Progenitor Virus Research Articles

Development and Characterization of a Mouse Model for Marburg Hemorrhagic Fever

The lack of a mouse model has hampered an understanding of the pathogenesis and immunity of Marburg hemorrhagic fever (MHF), the disease caused by marburgvirus (MARV), and has created a bottleneck in the development of antiviral therapeutics. Primary isolates of the filoviruses, i.e., ebolavirus (EBOV) and MARV, are not lethal to immunocompetent adult mice. Previously, pathological, virologic, and immunologic evaluation of a mouse-adapted EBOV, developed by sequential passages in suckling mice, identified many similarities between this model and EBOV infections in nonhuman primates. We recently demonstrated that serially passaging virus recovered from the liver homogenates of MARV-infected immunodeficient (SCID) mice was highly successful in reducing the time to death in these mice from 50 to 70 days to 7 to 10 days after challenge with the isolate MARV-Ci67, -Musoke, or -Ravn. In this study, we extended our findings to show that further sequential passages of MARV-Ravn in immunocompetent mice caused the MARV to kill BALB/c mice. Serial sampling studies to characterize the pathology of mouse-adapted MARV-Ravn revealed that this model is similar to the guinea pig and nonhuman primate MHF models. Infection of BALB/c mice with mouse-adapted MARV-Ravn caused uncontrolled viremia and high viral titers in the liver, spleen, lymph node, and other organs; profound lymphopenia; destruction of lymphocytes within the spleen and lymph nodes; and marked liver damage and thrombocytopenia. Sequencing the mouse-adapted MARV-Ravn strain revealed differences in 16 predicted amino acids from the progenitor virus, although the exact changes required for adaptation are unclear at this time. This mouse-adapted MARV strain can now be used to develop and evaluate novel vaccines and therapeutics and may also help to provide a better understanding of the virulence factors associated with MARV.

Molecular Characterization of Highly Pathogenic H5N1 Avian Influenza A Viruses Isolated from Raccoon Dogs in China

BackgroundThe highly pathogenic avian influenza H5N1 virus can infect a variety of animals and continually poses a threat to animal and human health. While many genotypes of H5N1 virus can be found in chicken, few are associated with the infection of mammals. Characterization of the genotypes of viral strains in animal populations is important to understand the distribution of different viral strains in various hosts. This also facilitates the surveillance and detection of possible emergence of highly pathogenic strains of specific genotypes from unknown hosts or hosts that have not been previously reported to carry these genotypes.Methodology/Principal FindingsTwo H5N1 isolates were obtained from lung samples of two raccoon dogs that had died from respiratory disease in China. Pathogenicity experiments showed that the isolates were highly pathogenic to chicken. To characterize the genotypes of these viruses, their genomic sequences were determined and analyzed. The genetic contents of these isolates are virtually identical and they may come from the same progenitor virus. Phylogenetic analysis indicated that the isolates were genetically closely related to genotype V H5N1 virus, which was first isolated in China in 2003, and were distinct from the dominant virus genotypes (e.g. genotype Z) of recent years. The isolates also contain a multibasic amino acid motif at their HA cleavage sites and have an E residue at position 627 of the PB2 protein similar to the previously-identified avian viruses.Conclusions/SignificanceThis is the first report that genotype V H5N1 virus is found to be associated with a mammalian host. Our results strongly suggest that genotype V H5N1 virus has the ability to cross species barriers to infect mammalian animals. These findings further highlight the risk that avian influenza H5N1 virus poses to mammals and humans, which may be infected by specific genotypes that are not known to infect these hosts.

Identification of the progenitors of Indonesian and Vietnamese avian influenza A (H5N1) viruses from southern China.

The transmission of highly pathogenic avian influenza H5N1 virus to Southeast Asian countries triggered the first major outbreak and transmission wave in late 2003, accelerating the pandemic threat to the world. Due to the lack of influenza surveillance prior to these outbreaks, the genetic diversity and the transmission pathways of H5N1 viruses from this period remain undefined. To determine the possible source of the wave 1 H5N1 viruses, we recently conducted further sequencing and analysis of samples collected in live-poultry markets from Guangdong, Hunan, and Yunnan in southern China from 2001 to 2004. Phylogenetic analysis of the hemagglutinin and neuraminidase genes of 73 H5N1 isolates from this period revealed a greater genetic diversity in southern China than previously reported. Moreover, results show that eight viruses isolated from Yunnan in 2002 and 2003 were most closely related to the clade 1 virus sublineage from Vietnam, Thailand, and Malaysia, while two viruses from Hunan in 2002 and 2003 were most closely related to viruses from Indonesia (clade 2.1). Further phylogenetic analyses of the six internal genes showed that all 10 of those viruses maintained similar phylogenetic relationships as the surface genes. The 10 progenitor viruses were genotype Z and shared high similarity (>/=99%) with their corresponding descendant viruses in most gene segments. These results suggest a direct transmission link for H5N1 viruses between Yunnan and Vietnam and also between Hunan and Indonesia during 2002 and 2003. Poultry trade may be responsible for virus introduction to Vietnam, while the transmission route from Hunan to Indonesia remains unclear.

Aviäre Influenza – die Sicht der Veterinärmedizin

Influenza A viruses, in particular the H5 and H7 subtypes, have caused epizootic diseases in poultry for a long time. Wild aquatic birds and shorebirds form the natural virus reservoir. All influenza virus subtypes and almost all possible haemagglutinin/neuraminidase combinations have been detected in wild birds, whereas relatively few have been detected in humans and other mammals. In 1997, the emerging and spreading of the highly pathogenic strain H5N1 within Asia was supported by lack of hygiene in commercial poultry units and by the existence of live bird markets. During autumn 2005, migratory birds have been accused for spreading the infection along their flyways to Europe including Switzerland. For early detection of introduction to Europe, many countries have initiated surveillance programs for avian influenza in wild birds. Vaccines against influenza A viruses are existing for birds and are widely used to protect domestic fowl in endemic regions of Asia as well as valuable birds in zoos worldwide. Subtype H5N1 could be the progenitor virus of a new pandemic influenza virus. Therefore, the World Organisation for Animal Health (OIE, Paris) as well as the Food and Agriculture Organisation of the United Nations (FAO, Rome) will need to increase their efforts to assist countries to combat the disease in the field.

Changes in viral protein function that accompany retroviral endogenization

Endogenous retroviruses (ERVs) are the remnants of ancient retroviral infections of germ cells and have been maintained in whole or part as heritable genomic elements. The last known endogenization events occurred several million years ago, and therefore stepwise analysis of retroviral endogenization has not been possible. A unique opportunity to study this process became available when a full-length ERV isolated from koalas (KoRV) was shown to have integrated into their germ line within the past 100 years. Even though KoRV shares 78% nucleotide identity with the exogenous and highly infectious gibbon ape leukemia virus (GALV), the infectivity of KoRV, like that of other ERVs, is substantially lower than that of GALV. Differences in the protein coding regions of KoRV that distinguish it from GALV were introduced into the GALV genome, and their functional consequences were assessed. We identified a KoRV gagpol L domain mutation as well as five residues present in the KoRV envelope (env) that, when substituted for the corresponding residues of GALV, resulted in vectors exhibiting substantially reduced titers similar to those observed with KoRV vectors. In addition, KoRV env protein lacks an intact CETTG motif that we have identified as invariant among highly infectious gammaretroviruses. Disruption of this motif in GALV results in vectors with reduced syncytia forming capabilities. Functional assessment of specific sequences that contribute to KoRV's attenuation from a highly infectious GALV-like progenitor virus has allowed the identification of specific modifications in the KoRV genome that correlate with its endogenization.

Fowl adenovirus (FAdV) serotype 4 causes depletion of B and T cells in lymphoid organs in specific pathogen-free chickens following experimental infection

In the present investigation flow cytometric analysis and immunohistochemistry were applied together for the first time to gain new insights into the interaction between virulent fowl adenoviruses (FAdV) and the immune system of chickens. As a model for virulent FAdV infections a FAdV-4 strain was used, known as the aetiological agent of Hepatitis-Hydropericardium syndrome (HHS) in broilers sometimes also named Angara Disease. Specified pathogen-free chickens (SPF) were divided into three different groups. Group I was infected at first day of life with an attenuated form of the virus obtained through continuous cell culture passage with the virulent virus and then re-infected 3 weeks later with the virulent progenitor virus. Group II was solely infected with the virulent virus at 3 weeks and group III served as a negative control. Following infection with the virulent virus a decrease of CD3 +, CD4 + and CD8 + cells was noticed in the spleen. This was accompanied by a decrease of CD4 + and CD8 + T-lymphocytes in the thymus. Those birds infected with the attenuated virus in first instance and challenged with the virulent virus did not show these pathological effects in the thymus. In the bursa of Fabricius a severe depletion of lymphocytes was observed by immunohistochemistry in birds, infected with the virulent virus. Taken together it can be concluded that an infection with FAdV-4 has profound effects on cells, of the humoral and cell-mediated immune responses. The effects are much more severe in the birds infected with the virulent virus only indicating that the preceding infection with the attenuated virus reduces significantly the adverse effects induced by the virulent virus.

H9N2 Influenza Viruses from Israeli Poultry: A Five-Year Outbreak

Since 2000, hundreds of H9N2 viruses have been isolated from all types of domestic birds. Although H9N2 is a low-pathogenicity virus, disease has been observed in all types of poultry in the field. Clinical signs ranged from very mild disease to high morbidity and mortality when the virus was associated with a secondary pathogen. Because of the wide range of the virus and the great losses it caused, initially a local vaccination program was implemented, but mass vaccination was quickly authorized. A local strain, isolated in 2002 was selected and is currently in use as an inactivated vaccine. An intensive operation is in progress to characterize the isolates. Several genes (hemagglutinin [HA], neuraminidase, nonstructural protein, nucleoprotein, and matrix) were sequenced, revealing three main groups: the first group included two isolates from 2000, the second group included isolates from 2001 to the beginning of 2003, and the third group included all isolates from 2003 to date. The differences between the second and third groups, in a part of the HA gene, ranged from 3.49% to 6.97% (average 4.57%) of the nucleotides. Similar differences were recorded in the other tested genes. These data could indicate the probable introduction of distinct progenitor viruses into the Israeli poultry population. Furthermore, sequencing of the HA protein of some Israeli isolates revealed the presence of L216 in the binding site; this finding was typical of the H9N2 viruses isolated from humans, which raises the possibility of an influence on host specificity and virulence.

Sequence variation in the Newcastle disease virus genome

Full-length genome sequences of five virulent and five avirulent strains of Newcastle disease virus isolated between 1998 and 2002 in Victoria and New South Wales, Australia were determined. Comparisons between these strains revealed that coding sequence variability in the haemagglutinin-neuraminidase (HN), matrix (M) and phosphoprotein (P) gene sequences appeared to be more variable than in the fusion (F), nucleocapsid (N) and RNA dependent-RNA replicase (L) genes. Sequence analysis of a number of other isolates made during the recent virulent NDV outbreaks, also identified the presence of a number of variants with altered F gene cleavage sites, which resulted in altered biological properties of those viruses. Quasispecies analysis of a number of field isolates indicated the presence of virulent virus in one particular isolate. Gene sequence analysis of the progenitor virus isolated in 1998 showed very little sequence variation when compared to that of a progenitor-like virus isolated in 2001, demonstrating that in the field, viral genome sequence variation appears to be biologically restricted to that of a consensus sequence.

Review of Bats and SARS

Bats have been identified as a natural reservoir for an increasing number of emerging zoonotic viruses, including henipaviruses and variants of rabies viruses. Recently, we and another group independently identified several horseshoe bat species (genus Rhinolophus) as the reservoir host for a large number of viruses that have a close genetic relationship with the coronavirus associated with severe acute respiratory syndrome (SARS). Our current research focused on the identification of the reservoir species for the progenitor virus of the SARS coronaviruses responsible for outbreaks during 2002-2003 and 2003-2004. In addition to SARS-like coronaviruses, many other novel bat coronaviruses, which belong to groups 1 and 2 of the 3 existing coronavirus groups, have been detected by PCR. The discovery of bat SARS-like coronaviruses and the great genetic diversity of coronaviruses in bats have shed new light on the origin and transmission of SARS coronaviruses.

More recent swine vesicular disease virus isolates retain binding to coxsackie–adenovirus receptor, but have lost the ability to bind human decay-accelerating factor (CD55)

Swine vesicular disease virus (SVDV) evolved from coxsackie B virus serotype 5 (CVB5) in the recent past, crossing the species barrier from humans to pigs. Here, SVDV isolates from early and recent outbreaks have been compared for their capacity to utilize the progenitor virus receptors coxsackie-adenovirus receptor (CAR) and decay-accelerating factor (DAF; CD55). Virus titre of CVB5 and SVDV isolates It'66 and UK'72 on human HeLa cells was reduced by pre-incubation with either anti-DAF or anti-CAR antibodies; however, recent SVDV isolates R1072, R1120 and SPA'93 did not infect HeLa cells lytically. CVB5 and SVDV infection of the pig cell line IB-RS-2 was inhibited completely by anti-CAR antibodies for all isolates, and no reduction was observed following pre-incubation of cells with anti-pig DAF antibodies. Expression of human DAF in the pig cell line IB-RS-2 enhanced the virus titre of early SVDV isolates by 25-fold, but had no effect on recent SVDV isolate titre. Binding of radiolabelled CVB5 to IB-RS-2 cells was increased seven- to eightfold by expression of human DAF and binding of early SVDV isolates was increased 1.2-1.3-fold, whereas no increase in binding by recent SVDV isolates was mediated by human DAF expression. Addition of soluble hDAF-Fc inhibited CVB5, but not SVDV, infection of pig cells. Pre-incubation of all viruses with soluble hCAR-Fc blocked infection of IB-RS-2 pig cells completely; titration of the amount of soluble hCAR-Fc required to block infection revealed that early isolate UK'72 was the least susceptible to inhibition, and the most recent isolate, SPA'93, was the most susceptible.

Dengue Emergence and Adaptation to Peridomestic Mosquitoes

Phylogenetic evidence suggests that endemic and epidemic dengue viruses (DENV), transmitted among humans by the anthropophilic mosquitoes Aedes aegypti and Ae. albopictus, emerged when ancestral, sylvatic DENV transmitted among nonhuman primates by sylvatic Aedes mosquitoes adapted to these peridomestic vectors. We tested this hypothesis by retrospectively examining evidence for adaptation of epidemic and endemic versus sylvatic strains of DENV-2 to Ae. albopictus and Ae. aegypti. First and second-generation offspring of mosquitoes from different geographic regions in the Americas and Southeast Asia were tested for their susceptibility to epidemic/endemic and sylvatic DENV-2 isolates from West Africa, Southeast Asia, and Oceania. Both Aedes species were highly susceptible (up to 100% infected) to endemic/epidemic DENV-2 strains after ingesting artificial blood meals but significantly less susceptible (as low as 0%) to sylvatic DENV-2 strains. Our findings support the hypothesis that adaptation to peridomestic mosquito vectors mediated dengue emergence from sylvatic progenitor viruses.

Analyses of genotypic diversity among North, South, and Central American isolates of sugarcane yellow leaf virus: evidence for Colombian origins and for intraspecific spatial phylogenetic variation.

We have analyzed the genotypic diversity of sugarcane yellow leaf virus (SCYLV) collected from North, South, and Central America by fingerprinting assays and selective cDNA cloning and sequencing. One group of isolates from Colombia, designated the C-population, has been identified as residing at the root node between a separable superpopulation structure of SCYLV and other members of the family Luteoviridae, indicating that the progenitor viruses of the North, South, and Central American isolates of the SCYLV superpopulation most likely arose from a C-population structure. From a model of intrafamilial evolution (F. Moonan et al., Virology 269:156-171, 2000), a prediction could be made that within the SCYLV species, the capacity of genomic sequence divergence would range from lowest in the capsid protein open reading frame 3 (ORF 3) to highest in a region spanning across the carboxy-terminal end of the RNA-dependent RNA polymerase ORF. We have demonstrated the validity and applicability of this intrafamilial model for the prediction of intraspecies SCYLV diversity. Analysis of spatial phylogenetic variation (SPV) within the SCYLV isolates could not be assessed by application of a "partial likelihoods assessed through optimization" (PLATO)-derived intraspecies model alone. However, application of a PLATO-derived intrafamilial model with the intraspecies-derived model allowed distinction of three forms of SPV. Two of the SPV forms identified correspond to the extremes in a continuum of sequence evolution displayed in a SCYLV superpopulation structure, and the third form was diagnostic of a C-population structure. The application of these types of models has value in terms of predicting the types of SCYLV intraspecies diversity that may exist worldwide, and in general, may be useful in application for more informed design of transgenes for use in the elicitation of homology-dependent virus resistance mechanisms in transgenic plants.

Virulent Newcastle disease in Australia: Molecular epidemiological analysis of viruses isolated prior to and during the outbreaks of 1998–2000

Gene sequence analysis of fusion (F) gene cleavage motifs and haemagglutinin-neuraminidase (HN) carboxyl-terminal extension sequences was used to analyse Newcastle disease viruses (NDV) associated with virulent outbreaks of the disease which occurred in New South Wales, Australia in 1998–2000. PCR fragments were amplified directly from diseased tissue or allantoic fluids and sequence analyses used for phylogenetic comparisons between these viruses and Australian reference NDV. F and HN gene sequence comparison showed a strong relationship to sequences derived from endemic Australian NDV rather than those of overseas viruses or wild bird isolates. Prior to notification of the 1998 outbreak, an NDV was isolated from chickens suffering respiratory disease that appeared to be the progenitor virus from which the virulent virus originated. In turn, these viruses are closely related to two previously isolated ‘ancestor’ viruses that have the same unique HN extension sequence.

Mutations of rubella virus vaccine TO-336 strain occurred in the attenuation process of wild progenitor virus

The sequences of the genomes in the TO-336 vaccine strain (TO-336vac) of rubella virus and its wild progenitor virus (TO-336wt) have been determined and compared with each other. There were 21 differences in the nucleotide sequences between the TO-336vac and the TO-336wt: 13 in the nonstructural protein open reading frame (NSP-ORF), five in the structural protein open reading frame (SP-ORF) and three in the untranslated regions (UTRs) (one in each three UTRs). These mutations resulted in amino acid substitutions at ten residues. Of the ten substitutions, eight were in NSP-ORF and two were in the SP-ORF. Of the eight substitutions in NSP-ORF, four (amino acids (aa) 320, 501, 573 and 704) were in the regions of unknown function, two (aa 1154 and 1159) were within the protease motif, and two (aa 1351 and 1559) were within the helicase motif. Both of the two residues (aa 890 and 954) in the SP-ORF were within the E1 gene. The predicted second structure of the 5′UTR of the TO-336vac was identical to that of TO-336wt. Comparing the TO-336 sequences with other four strains, Therien and M33 (wild viruses), and RA27/3 and Cendehill (vaccine viruses), the mutations responsible for attenuation are thought to differ with each vaccine strain. This is the first report of sequencing in a pair of live attenuated rubella vaccines and their wild-type parent.

Adaptation of wild-type measles virus to CD46 receptor usage.

Vaccine strains of measles virus (MV) use CD46 as receptor and downregulate CD46 from the surface of infected cells. MVs isolated and passaged on B-lymphoid cells (wild-type MVs) seem to use another receptor and do not downregulate CD46. In the present study, we found that isolation of MV on human or marmoset B-lymphoid cells did not alter the MV haemagglutinin (H) protein relative to that in the patient. The wild-type isolates were adapted to the human epithelial HEp-2 cell line or the monkey fibroblast Vero cell line. All HEp-2 cell adapted viruses and 1 out of 4 Vero cell adapted viruses acquired the capacity to use CD46 as receptor, as measured by their ability to infect murine cells expressing human CD46. Adaptation to CD46 receptor usage was coupled to substitution of amino acid 481 of the MV H protein from asparagine to tyrosine but not to CD46 downregulation. The present study demonstrates that CD46 receptor usage can be induced by adaptation of wild-type MV to cells that do not express a wild-type receptor and suggests that a similar mechanism acted on the progenitor viruses of the present MV vaccine strains during their isolation and attenuation.

Impact of recombinant baculovirus field applications on a nontarget heliothine parasitoid, Microplitis croceipes (Hymenoptera: Braconidae).

The kill times of two viruses infectious to the heliothine pest complex indigenous to Texas cotton have been significantly reduced by expressing a scorpion toxin gene. Autographa californica nucleopolyhedrovirus (NPV) and Helicoverpa zea NPV express the toxin only in permissive lepidopteran hosts. The toxin, however, could indirectly harm members of upper trophic levels that feed upon and parasitize infected larvae producing the toxin. In this study, the effects of recombinant and wild-type viruses on Microplitis croceipes (Cresson) were studied in cotton using Heliothis virescens (F.) (Lepidoptera: Noctuidae) as hosts. Two recombinant viruses, their two wild-type progenitor viruses, and untreated cotton served as the five treatments of study. Larvae were previously parasitized 2 and 4 d before being confined for 72 h to cotton terminals treated with field rates of virus or left untreated. The sexes of adult M. croceipes that emerged from the recovered H. virescens larvae were determined and their head capsule widths were measured. Polymerase chain reaction (PCR) searched their extracts for virus DNA. There were no differences in percentage emergence and sex ratios of parasitoids among recombinant, wild-type, and control treatments. Significantly more wasps emerged from the 4-d cohort, but these wasps were significantly smaller than wasps from the 2-d cohort regardless of treatment. Finally, PCR found only 15-25% of the recovered H. virescens larvae and none of the emergent M. croceipes had detectable levels of viral DNA. Recombinant and wild-type viruses had a similar, minimal impact on emergent wasps, and the probability of virus dispersal via parasitoids is low in the system tested.

Emerging cytopathic and antigenic simian immunodeficiency virus variants influence AIDS progression.

Genetic variants of human and simian immunodeficiency virus (HIV and SIV) that evolve during the course of infection and progression to AIDS are phenotypically and antigenically distinct from their progenitor viruses present at early stages of infection. However, it has been unclear how these late variants, which are typically T-cell tropic, cytopathic and resistant to neutralizing antibodies, influence the development of clinical AIDS. To address this, we infected macaques with cloned SIVs representing prototype variants from early-, intermediate- and late-stage infection having biological characteristics typical of viruses found at similar stages of HIV infection in humans. These studies demonstrate that sequential, phenotypic and antigenic variants represent viruses that have become increasingly fit for replication in the host, and our data support the hypothesis that emerging variants have increased pathogenicity and drive disease progression in SIV and HIV infection.

Immunological properties of plaque purified strains of live attenuated respiratory syncytial virus (RSV) for human vaccine

Respiratory syncytial virus (RSV) causes severe lower respiratory tract disease in infants, young children, and the elderly. Efforts to develop satisfactory live or inactivated vaccines have not yet been proven successful. Our research focuses on the development of four purified live attenuated RSV sub-type A human vaccine clones. Temperature sensitive (ts) and attenuated purified clones of either cold-adapted (ca) RSV or high-passage (hp) RSV were administered intra-nasally (i.n.) to BALB/c mice and tested for immunogenicity. All four clones produced significant anti-RSV F IgG 2a and IgG 1 titres in the sera of mice, RSV-specific neutralizing titres higher than those produced by their wild-type progenitor viruses, cytotoxic T-lymphocyte (CTL) activity, and total protection against wild-type (wt) viral challenge. These purified vaccine candidates await testing in humans to determine which contain the required balance between immunogenicity and attenuation.

Coresistance to zidovudine and foscarnet is associated with multiple mutations in the human immunodeficiency virus type 1 reverse transcriptase.

Human immunodeficiency virus type 1 (HIV-1) isolates obtained from a patient with AIDS were assessed for coresistance to foscarnet and zidovudine. An HIV-1 strain (AP20) coresistant to foscarnet and zidovudine was isolated after 20 months of continuous combination therapy. The reverse transcriptase (RT) gene of AP20 had 41 substitutions which were different from the HXB2-D sequence and 9 that were different from the sequence of its foscarnet-sensitive, zidovudine-resistant progenitor virus (AP6). Six of these mutations were nonpolymorphic (T39A, V108I, K166R, K219R, K223Q, and L228R). Both strains had the conventional mutations mediating zidovudine resistance. In vivo selection may result in HIV-1 strains that are coresistant to foscarnet and zidovudine, but coresistance appears to require a complex evolutionary path and multiple RT mutations.

A Point Mutation in the Sendai Virus Accessory C Proteins Attenuates Virulence for Mice, but Not Virus Growth in Cell Culture

A mutant Sendai virus (SeVMVC), which grows much better than its progenitor virus (SeVM) in cell culture, but, in strong contrast to SeVM, is totally avirulent for mice, has been described. SeVMVCcontains two amino acid substitutions relative to SeVM, namely, F170S in the C protein and E2050A in the L protein. We have examined which substitutions were responsible for the above phenotypes by exchanging the C gene of our reference strain Z with those of SeVH(another reference strain), SeVM, and SeVMVC, in turn. We have found that the F170S mutation in the CMVCprotein is responsible both for enhanced replication in cell culture and for avirulence in mice. Avirulence appeared to be due to restricted viral replication primarily after day 1, implicating some aspect of innate immunity in this process. The SeV C proteins thus appear to be required for multiple cycles of replication in mice.