Swine H1N1 Viruses Research Articles

Origin of internal genes of two strains of swine influenza A (H1N1) virus

Through the analysis of internal genes of viruses to understand whether the internal genes of two strains of swine influenza A(H1N1) virus contain the gene segment deriving from avian influenza A viruses, and whether the reassortment of the internal genes occurred between swine H1N1 and swine H9N2 viruses. Viruses were passaged in embryonated hen eggs and virion RNA was extracted from allantoic fluids and reverse transcribed to synthesize cDNA. cDNA was amplified by PCR and the PCR product was purified with a purification kit. Afterwards, RNA sequence analysis was performed by dideoxynucleotide chain termination and a cloning method. Finally, phylogenetic analysis of the sequencing data was performed with MegAlign (version 1.03). All the six, but PB2 gene segment internal gene segments were the same between A/swine/Beijing/1/2002(H1N1) and A/Swine/Beijing/3/2002(H1N1) viruses. Whereas all the six internal gene segments in two trains of swine (H1N1) virus were similar to those of swine H1N1 viruses, but different from those of classical strain of swine (H1N1) virus. Two strains of Beijing swine H1N1 influenza A virus were not reassortant, All the six internal gene segments were closely related to swine influenza A (H1N1) viruses.

H3N2 Influenza Virus Transmission from Swine to Turkeys, United States

In 1998, a novel H3N2 reassortant virus emerged in the United States swine population. We report the interspecies transmission of this virus to turkeys in two geographically distant farms in the United States in 2003. This event is of concern, considering the reassortment capacity of this virus and the susceptibility of turkey to infection by avian influenza viruses. Two H3N2 isolates, A/turkey/NC/16108/03 and A/turkey/MN/764/03, had 98.0% to 99.9% nucleotide sequence identity to each other in all eight gene segments. All protein components of the turkey isolates had 97% to 98% sequence identity to swine H3N2 viruses, thus demonstrating interspecies transmission from pigs to turkeys. The turkey isolates were better adapted to avian hosts than were their closest swine counterparts, which suggests that the viruses had already begun to evolve in the new host. The isolation of swine-like H3N2 influenza viruses from turkeys raises new concerns for the generation of novel viruses that could affect humans.

Emergence of distinct avian-like influenza A H1N1 viruses in pigs in Ireland and their reassortment with cocirculating H3N2 viruses

The influenza A H1N1 viruses, initially isolated from pigs in Ireland in 1991, were genetically distinct from previously identified human and swine H1N1 viruses, and appear to be the result of introduction of an avian virus distinct from those circulating in pigs in other European countries. The first Irish H3N2 swine isolates were genetically closely related to human H3N2 viruses. In contrast, H3N2 viruses isolated during the latter half of the 1990s were reassortants possessing internal genes similar to those of the H1N1 viruses, and both subtypes acquired amantadine resistance. These changes mimic those which occurred among swine influenza viruses in continental Europe during the 1980s, and emphasize the apparent advantage of properties associated with the avian internal genes and amantadine resistance for maintenance of the viruses in pigs.

Characterization of a swine-like reassortant H1N2 influenza virus isolated from a wild duck in the United States

An H1N2 influenza virus (A/Duck/North Carolina/91347/01) (Dk/NC) was isolated from a wild duck in the United States in 2001. Genetic analyses showed that this duck virus has the same human/classical swine/avian reassortant genotype as the H1N2 viruses that have been isolated from pigs and turkeys in the US since 1999. Phylogenetic analyses of each gene segment further confirmed that the Dk/NC virus is closely related to the domestic animal H1N2 isolates. In particular, Dk/NC is most closely related to a swine H1N2 virus also isolated in North Carolina. These two viruses and a phylogenetically-defined subset of additional swine H1N2 viruses share a common mutation in the Sb antigenic site on the hemagglutinin protein. The recovery of Dk/NC from a wild bird raises concerns for further widespread distribution of these H1N2 viruses via waterfowl migration.

Phylogenetic analysis of H1N2 isolates of influenza A virus from pigs in the United States

Twenty-four H1N2 influenza A viruses were newly isolated from pigs in the United States. These isolates originated from 19 farms in 9 different swine producing states between 1999 and 2001. All farms had clinical histories of respiratory problem and/or abortion. The viral isolates were characterized genetically to determine the origin of all eight gene segments. The results showed that all H1N2 isolates were reassortants of classical swine H1N1 and triple reassortant H3N2 viruses. The neuraminidase (NA) and PB1 genes of the H1N2 isolates were of human origin, while the hemagglutinin (HA), nucleoprotein (NP), matrix (M), non-structural (NS), PA and PB2 polymerase genes were of avian or swine origin. Fifteen of the 24 H1N2 isolates were shown to have a close phylogenic relationship and high amino acid homology with the first US isolate of H1N2 (A/SW/IN/9K035/99). The remaining nine isolates had a close phylogenic relationship with classical swine influenza H1N1 in the HA gene. All other genes including NA, M, NP, NS, PA, PB1 and PB2 showed a close phylogenic relationship with the H1N2 (A/SW/IN/9K035/99) strain and triple reassortant H3N2 viruses. However, PB1 genes of two isolates (A/SW/KS/13481-S/00, A/SW/KS/13481-T/00) were originated from avian influenza A virus lineage. These results suggest that although there are some variations in the HA genes, the H1N2 viruses prevalent in the US swine population are of a similar genetic lineage.

Detection and subtyping of swine influenza H1N1, H1N2 and H3N2 viruses in clinical samples using two multiplex RT-PCR assays

A total of 360 type A swine influenza virus-positive samples including cell culture isolates, nasal swabs or lung tissues along with 30 virus-negative samples were tested for the detection and subtyping of H1N1, H1N2 or H3N2 by two multiplex reverse transcription (RT)-PCR assays. The positive samples had been collected between 1999 and 2001 from pigs with respiratory diseases, and type A influenza virus was isolated and subtyped by hemagglutination inhibition (HI) test at the Minnesota Veterinary Diagnostic Laboratory (MVDL). Two multiplex RT-PCR assays specific for H1 and H3, and N1 and N2 were developed. RT-PCR products with unique sizes characteristic of each subtype of influenza A virus were sequenced, and the sequences were demonstrated to be specific for H1N1, H1N2 or H3N2. Genomic RNAs or DNAs from 12 common swine pathogens other than type A influenza viruses were not amplified when the PCR assays were performed with these primer sets. Positive amplification reaction could be visualized with RNA extracted from up to 10 −5 dilution of each reference virus with original infectivity titer of 10 5 TCID 50/ml. Of the 360 samples tested, swine influenza virus H1N1, H1N2 and H3N2 were identified in 200, 13 and 139 samples, respectively. The remaining eight samples were positive for both H1N1 and H3N2 viruses. The results of multiplex RT-PCR were 100% in agreement with those of virus isolation. These results demonstrate the usefulness of multiplex RT-PCR for detection and identification of influenza A virus subtypes. The results also indicate an increased occurrence of H1N2 in US swine population.

The emergence of novel swine influenza viruses in North America

Since 1997, novel viruses of three different subtypes and five different genotypes have emerged as agents of influenza among pigs in North America. The appearance of these viruses is remarkable because there were no substantial changes in the overall epidemiology of swine influenza in the United States and Canada for over 60 years prior to this time. Viruses of the classical H1N1 lineage were virtually the exclusive cause of swine influenza from the time of their initial isolation in 1930 through 1998. Antigenic drift variants of these H1N1 viruses were isolated in 1991–1998, but a much more dramatic antigenic shift occurred with the emergence of H3N2 viruses in 1997–1998. In particular, H3N2 viruses with genes derived from human, swine and avian viruses have become a major cause of swine influenza in North America. In addition, H1N2 viruses that resulted from reassortment between the triple reassortant H3N2 viruses and classical H1N1 swine viruses have been isolated subsequently from pigs in at least six states. Finally, avian H4N6 viruses crossed the species barrier to infect pigs in Canada in 1999. Fortunately, these H4N6 viruses have not been isolated beyond their initial farm of origin. If these viruses spread more widely, they will represent another antigenic shift for our swine population, and could pose a threat to the world's human population. Research on these novel viruses may offer important clues to the genetic basis for interspecies transmission of influenza viruses.

Isolation from turkey breeder hens of a reassortant H1N2 influenza virus with swine, human, and avian lineage genes.

Type A influenza viruses can infect a wide range of birds and mammals, but influenza in a particular species is usually considered to be species specific. However, infection of turkeys with swine H1N1 viruses has been documented on several occasions. This report documents the isolation of an H1N2 influenza virus from a turkey breeder flock with a sudden drop in egg production. Sequence analysis of the virus showed that it was a complex reassortant virus with a mix of swine-, human-, and avian-origin influenza genes. A swine influenza virus with a similar gene complement was recently reported from pigs in Indiana. Isolation and identification of the virus required the use of nonconventional diagnostic procedures. The virus was isolated in embryonated chicken eggs by the yolk sac route of inoculation rather than by the typical chorioallantoic sac route. Interpretation of hemagglutination-inhibition test results required the use of turkey rather than chicken red blood cells, and identification of the neuraminidase subtype required the use of alternative reference sera in the neuraminidase-inhibition test. This report provides additional evidence that influenza viruses can cross species and cause a disease outbreak, and diagnosticians must be aware that the variability of influenza viruses can complicate the isolation and characterization of new isolates.

Evolving H3N2 and emerging H1N2 swine influenza viruses in the United States

Background: Reassortant H3N2 influenza viruses have recently become established in swine populations throughout the United States. Viruses of two genotypes (a human and swine virus reassortant and a human, swine, and avian virus reassortant) were isolated from swine during the index disease outbreaks. Methods: To identify the genetic composition of the currently circulating viruses, we sequenced the genes of seven isolates from different infected herds. Results: Six viruses contained the human/swine/avian gene complex, but the complex was associated with three phylogenetically distinct human H3 haemagglutinins (HA). The remaining virus was an H1N2 reassortant containing seven genes similar to those of swine H3N2 viruses and an HA gene derived from a classical-swine H1N1 virus. The non-HA genes of this H1N2 virus were more similar to those of swine H3N2 viruses than to those of the recently reported H1N2 swine virus from Indiana [J. Clin. Microbiol. 38 (2000) 2453]. This finding suggests that each of the H1N2 viruses were derived through independent reassortment events. Conclusions: We conclude that viruses containing the avian-like genes are primarily responsible for the increased prevalence of H3N2 viruses throughout U.S. swine populations. H3N2 viruses have continued to spread and have undergone further reassortment with human and swine viruses resulting in the emergence of viruses with distinct antigenicity and subtype.

Antigenic and molecular heterogeneity in recent swine influenza A(H1N1) virus isolates with possible implications for vaccination policy

In order to explore the occurrence of antigenic drift in swine influenza A(H1N1) viruses and the match between epidemic and vaccine strains, 26 virus isolates from outbreaks of respiratory disease among finishing pigs in the Netherlands in the 1995/1996 season and reference strains from earlier outbreaks were examined using serological and molecular methods. In contrast to swine H3N2 viruses, no significant antigenic drift was observed in swine H1N1 viruses isolated from the late 1980s up to 1996 inclusive. However, a marked antigenic and genetic heterogeneity in haemagglutination inhibition tests and nucleotide sequence analyses was detected among the 26 recent swine H1N1 virus strains. Interestingly, the observed antigenic and molecular variants were not randomly distributed over the farms. This finding indicates independent introductions of different swine H1N1 virus variants at the various farms of the study and points to a marked difference between the epidemiologies of human and swine influenza viruses. The observed heterogeneity may hamper the control of swine influenza by vaccination and indicates that the efficacy of current swine influenza vaccines requires re-evaluation and that the antigenic reactivity of swine influenza viruses should be monitored on a regular basis.

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.

Virologic and serologic surveillance for human, swine and avian influenza virus infections among pigs in the north-central United States.

Summary. Influenza virus infection in pigs is both an animal health problem and a public health concern. As such, surveillance and characterization of influenza viruses in swine is important to the veterinary community and should be a part of human pandemic preparedness planning. Studies in 1976/1977 and 1988/1989 demonstrated that pigs in the U.S. were commonly infected with classical swine H1N1 viruses, whereas human H3 and avian influenza virus infections were very rare. In contrast, human H3 and avian H1 viruses have been isolated frequently from pigs in Europe and Asia over the last two decades. From September 1997 through August 1998, we isolated 26 influenza viruses from pigs in the north-central United States at the point of slaughter. All 26 isolates were H1N1 viruses, and phylogenetic analyses of the hemagglutinin and nucleoprotein genes from 11 representative viruses demonstrated that these were classical swine H1 viruses. However, monoclonal antibody analyses revealed antigenic heterogeneity among the HA proteins of the 26 viruses. Serologically, 27.7% of 2,375 pigs tested had hemagglutination-inhibiting antibodies against classical swine H1 influenza virus. Of particular significance, however, the rates of seropositivity to avian H1 (7.6%) and human H3 (8.0%) viruses were substantially higher than in previous studies.

Antigenic drift in swine influenza H3 haemagglutinins with implications for vaccination policy

In order to explore the occurrence of antigenic drift in swine influenza A(H3N2) virus, we examined virus strains from outbreaks of respiratory disease among finishing pigs in the Netherlands in 1996 and 1997 and from earlier outbreaks. In contrast to swine H3N2 strains from the 1980s, the recent isolates did not show significant cross-reactivity with human influenza A(H3N2) viruses from 1972–1975 in haemagglutination inhibition tests. These new strains form a separate branch in the phylogenetic tree of the HA1 parts of HA. We conclude that recently there has been considerable antigenic drift within the swine H3N2 viruses in the Netherlands and Belgium and recommend replacement of the A/Port Chalmers/1/73 (H3N2) strain in the current vaccine by a more recent swine H3N2 isolate.

Multiple genetic reassortment of avian and human influenza A viruses in European pigs, resulting in the emergence of an H1N2 virus of novel genotype.

Novel H1N2 influenza A viruses which were first detected in pigs in Great Britain in 1994 were examined antigenically and genetically to determine their origins and establish the potential mechanisms for genetic reassortment. The haemagglutinin (HA) of all swine H 1 N2 viruses examined was most closely related to, but clearly distinguishable both antigenically and genetically from, the HA of human H1N1 viruses which circulated in the human population during the early 1 980s. Phylogenetic analysis of the HA gene revealed that the swine H 1 N2 viruses formed a distinct branch on the human lineage and were probably introduced to pigs shortly after 1980. Following apparent transfer to pigs the HA gene underwent genetic variation resulting in the establishment and cocirculation of genetically and antigenically heterogeneous virus populations. Genetic analyses of the other RNA segments of all swine H1N2 viruses indicated that the neuraminidase gene was most closely related to those of early 'human-like' swine H3N2 viruses, whilst the RNA segments encoding PB2, PB1, PA, NP, M and NS were related most closely to those of avian viruses, which have been circulating recently in pigs in Northern Europe. The potential mechanisms and probable progenitor strains for genetic reassortment are discussed, but we propose that the swine H1N2 viruses examined originated following multiple genetic reassortment, initially involving human H1N1 and 'human-like' swine H3N2 viruses, followed by reassortment with 'avian-like' swine H1N1 virus. These findings suggest multiple reassortment and replication of influenza viruses may occur in pigs many years before their detection as clinical entities.

Continued Evolution of H1N1 and H3N2 Influenza Viruses in Pigs in Italy

Swine influenza viruses possessing avian genes were first detected in Europe in 1979 (Scholtisseket al.,1983,Virology,129, 521–523) and continue to circulate in pigs in that region of the world. To characterize the molecular epidemiology of swine influenza viruses currently circulating in Europe, we used dot-blot hybridization and sequence analysis to determine the origin of the genes encoding the nonsurface proteins (“internal” genes) of 10 H1N1 and 11 H3N2 swine influenza viruses isolated in Italy between 1992 and 1995. All of the 126 genes examined were of avian origin; thus the currently circulating H3N2 strains which possess A/Port Chalmers/1/73-like surface glycoproteins appear to be descendants of the reassortant human–avian viruses that emerged between 1983 and 1985 in Italy. Sequence analysis of matrix (M), nonstructural, and nucleoprotein genes, as well as phylogenetic analysis of M gene showed that the H1N1 and H3N2 viruses from the pigs were closely related to recent isolates of the avian-like swine H1N1 influenza strain currently circulating in northern Europe and were distinguishable from the genes of viruses isolated from European swine in 1979. To evaluate the frequency of transmission of swine H1N1 and H3N2 viruses to man, we tested 123 human sera for hemagglutination-inhibiting antibodies against avian and mammalian H1N1 and H3N2 virus strains. Our findings indicate that swine influenza viruses possessing A/Port Chalmers/1/73-like hemagglutinin may have transmitted to approximately 20% of young persons under 20 years of age who had contact with pigs. Thus, H3N2 swine viruses, possibly possessing avian-derived internal genes, may be entering humans more often than was previously thought. We strongly recommend that pigs be regularly monitored as a potential early warning system for detection of future pandemic strains.

Large outbreak of swine influenza in southern Japan caused by reassortant (H1N2) influenza viruses: its epizootic background and characterization of the causative viruses.

In the winter of 1989 and the spring of 1990, there were large outbreaks of respiratory disease in two swine herds in Nagasaki Prefecture, southern Japan. Serological surveillance indicated that the majority of swine possessed antibodies to swine influenza virus H1 haemagglutinin and neuraminidase of early H3N2 influenza virus strains. Eight viruses were isolated from swine that showed typical clinical symptoms of influenza. The haemagglutinin and neuraminidase of these isolates were closely related to those of swine H1N1 and early human H3N2 viruses, respectively. At least two types of haemagglutinin antigens, distinguished by two monoclonal antibodies, were involved in the outbreaks. Evolutionary analyses indicated that the haemagglutinin gene of the H1N2 reassortants was closely related to those of a recent swine lineage (A/sw/HK/1/74 and A/sw/Ehime/1/80 viruses). However, the neuraminidase genes of the H1N2 reassortants were similar to those of swine N2 viruses which in turn are related to early human H3N2 viruses. A comparison of partial nucleotide sequences revealed that the six other genes of A/sw/Nagasaki/1/89 were derived from those of swine H1N1 virus.

Antigenic and genetic characteristics of H1N1 human influenza virus isolated from pigs in Japan

Two strains of influenza A virus were isolated from pigs in northern Japan in 1992. Serological tests showed that the haemagglutinin (HA) and neuraminidase (NA) antigens were more closely related to those of recent human H1N1 viruses than to those of swine H1N1 viruses. The HA and NA genes of isolate A/sw/Obihiro/5/92 were shown to be closely related to those of current human H1N1 viruses. Evolutionary trees constructed from nucleotide sequences showed that the HA and NA genes of A/sw/Obihiro/5/92 were apparently on a branch cluster containing human strains isolated between 1990 and 1992.

Genetic relatedness of the nucleoprotein (NP) of recent swine, turkey, and human influenza Avirus(H1N1) isolates

The sequences of nucleoprotein (NP) genes of recent human and turkey isolates of influenza A viruses, which serologically could be correlated to contemporary swine viruses, were determined. These sequences were closely related to the NPs of these swine viruses and they formed a separate branch on the phylogenetic tree. While the early swine virus from 1931 resembled the avian strains in consensus amino acids of the NP and in its ability to rescue NP ts mutants of fowl plague virus in chicken embryo cells, the later strains on that branch were different: at 15 positions they have their own amino acids and they rescued the NP ts mutants only poorly. Of the NPs of the human New Jersey/76 isolates analysed, one clustered with the recent H1N1 swine viruses of the U.S.A., the other one with contemporary human strains. Since the NP is one of the main determinants of species specificity it is concluded that, although the H1N1 swine isolates from the U.S.A. form their own branch in the phylogenetic tree, they can be transmitted to humans and turkeys, but they do not spread further in these populations and so far have not contributed to human pandemics. It is not very likely that they will do so in future, since its branch in the phylogenetic tree develops further away from the human and avian branch.

Evolution of influenza A virus nucleoprotein genes: implications for the origins of H1N1 human and classical swine viruses

A phylogenetic analysis of 52 published and 37 new nucleoprotein (NP) gene sequences addressed the evolution and origin of human and swine influenza A viruses. H1N1 human and classical swine viruses (i.e., those related to Swine/Iowa/15/30) share a single common ancestor, which was estimated to have occurred in 1912 to 1913. From this common ancestor, human and classical swine virus NP genes have evolved at similar rates that are higher than in avian virus NP genes (3.31 to 3.41 versus 1.90 nucleotide changes per year). At the protein level, human virus NPs have evolved twice as fast as classical swine virus NPs (0.66 versus 0.34 amino acid change per year). Despite evidence of frequent interspecies transmission of human and classical swine viruses, our analysis indicates that these viruses have evolved independently since well before the first isolates in the early 1930s. Although our analysis cannot reveal the original host, the ancestor virus was avianlike, showing only five amino acid differences from the root of the avian virus NP lineage. The common pattern of relationship and origin for the NP and other genes of H1N1 human and classical swine viruses suggests that the common ancestor was an avian virus and not a reassortant derived from previous human or swine influenza A viruses. The new avianlike H1N1 swine viruses in Europe may provide a model for the evolution of newly introduced avian viruses into the swine host reservoir. The NPs of these viruses are evolving more rapidly than those of human or classical swine viruses (4.50 nucleotide changes and 0.74 amino acid change per year), and when these rates are applied to pre-1930s human and classical swine virus NPs, the predicted date of a common ancestor is 1918 rather than 1912 to 1913. Thus, our NP phylogeny is consistent with historical records and the proposal that a short time before 1918, a new H1N1 avianlike virus entered human or swine hosts (O. T. Gorman, R. O. Donis, Y. Kawaoka, and R. G. Webster, J. Virol. 64:4893-4902, 1990). This virus provided the ancestors of all known human influenza A virus genes, except for HA, NA, and PB1, which have since been reassorted from avian viruses. We propose that during 1918 a virulent strain of this new avianlike virus caused a severe human influenza pandemic and that the pandemic virus was introduced into North American swine populations, constituting the origin of classical swine virus.

Evolution of influenza A virus PB2 genes: implications for evolution of the ribonucleoprotein complex and origin of human influenza A virus

Phylogenetic analysis of 20 influenza A virus PB2 genes showed that PB2 genes have evolved into the following four major lineages: (i) equine/Prague/56 (EQPR56); (ii and iii) two distinct avian PB2 lineages, one containing FPV/34 and H13 gull virus strains and the other containing North American avian and recent equine strains; and (iv) human virus strains joined with classic swine virus strains (i.e., H1N1 swine virus strains related to swine/Iowa/15/30). The human virus lineage showed the greatest divergence from its root relative to other lineages. The estimated nucleotide evolutionary rate for the human PB2 lineage was 1.82 x 10(-3) changes per nucleotide per year, which is within the range of published estimates for NP and NS genes of human influenza A viruses. At the amino acid level, PB2s of human viruses have accumulated 34 amino acid changes over the past 55 years. In contrast, the avian PB2 lineages showed much less evolution, e.g., recent avian PB2s showed as few as three amino acid changes relative to the avian root. The completion of evolutionary analyses of the PB1, PB2, PA and NP genes of the ribonucleoprotein (RNP) complex permits comparison of evolutionary pathways. Different patterns of evolution among the RNP genes indicate that the genes of the complex are not coevolving as a unit. Evolution of the PB1 and PB2 genes is less correlated with host-specific factors, and their proteins appear to be evolving more slowly than NP and PA. This suggests that protein functional constraints are limiting the evolutionary divergence of PB1 and PB2 genes. The parallel host-specific evolutionary pathways of the NP and PA genes suggest that these proteins are coevolving in response to host-specific factors. PB2s of human influenza A viruses share a common ancestor with classic swine virus PB2s, and the pattern of evolution suggests that the ancestor was an avian virus PB2. This same pattern of evolution appears in the other genes of the RNP complex. Antigenic studies of HA and NA proteins and sequence comparisons of NS and M genes also suggest a close ancestry for these genes in human and classic swine viruses. From our review of the evolutionary patterns of influenza A virus genes, we propose the following hypothesis: the common ancestor to current strains of human and classic swine influenza viruses predated the 1918 human pandemic virus and was recently derived from the avian host reservoir.