Classical Swine H1N1 Viruses Research Articles

Antigenically Diverse Swine Origin H1N1 Variant Influenza Viruses Exhibit Differential Ferret Pathogenesis and Transmission Phenotypes.

Influenza A(H1) viruses circulating in swine represent an emerging virus threat, as zoonotic infections occur sporadically following exposure to swine. A fatal infection caused by an H1N1 variant (H1N1v) virus was detected in a patient with reported exposure to swine and who presented with pneumonia, respiratory failure, and cardiac arrest. To understand the genetic and phenotypic characteristics of the virus, genome sequence analysis, antigenic characterization, and ferret pathogenesis and transmissibility experiments were performed. Antigenic analysis of the virus isolated from the fatal case, A/Ohio/09/2015, demonstrated significant antigenic drift away from the classical swine H1N1 variant viruses and H1N1 pandemic 2009 viruses. A substitution in the H1 hemagglutinin (G155E) was identified that likely impacted antigenicity, and reverse genetics was employed to understand the molecular mechanism of antibody escape. Reversion of the substitution to 155G, in a reverse genetics A/Ohio/09/2015 virus, showed that this residue was central to the loss of hemagglutination inhibition by ferret antisera raised against a prototypical H1N1 pandemic 2009 virus (A/California/07/2009), as well as gamma lineage classical swine H1N1 viruses, demonstrating the importance of this residue for antibody recognition of this H1 lineage. When analyzed in the ferret model, A/Ohio/09/2015 and another H1N1v virus, A/Iowa/39/2015, as well as A/California/07/2009, replicated efficiently in the respiratory tract of ferrets. The two H1N1v viruses transmitted efficiently among cohoused ferrets, but respiratory droplet transmission studies showed that A/California/07/2009 transmitted through the air more efficiently. Preexisting immunity to A/California/07/2009 did not fully protect ferrets from challenge with A/Ohio/09/2015.IMPORTANCE Human infections with classical swine influenza A(H1N1) viruses that circulate in pigs continue to occur in the United States following exposure to swine. To understand the genetic and virologic characteristics of a virus (A/Ohio/09/2015) associated with a fatal infection and a virus associated with a nonfatal infection (A/Iowa/39/2015), we performed genome sequence analysis, antigenic testing, and pathogenicity and transmission studies in a ferret model. Reverse genetics was employed to identify a single antigenic site substitution (HA G155E) responsible for antigenic variation of A/Ohio/09/2015 compared to related classical swine influenza A(H1N1) viruses. Ferrets with preexisting immunity to the pandemic A(H1N1) virus were challenged with A/Ohio/09/2015, demonstrating decreased protection. These data illustrate the potential for currently circulating swine influenza viruses to infect and cause illness in humans with preexisting immunity to H1N1 pandemic 2009 viruses and a need for ongoing risk assessment and development of candidate vaccine viruses for improved pandemic preparedness.

Influenza A virus infection in Japanese wild boars (Sus scrofa leucomystax).

Serum samples were collected from 385 wild boars between 2010 and 2013 to examine the seroprevalence of influenza A virus (IAV) in Japan. Antibodies against IAV were identified using a commercial kit in 13 wild boars (3.4%). To identify the serotypes, positive sera were examined by virus-neutralization test using representative serotypes and strains. Three wild boars in Yamaguchi and four in Tochigi showed the highest antibody titers against the pandemic H1N1 2009 virus and classical swine H1N1 virus strains, respectively. These data indicate that wild boars may have close contact with humans and domestic pigs and therefore that there is potential for IAVs to reassort in wild boars as they have been shown to do in pigs.

Spillback transmission of European H1N1 avian-like swine influenza viruses to turkeys: A strain-dependent possibility?

In 1979, an avian influenza virus of the H1N1 subtype began to circulate in European swine herds, rapidly replacing classical swine H1N1 viruses. Spill-back transmissions to turkeys were recorded occasionally, but they might have been underreported due to the asymptomatic nature of the infection and the lack of specific surveillance. In our study, we evaluated the infectivity and transmissibility in turkeys of seven strains of H1N1 avian-like swine viruses isolated from 1979 to 2006, and compared them with their closest progenitor A/duck/Bavaria/1/77 (H1N1), to establish whether the adaptation to pigs has gradually decreased their fitness in turkeys. Our data indicate that the circulation of European H1N1 in pigs might have impaired the possibility of infecting turkeys. Nevertheless, the two swine-origin strains, which showed the ability to replicate and transmit in turkeys, possess typical swine-like genetic traits, not different from the rest of the tested isolates, suggesting replication of avian-like swine H1N1 viruses in turkeys as a strain-dependent polygenic feature.

Identification of a linear epitope on the haemagglutinin protein of pandemic A/H1N1 2009 influenza virus using monoclonal antibodies

A novel influenza A/H1N1 virus, emerging from Mexico and the United States in the spring of 2009, caused the pandemic human infection of 2009-2010. The haemagglutinin (HA) glycoprotein is the major surface antigen of influenza A virus and plays an important role in viral infection. In this study, three hybridoma cell lines secreting specific monoclonal antibodies (Mabs) against the HA protein of pandemic influenza A/H1N1 2009 virus were generated with the recombinant plasmid pCAGGS-HA as an immunogen. Using Pepscan analysis, the binding sites of these Mabs were identified in a linear region of the HA protein. Further, refined mapping was conducted using truncated peptides expressed as GST-fusion proteins in E. coli. We found that the (250)VPRYA(254) motif was the minimal determinant of the linear epitope that could be recognized by the Mabs. Alignment with sequences from the databases showed that the amino acid residues of this epitope were highly conserved among all pandemic A/H1N1 2009 viruses as well as the classical swine H1N1 viruses isolated to date. These results provide additional insights into the antigenic structure of the HA protein and virus-antibody interactions at the amino acid level, which may assist in the development of specific diagnostic methods for influenza viruses.

Circulation of classical swine influenza virus in Europe between the wars?

The complete genomes of two swine influenza viruses from England were sequenced. Phylogenetic analysis revealed classical swine H1N1 viruses, one of which, A/swine/London, is closely related to virus strains of the early 1930s. Both strains are also antigenically related to A/swine/Iowa/15/1930, the strain originally isolated by Richard Shope. The source of A/swine/London is unknown, but its relationship to early classical swine influenza viruses suggests that the emergence of these viruses in Europe has to be antedated by 15-20 years.

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.

Co-circulation of pandemic 2009 H1N1, classical swine H1N1 and avian-like swine H1N1 influenza viruses in pigs in China

The pandemic A/H1N1 influenza viruses emerged in both Mexico and the United States in March 2009, and were transmitted efficiently in the human population. They were transmitted occasionally from humans to other mammals including pigs, dogs and cats. In this study, we report the isolation and genetic analysis of novel viruses in pigs in China. These viruses were related phylogenetically to the pandemic 2009 H1N1 influenza viruses isolated from humans and pigs, which indicates that the pandemic virus is currently circulating in swine populations, and this hypothesis was further supported by serological surveillance of pig sera collected within the same period. Furthermore, we isolated another two H1N1 viruses belonging to the lineages of classical swine H1N1 virus and avian-like swine H1N1 virus, respectively. Multiple genetic lineages of H1N1 viruses are co-circulating in the swine population, which highlights the importance of intensive surveillance for swine influenza in China.

Pandemic 2009 H1N1 vaccine protects against 1918 Spanish influenza virus

The 1918 influenza A virus caused the most devastating pandemic, killing approximately 50 million people worldwide. Immunization with 1918-like and classical swine H1N1 virus vaccines results in cross-protective antibodies against the 2009 H1N1 pandemic influenza, indicating antigenic similarities among these viruses. In this study, we demonstrate that vaccination with the 2009 pandemic H1N1 vaccine elicits 1918 virus cross-protective antibodies in mice and humans, and that vaccination or passive transfer of human-positive sera reduced morbidity and conferred full protection from lethal challenge with the 1918 virus in mice. The spread of the 2009 H1N1 influenza virus in the population worldwide, in addition to the large number of individuals already vaccinated, suggests that a large proportion of the population now have cross-protective antibodies against the 1918 virus, greatly alleviating concerns and fears regarding the accidental exposure/release of the 1918 virus from the laboratory and the use of the virus as a bioterrorist agent.

Swine Influenza A Vaccines, Pandemic (H1N1) 2009 Virus, and Cross-Reactivity

To the Editor: Since its first emergence in the human population in spring 2009 (1–3) infections with pandemic (H1N1) 2009 virus have been reported in pigs, turkeys, and some carnivore species (4,5). The pandemic (H1N1) 2009 virus can be experimentally transmitted between pigs (6). The reported transmissibility of the virus raises the question as to whether authorized swine influenza vaccine strains may be cross-reactive to pandemic (H1N1) 2009 virus. Kyriakis et al. (7) investigated the cross-reactivity of 66 pig serum samples from different infection and vaccination trials and reported cross-reactions between the avian-like H1N1 viruses circulating in the European pig population (avH1N1) and the classical swine H1N1 viruses (cH1N1) with pandemic (H1N1) 2009 virus by hemagglutination inhibition assay. To investigate this cross-reactivity in more detail, a neutralization test was applied in the study we report here. A serial dilution of serum samples was prepared (log4). All virus strains were adjusted to 100 fifty-percent tissue culture infectious doses. This working dilution of virus was mixed with serum dilutions and incubated 1 hour at 37°C. Madin-Darby bovine kidney monolayers were infected with the neutralization mixtures. After 48 hours of incubation, cells were fixed with acetone (4°C–8°C) and investigated by indirect immunofluorescent assay. Finally, the 50% neutralization titer was calculated. Hyperimmune serum samples were established by using a 4-fold vaccination of pigs with antigens of H1N1 vaccine strains (A/New Jersey/8/1976, A/sw/Netherlands/25/1980, A/sw/IDT/Re230/1992, A/sw/Haselunne/IDT2617/2003), and a strain of pandemic (H1N1) 2009 virus (A/Hamburg/7/2009) by using Freund adjuvant. Blood samples were taken 14 days after last immunization. A vaccine containing the pandemic (H1N1) 2009 virus was produced. Swine influenza vaccines available in central Europe and the newly produced vaccine containing pandemic (H1N1) 2009 virus (A/Hamburg/7/2009) were administered to pigs (2-fold vaccination with 1–2 mL of the vaccine 21–28 days apart intramuscularly). Blood was withdrawn 7 days after second administration. In addition, an experimental aerosol infection was conducted by using the parental strain of the most recent avH1N1 strain contained in a European swine influenza vaccine (A/sw/Haselunne/IDT2617/2003). Blood samples were taken 10 days after infection. The investigation of the hyperimmune serum samples detected neutralizing activity between the pandemic (H1N1) 2009 virus and European avH1N1 vaccine strains (A/sw/Netherlands/25/1980, A/sw/IDT/Re230/1992, A/sw/Haselunne/IDT2617/2003), as well as with the cH1N1 strain A/New Jersey/8/1976 (Fort Dix reassortant). The hyperimmune serum established against pandemic (H1N1) 2009 virus also showed cross-reactivity with European avH1N1 virus. The reactions against several strains of the pandemic virus were similar, reflecting high titers against pandemic (H1N1) 2009 virus but also cross-reactions with hyperimmune serum samples of all swine influenza A virus H1N1 vaccine strains (Appendix Table). The bivalent vaccines induced high titers of neutralizing antibodies against avH1N1 virus and human-like H3N2 virus (huH3N2). Only a low number of pigs reacted with H1N2 virus whereas the trivalent vaccine induced high neutralizing activity in serum samples of all vaccinated pigs. The vaccines induced neutralizing antibodies against pandemic (H1N1) 2009 virus. The titers were lower in comparison to those obtained for avH1N1 and not all pigs responded. The reactions were best for the vaccines containing mineral oil. Pigs vaccinated with the trivalent vaccine with carbomer adjuvant showed almost no antibodies against pandemic (H1N1) 2009 virus, although the vaccine strain reacted well in hyperimmunization tests. A vaccine batch of the trivalent vaccine was produced that contained mineral oil instead of carbomer. All pigs vaccinated with the trivalent vaccine with mineral oil had antibodies against the pandemic (H1N1) 2009 virus (data not shown). At the same time, efficacy trials with all authorized vaccines were conducted (8; T.W. Vahlenkamp, pers. comm.) in which all vaccines including the trivalent vaccine with carbomer adjuvant showed a comparable level of protection (limited period of viral shedding). Mineral oil adjuvants can induce severe distress in pig herds due to their limited safety. Despite cross-reactivity between avH1N1 and cH1N1 with pandemic (H1N1) 2009 virus, the highest degree of cross-neutralization was achieved by the vaccine containing pandemic (H1N1) virus strain. Proof of cross-reactivity was also reflected in the infection trial. Pigs infected with avH1N1 responded to avH1N1 as well as to pandemic (H1N1) 2009 virus. All results were additionally confirmed by hemagglutination inhibition assay (data not shown). Furthermore, 1,559 pig serum samples from 195 German pig herds collected from mid-June through mid-September 2009 were tested in routine diagnostics by hemagglutination inhibition assay. All reflected almost similar results for avH1N1 and the pandemic (H1N1) 2009 virus (seroprevalences for individual pigs were pandemic [H1N1] 2009 virus 52%, avH1N1 53%, huH1N2 28%, and huH3N2 52%; for pig herds pandemic [H1N1] 2009 virus 46%, avH1N1 46%, huH1N2 24%, and huH3N2 45%). These results suggest cross-reactivity between porcine H1N1 viruses and pandemic (H1N1) 2009 virus. Despite this cross-reactivity, a vaccine consisting of pandemic (H1N1) 2009 virus is superior in terms of efficacy in comparison with vaccines already authorized in Europe.

Prior infection with classical swine H1N1 influenza viruses is associated with protective immunity to the 2009 pandemic H1N1 virus.

Please cite this paper as: Kash et al. (2010) Prior infection with classical swine H1N1 influenza viruses is associated with protective immunity to the 2009 pandemic H1N1 virus. Influenza and Other Respiratory Viruses 4(3), 121–127. Background The 2009 H1N1 pandemic emerged even though seasonal H1N1 viruses have circulated for decades. Epidemio‐logical evidence suggested that the current seasonal vaccine did not offer significant protection from the novel pandemic, and that people over the age of 50 might were less susceptible to infection. Objectives In a mouse challenge study with the 2009 pandemic H1N1 virus, we evaluated protective immune responses elicited by prior infection with human and swine influenza A viruses. Results Mice infected with A/Mexico/4108/2009 (Mex09) showed significant weight loss and 40% mortality. Prior infection with a 1976 classical swine H1N1 virus resulted in complete protection from Mex09 challenge. Prior infection with either a 2009 or a 1940 seasonal H1N1 influenza virus provided partial protection and a >100‐fold reduction in viral lung titers at day 4 post‐infection. Conclusions These findings indicate that in experimental animals recently induced immunity to 1918‐derived H1N1 seasonal influenza viruses, and to a 1976 swine influenza virus, afford a degree of protection against the 2009 pandemic virus. Implications of these findings are discussed in the context of accumulating data suggesting partial protection of older persons during the 2009 pandemic.

Protection of Mice against Lethal Challenge with 2009 H1N1 Influenza A Virus by 1918-Like and Classical Swine H1N1 Based Vaccines

The recent 2009 pandemic H1N1 virus infection in humans has resulted in nearly 5,000 deaths worldwide. Early epidemiological findings indicated a low level of infection in the older population (>65 years) with the pandemic virus, and a greater susceptibility in people younger than 35 years of age, a phenomenon correlated with the presence of cross-reactive immunity in the older population. It is unclear what virus(es) might be responsible for this apparent cross-protection against the 2009 pandemic H1N1 virus. We describe a mouse lethal challenge model for the 2009 pandemic H1N1 strain, used together with a panel of inactivated H1N1 virus vaccines and hemagglutinin (HA) monoclonal antibodies to dissect the possible humoral antigenic determinants of pre-existing immunity against this virus in the human population. By hemagglutinination inhibition (HI) assays and vaccination/challenge studies, we demonstrate that the 2009 pandemic H1N1 virus is antigenically similar to human H1N1 viruses that circulated from 1918–1943 and to classical swine H1N1 viruses. Antibodies elicited against 1918-like or classical swine H1N1 vaccines completely protect C57B/6 mice from lethal challenge with the influenza A/Netherlands/602/2009 virus isolate. In contrast, contemporary H1N1 vaccines afforded only partial protection. Passive immunization with cross-reactive monoclonal antibodies (mAbs) raised against either 1918 or A/California/04/2009 HA proteins offered full protection from death. Analysis of mAb antibody escape mutants, generated by selection of 2009 H1N1 virus with these mAbs, indicate that antigenic site Sa is one of the conserved cross-protective epitopes. Our findings in mice agree with serological data showing high prevalence of 2009 H1N1 cross-reactive antibodies only in the older population, indicating that prior infection with 1918-like viruses or vaccination against the 1976 swine H1N1 virus in the USA are likely to provide protection against the 2009 pandemic H1N1 virus. This data provides a mechanistic basis for the protection seen in the older population, and emphasizes a rationale for including vaccination of the younger, naïve population. Our results also support the notion that pigs can act as an animal reservoir where influenza virus HAs become antigenically frozen for long periods of time, facilitating the generation of human pandemic viruses.

Genetic characterization of H1N1 swine influenza A viruses isolated in eastern China

Three influenza H1N1 viruses were isolated in 2005 from pigs with respiratory disease on a farm in eastern China. The three isolates were characterized to determine their probable origin. Each of the eight genes of the isolates was most closely related to the corresponding gene from the classical swine H1N1 virus. Also, phylogenetic analysis further confirmed that each of the eight genes of the isolates was closely related to the classical swine H1N1 viruses, especially those isolated in China. The HA1 proteins of the three isolates were identical to that of A/Swine/Guangdong/ 1/01, a virus isolated in 2001 in China, even though three nucleotide differences were observed. These results further support the concept that swine can serve as a reservoir of genetically stable influenza viruses.

Different Evolutionary Trajectories of European Avian-Like and Classical Swine H1N1 Influenza A Viruses

In 1979, a lineage of avian-like H1N1 influenza A viruses emerged in European swine populations independently from the classical swine H1N1 virus lineage that had circulated in pigs since the Spanish influenza pandemic of 1918. To determine whether these two distinct lineages of swine-adapted A/H1N1 viruses evolved from avian-like A/H1N1 ancestors in similar ways, as might be expected given their common host species and origin, we compared patterns of nucleotide and amino acid change in whole genome sequences of both groups. An analysis of nucleotide compositional bias across all eight genomic segments for the two swine lineages showed a clear lineage-specific bias, although a segment-specific effect was also apparent. As such, there appears to be only a relatively weak host-specific selection pressure. Strikingly, despite each lineage evolving in the same species of host for decades, amino acid analysis revealed little evidence of either parallel or convergent changes. These findings suggest that although adaptation due to evolutionary lineages can be distinguished, there are functional and structural constraints on all gene segments and that the evolutionary trajectory of each lineage of swine A/H1N1 virus has a strong historical contingency. Thus, in the context of emergence of an influenza A virus strain via a host switch event, it is difficult to predict what specific polygenic changes are needed for mammalian adaptation.

Genetic characterization of novel reassortant H1N2 influenza A viruses isolated from pigs in southeastern China

Summary.In December 2004, three influenza H1N2 viruses were isolated from lung samples of pigs that had died from respiratory disease on a farm in southeastern China. To determine the genetic characterization and probable origin, one of the three isolates, A/Swine/Zhejiang/1/2004 (Sw/ZJ/1/2004), was genetically analyzed. Sw/ZJ/1/2004 was a reassortant with an NA gene most closely related to the corresponding gene from a human-like H3N2 virus circulating in 1995. The remaining seven genes were most closely related to those from the classical swine H1N1 virus. Sw/ZJ/1/2004 appeared to be a novel reassortant H1N2 virus that was genetically distinguishable from other H1N2 viruses found in pigs worldwide. The isolation of Sw/ZJ/1/2004 provided further evidence for pigs serving as a “mixing vessel” for the generation of new reassortant genotypes of influenza viruses and emphasizes the importance of reinforcing influenza virus surveillance in pigs in China.

Multiple lineages of antigenically and genetically diverse influenza A virus co-circulate in the United States swine population

Before the isolation of H3N2 viruses in 1998, swine influenza in the United States was an endemic disease caused exclusively by classical-swine H1N1 viruses. In this study we determined the antigenic and phylogenetic composition of a selection of currently circulating strains and revealed that, in contrast to the situation pre-1998, the swine population in the United States is now a dynamic viral reservoir containing multiple viral lineages. H3N2 viruses still circulate and representatives of each of two previously identified phylogenetic groups were isolated. H1N1 and H1N2 viruses were also identified. In addition to the genotypic diversity present, there was also considerable antigenic diversity seen. At least three antigenic profiles of H1 viruses were noted and all of the recent H3N2 viruses reacted poorly, if at all, to the index A/swine/Texas/4199-2/98 H3N2 antiserum in hemagglutination inhibition assays. The influenza reservoir in the United States swine population has thus gone from a stable single viral lineage to one where genetically and antigenically heterogeneic viruses co-circulate. The growing complexity of influenza at this animal–human interface and the presence of viruses with a seemingly high affinity for reassortment makes the United States swine population an increasingly important reservoir of viruses with human pandemic potential.

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.

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.