evident primarily for the F and L proteins and segments in the C-terminal region of N. The NS1, NS2, M2-1, and M2-2 proteins of RSV have no counterparts in the family Paramyxoviridae, and an SH protein (which is found in all members of Pneumovirinae) is present only in a few members of the family Paramyxoviridae.

As used herein, a bacterial artificial chromosome (BAC) is a nucleic acid (preferably DNA) that is used as a vector to clone foreign nucleic acid segm ents ( prefera bly D NA), that is introduced for cloni ng i nto a bacteri um where it is maintai ned as a plasmid, a nd into which a segment of a foreign nucleic acid such as foreign D NA may be spliced (i nserted) for cloning As used herein, a "vector" is a nucleic acid such as a DNA molecule to which heterologous nucleic acid such as DNA may be operatively linked so as to bring about replication of the heterologous nucleic acid. Vectors are conventionally used to deliver nucleic molecules to cells, including E. coli cells that are typically used in a majority of cloning application.

Construction of novel wild-type Pneumovirus reverse genetics systems:

New reverse genetics systems have been developed by the Molecular Virology group at the Research Center for Emerging Infections and Zoonoses (RIZ), Hannover, and its improved use has been demonstrated for wild-type human respiratory syncytial virus (HRSV) A and B strain (HRSV-B-9671, HRSV-A-0594). The vector used in the reverse genetic system herein uses a BAC (pSMART BAC, Lucigen Technologies) which has been modified according to the invention to facilitate wild-type RSV strain reverse genetics by removal of a second Notl restriction endonuclease cleavage site in the multiple cloning site (MCS) and insertion of essential 5' and 3' flanking sequences (T7 promoter, hammerhead ribozyme, hepatitis delta ribozyme (HDR), T7 terminator sequences) using two BamHI restriction enzyme endonuclease sites present in the MCS. RSV sequences are inserted into the BAC vector using the unique Notl restriction cleavage site in the BAC MCS and a Kasl restriction cleavage site present in the HDR. Exemplary use of the novel genetic system has been provided by the here disclosed RSV reverse genetics system as demonstrated with sequences derived from recent wild-type RSV A (0594) and B (9671).

These are examples of notoriously difficult to maintain wild-type RSV strains containing unique duplications in the G protein as opposed to using a hybrid virus, based on for example the laboratory adapted A2 strain which is no longer circulating in humans, wherein which the F gene has been replaced by the equivalent gene from the line 19 strain to accommodate replication (Virology. 2012 Dec 5; 434(1): 129-136, [also described in US 20160030549 and US 20120264217]). In these last 3 publications, each RSV gene is flanked by restriction endonuclease cleavage sites to allow for easy manipulation of any gene, which however does not allow for studying G gene duplications as opposed to the reverse genetic system as provided herein. Furthermore, these publications strive to avoid genetic reversion towards undesirable wild-type viral sequences.

The use of the modified pSMART BAC as provided herein enables the recovery of higher DNA yields than the vector backbone pKBS3. The BAC Cloning System used herein contains both an ori2 (oriS) origin of replication allowing for a single copy state in bacteria but also an oriV medium-copy origin of replication, which is activated in the presence of the TrfA replication protein. TrfA is expressed in BAC-Optimized Replicator v2.0 bacteria under the control of the araC-Pe_AD promoter. Thus, expression of L-arabinose as a supplement to the transformed bacterial culture induces expression of TrfA, which in turn activates oriV, enabling the BAC to replicate to up to 50 copies per cell. Surprisingly however, despite the high copy number, no loss of stability of modified pSMART BAC vectors containing the full-length RSV antigenome has been observed following the addition of L-arabinose induction solution to transformed cells.

The inventors have designed and constructed BAC clones containing an authentic copy of the full-length antigenome of representative wild-type strains of RSV. The inventors specifically decided to retain an authentic RSV wild-type sequence without any artificially introduced restriction enzyme sites that separate the individual RSV genes as described above. As such this precludes the use of artificially introduced restriction endonuclease cleavage sites which would alter the virus sequence. Instead construction of the BAC clones relied on naturally occurring unique restriction enzymes sites and the use of NEB HiFi assembly to rapidly assemble the viral antigenome by combining shorter DNA fragments into longer stretches of DNA which could be cloned between two naturally occurring restriction enzyme sites. This enables the rescue of recombinant RSVs which have an identical sequence to the parental non-recombinant RSV, thus allowing for the mechanistic study of single point mutations which confers resistance to specific antibodies or antivirals.

The inventors use currently circulating wild-type RSVs as the source for their virus sequences for inclusion in the BAC system, as opposed to using older strains (A2) or hybrid strains (A2 + F gene of line 19) which are no longer circulating. Furthermore, the inventors integrate fully consecutive viral antigenome, wherein viral protein encoding nucleic acid sequences are not separated by artificially introduced and non-viral sequences encoding one or more restriction sites. Furthermore, BAC as provided herein can be amplified to higher copy numbers per bacterium without any loss of stability of the full-length BA, generating sufficient more DNA with viral sequences to make the system is work. The generation of novel recombinant respiratory syncytial viruses derived from clinical isolates now provides use as well-defined viruses in human challenge studies, vaccine development and assessment of the efficacy of therapeutic intervention strategies. In order to obtain a more representative HRSV strain for human challenge studies with the ability to attenuate the virus through point mutations or gene deletions or to test the efficacy and mode of action of vaccines or antivirals, the applicants-initiated studies aimed at generating recombinant HRSVs based on clinical isolates. The resulting full-length virus clones were designed without any artificially introduced restriction enzyme sites and had an identical sequence to the consensus HRSV sequence present in the patients.

Both HRSV strains were isolated from clinical swabs obtained from children hospitalized in Hannover with confirmed HRSV infections.

Deep sequencing was performed on RNA isolated directly from clinical swabs resulting in the generation of a consensus sequence for both stains (HRSV-A-0594) and (HRSV-B-9671). These sequences were found to be identical to analogous deep sequencing performed on viral RNA isolated following one passage on HEp-2 cells. The specific sequences of the leader and trailer regions were identified by 5' and 3' RACE.

HRSV-A-0594 was identified as an ONI genotype strain of HRSV containing a 72-nucleotide duplication in the C-terminal part of the G gene. This strain, originally discovered in Ontario, Canada in 2010, has rapidly spread worldwide in recent years and appears to have a selective advantage with respect to transmission compared to older lineages which are missing this duplication e.g. A2 or Long lineage strains.

HRSV-B-9671 was identified as a BA genotype strain of HRSV containing a 60-nucleotide duplication in the C-terminal part of the G gene. This strain was originally isolated in Buenos Aires in 1999 and have subsequently outcompeted older HRSV B strain lineages which lack this sequence duplication. In order to construct full-length clones, RNA was extracted from virus infected HEp-2 cells and reverse transcribed using Superscript IV (ThermoFisher) to produce cDNA. This was used as the template for PCRs to amplify genome segments.

Initially, construction was attempted in pBluescript using a combination of standard restriction digest and ligation protocols and HiFi assembly (NEB). However, due to instability issues in the G gene region, no stable full-length clones could be obtained for HRSV A. A full- length clone was obtained for HRSV-B-9671 but this was only stable under extreme selective pressures in E. coli, effectively blocking further use of recombinant RSV from the pBluescript vector amenable to rapid mechanistic translational studies.

Subsequently, full-length cDNA clones were constructed in a bacterial artificial chromosome (pSMART, Lucigen) in which a Notl site was removed using point mutagenesis to facilitate the use of a second Notl site and Kasl in cloning procedures. Essential flanking sequences necessary for virus rescue were first inserted using a synthetic DNA fragment. The full sequence of the HRSV-B-9671 antigenome present in a pBluescript vector was inserted into pSMART via standard digest and ligation protocols. Similarly, a partial sequence of HRSV-A- 0594 in which the G gene region was missing was inserted into pSMART-BAC using analogous techniques. The missing HRSV A sequence was inserted using a 4 fragment HiFi assembly between two native Nhel restriction enzyme sites. Both clones have proven to be extremely stable and the use of the pSMART vector has enabled higher plasmid DNA yields than standard BAC vectors would allow for e.g. pBeloBACll

Essential flanking sequences necessary for virus rescue include a T7 promoter is present at the 5' end in order to initiate transcription by T7 DNA dependent RNA polymerase (refs: Radeke et a I, 1995; Schnell et al, 1994). This is followed by 'GGG' which has been shown to increase the efficiency of transcription. A hammerhead ribozyme has been inserted before the leader sequence in order to ensure an authentic 5' end of the viral genome by catalyzing the removal of additional nucleotides. This improves the efficiency of virus rescue.

A hepatitis delta ribozyme (HDV) was inserted following the trailer sequence of the HRSV antigenome in order to ensure the generation of an authentic 3'end of the viral genome. Two T7 terminator sequences were inserted after the HDV to ensure detachment of the T7 DNA dependent RNA polymerase.

Additional full-length cDNA clones have been generated using HiFi assembly (NEB) which stably express either EGFP, dTOM or luciferase from an additional transcription unit inserted between the P and M genes.

Additional plasmids essential for virus rescue have been generated by cloning the open reading frame of the HRSV A-0594 or HRSV-B-9671 N, P, M2-1 and L proteins into the eukaryotic expression vector pcDNA3.1 which has a CMV promoter to enable high level expression of the protein of interest in mammalian cells. A minimal Kozak sequence was inserted before the start codon to ensure optimal expression of RSV proteins.

Virus rescue protocols were similar to previously reported protocols. Briefly 1 x 10⁶ HEp-2 cells were seeded into each well of a 6 well tray. After 24hrs, media was removed, and cells were infected for one hour with the vaccinia virus MVA strain expressing T7 DNA dependent RNA polymerase. Alternatively, a plasmid expressing a codon optimized form of T7 DNA dependent RNA polymerase was transfected into cells. Cells were transfected with pSM- HRSV-A-0594 (1.6 pg) or pSM-HRSV-B-9671 (1.6 pg) together with pcDNA3.1 plasmids expressing the N (1.6 pg), P (1.2 pg), M2-1 (0.8 pg) and L (0.4 pg) proteins using lipofectamine 3000. After addition of Optimem to each well, trays were transferred to the 37°C incubator overnight and the next day, media in each well was replaced.

Recombinant viruses have been rescued between 5 and 7 days post -transfection in HEp-2 cells with high efficiency. This system allows expedient rescue recombinant HRSVs with specific attenuating point mutations and/or gene deletions. In order to optimize virus growth (a problem with clinical isolates), protocols for virus harvesting and stabilization have been optimized. Upon observation of extensive cytopathic effects, cells were scraped into the medium using a cell scraper and this was transferred to sterile 50ml tube and centrifuged at 3000RPM for 5 minutes at 4°C. Supernatant was transferred to a fresh, sterile 50ml tube and an equivalent volume of stabilization solution was added and placed on ice. The stabilization solution consisted of 40% glycerol/10% FBS (Gupta et al, Vaccine, 1996; Ke Z, HRSV PhD, Hong Kong University, 2014). The cell pellet was resuspended in 3mls of Optimem (taken from the tube containing supernatant virus with added stabilizer). 1ml of this cell-associated fraction was transferred to each of three cryovials, and flash frozen in liquid nitrogen. The three cryovials were defrosted rapidly in a 37°C waterbath. The samples were flash frozen once more in liquid nitrogen and then defrost again at 37°C. The cell associated fraction was transferred to a 15ml tube and spun at 3000RPM for 5 minutes at 4°C. The 3mls of supernatant was added back into the stabilized supernatant HRSV kept on ice and mixed briefly and aliquoted into cryovials which were flash frozen in liquid nitrogen and transferred to long-term storage in a -150°C freezer.

Applications of recombinant viruses (only translational aspects are listed)

For use a well-defined virus for human challenge studies in which it is essential that a virus is used which is representative of currently circulating strains so that any point mutations conferring escape from antibodies and/or antivirals can be mechanistically investigated via the generation of recombinant viruses.

Used as the basis for vaccine development via the insertion of point mutations and/or gene deletions. This has been done previously for the lab-adapted A2 strain (isolated in 1961) but this virus has proved to be over-attenuated in human vaccination experiments.

Use as a challenge virus in assessment of the efficacy of antivirals or vaccines to ensure that such experiments use a recombinant strain representative of currently circulating wild-type strains.

Testing efficacy of recombinant wild-type HRSVs for use in development of therapeutic intervention strategies

We generate rHRSVs with and without mutations conferring resistance to the monoclonal antibody Synagis (Palivizumab, Medlmmune) or a broad class of HRSV polymerase inhibitors and test these in an in vitro phenotypic read-out assay. Synagis (Palivizumab, Medlmmune) is a humanized monoclonal antibody (IgG) directed against an epitope between amino acids 258 and 275 in the A antigenic site of the F glycoprotein of HRSV. This antibody has been approved by the European Medicines Agency for use in Europe since August 13th, 1999. Specific mutations (K272E/Q. and S275F/L in antigenic site A, a highly conserved region on the RSV F protein), shown previously to enable escape from Palivizumab (Zhu et al, 2011), can be inserted individually into the F glycoprotein gene of full-length antigenomic clones of HRSV A and B strains. Additional rHRSV-A-0594 and rHRSV-B-9671 can be generated which contain a single point mutation (Y1631H) in the polymerase gene which has been shown previously to confer resistance to a number of polymerase inhibitors (Fearns and Deval, 2016). This is achieved using established protocols including NEBuilder HiFi DNA Assembly (NEB) or BAC mutagenesis via Red^®/ET^® Recombination (Gene Bridges GmbH) which enable single mutations to be rapidly introduced into our BAC vectors as provided herein, a technique which is not possible with standard low copy or high copy plasmid vectors which encode the full-length antigenome of a pneumovirus. The introduction of point mutations into BAC clones using NEBuilder HiFi DNA Assembly relies on the error free joining of overlapping DNA fragments which are amplified by primers which contain complementary mismatches at the site of the desired mutation. The flanking sequences of the annealed DNA fragments contain an overlap of 20-25 nucleotides with the sites of unique restriction sites in the BAC vector. This facilitates ligation of the single DNA fragment containing the desired mutations into a BAC vector which has been linearized by pre-digestion with these restriction enzymes. Alternatively, one or more mutations can be introduced into a BAC clone using Red/ET Recombination (El-mediated recombination) in E. coli. This is based on homologous recombination between a linear DNA fragment and a region of the BAC clone in which mutation(s) are required. This is mediated by the expression in E. coli of the phage-derived protein pairs RecE (an exonuclease) and RecT (DNA annealing protein) which mediate recombination between the BAC clone and the linear DNA fragment at homology regions of approximately 50bp at the flanking regions.

Recombinant wild-type and mutant viruses are rescued following transfection of HEp-2 cells with full-length antigenomic HRSV A or B clones together with helper plasmids expressing HRSV N, P, M2-1 and L proteins and an additional plasmid expressing codon optimized T7 DNA dependent RNA polymerase. Stocks of rescued viruses are prepared by passage on HEp-2 cells and virus stocks are completely sequenced to ensure retention of the introduced mutation in the F glycoprotein or in the polymerase gene.

For phenotypic HRSV analysis, Hep-2 cells are inoculated with rHRSV-A-0594 or rHRSV-B-9671 strains expressing renilla luciferase with and without the listed point mutations and subsequently incubated in presence of a range of Synagis or HRSV polymerase inhibitor concentrations, until the optimal virus signal is achieved in control cultures without inhibitor. Following detection of the maximal virus signal and the signals at each of the antiviral compound concentrations, 50% or 90% inhibitory concentrations are calculated from the percentages inhibition of the maximal signal. The efficiencies with which rHRSVs with specific point mutations are able to escape from inhibition are examined using a luciferase readout assay.

Additional references

Zhu Q, McAuliffe JM, Patel NK, Palmer-Hill FJ, Yang C, Liang B, Su L, Zhu W, Wachter L,

Wilson S et al (2011). Analysis of Respiratory Syncytial Virus Preclinical and Clinical Variants Resistant to Neutralization by Monoclonal Antibodies Palivizumab and/or Motavizumab. Journal of Infectious Disease 203: 674-682.

Fearns R, Deval J (2016). New antiviral approaches for respiratory syncytial virus and other mononegaviruses: Inhibiting the RNA polymerase. Antiviral Research 134: 63-76.

Sidhu MS, Chan J, Kaelin K, Spielhofer P, Radecke F, Schneider H, Masurekar M, Dowling PC, Billeter MA, Udem SA. 1995. Rescue of synthetic measles virus minireplicons: measles genomic termini direct efficient expression and propagation of a reporter gene. Virology. 208: 800-807.

Radecke F, Spielhofer P, Schneider H, Kaelin K, Huber M, Dotsch C, Christiansen G, Billeter MA. 1995. Rescue of measles viruses from cloned DNA. EMBO Journal. 14(23):5773-5784. Price SR, Ito N, Oubridge C, Avis JM, Nagai K. 1995. Crystallization of RNA-protein complexes. I. Methods for the large-scale preparation of RNA suitable for crystallographic studies. Journal of Molecular Biology. 249:398-408.

Yun T, Park A, Hill TE, Pernet O, Beaty SM, Juelich TL, Smith JK, Zhang L, Wang YE, Vigant F, Gao J, Wu P, Lee B, Freiberg AN. 2015. Efficient reverse genetics reveals genetic determinants of budding and fusogenic differences between Nipah and Hendra viruses and enables real time monitoring of viral spread in small animal models of henipavirus infection. Journal of Virology. 89(2):1242-1253.

Claims

1 A nucleic acid comprising a bacterial artificial chromosome additionally provided with DNA dependent RNA-polymerase promotor/terminator nucleic acid sequences, said nucleic acid also comprising a nucleic acid encoding a hammerhead ribozyme, said nucleic acid also comprising a nucleic acid encoding an antigenome of an enveloped, non-segmented negative-strand RNA virus classified in the family Pneumoviridae, said nucleic acid also comprising a nucleic acid encoding a hepatitis delta virus ribozyme.

2 A nucleic acid according to claim 1 wherein said virus is an Orthopneumovirus.

3 A nucleic acid according to claim 2 wherein said virus is selected from the group of human or ruminant respiratory syncytial viruses.

4 A nucleic acid according to claim 3 wherein said virus is a human respiratory syncytial virus (HRSV).

5 A nucleic acid according to claim 4 wherein said virus is wild-type HRSV-A.

6 A nucleic acid according to claim 5 wherein said virus has a duplication in the gene encoding the G-protein.

7 A nucleic acid according to claim 6 wherein said virus has a nucleic acid sequence homology of at least 80%, preferably at least 90%, more preferably at least 95%, most preferably of at least 99% with the nucleic acid of SEQ ID NO: 1.

8 A nucleic acid according to claim 6 wherein said virus is HRSV-A-0594 with SEQ ID NO: 1.

9 A nucleic acid according to claim 4 wherein said virus is wild-type HRSV-B.

10 A nucleic acid according to claim 9 wherein said virus has a duplication in the gene encoding the G-protein.

11 A nucleic acid according to claim 10 wherein said virus has a nucleic acid

sequence homology of at least 80%, preferably at least 90%, more preferably at least 95%, most preferably of at least 99% with the nucleic acid of SEQ ID NO: 13.

12 A nucleic acid according to claim 11 wherein said virus is HRSV-B-9671 with SEQ ID NO: 13

13 A nucleic acid according to claim 3 wherein said virus is bovine respiratory

syncytial virus (BRSV). 14 A nucleic acid according to claim 3 wherein said virus is ovine respiratory syncytial virus (ORSV).

15 A nucleic acid according to claim 1 wherein said virus is a Metapneumovirus.

16 A nucleic acid according to claim 15 wherein said virus is human

metapneumovirus (HMPV).

17 A nucleic acid according to claim 15 wherein said virus is avian

metapneumovirus.

18 A nucleic acid according to claim 15 to 17 wherein said virus has a duplication in the gene encoding the G-protein.

19 A cell comprising a nucleic acid according to anyone of claims 1 to 18.

20 A prokaryote cell according to claim 19.

21 A cell according to claim 20 additionally provided with a nucleic acid encoding a TrfA replication protein.

22 A eukaryote cell according to claim 19.

23 A cell according to claim 22 additionally provided with a nucleic acid encoding a DNA dependent RNA polymerase.

24 A cell according to claim 22 or 23 additionally provided with a nucleic acid

encoding at least one protein selected from the group of N, P, M2-1 and L proteins from a virus classified in the family Pneumoviridae.

25 A virus obtainable by rescue from a cell according to claim 24.

26 Use of a virus according to claim 25 in assessment of binding of an antibody.

27 Use of a virus according to claim 25 in assessment of efficacy of a vaccine.

28 Use of a virus according to claim 25 in assessment of efficacy of an antiviral agent.

29 Use of a virus according to claim 25 in assessment of virus entry in a eukaryote cell.

30 Use of a virus according to claim 25 in assessment of a T-helper cell response.