Murine rotavirus genes encoding outer capsid proteins VP4 and VP7 are not major determinants of host range restriction and virulence.

Simian rotavirus (RRV) and murine rotavirus (EDIM-RW) differ dramatically in the oral inoculum required to cause diarrheal disease in neonatal mouse pups and in their ability to spread and cause disease in uninoculated littermates. A genetic approach was used to explore the molecular basis of these differences. Reassortant viruses were produced in vivo by coinfecting infant mice with RRV and EDIM-RW. Reassortant viruses were isolated by plaque purification of progeny virus obtained from mouse pup intestines on MA104 cells. The plaque-purified reassortants were evaluated for 50% diarrhea dose (DD50) and for the ability to spread and cause diarrhea in uninoculated littermates. The parental RRV strain had a DD50 of 10(5) PFU per animal, while the EDIM-RW parental strain had a DD50 of less than 1 PFU per animal. RRV never spreads from inoculated to uninoculated littermates and causes disease. Twenty-three reassortants were tested. Of great interest were the reassortants D1/5 and C3/2, which derived genes 4 and 7 (encoding VP4 and VP7) from RRV. These viruses had a DD50 similar or identical to that of EDIM-RW and spread efficiently from inoculated mouse pups to uninoculated pups. We conclude that the major outer capsid proteins VP4 and VP7 are not primarily responsible for virulence or host range restriction in the mouse model using a homologous murine rotavirus.     

Influenza—an emerging and re-emerging disease

Influenza A viruses continue to emerge from the aquatic avian reservoir and cause pandemics. There are periodic exchanges of influenza virus genes or whole viruses between avians and other species giving rise to pandemics of diseases in humans, lower animals and birds. It is hypothesized that pigs are an intermediate host and that China is an epicenter for the evolution of human pandemic strains. However, the transmission of avian influenza viruses to pigs in Europe in 1979 and detection of reassortants with human influenza genes in pigs raises the question of whether the next pandemic of influenza will emerge in Europe!     

Efficacy of production of reassortant of epidemic strains and cold-adapted influenza viruses in chicken embryo and MDCK cells

Reassortant strains for modern live influenza vaccines are prepared using growing chicken embryos. It is very important to switch manufacture of influenza vaccines from chicken embryos to cell cultures, especially due to the threat of future pandemic, when there will be need of big quantities of vaccine for immunization of all age groups. Efficacy of production of reassortant strains with 6:2 vaccine formulation of genome (6 internal genes from the donor of attenuation and 2 genes coding external antigens--hemagglutinin and neuraminidase--from epidemic strain) in MDCK cell culture, using standard techniques employed for production of the vaccine in chicken embryos, was studied. It was shown that yield frequency of aforementioned reassortants of influenza A viruses did not exceed 5.7% whereas in chicken embryos vaccine 6:2 reassortants were isolated with frequency of 4%. For influenza B viruses, yield of 6:2 reassortants in growing chicken embryos exceeded 67% whereas in MDCK cell culture we were unable to produce clones with required genome composition. Thus, existing method while effective for production of vaccine reassortants in chicken embryos is low effective for isolation of 6:2 reassortants in MDCK cell culture. Fundamentally new techniques are needed for production of reassortant strains for live influenza vaccine in cell culture.     

Genetic mapping of lymphocytic choriomeningitis virus pathogenicity: virulence in guinea pigs is associated with the L RNA segment.

The Armstrong CA 1371 (ARM) and WE strains of lymphocytic choriomeningitis virus (LCMV) differ in the ability to produce disease in adult guinea pigs. Infection with the ARM strain is not lethal, even at high virus doses (greater than 10,000 PFU), whereas the WE strain causes 100% mortality even at low doses (less than 10 PFU). To determine the genetic basis of this virulence, intertypic reassortants were made between the ARM and WE strains of LCMV. The two reassortants with the genotypes WE/ARM (L segment of WE and S segment of ARM) and ARM/WE (L segment of ARM and S segment of WE) were tested for their pathogenicity in guinea pigs. The ARM/WE reassortant was avirulent like the ARM/ARM parental strain. Minimal viral replication was observed in organs of guinea pigs inoculated with 10(2) or 10(5) PFU of ARM/ARM or ARM/WE, and all animals survived. In contrast, the WE/ARM reassortant was highly virulent like the WE/WE parental strain and killed all of the infected animals. High levels of viral replication were observed in guinea pigs infected with the latter two strains. In contrast to these in vivo observations, both the parental strains and the ARM/WE or WE/ARM reassortants had similar growth potential in cultured guinea pig fibroblasts. Thus, the L RNA segment of LCMV WE is important for viral replication in vivo and is associated with fatal acute disease after infection of adult guinea pigs.     

Strains from both Theiler's virus subgroups encode a determinant for demyelination.

The GDVII strain and other members of the GDVII subgroup of Theiler's murine encephalomyelitis viruses (TMEV) cause an acute lethal neuronal infection in mice, whereas the DA strain and other members of the TO subgroup of TMEV cause a chronic demyelinating disease associated with a persistent virus infection. We used GDVII/DA chimeric infectious cDNAs to produce intratypic recombinant viruses in order to clarify reasons for the TMEV subgroup-specific difference in demyelinating activity. We found that both the GDVII and DA strains contain a genetic determinant(s) for demyelinating activity. No demyelination occurs following GDVII strain inoculation because this strain produces an early neuronal disease that kills mice before white matter disease and persistent infection can occur.     

Neuroattenuation of an avirulent bunyavirus variant maps to the L RNA segment.

The derivation and characterization of a neuroattenuated reassortant clone (RFC 25/B.5) of California serogroup bunyavirus was described previously (M. J. Endres, A. Valsamakis, F. Gonzalez-Scarano, and N. Nathanson, J. Virol. 64:1927-1933, 1990). To map the RNA segment responsible for this attenuation, a panel of reassortants was constructed between the attenuated clone B.5 (genotype TLL) and a virulent clone (B1-1a) of reciprocal genotype (LTT). Parent viruses and clones representing all of the six possible reassortants were examined for neurovirulence by intracerebral injection in adult mice. Reassortants bearing the large RNA segment from the virulent parent were almost as virulent as the virulent parent virus, while reassortants bearing the large RNA segment from the avirulent parent virus exhibited low or intermediate virulence. These results indicate that the large RNA segment is the major determinant of neuroattenuation of clone B.5. In addition to its neuroattenuation, clone B.5 was temperature sensitive and exhibited an altered plaque morphology. These phenotypes also segregated with the large RNA segment. The importance of the large RNA segment (which encodes the viral polymerase) in neurovirulence contrasts with prior studies which indicate that the ability to cause lethal encephalitis after peripheral injection of suckling mice (neuroinvasiveness) is primarily determined by the middle-sized RNA segment, which encodes the viral glycoproteins.     

Growth and Pathogenic Potential of Naturally Selected Reassortants after Coinfection with Pandemic H1N1 and Highly Pathogenic Avian Influenza H5N1 Viruses

Coinfection of ferrets with H5N1 and pH1N1 viruses resulted in two predominate genotypes in the lungs containing surface genes of highly pathogenic avian influenza H5N1 virus in the backbone of pandemic H1N1 2009 (pH1N1). Compared to parental strains, these reassortants exhibited increased growth and virulence in vitro and in mice but failed to be transmitted indirectly to naive contact ferrets. Thus, this demonstrates a possible natural reassortment following coinfection as well as the pathogenicity of the potential reassortants.     

Virulence of La Crosse virus is under polygenic control.

To identify which RNA segments of the California serogroup bunyaviruses determine virulence, we prepared reassortant viruses by coinfecting BHK-21 cells with two wild-type parents, La Crosse/original and Tahyna/181-57 viruses, which differed about 30,000-fold in virulence. The progeny clones were screened by polyacrylamide gel electrophoresis to ascertain the phenotype of the M and S RNA segments, and RNA-RNA hybridization was used to determine the genotype of selected clones. Two or three clones of each of the six possible reassortant genotypes were characterized quantitatively for neuroinvasiveness by determining the PFU/50% lethal dose (LD50) ratio after subcutaneous injection into suckling mice. The reassortants fell into two groups. (i) Six of seven reassortants with a La Crosse M RNA segment were as virulent as the parent La Crosse virus (about 1 PFU/LD50); the one exception was strikingly different (about 1,000 PFU/LD50) and probably represents a spontaneous mutant. (ii) The seven reassortants with a Tahyna M RNA segment were about 10-fold more virulent than the parent Tahyna virus (median 1,600 PFU/LD50 for reassortants and 16,000 PFU/LD50 for Tahyna virus). A comparative pathogenesis study in suckling mice of one reassortant virus and the parent Tahyna virus confirmed the greater neuroinvasiveness of the reassortant virus. From these data it was concluded that the M RNA segment was the major determinant of virulence, but that the other two gene segments could modulate the virulence of a nonneuroinvasive California serogroup virus.     

Characterization of the Candiru antigenic complex (Bunyaviridae: Phlebovirus), a highly diverse and reassorting group of viruses affecting humans in tropical America

The genus Phlebovirus of the family Bunyaviridae consists of approximately 70 named viruses, currently assigned to nine serocomplexes (species) based on antigenic similarities. Sixteen other named viruses that show little serologic relationship to the nine recognized groups are also classified as tentative species in the genus. In an effort to develop a more precise classification system for phleboviruses, we are attempting to sequence most of the named viruses in the genus with the goal of clarifying their phylogenetic relationships. In this report, we describe the serologic and phylogenetic relationships of 13 viruses that were found to be members of the Candiru serocomplex; 6 of them cause disease in humans. Analysis of full genome sequences revealed branching inconsistencies that suggest five reassortment events, all involving the M segment, and thus appear to be natural reassortants. This high rate of reassortment illustrates the inaccuracy of a classification system based solely on antigenic relationships.     

Structural and nonstructural protein genome regions of eastern equine encephalitis virus are determinants of interferon sensitivity and murine virulence

Eastern equine encephalitis virus (EEEV) causes sporadic epidemics of human and equine disease in North America, but South American strains have seldom been associated with human neurologic disease or mortality, despite serological evidence of infection. In mice, most North American and South American strains of EEEV produce neurologic disease that resembles that associated with human and equine infections. We identified a South American strain that is unable to replicate efficiently in the brain or cause fatal disease in mice yet produces 10-fold higher viremia than virulent EEEV strains. The avirulent South American strain was also sensitive to human interferon (IFN)-alpha, -beta, and -gamma, like most South American strains, in contrast to North American strains that were highly resistant. To identify genes associated with IFN sensitivity and virulence, infectious cDNA clones of a virulent North American strain and the avirulent South American strain were constructed. Two reciprocal chimeric viruses containing swapped structural and nonstructural protein gene regions of the North American and South American strains were also constructed and found to replicate efficiently in vitro. Both chimeras produced fatal disease in mice, similar to that caused by the virulent North American strain. Both chimeric viruses also exhibited intermediate sensitivity to human IFN-alpha, -beta, and -gamma compared to that of the North American and South American strains. Virulence 50% lethal dose assays and serial sacrifice experiments further demonstrated that both structural and nonstructural proteins are important contributors to neurovirulence and viral tissue tropism. Together, the results of this study emphasize the complex and important influences of structural and nonstructural protein gene regions on EEEV virulence.     

High growth reassortant influenza vaccine viruses: new approaches to their control.

When a new strain of an influenza virus is required to be incorporated into influenza vaccine, attempts are made to recombine such strains with laboratory adapted viruses, which will grow to high titre in order to improve the yield of the vaccine strain. It is important that such high growth reassortant vaccine strains are not contaminated with genes coding for the antigenic determinants of the high growth laboratory strain. We describe the characterization of two recent high growth reassortants and the application of the polymerase chain reaction to ensure their genetic identity and purity.     

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.     

The rhesus rotavirus gene encoding VP4 is a major determinant in the pathogenesis of biliary atresia in newborn mice

Biliary atresia (BA) is a devastating disease of childhood for which increasing evidence supports a viral component in pathogenesis. The murine model of BA is induced by perinatal infection with rhesus rotavirus (RRV) but not with other strains of rotavirus, such as TUCH. To determine which RRV gene segment(s) is responsible for pathogenesis, we used the RRV and TUCH strains to generate a complete set of single-gene reassortants. Eleven single-gene "loss-of-function" reassortants in which a TUCH gene replaced its RRV equivalent and 11 single-gene "gain-of-function" reassortants in which an RRV gene replaced its TUCH equivalent were generated. Newborn BALB/c mice were inoculated with the reassortants and were monitored for biliary obstruction and mortality. In vitro, the ability to bind to and replicate within cholangiocytes was analyzed. Infection of mice with the "loss-of-function" reassortant R(T(VP4)), where gene 4 from TUCH was placed on an RRV background, eliminated the ability of RRV to cause murine BA. In a reciprocal fashion, the "gain-of-function" reassortant T(R(VP4)) resulted in murine BA with 88% mortality. Compared with those for RRV, R(T(VP4)) binding and titers in cholangiocytes were significantly attenuated, while T(R(VP4)) binding and titers were significantly increased over those for TUCH. Reassortants R(T(VP3)) and T(R(VP3)) induced an intermediate phenotype. RRV gene segment 4 plays a significant role in governing tropism for the cholangiocyte and the ability to induce murine BA. Gene segment 3 did not affect RRV infectivity in vitro but altered its in vivo effect.     

Genetic compatibility and virulence of reassortants derived from contemporary avian H5N1 and human H3N2 influenza A viruses

The segmented structure of the influenza virus genome plays a pivotal role in its adaptation to new hosts and the emergence of pandemics. Despite concerns about the pandemic threat posed by highly pathogenic avian influenza H5N1 viruses, little is known about the biological properties of H5N1 viruses that may emerge following reassortment with contemporary human influenza viruses. In this study, we used reverse genetics to generate the 63 possible virus reassortants derived from H5N1 and H3N2 viruses, containing the H5N1 surface protein genes, and analyzed their viability, replication efficiency, and mouse virulence. Specific constellations of avian-human viral genes proved deleterious for viral replication in cell culture, possibly due to disruption of molecular interaction networks. In particular, striking phenotypes were noted with heterologous polymerase subunits, as well as NP and M, or NS. However, nearly one-half of the reassortants replicated with high efficiency in vitro, revealing a high degree of compatibility between avian and human virus genes. Thirteen reassortants displayed virulent phenotypes in mice and may pose the greatest threat for mammalian hosts. Interestingly, one of the most pathogenic reassortants contained avian PB1, resembling the 1957 and 1968 pandemic viruses. Our results reveal the broad spectrum of phenotypes associated with H5N1/H3N2 reassortment and a possible role for the avian PB1 in the emergence of pandemic influenza. These observations have important implications for risk assessment of H5N1 reassortant viruses detected in surveillance programs.     

A Single Amino Acid in the M1 Protein Responsible for the Different Pathogenic Potentials of H5N1 Highly Pathogenic Avian Influenza Virus Strains

Two highly pathogenic avian influenza virus strains, A/duck/Hokkaido/WZ83/2010 (H5N1) (WZ83) and A/duck/Hokkaido/WZ101/2010 (H5N1) (WZ101), which were isolated from wild ducks in Japan, were found to be genetically similar, with only two amino acid differences in their M1 and PB1 proteins at positions 43 and 317, respectively. We found that both WZ83 and WZ101 caused lethal infection in chickens but WZ101 killed them more rapidly than WZ83. Interestingly, ducks experimentally infected with WZ83 showed no or only mild clinical symptoms, whereas WZ101 was highly lethal. We then generated reassortants between these viruses and found that exchange of the M gene segment completely switched the pathogenic phenotype in both chickens and ducks, indicating that the difference in the pathogenicity for these avian species between WZ83 and WZ101 was determined by only a single amino acid in the M1 protein. It was also found that WZ101 showed higher pathogenicity than WZ83 in mice and that WZ83, whose M gene was replaced with that of WZ101, showed higher pathogenicity than wild-type WZ83, although this reassortant virus was not fully pathogenic compared to wild-type WZ101. These results suggest that the amino acid at position 43 of the M1 protein is one of the factors contributing to the pathogenicity of H5N1 highly pathogenic avian influenza viruses in both avian and mammalian hosts.     

Association of the reovirus S1 gene with serotype 3-induced biliary atresia in mice.

A panel of serotype 3 (T3) reovirus strains was screened to determine their relative capacities to cause lethal infection and hepatobiliary disease following peroral inoculation in newborn mice. A wide range of 50% lethal doses (LD50s) was apparent after peroral inoculation of the different virus strains. Two of the strains, T3 Abney and T3 clone 31, caused mice to develop the oily fur syndrome associated with biliary atresia. The capacity to cause biliary atresia was not related to the capacity to cause lethal infection, however, because the LD50s of T3 Abney and T3 clone 31 were grossly disparate. Examination of liver and bile duct tissues revealed histopathologic evidence of biliary atresia and hepatic necrosis in T3 Abney-infected mice but not in mice inoculated with a T3 strain of similar virulence or with the hepatotropic T1 Lang strain. The consistency with which T3 Abney-infected mice developed biliary atresia-associated oily fur syndrome permitted us to determine the viral genetic basis of reovirus-induced biliary atresia. Analysis of reassortant viruses isolated from an in vitro coinfection with T3 Abney and T1 Lang indicated a strong association of the hepatobiliary disease-producing phenotype with the T3 Abney S1 gene, which encodes the viral cell attachment protein, sigma 1. Amino acid residues within the sigma 1 protein that were unique to disease-producing T3 strains were identified by comparative sequence analysis. Specific changes exist within two regions of the protein, one of which is thought to be involved in binding to host cell receptors. We hypothesize that changes within this region of the protein are important in determining the tropism of this virus for bile-ductular epithelium.     

Evolution and molecular epidemiology of H9N2 influenza A viruses from quail in southern China, 2000 to 2005

H9N2 influenza viruses have become established and maintain long-term endemicity in terrestrial poultry in Asian countries. Occasionally these viruses transmit to other mammals, including humans. Increasing epidemiological and laboratory findings suggest that quail may be an important host, as they are susceptible to different subtypes of influenza viruses. To better understand the role of quail in influenza virus ecology and evolution, H9N2 viruses isolated from quail during 2000 to 2005 were antigenically and genetically characterized. Our results showed that H9N2 viruses are prevalent year-round in southern China and replicate mainly asymptomatically in the respiratory tract of quail. Genetic analysis revealed that both the G1-like and Ck/Bei-like H9N2 lineages were cocirculating in quail since 2000. Phylogenetic analyses demonstrated that most of the isolates tested were double- or multiple-reassortant variants, with four G1-like and 16 Ck/Bei-like genotypes recognized. A novel genotype of G1-like virus became predominant in quail since 2003, while multiple Ck/Bei-like genotypes were introduced into quail, wherein they incorporated G1-like gene segments, but none of them became established in this host. Those Ck/Bei-like reassortants generated in quail have then been introduced into other poultry. These complex interactions form a two-way transmission system between quail and other types of poultry. The present study provides evidence that H9N2 and H5N1 subtype viruses have also exchanged gene segments to generate currently circulating reassortants of both subtypes that have pandemic potential. Continuing influenza virus surveillance in poultry is critical to understanding the genesis and emergence of potentially pandemic strains in this region.     

Properties of reassortants of human and avian influenza viruses. Reproduction in MDCK cells at suboptimum temperatures

A series of reassortants has been constructed by crossing of UV-inactivated avian influenza virus of H3N8 subtype and live human influenza virus of H1N1 subtype, adapted to growth in continuous canine kidney cell line (MDCK). The analysis of RNA duplexes has shown that the reassortants contain HA gene of avian influenza virus whereas the other genes belong to human parent virus. The reassortants were efficiently reproduced in MDCK cells at low temperature (limiting for the avian parent virus). The data suggest that the avian virus HA gene does not hamper the reproduction of reassortant viruses in mammalian cells under the conditions unfavorable for the multiplication of avian influenza subtype H3N8 viruses.     

Reassortant H9N2 influenza viruses containing H5N1-like PB1 genes isolated from black-billed magpies in Southern China

H9N2 influenza A viruses have become endemic in different types of terrestrial poultry and wild birds in Asia, and are occasionally transmitted to humans and pigs. To evaluate the role of black-billed magpies (Pica pica) in the evolution of influenza A virus, we conducted two epidemic surveys on avian influenza viruses in wild black-billed magpies in Guangxi, China in 2005 and characterized three isolated black-billed magpie H9N2 viruses (BbM viruses). Phylogenetic analysis indicated that three BbM viruses were almost identical with 99.7 to 100% nucleotide homology in their whole genomes, and were reassortants containing BJ94-like (Ck/BJ/1/94) HA, NA, M, and NS genes, SH/F/98-like (Ck/SH/F/98) PB2, PA, and NP genes, and H5N1-like (Ck/YN/1252/03, clade 1) PB1 genes. Genetic analysis showed that BbM viruses were most likely the result of multiple reassortments between co-circulating H9N2-like and H5N1-like viruses, and were genetically different from other H9N2 viruses because of the existence of H5N1-like PB1 genes. Genotypical analysis revealed that BbM viruses evolved from diverse sources and belonged to a novel genotype (B46) discovered in our recent study. Molecular analysis suggested that BbM viruses were likely low pathogenic reassortants. However, results of our pathogenicity study demonstrated that BbM viruses replicated efficiently in chickens and a mammalian mouse model but were not lethal for infected chickens and mice. Antigenic analysis showed that BbM viruses were antigenic heterologous with the H9N2 vaccine strain. Our study is probably the first report to document and characterize H9N2 influenza viruses isolated from black-billed magpies in southern China. Our results suggest that black-billed magpies were susceptible to H9N2 influenza viruses, which raise concerns over possible transmissions of reassortant H9N2 viruses among poultry and wild birds.