Isolation of a recombinant influenza virus (Hsw1N2) from swine in Japan

Summary Outbreaks of swine influenza were first observed in Japan in 1978. A number of influenza viruses were isolated from diseased swine. Almost all viruses isolated were swine influenza virus (Hsw1N1) but two viruses isolated from the nasal swabs of swine showing clinical signs of influenza in the Kanagawa prefecture were characterized antigenically as Hsw1N2. Analysis of swine sera showed that influenza virus Hsw1N2 was epidemic in the farm from which the virus had been isolated. The new virus (Hsw1N2) seems to have been produced by recombination between swine influenza virus (Hsw1N1) and Hong Kong influenza virus (H3N2).     

Novel influenza A viruses isolated from Canadian feral ducks: including strains antigenically related to swine influenza (Hsw1N1) viruses.

Twelve influenza A viruses, antigenically related to the Ho, H1 and Hsw1 subtypes, were isolated from cloacal samples of feral ducks in Canada. Antigenic comparisons showed that these viruses were most closely related to the recent HSW1N1 isolates from man and pigs, whereas in vivo pathogenicity tests revealed differences between the Hsw1N1 viruses from the ducks and those from humans and pigs. Antigenic characterization of 94 additional influenza A viruses from the ducks showed four haemagglutinin subtypes (Hav1, Hav4, Hav5 and Hav7), an unclassified haemagglutinin, and six neuraminidase subtypes (N1, N2, Neq2, Nav1, Nav2 and Nav5) in various combinations, some of which are novel and have not previously been reported. Three of these duck influenza viruses possessed a haemagglutinin antigenically related to that of classical fowl plaque virus. A much higher percentage of virus isolations were from juvenile ducks (18.5%) than from adults (5%). All of the ducks, from which viruses were isolated, appeared healthy at the time of sampling. Serological studies on a limited number of humans and domestic birds living in close proximity to the Canadian ducks revealed no evidence of interspecies transmission. Our findings suggest that these birds serve as a substantial reservoir of antigenically diverse influenza viruses, including isolates antigenically related to the current human and animal influenza viruses. This reservoir in nature may be perpetuated by a cycle involving annual infection of juvenile birds followed by transmission to the remaining susceptible birds until the next congregation during the breeding season.     

Further studies in man of hsw1n1 influenza viruses

Two subpopulations of antigenically different HswlNl influenza viruses, cloned from ‘swine’ New Jersey virus 1976, were individually inoculated into antibody-free volunteers. One clone contained a haemagglutinin so far seen only in swine viruses prevalent in 1971 and after, the other a haemagglutinin of earlier strains dating back to at least 1957. Each of the viruses was infectious for man and intermediate in human virulence between a wild human virus and swine pathogens of 1966 and 1967, which had earlier been tested in man. Antigenically comparable clones segregated from viruses recovered in Wisconsin from a pig and a human contact, respectively, were also infectious for man; however, they were less virulent than their New Jersey counterparts. Differences between the Wisconsin clones themselves were small, but there was an indication of a relationship between human passage and human virulence. There was no evidence of inherently greater human virulence in the newer Hsw 1N1 serotype as compared with the earlier serotype. Hence, recent detection of swine influenza-like viruses in man is unlikely to be the consequence of a host-range mutation concurrent with an antigenic mutation. We believe that the Hsw1N1 viruses recovered from the human influenza outbreak at Fort Dix, New Jersey, and from recent single human infections were wholly derived from enzootic swine viruses that underwent limited human adaptation through man-to-man passage.     

Genetic reassortment of influenza A viruses in the intestinal tract of ducks

Genetic reassortment between influenza A viruses was shown to occur in the intestinal tract of ducks during mixed infection; this phenomenon was examined both in naturally and laboratory-infected ducks. Studies on cloacal samples from Canadian feral ducks demonstrated that 7% of these samples contained two or more antigenically distinguishable influenza viruses, indicating that mixed infections occur rather frequently in nature. The RNAs of multiple viruses isolated from one cloacal sample were separated by polyacrylamide gel electrophoresis; the RNA migration patterns showed that this sample contained a mixture of viruses, including antigenically identical viruses with heterogeneous RNAs. To determine if genetic reassortment may be responsible for the RNA heterogeneity, laboratory ducks were infected with two antigenically distinct avian influenza viruses (Hsw1N1 and Hav1Nav2). Antigenic recombinants (Hav1N1) were readily isolated, without selective antibody pressure, from the feces of these mixedly infected ducks. Comparisons between the RNA migration patterns of the parental viruses and multiple isolates from the feces showed that antigenically identical isolates (as the Hav1N1 recombinants) possessed different gene constellations. These findings show that genetic reassortment between viruses occurs readily in the duck's intestinal tract. Other parameters of virus infection in ducks were also examined. Juvenile ducks, inoculated with influenza virus, shed virus in their feces for 30 days, produced low levels of circulating antibodies (HI titer of 1:20), and were resistant to challenge with homologous virus. These studies illustrate the mechanism by which genetically diverse influenza A viruses may evolve in nature.     

Swine influenza viruses isolated in 1976 from man and pig contain two coexisting subpopulations with antigenically distinguishable hemagglutinins.

Hsw1N1 viruses isolated from recruits at Fort Dix, New Jersey, in 1976 were compared by hemagglutination inhibition (HI) and neuraminidase inhibition (NI) tests with HswlN1 viruses isolated from pigs from 1930 to 1976. Viruses isolated in 1976 from pigs and man were found to consist of two subpopulations with antigenically distinguishable hemagglutinins (HA). The HA of one subpopulation of 1976 viruses is similar to that of A/swine/Wisconsin/1/67 and has been identified on virus isolates dating back to at least 1957. The HA of the other subpopulation of 1976 viruses is similar to that of several other viruses isolated from 1971 to 1976, but has not yet been found on any North American isolate from before 1971 although it exhibits a significant cross-reactivity with the early isolate A/swine/Cambridge/39. Evidence is presented that the two antigenically distinguishable subpopulations coexisted in some cases in the infected host, including recruits at Fort Dix, a farmhand in Wisconsin, and an infected pig to which the Wisconsin farmhand was exposed. The results strongly support the likelihood that an influenza-infected pig was the initial source of virus containing the hemagglutinin genes present in the viruses ultimately isolated from recruits infected at Fort Dix, New Jersey.     

Comparative characteristics of hemagglutinins of influenza A viruses with antigenic structure Hsw1N1 isolated from man and animals

Competitive radioimmunoassay was used to study the antigenic composition of hemagglutinin of Hsw1N1 viruses isolated from man in comparison with hemagglutinin Hsw1 of influenza virus of swine and ducks. The data of oligonucleotide analysis of the 4th RNA segment coding for hemagglutinin in these viruses are presented. It has been shown that in Alma-Ata, 1984-1985, influenza viruses Hsw1N1 were isolated with the antigenic structure of hemagglutinin and with the hemagglutinin gene identical with those of the classical influenza virus of swine A/Swine/Iowa/15/30 but differing from virus A/New Jersey/8/76.     

Classical swine H1N1 influenza viruses confer cross protection from swine-origin 2009 pandemic H1N1 influenza virus infection in mice and ferrets

The hemagglutinin of the 2009 pandemic H1N1 influenza virus is a derivative of and is antigenically related to classical swine but not to seasonal human H1N1 viruses. We compared the A/California/7/2009 (CA/7/09) virus recommended by the WHO as the reference virus for vaccine development, with two classical swine influenza viruses A/swine/Iowa/31 (sw/IA/31) and A/New Jersey/8/1976 (NJ/76) to establish the extent of immunologic cross-reactivity and cross-protection in animal models. Primary infection with 2009 pandemic or NJ/76 viruses elicited antibodies against the CA/7/09 virus and provided complete protection from challenge with this virus in ferrets; the response in mice was variable and conferred partial protection. Although ferrets infected with sw/IA/31 virus developed low titers of cross-neutralizing antibody, they were protected from pulmonary replication of the CA/7/09 virus. The data suggest that prior exposure to antigenically related H1N1 viruses of swine-origin provide some protective immunity against the 2009 pandemic H1N1 virus.     

Further genetic evidence for maintenance of early Hong Kong-like influenza A(H3N2) strains in swine until 1976

The oligonucleotide map of the whole RNA of A/swine/Hong Kong/3/76 (H3N2) virus showed a relationship to, but considerable differences from, the maps of human isolates of influenza A(H3N2) with which this isolate antigenically cross-reacted. Comparison of the individual oligonucleotide spots of whole virus RNAs confirmed previous results of antigenic analysis that A/swine/Hong Kong/3/76 virus was more similar to an early A/Hong Kong/68 virus than to later H3N2 viruses which circulated in man during 1976. On the other hand, another swine isolate, A/swine/Hong Kong/4/76, showed a quite similar oligonucleotide map to that of the contemporary prevalent A/Victoria/75-like viruses to which this strain was reported to be antigenically similar. Comparison of oligonucleotide maps of individual RNA segments indicated that all genes of the A/swine/Hong Kong/3/76 virus were derived from a human H3N2 virus. The findings provide biochemical evidence that A/swine/Hong Kong/3/76 virus represents a 1968 Hong Kong-like virus that underwent genetic mutation without extensive change of its antigenicity during maintenance in the swine population.     

Antigenic and genetic characterization of a European avian-like H1N1 swine influenza virus from a boy in China in 2011

Cross-species transmission of influenza A viruses from swine to human occurs occasionally. In 2011, an influenza A H1N1 virus, A/Jiangsu/ALS1/2011 (JS/ALS1/2011), was isolated from a boy who suffered from severe pneumonia in China. The virus is closely related antigenically and genetically to avian-like swine H1N1 viruses that have recently been circulating in pigs in China and that were initially detected in European pig populations in 1979. The isolation of JS/ALS1/2011 provides additional evidence that swine influenza viruses can occasionally infect humans and emphasizes the importance of reinforcing influenza virus surveillance in both pigs and humans.     

Neuraminidase inhibitor susceptibility of swine influenza A viruses isolated in Germany between 1981 and 2008

European swine influenza A viruses donated the matrix protein 2 as well as the neuraminidase (NA) gene to pandemic influenza A (H1N1) viruses that emerged in 2009. As a result, the latter became amantadine resistant and neuraminidase inhibitor (NAI) susceptible. These recent developments reflecting the close connection between influenza A virus infection chains in humans and pigs urge an antiviral surveillance within swine influenza A viruses. Here, NAI susceptibility of 204 serologically typed swine influenza A viruses of subtypes H1N1, H1N2, and H3N2 circulating in Germany between 1981 and 2008 was analyzed in chemiluminescence-based NA inhibition assays. Mean 50% inhibitory concentrations of oseltamivir and zanamivir indicate a good drug susceptibility of tested viruses. As found for human isolates, the oseltamivir and zanamivir susceptibility was subtype-specific. So, swine influenza A (H1N1) viruses were just as susceptible to oseltamivir as to zanamivir. In contrast, swine H1N2 and H3N2 influenza A viruses were more sensitive to oseltamivir than to zanamivir. Furthermore, reduction in plaque size and virus spread by both drugs was tested with selected H1N1 and H1N2 isolates in MDCK cells expressing similar amounts of α2.3- and α2.6-linked sialic acid receptors. Data obtained in cell culture-based assays for H1N1 isolates correlated with that from enzyme inhibition assays. But, H1N2 isolates that are additionally glycosylated at Asn158 and Asn163 near the receptor-binding site of hemagglutinin (HA) were resistant to both NAI in MDCK cells. Possibly, these additional HA glycosylations cause a misbalance between HA and NA function that hampers or abolishes NAI activity in cells.     

Laboratory characterization of a swine influenza virus isolated from a fatal case of human influenza.

A swine influenza virus-like type A (H1N1) virus, designated A/Wisconsin/3523/88, was isolated in September 1988 from a Wisconsin woman who had died with primary viral pneumonia. Antigenic analyses with hemagglutinin-specific monoclonal antibodies and postinfection ferret serum indicated that the hemagglutinin of A/Wisconsin/3523/88 was antigenically closely related to viruses currently circulating in swine. Genetic analysis of the A/Wisconsin/3523/88 virus by RNA fingerprinting and partial RNA sequence analysis of seven of the eight segments indicated that the genome of the human isolate was similar to that of enzootic swine viruses. These laboratory data supported the epidemiologic findings that this human infection occurred by transmission of an enzootic swine influenza virus and that the virus showed no major genetic changes potentially related to increased pathogenesis.     

Hemagglutinin mutants of swine influenza virus differing in replication characteristics in their natural host.

In two mutant clones (L and H) of A/NJ/11/76 (Hsw 1N1) influenza viruses which differ slightly antigenically and markedly in replication characteristics in chicken embryos and Madin Darby canine kidney cells, these pleiotropic differences are mediated by mutation in the hemagglutinin gene (E. D. Kilbourne, Proc. Natl. Acad. Sci. U.S.A. 75:6258--6262, 1978). Experimental infection of swine with either the mutant L and H clones or recombinant viruses differing genetically only with respect to the presence of L or H hemagglutinin demonstrated greater infectivity for the natural host of viruses bearing the L hemagglutinin. Introduction of the L but not the H hemagglutinin gene into the human influenza virus A/PR/8/34 rendered it infective for swine. Both L and H variants were isolated from pigs naturally infected with contemporary swine influenza viruses when selective conditions for the suppression of the more prevalent L mutant were employed. The L and H mutants of swine influenza virus are yet another example of viral dimorphism in nature and probably are not mere artifacts of laboratory selection. In any event, the frequent apparent allelic appearance of the two forms suggests frequent mutation and/or reversion involving a point mutation in the hemagglutinin gene. The present studies demonstrate the importance of a single gene in the pathogenesis of an influenza viral infection in its natural host.     

Genetic analysis of two influenza A (H1) swine viruses isolated from humans in Thailand and the Philippines

Influenza viruses A/Philippines/341/2004 (H1N2) and A/Thailand/271/2005 (H1N1) were isolated from two males, with mild influenza providing evidence of sporadic human infection by contemporary swine influenza. Both viruses were antigenically and genetically distinct from influenza A (H1N1 and H1N2) viruses that have circulated in the human population. Genetic analysis of the haemagglutinin genes found these viruses to have the highest degree of similarity to the classical swine H1 viruses circulating in Asia and North America. The neuraminidase gene and the internal genes were found to be more closely related to viruses circulating in European swine, which appear to have undergone multiple reassorting events. Although transmission of swine influenza to humans appears to be a relatively rare event, swine have been proposed as the intermediate host in the generation of potential pandemic influenza virus that may have the capacity to cause human epidemics resulting in high morbidity and mortality.     

Origin of the hemagglutinin gene of H3N2 influenza viruses from pigs in China

Influenza viruses of the H3N2 subtype similar to Aichi/2/68 and Victoria/3/75 persist in pigs many years after their antigenic counterparts have disappeared from humans (Shortridge et al. (1977). Science 19, 1454-1455). To provide information on the mechanism of conservation of these influenza viruses in pigs, the hemagglutinin (HA) of four isolates from swine derived from Taiwan and Southern China were analyzed antigenically and genetically. The reactivity pattern of these viruses with a panel of monoclonal antibodies indicates that the HAs of these swine viruses were antigenically closely related to duck H3 and early human H3 viruses. Sequence analysis of the H3 genes from three swine viruses revealed that the swine H3 genes are more closely related to the duck genes than to early human H3 virus (A/Aichi/2/68). The degree of sequence homology of these genes is extremely high (more than 96.5%). Furthermore, the deduced amino acid sequence of the three swine HAs at residues 226 to 228 in the proposed receptor-binding site is Gln-Ser-Gly and is common with the majority of avian influenza viruses. These findings indicate that these H3 viruses may have been introduced into pigs from ducks. The HA gene of the fourth swine influenza virus from Southern China was genetically equally related to avian and early human H3 strains although the sequence through the receptor-binding pocket (226-228) was typical of a human H3 virus, suggesting that either this swine HA gene was derived from ducks or an early human H3 virus was introduced into the pig population where the virus accumulated substantial mutations. The present strains revealed genetic heterogeneity of swine H3 influenza viruses in nature.     

Antigenic and genetic characterization of swine influenza A (H1N1) viruses isolated from pneumonia patients in The Netherlands

It is generally believed that pigs can serve as an intermediate host for the transmission of avian influenza viruses to humans or as mixing vessels for the generation of avian-human reassortant viruses. Here we describe the antigenic and genetic characterization of two influenza A (H1N1) viruses, which were isolated in The Netherlands from two patients who suffered from pneumonia. Both viruses proved to be antigenically and genetically similar to avian-like swine influenza A (H1N1) viruses which currently circulate in European pigs. It is concluded that European swine H1N1 viruses can infect humans directly, causing serious disease without the need for any reassortment event.     

猪型(H1N1)流感病毒血凝素和神经氨酸酶基因来源的研究

目的 研究2002年我国内地从猪群中分离的猪型(H1N1)毒株HA和NA基因来源。及其使猪致病的原因。方法 用PCR扩增目的基因，用P^GEM-T Easy Vector，4℃过夜连接，重组质粒转入DH-10B细菌，筛选阳性菌落，酶切鉴定，送六合通公司自动测序，并作进化树分析。结果 3株猪型(H1N1)病毒的HA和NA基因属猪型(H1N1)流感病毒，而不同于其他禽或人的H1N1亚型流感病毒。2002年猪型毒株由1991年猪型毒株演变而来。近来我国内地猪群中猪型毒株活动增强，其对猪能致病是由于病毒粒HA和NA蛋白抗原性发生变异所造成。结论 3株猪型病毒的HA和NA基因来源于猪型(H1N1)毒株。近来猪型毒株对猪具有致病性和活动增强是由于其HA和NA蛋白分子上氨基酸序列发生替换所造成。     

Antigenic and biochemical analysis of influenza “A” H3N2 viruses isolated from pigs

Summary Four influenza A-H3N2 viruses isolated in pigs from different herds in Central Italy in the period 1981/82 have been antigenically and biochemically analysed. Three of them A/Sw/Italy/2/81, A/Sw/Italy/7/81, A/Sw/Italy/8/82 were found to be serologically related to A/Bangkok/1/79 (H3N2). These three viruses were shown to have an identical electrophoretic pattern, as regards virus induced polypeptides and were clearly distinguishable from the virus A/Sw/Italy/6/81 which was antigenically related to A/England/42/72 (H3N2) and A/Sw/Taiwan/7310/70 as shown by specific monoclonal and polyclonal antisera.The observed biochemical variations underline the importance of the changes occurring by genetic reassortments or mutations in human influenza viruses, during their maintenance in pigs.With 3 Figures     

Genetic analysis and antigenic characterization of swine origin influenza viruses isolated from humans in the United States, 1990-2010

Swine influenza viruses (SIV) have been recognized as important pathogens for pigs and occasional human infections with swine origin influenza viruses (SOIV) have been reported. Between1990 and 2010, a total of twenty seven human cases of SOIV infections have been identified in the United States. Six viruses isolated from1990 to 1995 were recognized as classical SOIV (cSOIV) A(H1N1). After 1998, twenty-one SOIV recovered from human cases were characterized as triple reassortant (tr_SOIV) inheriting genes from classical swine, avian and human influenza viruses. Of those twenty-one tr_SOIV, thirteen were of A(H1N1), one of A(H1N2), and seven of A(H3N2) subtype. SOIV characterized were antigenically and genetically closely related to the subtypes of influenza viruses circulating in pigs but distinct from contemporary influenza viruses circulating in humans. The diversity of subtypes and genetic lineages in SOIV cases highlights the importance of continued surveillance at the animal-human interface.     

Annual examination of influenza virus infection among pigs in Miyagi prefecture, Japan: the appearance of Hsw1N1 virus.

Annual dissemination of swine and Hong Kong influenza viruses among hogs in Miyagi prefecture, Japan, was examined by virus recovery and serological tests. The serological examination revealed that two distinct influenza A viruses, H3N2 and Hsw1N1, had caused dual epidemic in pigs since mid-1977. Some individual sera contained antibodies against both strains. Although positive sera against Hong Kong or swine influenza viruses appeared in the serosurvey throughout the year, the positive ratios indicated a peak in the early spring of 1978. During this high prevalence period, positive sera against the H3N2 strain were found throughout the prefecture, but high incidences of Hsw1N1 virus were found only in limited areas to the north-east of Sendai City. Four strains of influenza A virus were isolated from bronchial swabs collected from hogs in April, 1978. These strains were identified as Hsw1N1 viruses and were closely related to the A/New Jersey/8/76 strain.     

Further evidence for infection of pigs with human-like H1N1 influenza viruses in China

Classical swine and avian-like H1N1 influenza viruses were reported widely in swine population worldwide, but human-like H1N1 swine viruses were reported occasionally. In 2006, a human-like H1N1 swine virus (A/swine/Guangdong/96/06) was isolated from pigs in Guangdong province, which was reported in China for the first time. To get further evidence for infection of pigs with human-like H1N1 influenza viruses, we analyzed eight gene segments of three human-like swine H1N1 viruses (A/swine/Guangdong/96/06, A/swine/Tianjin/01/04 and A/swine/Henan/01/06) isolated in China. All the eight genes of the three viruses are highly homologous to recent (about 2000) and early (1980s) human H1N1 influenza viruses, respectively. Phylogenetic analyses revealed that A/Swine/Guangdong/96/06 was directly derived from about 2000 human H1N1 influenza viruses, while A/swine/Tianjin/01/04 and A/swine/Henan/01/06 seemed to be descendants of human H1N1 viruses circulating in 1980s. Seroprevalence of our isolate (A/swine/Guangdong/96/06) confirmed the presence of human-like H1N1 virus in pigs in China. Existence of these influenza viruses, especially older viruses (A/swine/Tianjin/01/04 and A/swine/Henan/01/06), indicates that human-like H1N1 influenza viruses may remain invariant for long periods in pigs and provides the evidence that pigs serve as reservoirs of older influenza viruses for human pandemics.