Mechanisms controlling virulence thresholds of mixed viral populations

The propensity of RNA viruses to revert attenuating mutations contributes to disease and complicates vaccine development. Despite the presence of virulent revertant viruses in some live-attenuated vaccines, disease from vaccination is rare. This suggests that in mixed viral populations, attenuated viruses may limit the pathogenesis of virulent viruses, thus establishing a virulence threshold. Here we examined virulence thresholds using mixtures of virulent and attenuated viruses in a transgenic mouse model of poliovirus infection. We determined that a 1,000-fold excess of the attenuated Sabin strain of poliovirus was protective against disease induced by the virulent Mahoney strain. Protection was induced locally, and inactivated virus conferred protection. Treatment with a poliovirus receptor-blocking antibody phenocopied the protective effect of inactivated viruses in vitro and in vivo, suggesting that one mechanism controlling virulence thresholds may be competition for a viral receptor. Additionally, the type I interferon response reduces poliovirus pathogenesis; therefore, we examined virulence thresholds in mice lacking the alpha/beta interferon receptor. We found that the attenuated virus was virulent in immunodeficient mice due to the enhanced replication and reversion of attenuating mutations. Therefore, while the type I interferon response limits the virulence of the attenuated strain by reducing replication, protection from disease conferred by the attenuated strain in immunocompetent mice can occur independently of replication. Our results identified mechanisms controlling the virulence of mixed viral populations and indicate that live-attenuated vaccines containing virulent virus may be safe, as long as virulent viruses are present at levels below a critical threshold.     

Canine distemper virus matrix protein influences particle infectivity, particle composition, and envelope distribution in polarized epithelial cells and modulates virulence

In paramyxoviruses, the matrix (M) protein mediates the interaction between the envelope and internal proteins during particle assembly and egress. In measles virus (MeV), M mutations, such as those found in subacute sclerosing panencephalitis (SSPE) strains, and differences in vaccine and wild-type M proteins can affect the strength of interaction with the envelope glycoproteins, assembly efficiency, and spread. However, the contribution of the M protein to the replication and pathogenesis of the closely related canine distemper virus (CDV) has not been characterized. To this end this, we generated a recombinant wild-type CDV carrying a vaccine strain M protein. The recombinant virus retained the parental growth phenotype in VerodogSLAMtag cells, but displayed an increased particle-to-infectivity ratio very similar to that of the vaccine strain, likely due to inefficient H protein incorporation. Even though infectious virus was released only from the apical surface, consistent with the release polarity of the wild-type CDV strain, envelope protein distribution in polarized epithelial cells reproduced the bipolar pattern seen in vaccine strain-infected cells. Most notably, the chimeric virus was completely attenuated in ferrets and caused only a mild and transient leukopenia, indicating that the differences in particle infectivity and envelope protein sorting mediated by the vaccine M protein contribute importantly to vaccine strain attenuation.     

Envelope protein glycosylation status influences mouse neuroinvasion phenotype of genetic lineage 1 West Nile virus strains

The introduction of West Nile virus (WNV) into North America has been associated with relatively high rates of neurological disease and death in humans, birds, horses, and some other animals. Previous studies identified strains in both genetic lineage 1 and genetic lineage 2, including North American isolates of lineage 1, that were highly virulent in a mouse neuroinvasion model, while other strains were avirulent or significantly attenuated (D. W. C. Beasley, L. Li, M. T. Suderman, and A. D. T. Barrett, Virology 296:17-23, 2002). To begin to elucidate the basis for these differences, we compared a highly virulent New York 1999 (NY99) isolate with a related Old World lineage 1 strain, An4766 (ETH76a), which is attenuated for mouse neuroinvasion. Genomic sequencing of ETH76a revealed a relatively small number of nucleotide (5.1%) and amino acid (0.6%) differences compared with NY99. These differences were located throughout the genome and included five amino acid differences in the envelope protein gene. Substitution of premembrane and envelope genes of ETH76a into a NY99 infectious clone backbone yielded a virus with altered in vitro growth characteristics and a mouse virulence phenotype comparable to ETH76a. Further site-specific mutagenesis studies revealed that the altered phenotype was primarily mediated via loss of envelope protein glycosylation and that this was associated with altered stability of the virion at mildly acidic pH. Therefore, the enhanced virulence of North American WNV strains compared with other Old World lineage 1 strains is at least partly mediated by envelope protein glycosylation.     

Canine distemper virus epithelial cell infection is required for clinical disease but not for immunosuppression

To characterize the importance of infection of epithelial cells for morbillivirus pathogenesis, we took advantage of the severe disease caused by canine distemper virus (CDV) in ferrets. To obtain a CDV that was unable to enter epithelial cells but retained the ability to enter immune cells, we transferred to its attachment (H) protein two mutations shown to interfere with the interaction of measles virus H with its epithelial receptor, human nectin-4. As expected for an epithelial receptor (EpR)-blind CDV, this virus infected dog and ferret epithelial cells inefficiently and did not cause cell fusion or syncytium formation. On the other hand, the EpR-blind CDV replicated in cells expressing canine signaling lymphocyte activation molecule (SLAM), the morbillivirus immune cell receptor, with similar kinetics to those of wild-type CDV. While ferrets infected with wild-type CDV died within 12 days after infection, after developing severe rash and fever, animals infected with the EpR-blind virus showed no clinical signs of disease. Nevertheless, both viruses spread rapidly and efficiently in immune cells, causing similar levels of leukopenia and inhibition of lymphocyte proliferation activity, two indicators of morbillivirus immunosuppression. Infection was documented for airway epithelia of ferrets infected with wild-type CDV but not for those of animals infected with the EpR-blind virus, and only animals infected with wild-type CDV shed virus. Thus, epithelial cell infection is necessary for clinical disease and efficient virus shedding but not for immunosuppression.     

The HA and NS Genes of Human H5N1 Influenza A Virus Contribute to High Virulence in Ferrets

Highly pathogenic H5N1 influenza A viruses have spread across Asia, Europe, and Africa. More than 500 cases of H5N1 virus infection in humans, with a high lethality rate, have been reported. To understand the molecular basis for the high virulence of H5N1 viruses in mammals, we tested the virulence in ferrets of several H5N1 viruses isolated from humans and found A/Vietnam/UT3062/04 (UT3062) to be the most virulent and A/Vietnam/UT3028/03 (UT3028) to be avirulent in this animal model. We then generated a series of reassortant viruses between the two viruses and assessed their virulence in ferrets. All of the viruses that possessed both the UT3062 hemagglutinin (HA) and nonstructural protein (NS) genes were highly virulent. By contrast, all those possessing the UT3028 HA or NS genes were attenuated in ferrets. These results demonstrate that the HA and NS genes are responsible for the difference in virulence in ferrets between the two viruses. Amino acid differences were identified at position 134 of HA, at positions 200 and 205 of NS1, and at positions 47 and 51 of NS2. We found that the residue at position 134 of HA alters the receptor-binding property of the virus, as measured by viral elution from erythrocytes. Further, both of the residues at positions 200 and 205 of NS1 contributed to enhanced type I interferon (IFN) antagonistic activity. These findings further our understanding of the determinants of pathogenicity of H5N1 viruses in mammals.     

Canine distemper virus uses both the anterograde and the hematogenous pathway for neuroinvasion

Canine distemper virus (CDV), a member of the Morbillivirus genus that also includes measles virus, frequently causes neurologic complications, but the routes and timing of CDV invasion of the central nervous system (CNS) are poorly understood. To characterize these events, we cloned and sequenced the genome of a neurovirulent CDV (strain A75/17) and produced an infectious cDNA that expresses the green fluorescent protein. This virus fully retained its virulence in ferrets: the course and signs of disease were equivalent to those of the parental isolate. We observed CNS invasion through two distinct pathways: anterogradely via the olfactory nerve and hematogenously through the choroid plexus and cerebral blood vessels. CNS invasion only occurred after massive infection of the lymphatic system and spread to the epithelial cells throughout the body. While at early time points, mostly immune and endothelial cells were infected, the virus later spread to glial cells and neurons. Together, the results suggest similarities in the timing, target cells, and CNS invasion routes of CDV, members of the Morbillivirus genus, and even other neurovirulent paramyxoviruses like Nipah and mumps viruses.     

Genome sequence of Brucella abortus vaccine strain S19 compared to virulent strains yields candidate virulence genes

The Brucella abortus strain S19, a spontaneously attenuated strain, has been used as a vaccine strain in vaccination of cattle against brucellosis for six decades. Despite many studies, the physiological and molecular mechanisms causing the attenuation are not known. We have applied pyrosequencing technology together with conventional sequencing to rapidly and comprehensively determine the complete genome sequence of the attenuated Brucella abortus vaccine strain S19. The main goal of this study is to identify candidate virulence genes by systematic comparative analysis of the attenuated strain with the published genome sequences of two virulent and closely related strains of B. abortus, 9-941 and 2308. The two S19 chromosomes are 2,122,487 and 1,161,449 bp in length. A total of 3062 genes were identified and annotated. Pairwise and reciprocal genome comparisons resulted in a total of 263 genes that were non-identical between the S19 genome and any of the two virulent strains. Amongst these, 45 genes were consistently different between the attenuated strain and the two virulent strains but were identical amongst the virulent strains, which included only two of the 236 genes that have been implicated as virulence factors in literature. The functional analyses of the differences have revealed a total of 24 genes that may be associated with the loss of virulence in S19. Of particular relevance are four genes with more than 60 bp consistent difference in S19 compared to both the virulent strains, which, in the virulent strains, encode an outer membrane protein and three proteins involved in erythritol uptake or metabolism.     

Replicase complex genes of Semliki Forest virus confer lethal neurovirulence

Semliki Forest virus (SFV) is a mosquito-transmitted pathogen of small rodents, and infection of adult mice with SFV4, a neurovirulent strain of SFV, leads to lethal encephalitis in a few days, whereas mice infected with the avirulent A7(74) strain remain asymptomatic. In adult neurons, A7(74) is unable to form virions and hence does not reach a critical threshold of neuronal damage. To elucidate the molecular mechanisms of neurovirulence, we have cloned and sequenced the entire 11,758-nucleotide genome of A7(74) and compared it to the highly neurovirulent SFV4 virus. We found several sequence differences and sought to localize determinants conferring the neuropathogenicity by using a panel of chimeras between SFV4 and a cloned recombinant, rA774. We first localized virulence determinants in the nonstructural region by showing that rA774 structural genes combined with the SFV4 nonstructural genome produced a highly virulent virus, while a reciprocal recombinant was asymptomatic. In addition to several amino acid mutations in the nonstructural region, the nsp3 gene of rA774 displayed an opal termination codon and an in-frame 21-nucleotide deletion close to the nsp4 junction. Replacement in rA774 of the entire nsp3 gene with that of SFV4 reconstituted the virulent phenotype, whereas an arginine at the opal position significantly increased virulence, leading to clinical symptoms in mice. Completion of the nsp3 deletion in rA774 did not increase virulence. We conclude that the opal codon and amino acid mutations other than the deleted residues are mainly responsible for the attenuation of A7(74) and that the attenuating determinants reside entirely in the nonstructural region.     

水貂犬瘟热动物模型的建立

目的建立水貂犬瘟热动物模型,并利用水貂犬瘟热模型评价不同犬瘟热强毒株的毒力,为水貂犬瘟热病毒疫苗的研究奠定基础。方法从猴、藏獒、犬的病料中分离犬瘟热病毒,测定犬瘟热病毒的毒力,并进行传代培养。利用犬的犬瘟热动物模型筛选稳定的犬瘟热强毒株,进行水貂犬瘟热动物模型的建立及其毒力评估。结果筛选出了稳定的犬瘟热强毒株并进行了家犬动物实验,同时表现出了强烈的临床症状,并利用不同的代次毒进行了犬瘟热动物模型的建立。结论成功建立了犬瘟热动物模型并对不同来源的犬瘟热病毒毒力进行了评估。     

N-linked glycosylation status of classical swine fever virus strain Brescia E2 glycoprotein influences virulence in swine

E2 is one of the three envelope glycoproteins of classical swine fever virus (CSFV). Previous studies indicate that E2 is involved in several functions, including virus attachment and entry to target cells, production of antibodies, induction of protective immune response in swine, and virulence. Here, we have investigated the role of E2 glycosylation of the highly virulent CSFV strain Brescia in infection of the natural host. Seven putative glycosylation sites in E2 were modified by site-directed mutagenesis of a CSFV Brescia infectious clone (BICv). A panel of virus mutants was obtained and used to investigate whether the removal of putative glycosylation sites in the E2 glycoprotein would affect viral virulence/pathogenesis in swine. We observed that rescue of viable virus was completely impaired by removal of all putative glycosylation sites in E2 but restored when mutation N185A reverted to wild-type asparagine produced viable virus that was attenuated in swine. Single mutations of each of the E2 glycosylation sites showed that amino acid N116 (N1v virus) was responsible for BICv attenuation. N1v efficiently protected swine from challenge with virulent BICv at 3 and 28 days postinfection, suggesting that glycosylation of E2 could be modified for development of classical swine fever live attenuated vaccines.     

Increased virulence of a mouse-adapted variant of influenza A/FM/1/47 virus is controlled by mutations in genome segments 4, 5, 7, and 8.

To cause disease, influenza virus must possess several genetically determined abilities that mediate stages in pathogenesis. The virulent mouse-adapted variant A/FM/1/47-MA (FM-MA), derived from the avirulent A/FM/1/47 (FM) strain, had acquired mutations in genes that control virulence. The purpose of this study was to identify those genes that had mutated to result in increased virulence and to obtain viruses that differed in virulence because of differences in individual genome segments. The genes that had mutated to increase virulence were initially identified by genetic analysis of reassortants obtained by crossing FM-MA with the avirulent strain A/HK/1/68 (HK). FM-MA genome segments 4, 5, 7, and 8 were significantly associated with virulence, as determined by using the Wilcoxon ranked sum analysis. The role of FM-MA segments 4, 7, and 8 was confirmed by reintroduction of these genes into the parental strain, which also provided virus strains that differed in virulence because of mutations in individual genome segments. Segments 4, 7, and 8 were responsible for a 10(3.6)-fold increase in virulence that was proportioned 10(2.2)-, 10(0.7)-, and 10(0.8)-fold, respectively. The role of segment 5 could not be confirmed on transfer back into the parental strain because of reversion during preparation of such reassortants. The incidence of reversion was shown to be significantly associated with culturing of FM-MA in chicken embryo cells but was not associated with growth in MDCK cells. The genetic analysis of FM-MA suggests that adaptation to increased virulence is an incremental process that involves the acquisition of mutations in multiple genes, each of which plays an individual role in pathogenesis. The structural and functional properties of segments 4, 7, and 8 that control the virulence of FM-MA can now be determined by using viruses that differ in virulence because of mutations in these individual genome segments.     

Genetic basis of the neurovirulence of pseudorabies virus.

Lomniczi et al. (J. Virol. 49:970-979, 1984) have shown previously that two attenuated vaccine strains of pseudorabies virus have a similar deletion in the short unique (US) region of the genome. The region which is deleted normally codes for several translationally competent mRNAs. As expected, these mRNAs are not formed in the cells infected with the vaccine strains. The function specified by these mRNAs is thus not necessary for growth in cell culture. Using intracerebral inoculation of 1-day-old chicks as a test system, we have attempted to determine whether a gene within the region that is missing from the attenuated strains specifies functions that are required for the expression of virulence. An analysis of recombinants between the Bartha vaccine strain and a virulent pseudorabies virus strain (having or lacking a thymidine kinase gene [TK+ or TK-]) revealed the following. None of the recombinant plaque isolates that were either TK- or which had a deletion in the US was virulent. Not all recombinant plaque isolates which were both TK+ and had an intact US were virulent. These results indicate that both thymidine kinase activity and an intact US were necessary but not sufficient for the expression of virulence. Marker rescue experiments involving cotransfection of the Bartha strain DNA and a restriction fragment spanning the region of the genome that was missing from the Bartha strain resulted in the isolation of virions to which an intact US had been restored. These virions were not virulent but had an improved ability to replicate in the brains of chicks compared with that of the parental nonrescued Bartha strain. Our results show that genes in the US region, which are missing from the Bartha strain, were necessary for virulence but that this strain was also defective in other genes required for the expression of virulence. Thus, the virulence of pseudorabies virus, as measured by intracerebral inoculation into chicks, appears to be controlled multigenically.     

Identification of VP1/2A and 2C as virulence genes of hepatitis A virus and demonstration of genetic instability of 2C

Fourteen different chimeric virus genomes were constructed from two infectious cDNA clones encoding a virulent and an attenuated isolate, respectively, of the HM175 strain of hepatitis A virus. The ability of each recombinant virus to infect tamarins and to cause acute hepatitis was determined. Comparisons of the genotype and phenotype of each virus suggested that VP1/2A and 2C genes were responsible for virulence. The 2C gene derived from the attenuated parent virus was unstable, and one or more mutations arose in this gene during the first passage in tamarins.     

Avian influenza (H5N1) viruses isolated from humans in Asia in 2004 exhibit increased virulence in mammals

The spread of highly pathogenic avian influenza H5N1 viruses across Asia in 2003 and 2004 devastated domestic poultry populations and resulted in the largest and most lethal H5N1 virus outbreak in humans to date. To better understand the potential of H5N1 viruses isolated during this epizootic event to cause disease in mammals, we used the mouse and ferret models to evaluate the relative virulence of selected 2003 and 2004 H5N1 viruses representing multiple genetic and geographical groups and compared them to earlier H5N1 strains isolated from humans. Four of five human isolates tested were highly lethal for both mice and ferrets and exhibited a substantially greater level of virulence in ferrets than other H5N1 viruses isolated from humans since 1997. One human isolate and all four avian isolates tested were found to be of low virulence in either animal. The highly virulent viruses replicated to high titers in the mouse and ferret respiratory tracts and spread to multiple organs, including the brain. Rapid disease progression and high lethality rates in ferrets distinguished the highly virulent 2004 H5N1 viruses from the 1997 H5N1 viruses. A pair of viruses isolated from the same patient differed by eight amino acids, including a Lys/Glu disparity at 627 of PB2, previously identified as an H5N1 virulence factor in mice. The virus possessing Glu at 627 of PB2 exhibited only a modest decrease in virulence in mice and was highly virulent in ferrets, indicating that for this virus pair, the K627E PB2 difference did not have a prevailing effect on virulence in mice or ferrets. Our results demonstrate the general equivalence of mouse and ferret models for assessment of the virulence of 2003 and 2004 H5N1 viruses. However, the apparent enhancement of virulence of these viruses in humans in 2004 was better reflected in the ferret.     

Effects of Naturally Occurring Six- and Twelve-Nucleotide Inserts on Newcastle Disease Virus Replication and Pathogenesis

Newcastle disease virus (NDV) isolates contain genomes of 15,186, 15,192 or 15,198 nucleotides (nt). The length differences reflect a 6-nt insert in the 5′ (downstream) non-translated region (NTR) of the N gene (15,192-nt genome) or a 12-nt insert in the ORF encoding the P and V proteins (causing a 4-amino acid insert; 15,198-nt genome). We evaluated the role of these inserts in the N and P genes on viral replication and pathogenicity by inserting them into genomes of two NDV strains that have natural genome lengths of 15,186 nt and represent two different pathotypes, namely the mesogenic strain Beaudette C (BC) and the velogenic strain GB Texas (GBT). Our results showed that the 6-nt and 12-nt inserts did not detectably affect N gene expression or P protein function. The inserts had no effect on the replication or virulence of the highly virulent GBT strain but showed modest degree of attenuation in mesogenic strain BC. We also deleted a naturally-occurring 6-nt insertion in the N gene from a highly virulent 15,192-nt genome-length virus, strain Banjarmasin. This resulted in reduced replication in vitro and reduced virulence in vivo. Thus, although these inserts had no evident effect on gene expression, protein function, or replication in vivo, they did affect virulence in two of the three tested strains.     

Receptor (SLAM [CD150]) recognition and the V protein sustain swift lymphocyte-based invasion of mucosal tissue and lymphatic organs by a morbillivirus

Experimental infections of ferrets with canine distemper virus (CDV) recapitulate many hallmarks of measles: rash, high fever, viremia, depression of delayed-type hypersensitivity responses, lowered leukocyte counts, and reduced lymphocyte proliferation activity. To understand how a morbillivirus invades the host and causes immunosuppression, we generated CDV either unable to recognize one of the receptors or incapable of expressing either one or both of the candidate interferon antagonist proteins V and C. Variants of these viruses expressing green fluorescent protein were also generated. Striking similarities between CDV infection of ferrets and human immunodeficiency virus host invasion were documented: first, massive early replication in the gut-associated lymphatic tissue, including intestinal Peyer's patches, followed by extensive infection of lymphatic organs, including thymus and circulating lymphocytes. Moreover, T cells were selectively depleted. Thus, CDV takes advantage of mucosal surfaces for host invasion and lymphocytes for swift dissemination. A CDV unable to recognize the signaling lymphocytic activation molecule (SLAM [CD150]) that is expressed in lymphocytes and other immune cells did not spread. A V-defective CDV multiplied with reduced efficiency in lymphocytes and did not inhibit the interferon and cytokine responses. Protein C affected the severity of rash and digestive symptoms elicited by V-defective CDV, but it was dispensable for the invasion of the lymphatic organs. These findings prove formally that SLAM recognition is necessary for morbillivirus virulence. They also reveal how two viral proteins affect pathogenesis: V sustains the swift lymphocyte-based invasion of mucosal tissue and lymphatic organs, whereas C sustains subsequent infection phases.     

Genetic reassortment of infectious bursal disease virus in nature

Infectious bursal disease virus (IBDV), a double-stranded RNA virus, is a member of the Birnaviridae family. Four pathotypes of IBDV, attenuated, virulent, antigenic variant, and very virulent (vvIBDV), have been identified. We isolated and characterized the genomic reassortant IBDV strain ZJ2000 from severe field outbreaks in commercial flocks. Full-length genomic sequence analysis showed that ZJ2000 is a natural genetic reassortant virus with segments A and B derived from attenuated and very virulent strains of IBDV, respectively. ZJ2000 exhibited delayed replication kinetics as compared to attenuated strains. However, ZJ2000 was pathogenic to specific pathogen free (SPF) chickens and chicken embryos. Similar to a standard virulent IBDV strain, ZJ2000 caused 26.7% mortality, 100% morbidity, and severe bursal lesions at both gross and histopathological levels. Taken together, our data provide direct evidence for genetic reassortment of IBDV in nature, which may play an important role in the evolution, virulence, and host range of IBDV. Our data also suggest that VP2 is not the sole determinant of IBDV virulence, and that the RNA-dependent RNA polymerase protein, VP1, may play an important role in IBDV virulence. The discovery of reassortant viruses in nature suggests an additional risk of using live IBDV vaccines, which could act as genetic donors for genome reassortment.