Ultrastructural study of myxoma virus morphogenesis.

Poxviruses are among the largest and most complex viruses known. Vaccinia virus, the prototype of the family Poxviridae, has been studied much more than myxoma virus. The aim of this work was to have a better knowledge about myxoma virus morphogenesis. The characterization of the main stages of MV morphogenesis was achieved by ultrastructural and immunological analysis. Specific antibodies were raised against M022L and M071L, two envelope proteins of extracellular enveloped virus and intracellular mature virus, respectively. The main stages of assembly were similar to those seen with other poxviruses, and the duration of the whole replication cycle was estimated to be around 16 h, longer than what was described for vaccinia virus. Morphological changes of infected cells were associated with the development of long cellular projections and enlarged microvilli. Intracellular enveloped viruses are associated with the cytoskeleton to move through the cell. Unlike earlier studies, as many cell-associated enveloped viruses as intracellular enveloped viruses were observed in relation with specialized microvilli, although these structures were rarely noticed. Finally, an unusual spreading process was observed, which uses cytoplasmic corridors.

A novel polyomavirus (goose hemorrhagic polyomavirus) is the agent of hemorrhagic nephritis enteritis of geese.

We have identified the etiological agent of hemorrhagic nephritis enteritis of geese (HNEG), a fatal disease of European geese. HNEG has been recognized in almost all goose breeding areas, with an epizootic pattern, and up to now, the infectious agent has remained unknown. In order to identify the causative agent, infected tissues from HNEG-affected geese were inoculated to 1-day-old goslings, which then developed clinical signs typical of HNEG. Tissue homogenates from these birds were subjected to Freon extraction followed by sucrose density gradient ultracentrifugation. The resulting main band was examined by electron microscopy and consisted of spherical, naked, papovavirus-like particles approximately 45 nm in diameter. The virus was isolated and propagated in goose kidney cell primary culture. Tissue- or culture-purified virus allowed the experimental reproduction of the disease in goslings. Random PCR amplification of viral nucleic acid produced a 1,175-bp fragment which was shown to be associated with field samples collected from geese affected by HNEG on commercial farms in France. Sequence analysis of the PCR product revealed a unique open reading frame, showing 63 to 72% amino acid similarity with the major capsid protein (VP1) of several polyomaviruses. Finally, based on phylogenetic analysis, we conclude that the causative agent of HNEG is closely related to but clearly distinct from other polyomaviruses; we thus have named this newly identified virus Goose hemorrhagic polyomavirus.

Development of an ELISA for detection of myxoma virus-specific rabbit antibodies: test evaluation for diagnostic applications on vaccinated and wild rabbit sera.

An enzyme-linked immunosorbent assay (ELISA) was developed and compared with 2 reference diagnostic tests (indirect immunofluorescence [IF] and complement fixation) to detect myxoma virus-specific antibodies in sera from 50 rabbits experimentally vaccinated with an attenuated strain of myxoma virus or with a Shope fibroma virus. The ELISA was highly specific (100% specificity) and sensitive (100%, 21 days after homologous vaccination). In a comparison of the ELISA with the IF test in 128 wild rabbits from France, discrepant results were obtained in only 11 (8.6%) animals, which were positive with the ELISA and negative with the IF test. The higher sensitivity and the good specificity of the ELISA was confirmed in a serologic survey of 118 rabbits from 2 Kerguelen (Indian Ocean) islands, where the prevalence of myxomatosis varied considerably. The ELISA is an alternative serologic test for diagnosis, vaccine evaluation, and seroepidemiologic surveys of myxomatosis.

Evaluation of a nested reverse transcription-PCR assay based on the nucleoprotein gene for diagnosis of spontaneous and experimental bovine respiratory syncytial virus infections.

The first nested reverse transcription (RT)-PCR based on the nucleoprotein gene (n RT-PCR-N) of the bovine respiratory syncytial virus (BRSV) has been developed and optimized for the detection of BRSV in bronchoalveolar lavage fluid cells of calves. This test is characterized by a low threshold of detection (0.17 PFU/ml), which is 506 times lower than that obtained by an enzyme immunosorbent assay (EIA) test (RSV TESTPACK ABBOTT). During an experimental infection of 17 immunocompetent calves less than 3 months old, BRSV RNA could be detected up to 13 days after the onset of symptoms whereas isolation in cell culture was possible only up to 5 days. Compiling results obtained by conventional techniques (serology, antigen detection, and culture isolation) for 132 field samples collected from calves with acute respiratory signs revealed that n RT-PCR-N showed the highest diagnostic sensitivity and very good specificity. This n RT-PCR-N with its long period of detection during BRSV infection thus provides a valuable tool for diagnostic and epidemiological purposes.

Serp2, an inhibitor of the interleukin-1beta-converting enzyme, is critical in the pathobiology of myxoma virus.

Recently, myxoma virus was shown to encode an additional member of the serpin superfamily. The viral gene, called serp2, was cloned, and the Serp2 protein was shown to specifically bind to interleukin-1beta (IL-1beta)-converting enzyme (ICE), thus inhibiting the cleavage of pro-IL-1beta by the protease (F. Petit, S. Bertagnoli, J. Gelfi, F. Fassy, C. Boucraut-Baralon, and A. Milon, J. Virol. 70:5860-5866, 1996). Here, we address the role of Serp2 in the development of myxomatosis, a lethal infectious disease of the European rabbit. A Serp2 mutant myxoma virus was constructed by disruption of the single-copy serp2 gene and insertion of the Escherichia coli gpt gene serving as the selectable marker. A revertant virus was obtained by replacing the E. coli gpt gene by the intact serp2 open reading frame. The Serp2(-) mutant virus replicated with wild-type kinetics both in rabbit fibroblasts and a rabbit CD4(+) T-cell line (RL5). Moderate reduction of cell surface levels of major histocompatibility complex I was observed after infection with wild-type or Serp2(-) mutant myxoma virus, and both produced white pocks on the chorioallantoic membrane of the chick embryo. After the infection of European rabbits, the Serp2(-) mutant virus proved to be highly attenuated compared to wild-type myxoma virus, as demonstrated by the clinical course of myxomatosis and the survival rates of infected animals. Pathohistological examinations revealed that infection with wild-type myxoma virus resulted in a blockade of the inflammatory response at the vascular level. In contrast, rapid inflammatory reactions occurred upon infection with the Serp2(-) mutant virus. Furthermore, lymphocytes in lymph nodes derived from animals inoculated with Serp2 mutant virus were shown to rapidly undergo apoptosis. We postulate that the virulence of myxoma virus in the European rabbit can be partially attributed to an impairment of host inflammatory processes and to the prevention of apoptosis in lymphocytes. The weakening of host defense is directly linked to serp2 gene function and is likely to involve the inhibition of IL-1beta-converting-enzyme-dependent pathways.

Characterization of a myxoma virus-encoded serpin-like protein with activity against interleukin-1 beta-converting enzyme.

A genomic library of myxoma virus (MV) DNA, a leporipoxvirus that causes myxomatosis, was constructed and screened by in vitro transcription-translation. A clone was selected on the basis of its strong reactivity with MV antiserum. Analysis of the corresponding DNA sequence and the deduced amino acid sequence revealed an open reading frame coding for a 34-kDa protein with strong homologies to members of the serpin superfamily. The gene encoding this new protein, called serp2, was localized on the MV genome. Interestingly, this gene is deleted in an attenuated strain. We constructed a baculovirus vector to produce recombinant Serp2 protein and raised specific antisera that allowed the characterization of Serp2 expression during the MV cycle. The biological relevance of this new serpin from MV was monitored, and it was shown that Serp2 could inhibit human interleukin-1 beta-converting enzyme activity.

Protection against myxomatosis and rabbit viral hemorrhagic disease with recombinant myxoma viruses expressing rabbit hemorrhagic disease virus capsid protein.

Two myxoma virus-rabbit hemorrhagic disease virus (RHDV) recombinant viruses were constructed with the SG33 strain of myxoma virus to protect rabbits against myxomatosis and rabbit viral hemorrhagic disease. These recombinant viruses expressed the RHDV capsid protein (VP60). The recombinant protein, which is 60 kDa in size, was antigenic, as revealed by its reaction in immunoprecipitation with antibodies raised against RHDV. Both recombinant viruses induced high levels of RHDV- and myxoma virus-specific antibodies in rabbits after immunization. Inoculations by the intradermal route protected animals against virulent RHDV and myxoma virus challenges.

Protection of rabbits against rabbit viral haemorrhagic disease with a vaccinia-RHDV recombinant virus.

In order to protect domestic and wild rabbits against RVHD, we constructed a recombinant vaccinia-RHDV virus, using the Copenhagen strain of the vaccinia virus. This recombinant virus expressed the RHDV capsid protein (VP60). Analysis of the expressed product showed that the recombinant protein, which is 60 kDa in size, was antigenic as revealed by its reactions in immunoprecipitation and indirect immunofluorescence with the antibodies raised against RHDV. The recombinant virus induced high level of RHDV specific antibodies in rabbits following immunization. Inoculations by both the intradermal and oral routes allow protection of animals against a challenge with virulent RHDV.

Functional domains in the spike protein of transmissible gastroenteritis virus.

The coronavirus spike protein S is assumed to mediate essential biological functions, including recognition of target cells. Earlier studies from our and other groups identified two regions of the TGEV S (220K) protein possibly implicated in such functions. The first of these corresponds to the 224 amino acid N-terminal region which is deleted in PRCV, the respiratory variant of TGEV. We have examined the pathogenicity for the newborn piglet of a series of neutralization escape mutants encoding an S protein mutated in this region. Several amino acid changes were correlated with a dramatic loss of enterovirulence, thus indicating that crucial determinants are associated with this domain of S. The second region of potential relevance is the major neutralization domain. Baculovirus-vectored expression of 150 to 220 amino acid-long stretches encompassing this region, which is encoded by both TGEV and PRCV, was performed. The resultant recombinant proteins were shown to react with the cognate antibodies and to bind APN specifically, thus localizing the receptor-binding site on the S primary structure. Altogether these data lend support to the view that a domain of S protein structurally distinct from the receptor binding site is required for the virus to express its enteric tropism.

Overexpression of TGEV cell receptor impairs the production of virus particles.

The porcine aminopeptidase-N (pAPN) is the cellular receptor for the transmissible gastroenteritis virus (TGEV) due to the specific binding of the spike protein S to APN. In the present study, we performed both biological and biochemical experiments to analyze how the level of expression of a virus receptor can influence the viral protein biosynthesis and the virus production. We generated two swine testis cell clones overexpressing pAPN (ST-APN clones). These clones produced 10(4) less infectious virus than control ST cells. Plaque assays revealed a four-fold reduction of the diameter of the plaques in ST-APN cells compared to ST cells. Pulse-chase experiments revealed that S transport from the endoplasmic reticulum to the Golgi apparatus was not affected in ST-APN cells. Additionally, an anti-APN antibody was able to increase the virus released in the supernatant of ST-APN cells. Likewise, BHK clones expressing variable amounts of pAPN were shown to acquire TGEV susceptibility and to produce infectious particles as an inverse function of their level of pAPN expression. In contrast, MDCK clones expressing low or large amounts of pAPN failed to produce infectious particles. Taken together, these studies strongly suggest that overexpression of receptor, but also other(s) undetermined factor(s), can impair the production of viral particles.

Determinants essential for the transmissible gastroenteritis virus-receptor interaction reside within a domain of aminopeptidase-N that is distinct from the enzymatic site.

The swine-specific coronavirus transmissible gastroenteritis virus (TGEV) uses pig aminopeptidase-N (pAPN) as a cellular receptor. We showed that the human aminopeptidase-N (hAPN) cannot substitute for pAPN in this respect, although the two enzymes have 80% amino acid sequence identity. In order to map the TGEV binding site on pAPN, we constructed a series of APN cDNA chimeras between pAPN and hAPN and analyzed them for their capacity to confer infectivity. The region between residues 717 and 813 was found to be essential for infectivity. This region also contains the epitopes for three TGEV-blocking monoclonal antibodies directed against pAPN. These data support the view that the catalytic site and the TGEV receptor site are located in different domains. Moreover, APN inhibitors and mutations in the catalytic site had no obvious effect on permissiveness for virus, thus providing evidence that the APN enzymatic activity is not involved in the process of infection.

Genome organization of porcine epidemic diarrhoea virus.

In order to study the organization of the genome of porcine epidemic diarrhoea virus (PEDV), we constructed a cDNA library in a phage expression vector by using poly(A) RNA from PEDV-infected Vero cells. An anti-PEDV hyperimmune serum was used to probe the library. The first isolated clone mapped within the N gene and was subsequently used for rescreening the library. The selected clones allowed us to establish the sequence of the 3'-most 7.4 kb of the PEDV genome. Analysis of the cDNA sequences revealed a 3'-coterminal nested structure, which is typical of Coronaviridae and the presence of a hexameric sequence XUA(A/G)AC upstream of each coding region. The amino acid sequences deduced from four of the five ORFs identified showed the characteristic features of the structural proteins S, M, sM and N. Only one ORF located between the S and M genes was found to potentially encode a non-structural polypeptide. Our data lead us to conclude that PEDV is a member of Coronaviridae and belongs to the same genetic subset as TGEV, FIPV and HCV 229E.

Further characterization of aminopeptidase-N as a receptor for coronaviruses.

We recently reported that porcine aminopeptidase-N (pAPN) acts as a receptor for transmissible gastroenteritis virus (TGEV). In the present work, we addressed the question of whether TGEV tropism is determined only by the virus-receptor interaction. To this end, different non-permissive cell lines were transfected with the porcine APN cDNA and tested for their susceptibility to TGEV infection. The four transfected cell lines shown to express pAPN at their membrane became sensitive to infection. Two of these cell lines were found to be defective for the production of viral particles. This suggests that other factor(s) than pAPN expression may be involved in the production of infectious virions. The pAPN-transfected cells were also tested for their susceptibility to several viruses which have a close antigenic relationship to TGEV. So far, we failed to evidence permissivity to the feline infectious peritonitis coronavirus FIPV and canine coronavirus CCV. In contrast, we found clear evidence that porcine respiratory coronavirus PRCV, a variant of TGEV which replicates efficiently in the respiratory tract but to a very low extent in the gut, may also utilise APN to gain entry into the host cells. This suggests that the switch between TGEV and PRCV tropisms in vivo may involve other determinant(s) than receptor recognition.

Aminopeptidase N is a major receptor for the entero-pathogenic coronavirus TGEV.

Coronaviruses, like many animal viruses, are characterized by a restricted host range and tissue tropism. Transmissible gastroenteritis virus (TGEV), a major pathogen causing a fatal diarrhoea in newborn pig, replicates selectively in the differentiated enterocytes covering the villi of the small intestine. To investigate the molecular determinants of the infection, we characterized the surface molecule used by the virus for binding and entry into host cells. Here we report that aminopeptidase N, an ectoenzyme abundantly expressed at the apical membrane of the enterocytes, serves as a receptor for TGEV. Monoclonal antibodies were selected for their ability to block infection by TGEV of porcine cell lines. They recognized a brush-border membrane protein of M(r) 150K, which was identified as aminopeptidase N by amino-terminal sequencing. Two lines of evidence supported the view that the peptidase itself acts as a receptor. First, virions bound specifically to aminopeptidase N that was purified to homogeneity. Second, recombinant expression of aminopeptidase N conferred infectivity by TGEV to an otherwise non-permissive cell line.

Single amino acid changes in the viral glycoprotein M affect induction of alpha interferon by the coronavirus transmissible gastroenteritis virus.

Transmissible gastroenteritis virus, an enteropathogenic coronavirus of swine, is a potent inducer of alpha interferon (IFN-alpha) both in vitro and in vivo. Previous studies have shown that virus-infected fixed cells or viral suspensions were able to induce an early and strong IFN-alpha synthesis by naive lymphocytes. Two monoclonal antibodies directed against the viral membrane glycoprotein M (29,000; formerly E1) were found to markedly inhibit virus-induced IFN production, thus assigning to M protein a potential effector role in this phenomenon (B. Charley and H. Laude, J. Virol. 62:8-11, 1988). The present report describes the selection and characterization of a collection of 125 mutant viruses which escaped complement-mediated neutralization by two IFN induction-blocking anti-M protein monoclonal antibodies. Two of these mutants, designated H92 and dm49-4, were found to exhibit a markedly reduced interferogenic activity. IFN synthesis by lymphocytes incubated with purified suspensions of these mutants was 30- to 300-fold lower than that of the parental virus. The transcription of IFN-alpha genes following induction by each mutant was decreased proportionally, as evidenced by Northern (RNA) blot analysis. The sequence of the M gene of 20 complement-mediated neutralization-resistant mutants, including the 2 defective mutants, was determined by direct sequencing of genome RNA. Thirteen distinct amino acid changes were predicted, all located at positions 6 to 22 from the N terminus of the mature M protein and within the putative ectodomain of the molecule. Two substitutions, Thr-17 to Ile and Ser-19 to Pro, were assumed to generate the defective phenotypes of mutants dm49-4 and H92, respectively. The alteration of an Asn-Ser-Thr sequence in dm49-4 virus led to the synthesis of an M protein devoid of a glycan side chain, which suggests a possible involvement of this structure in IFN induction. Overall, these data supported the view that an interferogenic determinant resides in the N-terminal, exposed part of the molecule and provided further evidence for the direct role of M protein in the induction of IFN-alpha by transmissible gastroenteritis virus. The acronym VIP (viral interferogenic protein) is proposed as a designation for this particular class of proteins.

Molecular biology of transmissible gastroenteritis virus.

The causative agent (TGEV) of porcine transmissible gastroenteritis belongs to the Coronaviridae, a family of enveloped viruses with a positive, single-stranded RNA genome. Important progress has recently been made concerning the molecular biology of TGEV. The research work of our group has been focused on two main aspects: genome structure and functional domains of the envelope proteins. TGEV genomic RNA is organised into seven regions. The sequence of six of them, i.e. the 3' most 8300 nucleotides, has been established from cDNA clones. Three genes encoding the structural proteins, the peplomer protein E2, the transmembrane protein E1 and the nucleoprotein, have been identified. Additional open reading frames allowed for the prediction of four non-structural polypeptides, the role of which remains to be discovered. The remaining part of the genome (estimated length 20 kb) is thought to encode the polymerase. Expression of TGEV genes involves the production of six subgenomic mRNAs, which together with the virion RNA, form a 3' terminal nested set. The peplomer glycoprotein E2 (220 kDa) is 1431 residues long and highly glycosylated. Several domains were identified, including a C-terminal anchoring region and at least four major antigenic sites, which cluster in the amino half part of the molecule. Two sites containing most of the critical neutralisation determinants are highly conserved among TGEV strains. The glycoprotein E1 (29kDa) is mostly embedded in the membrane and plays a crucial role in the virion architecture. However, a short N-terminal domain protruding out of the particle mediates complement-dependent neutralisation, and induces alpha interferon synthesis, likely through a direct interaction with the lymphocyte membrane.

Four major antigenic sites of the coronavirus transmissible gastroenteritis virus are located on the amino-terminal half of spike glycoprotein S.

Four major antigenic sites have been delineated on the spike protein (S) of the porcine enteric coronavirus transmissible gastroenteritis virus (TGEV) in previous topological studies using monoclonal antibodies (MAbs). Correlation of these sites with the physical structure of the protein was achieved by use of different approaches. Recombinant pEX plasmids directing the synthesis of various fused S polypeptides were constructed. A hybrid protein containing nine S-specific residues (363 to 371) was shown to express site C epitopes. The other sites were localized through study of the antigenic activity of fragments generated by controlled cleavage of the native protein with different endopeptidases. Two identified cleavage products of 26K and 13K, immunoreactive to site A-B- and site D-specific MAbs respectively, could be aligned on the S primary structure according to N-terminal sequence data. This led us to propose that the major neutralization domain A-B is contained in a region of approximately 200 residues with residue 506 as its N boundary. Similarly, site D epitopes should be located within a stretch of 130 residues, starting at 82 residues from the N terminus. Point mutations identified by direct RNA sequencing of neutralization-resistant mutants were consistent with the proposed location of these sites.

Enteric coronavirus TGEV: partial sequence of the genomic RNA, its organization and expression.

The sequence of the 3'-most 8300 nucleotides of the genome RNA of the Purdue-115 strain of the transmissible gastroenteritis virus TGEV, a porcine coronavirus, was determined from cDNA clones. The available sequence corresponds to the part of the genome (total length greater than 20 kb) expressed through subgenomic mRNAs. The 5 subgenomic and the genomic RNA species detected in TGEV-infected cells form a 3'-coterminal 'nested' structure, a unique feature of Coronaviridae. The transcription initiation site of the TGEV subgenomic RNAs appears to involve the hexameric sequence 5'CTAAAC, which is present upstream from each coding region. In addition to the previously identified genes encoding the three structural proteins, E2, E1 and N, two regions, X1 and X2, corresponding to the non-overlapping portion of mRNAs 4 and 3, may code for so far unidentified non-structural polypeptides. The predicted X1 polypeptide (9.2 kDa) is highly hydrophobic. The sequence of the X2 region allows the translation of two non-overlapping products, i.e., X2a (7.7 kDa) and X2b (18.8 kDa). No RNA species liable to express the extreme 3' open reading frame X3 was found.