Does pasteurization inactivate bird flu virus in milk?

Recently, an outbreak of highly pathogenic avian influenza A (H5N1), which carries the clade 2.3.4.4b hemagglutinin (HA) gene and has been prevalent among North American bird populations since the winter of 2021, was reported in dairy cows in the United States. As of 24 May 2024, the virus has affected 63 dairy herds across nine states and has resulted in two human infections. The virus causes unusual symptoms in dairy cows, including an unexpected drop in milk production, and thick colostrum-like milk. Notably, The US Food and Drug Administration reported that around 20% of tested retail milk samples contained H5N1 viruses, with a higher percentage of positive results from regions with infected cattle herds. Data are scant regarding how effectively pasteurization inactivates the H5N1 virus in milk. Therefore, in this study, we evaluated the thermal stability of the H5 clade 2.3.4.4b viruses, along with one human H3N2 virus and other influenza subtype viruses, including H1, H3, H7, H9, and H10 subtype viruses. We also assessed the effectiveness of pasteurization in inactivating these viruses. We found that the avian H3 virus exhibits the highest thermal stability, whereas the H5N1 viruses that belong to clade 2.3.4.4b display moderate thermal stability. Importantly, our data provide direct evidence that the standard pasteurization methods used by dairy companies are effective in inactivating all tested subtypes of influenza viruses in raw milk. Our findings indicate that thermally pasteurized milk products do not pose a safety risk to consumers.

Continued evolution of the Eurasian avian-like H1N1 swine influenza viruses in China.

Animal influenza viruses continue to pose a threat to human public health. The Eurasian avian-like H1N1 (EA H1N1) viruses are widespread in pigs throughout Europe and China and have caused human infections in several countries, indicating their pandemic potential. To carefully monitor the evolution of the EA H1N1 viruses in nature, we collected nasal swabs from 103,110 pigs in 22 provinces in China between October 2013 and December 2019, and isolated 855 EA H1N1 viruses. Genomic analysis of 319 representative viruses revealed that these EA H1N1 viruses formed eight different genotypes through reassortment with viruses of other lineages circulating in humans and pigs, and two of these genotypes (G4 and G5) were widely distributed in pigs. Animal studies indicated that some strains have become highly pathogenic in mice and highly transmissible in ferrets via respiratory droplets. Moreover, two-thirds of the EA H1N1 viruses reacted poorly with ferret serum antibodies induced by the currently used H1N1 human influenza vaccine, suggesting that existing immunity may not prevent the transmission of the EA H1N1 viruses in humans. Our study reveals the evolution and pandemic potential of EA H1N1 viruses and provides important insights for future pandemic preparedness.

A Eurasian avian-like H1N1 swine influenza reassortant virus became pathogenic and highly transmissible due to mutations in its PA gene.

Previous studies have shown that the Eurasian avian-like H1N1 (EA H1N1) swine influenza viruses circulated widely in pigs around the world and formed multiple genotypes by acquiring non-hemagglutinin and neuraminidase segments derived from other swine influenza viruses. Swine influenza control is not a priority for the pig industry in many countries, and it is worrisome that some strains may become more pathogenic and/or transmissible during their circulation in nature. Our routine surveillance indicated that the EA H1N1 viruses obtained different internal genes from different swine influenza viruses and formed various new genotypes. In this study, we found that a naturally isolated swine influenza reassortant, A/swine/Liaoning/265/2017 (LN265), a representative strain of one of the predominant genotypes in recent years, is lethal in mice and transmissible in ferrets. LN265 contains the hemagglutinin, neuraminidase, and matrix of the EA H1N1 virus; the basic polymerase 2, basic polymerase 1, acidic polymerase (PA), and nucleoprotein of the 2009 H1N1 pandemic virus; and the nonstructural protein of the North American triple-reassortment H1N2 virus. By generating and testing a series of reassortants and mutants, we found that four gradually accumulated mutations in PA are responsible for the increased pathogenicity and transmissibility of LN265. We further revealed that these mutations increase the messenger RNA transcription of viral proteins by enhancing the endonuclease cleavage activity and viral RNA-binding ability of the PA protein. Our study demonstrates that EA H1N1 swine influenza virus became pathogenic and transmissible in ferrets by acquiring key mutations in PA and provides important insights for monitoring field strains with pandemic potential.

Immune Escape Adaptive Mutations in Hemagglutinin Are Responsible for the Antigenic Drift of Eurasian Avian-Like H1N1 Swine Influenza Viruses.

The continuous antigenic variation of influenza A viruses remains a major hurdle for vaccine selection; however, the molecular determinants and mechanisms of antigenic change remain largely unknown. In this study, two escape mutants were generated by serial passages of the Eurasian avian-like H1N1 swine influenza virus (EA H1N1 SIV) A/swine/Henan/11/2005 (HeN11) in the presence of two neutralizing monoclonal antibodies (mAbs) against the hemagglutinin (HA) protein, which were designated HeN11-2B6-P5 and HeN11-4C7-P8, respectively. The HeN11-2B6-P5 mutant simultaneously harbored the N190D and I230M substitutions in HA, whereas HeN11-4C7-P8 harbored the M269R substitution in HA (H3 numbering). The effects of each of these substitutions on viral antigenicity were determined by measuring the neutralization and hemagglutination inhibition (HI) titers with mAbs and polyclonal sera raised against the representative viruses. The results indicate that residues 190 and 269 are key determinants of viral antigenic variation. In particular, the N190D mutation had the greatest antigenic impact, as determined by the HI assay. Further studies showed that both HeN11-2B6-P5 and HeN11-4C7-P8 maintained the receptor-binding specificity of the parent virus, although the single mutation N190D decreased the binding affinity for the human-type receptor. The replicative ability in vitro of HeN11-2B6-P5 was increased, whereas that of HeN11-4C7-P8 was decreased. These findings extend our understanding of the antigenic evolution of influenza viruses under immune pressure and provide insights into the functional effects of amino acid substitutions near the receptor-binding site and the interplay among receptor binding, viral replication, and antigenic drift. IMPORTANCE The antigenic changes that occur continually in the evolution of influenza A viruses remain a great challenge for the effective control of disease outbreaks. Here, we identified three amino acid substitutions (at positions 190, 230, and 269) in the HA of EA H1N1 SIVs that determine viral antigenicity and result in escape from neutralizing monoclonal antibodies. All three of these substitutions have emerged in nature. Of note, residues 190 and 230 have synergistic effects on receptor binding and antigenicity. Our findings provide a better understanding of the functional effects of amino acid substitutions in HA and their consequences for the antigenic drift of influenza viruses.

Robustness of the Ferret Model for Influenza Risk Assessment Studies: a Cross-Laboratory Exercise.

Past pandemic influenza viruses with sustained human-to-human transmissibility have emerged from animal influenza viruses. Employment of experimental models to assess the pandemic risk of emerging zoonotic influenza viruses provides critical information supporting public health efforts. Ferret transmission experiments have been utilized to predict the human-to-human transmission potential of novel influenza viruses. However, small sample sizes and a lack of standardized protocols can introduce interlaboratory variability, complicating interpretation of transmission experimental data. To assess the range of variation in ferret transmission experiments, a global exercise was conducted by 11 laboratories using two common stock H1N1 influenza viruses with different transmission characteristics in ferrets. Parameters known to affect transmission were standardized, including the inoculation route, dose, and volume, as well as a strict 1:1 donor/contact ratio for respiratory droplet transmission. Additional host and environmental parameters likely to affect influenza transmission kinetics were monitored and analyzed. The overall transmission outcomes for both viruses across 11 laboratories were concordant, suggesting the robustness of the ferret model for zoonotic influenza risk assessment. Among environmental parameters that varied across laboratories, donor-to-contact airflow directionality was associated with increased transmissibility. To attain high confidence in identifying viruses with moderate to high transmissibility or low transmissibility under a smaller number of participating laboratories, our analyses support the notion that as few as three but as many as five laboratories, respectively, would need to independently perform viral transmission experiments with concordant results. This exercise facilitates the development of a more homogenous protocol for ferret transmission experiments that are employed for the purposes of risk assessment. IMPORTANCE Following detection of a novel virus, rapid characterization efforts (both in vitro and in vivo) are undertaken at numerous laboratories worldwide to evaluate the relative risk posed to human health. Aggregation of these data are critical, but the use of nonstandardized protocols can make interpretation of divergent results a challenge. For evaluation of virus transmissibility, a multifactorial trait which can only be evaluated in vivo, identifying intrinsic levels of variability between groups can improve the utility of these data, as well as ensure that experiments are performed with sufficient replication to ensure high confidence in compiled results. Using the ferret transmission model and two influenza A viruses, we conducted a multicenter standardization exercise to improve the interpretation of transmission data generated during risk assessment activities; this exercise serves as a model for future efforts employing both in vitro and in vivo models against possible pandemic pathogens.

A single amino acid at position 158 in haemagglutinin affects the antigenic property of Eurasian avian-like H1N1 swine influenza viruses.

Influenza viruses have been posing a great threat to public health and animal industry. The developed vaccines have been widely used to reduce the risk of potential pandemic; however, the ongoing antigenic drift makes influenza virus escape from host immune response and hampers vaccine efficacy. Until now, the genetic basis of antigenic variation remains largely unknown. In this study, we used A/swine/Guangxi/18/2011 (GX/18) and A/swine/Guangdong/104/2013 (GD/104) as models to explore the molecular determinant for antigenic variation of Eurasian avian-like H1N1 (EA H1N1) swine influenza viruses (SIVs) and found that the GD/104 virus exhibited 32- to 64-fold lower antigenic cross-reactivity with antibodies against GX/18 virus. Therefore, we generated polyclonal antibodies against GX/18 or GD/104 virus and a monoclonal antibody (mAb), named mAb102-95, targeted to the haemagglutinin (HA) protein of GX/18 virus and found that a single amino acid substitution at position 158 in HA protein substantially altered the antigenicity of the virus. The reactivity of GX/18 virus containing G158E mutation with the mAb102-95 decreased eightfold than that of the parental strain. Contrarily, the reactivity of GD/104 virus bearing E158G mutation with the mAb102-95 increased by 32 times as compared with that of the parental virus. Structural analysis showed that the amino acid mutation from G to E was accompanied with the R group changing from -H to -(CH2 )2 -COOH. The induced steric effect and increased hydrophilicity of HA protein surface probably jointly contributed to the antigenic drift of EA H1N1 SIVs. Our study provides experimental evidence that G158E mutation in HA protein affects the antigenic property of EA H1N1 SIVs and widens our horizon on the antigenic drift of influenza virus.

Continued evolution of H6 avian influenza viruses isolated from farms in China between 2014 and 2018.

H6 avian influenza virus (AIV) is one of the most prevalent AIV subtypes in the world. Our previous studies have demonstrated that H6 AIVs isolated from live poultry markets pose a potential threat to human health. In recent years, increasing number of H6 AIVs has been constantly isolated from poultry farms. In order to understand the biological characteristics of H6 AIVs in the context of farms, here, we analyzed the phylogenetic relationships, antigenicity, replication in mice and receptor binding properties of H6 AIVs isolated from farms in China between 2014 and 2018. Phylogenetic analysis showed that 19 different genotypes were formed among 20 representative H6 viruses. Notably, the internal genes of these H6 viruses exhibited complicated relationships with different subtypes of AIVs worldwide, indicating that these viruses are the products of complex and frequent reassortment events. Antigenic analysis revealed that 13 viruses tested were divided into three antigenic groups. 10 viruses examined could all replicate in the respiratory organs of infected mice without prior adaptation. Receptor binding analysis demonstrated that some of the H6 AIVs bound to both α-2, 3-linked glycans (avian-type receptor) and α-2, 6-linked glycans (human-type receptor), thereby posing a potential threat to human health. Together, these findings revealed the prevalence, complicated genetic evolution, diverse antigenicity, and dual receptor binding specificity of H6 AIVs in the settings of poultry farms, which emphasize the importance to continuously monitor the evolution and biological properties of H6 AIVs in nature.

Fur Seal Feces-Associated Circular DNA Virus Identified in Pigs in Anhui, China.

Fur seal feces-associated circular DNA virus (FSfaCV) is an unclassified circular replication-associated protein (Rep)-encoding single-stranded (CRESS) DNA virus that has been detected in mammals (fur seals and pigs). The biology and epidemiology of the virus remain largely unknown. To investigate the virus diversity among pigs in Anhui Province, China, we pooled 600 nasal samples in 2017 and detected viruses using viral metagenomic methods. From the assembled contigs, 12 showed notably high nucleotide acid sequence similarities to the genome sequences of FSfaCVs. Based on these sequences, a full-length genome sequence of the virus was then obtained using overlapping PCR and sequencing, and the virus was designated as FSfaCV-CHN (GenBank No. MK462122). This virus shared 91.3% and 90.9% genome-wide nucleotide sequence similarities with the New Zealand fur seal strain FSfaCV-as50 and the Japanese pig strain FSfaCV-JPN1, respectively. It also clustered with the two previously identified FSfaCVs in a unique branch in the phylogenetic tree based on the open reading frame 2 (ORF2), Rep-coding gene, and the genome of the reference CRESS DNA viruses. Further epidemiological investigation using samples collected in 2018 showed that the overall positive rate for the virus was 56.4% (111/197) in Anhui Province. This is the first report of FSfaCVs identified in pigs in China, and further epidemiological studies are warranted to evaluate the influence of the virus on pigs.

Susceptibility of ferrets, cats, dogs, and other domesticated animals to SARS-coronavirus 2.

Severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2) causes the infectious disease COVID-19 (coronavirus disease 2019), which was first reported in Wuhan, China, in December 2019. Despite extensive efforts to control the disease, COVID-19 has now spread to more than 100 countries and caused a global pandemic. SARS-CoV-2 is thought to have originated in bats; however, the intermediate animal sources of the virus are unknown. In this study, we investigated the susceptibility of ferrets and animals in close contact with humans to SARS-CoV-2. We found that SARS-CoV-2 replicates poorly in dogs, pigs, chickens, and ducks, but ferrets and cats are permissive to infection. Additionally, cats are susceptible to airborne transmission. Our study provides insights into the animal models for SARS-CoV-2 and animal management for COVID-19 control.

A Single Amino Acid at Position 431 of the PB2 Protein Determines the Virulence of H1N1 Swine Influenza Viruses in Mice.

Genetic reassortments occurred continuously among multiple subtypes or genotypes of influenza viruses prevalent in pigs. Of note, some reassortant viruses bearing the internal genes of the 2009 pandemic H1N1 (2009/H1N1) virus sporadically caused human infection, which highlights their potential threats to human public health. In this study, we performed phylogenetic analysis on swine influenza viruses (SIVs) circulating in Liaoning Province, China. A total of 22 viruses, including 18 H1N1 and 4 H1N2 viruses, were isolated from 5,750 nasal swabs collected from pigs in slaughterhouses from 2014 to 2016. H1N1 viruses formed four genotypes, which included Eurasian avian-like H1N1 (EA H1N1) and double/triple reassortant H1N1 derived from EA H1N1, 2009/H1N1, and triple reassortant H1N2 (TR H1N2) viruses. H1N1 SIVs with different genotypes and even those within the same genotypes represented different pathogenicities in mice. We further characterized two naturally isolated H1N1 SIVs that had similar viral genomes but differed substantially in their virulence in mice and found that a single amino acid at position 431 in the basic polymerase 2 (PB2) protein significantly affected the viral replication capacity and virulence of these two viruses. Taken together, our findings revealed the diverse genomic origins and virulence of the SIVs prevalent in Liaoning Province during 2014 to 2016, which highlights that continuous surveillance is essential to monitor the evolution of SIVs. We identified a naturally occurring amino acid mutation in the PB2 protein of H1N1 SIVs that impacts the viral replication and virulence in mice by altering the viral polymerase activity.IMPORTANCE The frequent reassortment among different influenza viruses in pigs adds complexity to the epidemiology of swine influenza. The diverse viral virulence phenotypes underline the need to investigate the possible genetic determinants for evaluating the pandemic potential to human public health. Here, we found that multiple genotypes of influenza viruses cocirculate in the swine population in Liaoning Province, China. Furthermore, we pinpointed a single amino acid at position 431 in the PB2 protein which plays a critical role in the virulence of H1N1 viruses in mice and found that the alteration of viral polymerase activities is the cause of the different virulence. Our study further indicated that the virulence of influenza virus is a polygenic trait, and the newly identified virulence-related residue in the PB2 provides important information for broadening knowledge on the genetic basis of viral virulence of influenza viruses.

Rapid Evolution of H7N9 Highly Pathogenic Viruses that Emerged in China in 2017.

H7N9 low pathogenic influenza viruses emerged in China in 2013 and mutated to highly pathogenic strains in 2017, resulting in human infections and disease in chickens. To control spread, a bivalent H5/H7 inactivated vaccine was introduced in poultry in September 2017. To monitor virus evolution and vaccine efficacy, we collected 53,884 poultry samples across China from February 2017 to January 2018. We isolated 252 H7N9 low pathogenic viruses, 69 H7N9 highly pathogenic viruses, and one H7N2 highly pathogenic virus, of which two low pathogenic and 14 highly pathogenic strains were collected after vaccine introduction. Genetic analysis of highly pathogenic strains revealed nine genotypes, one of which is predominant and widespread and contains strains exhibiting high virulence in mice. Additionally, some H7N9 and H7N2 viruses carrying duck virus genes are lethal in ducks. Thus, although vaccination reduced H7N9 infections, the increased virulence and expanded host range to ducks pose new challenges.

Emergence of H3N8 equine influenza virus in donkeys in China in 2017.

Equine influenza virus is a major respiratory pathogen in horses. Although both horses and donkeys belong to the genus Equus, donkey infection with influenza viruses is rare. In March 2017, an influenza outbreak occurred in donkeys in Shandong province, China. The causative virus, A/donkey/Shandong/1/2017(H3N8), was isolated from a dead donkey. Genetic analysis indicated that the virus originated from influenza A (H3N8) clade 2 of the Florida sub-lineage that has been circulating in Asian equine populations. Comparison of the deduced amino acid sequence of the HA gene of this causative virus with that of the A/equine/Richmond/1/2007 vaccine strain showed that substitutions had occurred in the antigenic regions A, B, and C. This study provides insight into the currently circulating and newly emerging H3N8 strains in donkeys in China.

H7N9 virulent mutants detected in chickens in China pose an increased threat to humans.

Certain low pathogenic avian influenza viruses can mutate to highly pathogenic viruses when they circulate in domestic poultry, at which point they can cause devastating poultry diseases and severe economic damage. The H7N9 influenza viruses that emerged in 2013 in China had caused severe human infections and deaths. However, these viruses were nonlethal in poultry. It is unknown whether the H7N9 viruses can acquire additional mutations during their circulation in nature and become lethal to poultry and more dangerous for humans. Here, we evaluated the evolution of H7N9 viruses isolated from avian species between 2013 and 2017 in China and found 23 different genotypes, 7 of which were detected only in ducks and were genetically distinct from the other 16 genotypes that evolved from the 2013 H7N9 viruses. Importantly, some H7N9 viruses obtained an insertion of four amino acids in their hemagglutinin (HA) cleavage site and were lethal in chickens. The index strain was not lethal in mice or ferrets, but readily obtained the 627K or 701N mutation in its PB2 segment upon replication in ferrets, causing it to become highly lethal in mice and ferrets and to be transmitted efficiently in ferrets by respiratory droplet. H7N9 viruses bearing the HA insertion and PB2 627K mutation have been detected in humans in China. Our study indicates that the new H7N9 mutants are lethal to chickens and pose an increased threat to human health, and thus highlights the need to control and eradicate the H7N9 viruses to prevent a possible pandemic.

Immune efficacy of an adenoviral vector-based swine influenza vaccine against antigenically distinct H1N1 strains in mice.

Avian-like H1N1 swine influenza viruses are prevalent in pigs and have occasionally crossed the species barrier and infected humans, which highlights the importance of preventing swine influenza. Human adenovirus serotype 5 (Ad5) has been tested in human influenza vaccine clinical trials and has exhibited a reliable safety profile. Here, we generated a replication-defective, recombinant adenovirus (designated as rAd5-avH1HA) expressing the hemagglutinin gene of an avian-like H1N1 virus (A/swine/Zhejiang/199/2013, ZJ/199/13). Using a BALB/c mouse model, we showed that a two-dose intramuscular administration of recombinant rAd5-avH1HA induced high levels of hemagglutination inhibition antibodies and prevented homologous and heterologous H1N1 virus-induced weight loss, as well as viral replication in the nasal turbinates and lungs of mice. Furthermore, a prime-boost immunization strategy trial with a recombinant plasmid (designated as pCAGGS-HA) followed by rAd5-avH1HA vaccine provided effective protection against homologous and heterologous H1N1 virus infection in mice. These results indicate that rAd5-avH1HA is an efficacious genetically engineered vaccine candidate against H1N1 swine influenza. Future studies should examine its immune efficacy in pigs.

A Single-Amino-Acid Substitution at Position 225 in Hemagglutinin Alters the Transmissibility of Eurasian Avian-Like H1N1 Swine Influenza Virus in Guinea Pigs.

Efficient transmission from human to human is the prerequisite for an influenza virus to cause a pandemic; however, the molecular determinants of influenza virus transmission are still largely unknown. In this study, we explored the molecular basis for transmission of Eurasian avian-like H1N1 (EAH1N1) swine influenza viruses by comparing two viruses that are genetically similar but differ in their transmissibility in guinea pigs: the A/swine/Guangxi/18/2011 virus (GX/18) is highly transmissible by respiratory droplet in guinea pigs, whereas the A/swine/Heilongjiang/27/2012 virus (HLJ/27) does not transmit in this animal model. We used reverse genetics to generate a series of reassortants and mutants in the GX/18 background and tested their transmissibility in guinea pigs. We found that a single-amino-acid substitution of glycine (G) for glutamic acid (E) at position 225 (E225G) in the HA1 protein completely abolished the respiratory droplet transmission of GX/18, whereas the substitution of E for G at the same position (G225E) in HA1 enabled HLJ/27 to transmit in guinea pigs. We investigated the underlying mechanism and found that viruses bearing 225E in HA1 replicated more rapidly than viruses bearing 225G due to differences in assembly and budding efficiencies. Our study indicates that the amino acid 225E in HA1 plays a key role in EAH1N1 swine influenza virus transmission and provides important information for evaluating the pandemic potential of field influenza virus strains.IMPORTANCE Efficient transmission among humans is a prerequisite for a novel influenza virus to cause a human pandemic. Transmissibility of influenza viruses is a polygenic trait, and understanding the genetic determinants for transmissibility will provide useful insights for evaluating the pandemic potential of influenza viruses in the field. Several amino acids in the hemagglutinin (HA) protein of influenza viruses have been shown to be important for transmissibility, usually by increasing virus affinity for human-type receptors. In this study, we explored the genetic basis of the transmissibility difference between two Eurasian avian-like H1N1 (EAH1N1) swine influenza viruses in guinea pigs and found that the amino acid glutamic acid at position 225 in the HA1 protein plays a critical role in the transmission of EAH1N1 virus by increasing the efficiency of viral assembly and budding.

Protective efficacy of an inactivated Eurasian avian-like H1N1 swine influenza vaccine against homologous H1N1 and heterologous H1N1 and H1N2 viruses in mice.

Eurasian avian-like H1N1 (EA H1N1) swine influenza viruses are prevalent in pigs in Europe and Asia, but occasionally cause human infection, which raises concern about their pandemic potential. Here, we produced a whole-virus inactivated vaccine with an EA H1N1 strain (A/swine/Guangxi/18/2011, SW/GX/18/11) and evaluated its efficacy against homologous H1N1 and heterologous H1N1 and H1N2 influenza viruses in mice. A strong humoral immune response, which we measured by hemagglutination inhibition (HI) and virus neutralization (VN), was induced in the vaccine-inoculated mice upon challenge. The inactivated SW/GX/18/11 vaccine provided complete protection against challenge with homologous SW/GX/18/11 virus in mice and provided effective protection against challenge with heterologous H1N1 and H1N2 viruses with distinctive genomic combinations. Our findings suggest that this EA H1N1 vaccine can provide protection against both homologous H1N1 and heterologous H1N1 or H1N2 virus infection. As such, it is an excellent vaccine candidate to prevent H1N1 swine influenza.

Prevalence, genetics, and transmissibility in ferrets of Eurasian avian-like H1N1 swine influenza viruses.

Pigs are important intermediate hosts for generating novel influenza viruses. The Eurasian avian-like H1N1 (EAH1N1) swine influenza viruses (SIVs) have circulated in pigs since 1979, and human cases associated with EAH1N1 SIVs have been reported in several countries. However, the biologic properties of EAH1N1 SIVs are largely unknown. Here, we performed extensive influenza surveillance in pigs in China and isolated 228 influenza viruses from 36,417 pigs. We found that 139 of the 228 strains from pigs in 10 provinces in China belong to the EAH1N1 lineage. These viruses formed five genotypes, with two distinct antigenic groups, represented by A/swine/Guangxi/18/2011 and A/swine/Guangdong/104/2013, both of which are antigenically and genetically distinct from the current human H1N1 viruses. Importantly, the EAH1N1 SIVs preferentially bound to human-type receptors, and 9 of the 10 tested viruses transmitted in ferrets by respiratory droplet. We found that 3.6% of children (≤10 y old), 0% of adults, and 13.4% of elderly adults (≥60 y old) had neutralization antibodies (titers ≥40 in children and ≥80 in adults) against the EAH1N1 A/swine/Guangxi/18/2011 virus, but none of them had such neutralization antibodies against the EAH1N1 A/swine/Guangdong/104/2013 virus. Our study shows the potential of EAH1N1 SIVs to transmit efficiently in humans and suggests that immediate action is needed to prevent the efficient transmission of EAH1N1 SIVs to humans.

Two different genotypes of H1N2 swine influenza virus isolated in northern China and their pathogenicity in animals.

During 2006 and 2007, two swine-origin triple-reassortant influenza A (H1N2) viruses were isolated from pigs in northern China, and the antigenic characteristics of the hemagglutinin protein of the viruses were examined. Genotyping and phylogenetic analyses demonstrated different emergence patterns for the two H1N2 viruses, Sw/Hebei/10/06 and Sw/Tianjin/1/07. Sequences for the other genes encoding the internal proteins were compared with the existing data to determine their origins and establish the likely mechanisms of genetic reassortment. Sw/Hebei/10/06 is an Sw/Indiana/9K035/99-like virus, whereas Sw/Tianjin/1/07 represents a new H1N2 genotype with surface genes of classic swine and human origin and internal genes originating from the Eurasian avian-like swine H1N1 virus. Six-week-old female BALB/c mice infected with the Sw/HeB/10/06 and Sw/TJ/1/07 viruses showed an average weight loss of 12.8% and 8.1%, respectively. Healthy six-week-old pigs were inoculated intranasally with either the Sw/HeB/10/06 or Sw/TJ/1/07 virus. No considerable changes in the clinical presentation were observed post-inoculation in any of the virus-inoculated groups, and the viruses effectively replicated in the nasal cavity and lung tissue. Based on the results, it is possible that the new genotype of the swine H1N2 virus that emerged in China may become widespread in the swine population and pose a potential threat to public health.

Novel triple reassortant H1N2 influenza viruses bearing six internal genes of the pandemic 2009/H1N1 influenza virus were detected in pigs in China.

BACKGROUND: 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. Transmissions of the pandemic 2009/H1N1 virus from humans to poultry and other species of mammals were reported from several continents during the course of the 2009 H1N1 pandemic. Reassortant H1N1, H1N2, and H3N2 viruses containing genes of the pandemic 2009/H1N1 viruses appeared in pigs in some countries. STUDY DESIGN: In winter of 2012, a total of 2600 nasal swabs were collected from healthy pigs in slaughterhouses located throughout 10 provinces in China. The isolated viruses were subjected to genetic and antigenic analysis. Two novel triple-reassortant H1N2 influenza viruses were isolated from swine in China in 2012, with the HA gene derived from Eurasian avian-like swine H1N1, the NA gene from North American swine H1N2, and the six internal genes from the pandemic 2009/H1N1 viruses. The two viruses had similar antigenic features and some significant changes in antigenic characteristics emerged when compared to the previously identified isolates. CONCLUSION: We inferred that the novel reassortant viruses in China may have arisen from the accumulation of the three types of influenza viruses, which further indicates that swine herds serve as "mixing vessels" for influenza viruses. Influenza virus reassortment is an ongoing process, and our findings highlight the urgent need for continued influenza surveillance among swine herds.