Genetic characterization of H1N2 swine influenza virus isolated in China and its pathogenesis and inflammatory responses in mice

In 2009, two H1N2 influenza viruses were isolated from trachea swabs of pigs in Hubei in China. We compared these sequences with the other 18 complete genome sequences of swine H1N2 isolates from China during 2004 to 2010 and undertook extensive analysis of their evolutionary patterns. Six different genotypes – two reassortants between triple reassortant (TR) H3N2 and classical swine (CS) H1N1 virus, three reassortants between TR H1N2, Eurasian avian-like H1N1 swine virus and H9N2 swine virus, and one reassortant between H1N1, H3N2 human virus and CS H1N1 virus – were observed in these 20 swine H1N2 isolates. The TR H1N2 swine virus is the predominant genotype, and the two Hubei H1N2 isolates were located in this cluster. We also used a mouse model to examine the pathogenesis and inflammatory responses of the two isolates. The isolates replicated efficiently in the lung, and exhibited a strong inflammatory response, serious pathological changes and mortality in infected mice. Given the role that swine can play as putative “genetic mixing vessels” and the observed transmission of TR H1N2 in ferrets, H1N2 influenza surveillance in pigs should be increased to minimize the potential threat to public health.     

Reassortants in recent human influenza A and B isolates from South East Asia and Oceania

From 2000 to 2002, human influenza A and B viruses that were genetic reassortants of contemporary circulating human strains, were isolated in South East Asia and Oceania. Similar to reports from other regions, A(H1N2) isolates were found to be reassortants of circulating A(H3N2) viruses that had acquired only the haemagglutinin gene of an A(H1N1) virus. Some of these reassortants from Thailand and Singapore predate those previously recorded during the winter of 2001-2002 in Europe and the Middle East and may be precursors of these viruses. The B reassortants had a haemagglutinin similar to an earlier B strain, B/Shangdong/7/97 (B/Victoria/2/87-lineage) and a neuraminidase similar to the recently circulating B/Sichuan/379/99 virus (B/Yamagata/16/88-lineage). Despite the early occurrences of A(H1N2) reassortants and the extensive circulation of A(H1) viruses in South East Asia and Oceania during 2000-2001, these reassortant influenza A viruses have to date not been prominent unlike Europe and the Middle East where they were common in the 2001-2002 winter. In contrast the reassortant B viruses, which first emerged in this region in early 2002, rapidly became the predominant strains isolated from patients with influenza B in South East Asia and Oceania.     

Evolution of highly pathogenic avian influenza (H5N1) virus populations in Vietnam between 2007 and 2010

We report on the genetic analysis of 213 highly pathogenic avian influenza (HPAI) H5N1 viruses isolated from poultry in Vietnam between 2007 and 2010. Phylogenetic analyses of the viral genomes revealed 38 distinct viral genotypes, 29 were novel and 9 were reported in Vietnam or neighboring countries in recent years. Viruses from only six genotypes persisted beyond one season or year. Thus, most reassortant viruses were transient, suggesting that such genotypes lacked significant fitness advantages. Viruses with clade 2.3.2.1 HA were re-introduced into Vietnam in 2009 and their prevalence rose steeply towards the end of 2010. Clade 2.3.4-like viruses (genotype V) were predominant in northern Vietnam and caused the majority of zoonotic infections, whereas clade 1.1 (genotype Z) viruses were only detected in the Mekong delta region, in southern Vietnam. Antigenic analysis of representative viruses from the four clades indicated substantial drift.     

Intergenic HA-NA interactions in influenza A virus: postreassortment substitutions of charged amino acid in the hemagglutinin of different subtypes

In our previous studies influenza A virus reassortants having neuraminidase (NA) gene of A/USSR/90/77 (H1N1) strain and hemagglutinin (HA) genes of H3, H4 and H13 subtypes were shown to produce a low virus yield and to exhibit a strong tendency to virion aggregation. More detailed studies with the use of a H3N1 reassortant and its high-yield non-aggregating variants revealed that NA of A/USSR/90/77 strain is inefficient in the removal of the terminal sialic acid residues from the virion components, and that the inefficiency of NA may be compensated by mutations in HA gene leading to a decrease of the receptor-binding affinity (Kaverin, N.V. , Gambaryan, A.S., Bovin, N.V., Rudneva, I.A., Shilov, A.A., Khodova, O.M., Varich, N.L., Sinitsin, B.V., Makarova, N.L., Kaverin, N.V., 1998. Postreassortment changes in influenza virus hemagglutinin restoring HA-NA functional match, Virology 244, 315-321). The present report describes studies performed with the use of H2N1 and H4N1 reassortants having HA genes of A/Pintail/Primorie/695/76 (H2N3) and A/Duck/Czechoslovakia/56 (H4N6) strains respectively and NA gene of A/USSR/90/77 strain. The low-yield reassortants and their high-yield non-aggregating variants were studied in both direct and competitive binding assays with sialic acid-containing substrates. The non-aggregating variants were shown to have a decreased affinity as compared to the initial reassortants toward high-molecular-weight sialic acid-containing substrates. The sequencing of HA genes revealed that all non-aggregating variants of H2N1 and H4N1 reassortants had amino acid substitutions increasing the negative charge of the HA molecule in the vicinity of the receptor-binding pocket. The results suggest that the influenza virus reassortants containing low-functional NA undergo similar postreassortment changes irrespective of the HA subtype: their receptor-binding activity decreased due to negatively charged amino acid substitutions in the vicinity of the receptor-binding pocket.     

Postreassortment changes in a model system: HA–NA adjustment in an H3N2 avian–human reassortant influenza virus

Summary. In our previous studies we described the postreassortment changes in the hemagglutinin (HA) of H2N1, H3N1, H4N1 and H13N1 influenza A virus reassortants with HAs derived from avian viruses and low-functional neuraminidase (NA) of a human parent virus A/USSR/90/77 (H1N1). The changes involved amino acid substitutions that increased the negative local charge in the vicinity of the receptor-binding pocket and decreased the affinity of HA to sialic acid receptors. In the present report we describe the studies performed with H3N2 reassortant viruses having HA of A/Duck/Ukraine/1/63 (H3N8) virus and NA of A/Aichi/2/68 (H3N2) virus. Amino acid changes in the HA gene registered in virus variants selected in the course of serial passages resulted in a decrease in the affinity to sialic acid-containing substrates and cell receptors. However, the decrease was less expressed than in the reassortants containing the low-functional NA of N1 subtype described in our earlier studies, and the amino acid changes were not necessarily associated with an increase of negative charge. In one passage variant an amino acid substitution in NA was detected. The relevance of these results for the evolution of the H3N2 virus of the 1968 pandemic is discussed.     

Characterization of a newly emerged genetic cluster of H1N1 and H1N2 swine influenza virus in the United States

H1 influenza A viruses that were distinct from the classical swine H1 lineage were identified in pigs in Canada in 2003–2004; antigenic and genetic characterization identified the hemagglutinin (HA) as human H1 lineage. The viruses identified in Canadian pigs were human lineage in entirety or double (human–swine) reassortants. Here, we report the whole genome sequence analysis of four human-like H1 viruses isolated from U.S. swine in 2005 and 2007. All four isolates were characterized as triple reassortants with an internal gene constellation similar to contemporary U.S. swine influenza virus (SIV), with HA and neuraminidase (NA) most similar to human influenza virus lineages. A 2007 human-like H1N1 was evaluated in a pathogenesis and transmission model and compared to a 2004 reassortant H1N1 SIV isolate with swine lineage HA and NA. The 2007 isolate induced disease typical of influenza virus and was transmitted to contact pigs; however, the kinetics and magnitude differed from the 2004 H1N1 SIV. This study indicates that the human-like H1 SIV can efficiently replicate and transmit in the swine host and now co-circulates with contemporary SIVs as a distinct genetic cluster of H1 SIV.     

Gene analysis and phenotypic characteristic of highly-reproductive reassortants, containing the gene for bird influenza virus subtype H2 hemagglutinin

A series of reassortant clones with antigenic formulae H2N1 and H2N3 were produced by genetic reassortment performed with the use of an avian influenza virus, A/Pintail Duck/Primorie/695/76 (H2N3) and a high-yield reassortant strain X-67. Preliminary identification of the parent origin of NP and NS genes for 5 reassortants was performed by comparison of the mobilities of virus-specific proteins in polyacrylamide gel electrophoresis. The parent origin of genes of internal and nonstructural proteins for 3 reassortants was identified by partial sequencing. Although the genes of internal and nonstructural proteins of the reassortants originated from high-yield X-67 virus, only H2N3 reassortants were similar to the high-yield parent virus as concerns the level of the virus accumulation evaluated by hemagglutination titration and measurement of the virus protein content.     

Antigenic and genomic analyses of influenza A(H1N1) viruses from different regions of the world, February 1978 to March 1980.

Antigenic analysis of influenza A(H1N1) virus isolates by hemagglutination inhibition reactions with postinfection ferret sera and mouse monoclonal antibodies showed that, although the predominant virus in 1978 to 1979 had an A/Brazil/11/78-like hemagglutinin, several new antigenic variants could be detected which differed from those identified in 1977 to 1978. Most of the variants showed very minor changes and did not achieve epidemiological significance. Genome analysis by ribonucleic acid-ribonucleic acid hybridization, and supported by oligonucleotide mapping, indicated that nonrecombinant H1N1 viruses cocirculated in the world with recombinant H1N1 viruses, identified in 1978 to 1979. The results of our antigenic analysis and genomic analysis, in conjunction with genomic analysis by other authors, showed that recombinants with A/USSR/90/77-like and A/Brazil/11/78-like hemagglutinin had different genome compositions, indicating that H3N2 virus recombined separately with these different antigenic variants. Prevalence of the nonrecombinant and recombinant H1N1 viruses varied between regions of the world, and influenza A(H1N1) virus with a genotype almost identical to that of nonrecombinant A/Brazil/11/78 virus reappeared as a cause of local outbreaks in the United States in 1980 despite the predominance of recombinant H1N1 strains in the preceding winter.     

Avian influenza viruses in Korean live poultry markets and their pathogenic potential

We surveyed live-poultry markets in Korea in 2003 and isolated 9 H9N2, 6 H3N2, and 1 H6N1 influenza viruses. Antigenic and phylogenetic analyses showed that all 9 H9N2 isolates were of A/Chicken/Korea/25232-96006/96-like lineage (which caused disease in chickens in Korea in 1996) but were different from H9N2 viruses of southeastern China. They had at least 4 genotypes and replicated in chickens but not in mice. The H3N2 and H6N1 viruses were new to Korea and were probably reassortants of avian influenza viruses from southeastern China and recent Korean H9N2 viruses. All 8 segments of the H3N2 viruses formed a single phylogenetic cluster with 99.1 to 100% homology. The H3N2 viruses replicated in chickens and mice without preadaptation, but the H6N1 virus did not. Our results show an increasingly diverse pool of avian influenza viruses in Korea that are potential pandemic influenza agents.     

Phenotypic expression of HA-NA combinations in human-avian influenza A virus reassortants

Summary Human-avian and human-mammalian influenza A virus reassortant clones with the neuraminidase (NA) gene of the A/USSR/90/77 (H1N1) strain and hemagglutinin (HA) genes of H3, H4 and H13 subtypes had been shown in an earlier publication to produce low HA yields in the embryonated chicken eggs. The low HA titers had been shown to be due, at least in part, to the formation of virion clusters at 4°C; the clustering was removed by the treatment with bacterial neuraminidase [Rudneva et al., Arch. Virol (1993) 133: 437–450]. By serial passages of the reassortants in chick embryos non-aggregating variants were selected: the variants produced HA titers of the same order as A/USSR/90/77 parent virus. The assessment of the virus yields by the analysis of the partially purified virus preparations from fixed volumes of the allantoic fluid revealed that actual virion yields of the initial reassortants were lower than the yields of their passaged variants or of the parent viruses. The passaged variant of a reassortant possessing the HA gene of A/Duck/Ukraine/1/63 (H3N2) virus differed from the original (non-passaged) reassortant and from the parent A/Duck/Ukraine/1/63 virus in the reaction with a panel of monoclonal antibodies against H3 hemagglutinin. The data suggest that some HA-NA combinations may lead to an incomplete functional match between HA and NA and to the formation of low-yield reassortants, thus representing a possible limiting factor in the emergence of new HA-NA combinations in natural conditions.     

Identification of human H1N2 and human-swine reassortant H1N2 and H1N1 influenza A viruses among pigs in Ontario, Canada (2003 to 2005)

Since 2003, three novel genotypes of H1 influenza viruses have been recovered from Canadian pigs, including a wholly human H1N2 virus and human-swine reassortants. These isolates demonstrate that human-lineage H1N2 viruses are infectious for pigs and that viruses with a human PB1/swine PA/swine PB2 polymerase complex can replicate in pigs.     

Reassortment and evolution of current human influenza A and B viruses

During the 2001-2002 influenza season, human influenza A (H1N2) reassortant viruses were detected globally. The hemagglutinin (HA) of these H1N2 viruses was similar to that of the A/New Caledonia/20/99 (H1N1) vaccine strain both antigenically and genetically, while their neuraminidase (NA) was antigenically and genetically related to that of recent human influenza H3N2 reference viruses such as A/Moscow/10/99. All six internal genes of the H1N2 reassortants originated from an H3N2 virus. After being detected only in eastern Asia during the past 10 years, Influenza B/Victoria/2/87 lineage viruses reappeared in many countries outside of Asia in 2001. Additionally, reassortant influenza B viruses possessing an HA similar to that of B/Shandong/7/97, a recent B/Victoria/2/87 lineage reference strain, and an NA closely related to that of B/Sichuan/379/99, a recent B/Yamagata/16/88 lineage reference strain, were isolated globally and became the predominant influenza B epidemic strain. The current influenza vaccine is expected to provide good protection against H1N2 viruses because it contains A/New Caledonia/20/99 (H1N1) and A/Panama/2007/99 (H3N2) like viruses whose H1 HA or N2 NA are antigenically similar to those of recent circulating H1N2 viruses. On the other hand, widespread circulation of influenza B Victoria lineage viruses required inclusion of a strain from this lineage in influenza vaccines for the 2002-2003 season.     

Single genotype of measles virus is dominant whereas several genotypes of mumps virus are co-circulating

We have reported that in Japan measles virus strains have been classified into three distinct different genotypes (C1, D3 and D5) under the new international genotype classification since 1984. Similarly, mumps virus strains have been divided into two genotypes with three subtypes (B1, B2, B3, and D) under the proposed international classification since 1976. To differentiate these genotypes we developed a restriction fragment length polymorphism assay in the hemagglutinin (H) region for measles virus and in the hemagglutinin-neuraminidase (HN) region for mumps virus to facilitate the expanded molecular epidemiology. In the Sapporo 1995/1996 measles outbreak, all 26 strains were classified as D5. Among 32 samples from patients with measles from 1994 to 1997 in Tokyo, 28 were identified as D5 and four were D3; these D3 strains were ascertained as a same hospital acquired infection. Among 45 strains obtained in the Tokyo 1999 outbreak, 38 were D3 and the remaining seven were D5. The dominant genotype of measles in Tokyo has replaced from D5 to D3 similar to the Chicago1/89 strain. We obtained 220 samples from patients with mumps from 1993 to 1997 and they were classified into one strain of B1, 14 strains of B2, 151 strains of B3, and 54 strains of D. Therefore, we suggest that two or three subtypes of mumps virus are co-circulating with a different geographic pattern in genotype distribution, whereas a single measles virus genotype is dominantly observed, showing different epidemiological patterns.     

Deviation from the random distribution pattern of influenza A virus gene segments in reassortants produced under non-selective conditions

High-frequency reassortment of gene segments is characteristic for influenza viruses, and it is considered to be of significance for the origin of pandemic influenza. In order to analyze whether the segregation of genes in the reassortants is random, or it deviates from the random pattern, we inoculated embryonated chicken eggs simultaneously with two influenza viruses, A/WSN/33 (H1N1) and A/Duck/ Czechoslovakia/56 (H4N6), at a high multiplicity of infection. The virus yield was used for plaque cloning, and the genetic content of plaque isolates was determined by analysis of the mobility of virus-induced proteins in polyacrylamide gel (for NP and NS genes), partial sequencing (for M gene) and polymerase chain reaction analysis with strain-specific primers for the other genes. Out of 37 isolates, 27 were reassortants. The majority of the reassortants contained the HA gene of A/WSN/33 (H1N1) virus and the NP gene of A/Duck/Czechoslovakia/56 (H4N6) virus. The data demonstrate the previously unrecognized phenomenon of segment-specific deviation from the random distribution of parent genes in the reassortants. The results are discussed in connection with the problem of differential competition between influenza A virus gene segments in mixed infection and random versus non-random reassortment of gene segments under non-selective conditions.     

Genetic drift of norovirus genotype GII-4 in seven consecutive epidemic seasons in Hungary.

BACKGROUND: Noroviruses are common pathogens in human gastroenteritis outbreaks worldwide. They belong to a genetically diverse group of RNA viruses with multiple genogroups (G) and genotypes. Genotype GII-4 norovirus (Lordsdale) is the predominant agent in epidemics. OBJECTIVES: To investigate the genetic variation in GII-4 strains isolated during seven epidemic seasons in Hungary from November 2000 to June 2007. STUDY DESIGN: Using the prospective epidemiological surveillance of norovirus outbreaks in Hungary, GII-4 strains were selected for further genetic analysis. After phylogenetic analysis, RNA-polymerase (open reading frame 1; ORF1), capsid (ORF2) and the ORF1/ORF2 junction were analysed by RT-PCR and sequencing. RESULTS: Three hundred and seventy-seven (76.8%) of 491 confirmed norovirus outbreaks were caused by genotype GII-4. GII-4 was the predominant genotype in six of the seven epidemic seasons. Four main GII-4 variants--epidemic point mutants--(GII-4-2000, GII-4-2002, GII-4-2004 and GII-4-2006b) were detected, with each variant predominating in two consecutive epidemic seasons. CONCLUSIONS: Genotype GII-4 was confirmed as the predominant genetic type in epidemic norovirus seasons in Hungary. Genetic drift successfully promotes the re-emergence of GII-4 variants in the population. The elevated number of norovirus outbreaks in the population predicts the emergence of new GII-4 genetic variants as part of an international epidemic.     

Influenza A virus reassortants with surface glycoprotein genes of the avian parent viruses: effects of HA and NA gene combinations on virus aggregation

Summary A series of 33 human-avian and human-mammalian influenza virus reassortant clones possessing either HA or both HA and NA genes of the avian or mammalian virus was obtained by crosses of A/USSR/90/77 (H1N1) human virus with 5 avian and 1 mammalian influenza virus strains. All of the reasortants possessing NA genes of the H1N1 human parent virus and HA gene of an avian or mammalian parent virus had high values of infectivity/HA activity ratio. Since this feature could result from a limited virion aggregation, several reassortants were analyzed by velocity sucrose gradient centrifugation. In all cases tested, the reassortants of H3N1, H4N1, H10N1 and H13N1 composition were shown to be aggregated, whereas the preparations of the parent H1N1 virus and the reassortants possessing both HA and NA genes from the avian parents were represented mostly by single virions. The aggregates were formed at 4°C and dissociated at 37°C. The dissociation was blocked by an inhibitor of neuraminidase activity (2-deoxy-2,3-dehydro-N-acetyl-neuraminic acid). The dissociation was reversible since the virions reaggregated at 4°C; however, treatment with bacterial neuraminidase led to an irreversible dissociation of the aggregates. The tendency of the reassortants to aggregate correlates with an increased infectivity/HA ratio. No regular decrease in the neuraminidase activity in the virions of reassortants as compared to the parent H1N1 virus was revealed. The most likely explanation of the observed phenomenon seems to be an inefficient removal of sialic acid residues from the avian virus hemagglutinin by the human virus N1 neuraminidase.