Avian-like H1N1 Swine Influenza Virus Research Articles

Genetic characteristics of the first human infection with the G4 genotype eurasian avian-like H1N1 swine influenza virus in Shaanxi Province,China

Objective: To analyze the genetic characteristics of the first human infection with the G4 genotype of Eurasian avian H1N1 swine influenza virus (EA H1N1 SIV) in Shaanxi Province. Methods: The patient's throat swab samples were collected, and MDCK cells were inoculated for virus isolation to obtain the virus strain. The whole genome deep sequencing method was used to obtain the eight gene segments of the isolated strain. The nucleotide homology analysis was conducted through the Blast program in the GenBank database, and a phylogenetic tree was constructed to analyze the genetic characteristics of the virus. Results: The throat swab specimens of the case were confirmed as EA H1N1 SIV in the laboratory, and the isolated strain was named A/Shaanxi-Weicheng/1351/2022(H1N1v). Homology analysis found that the PB2, NP, HA, NA, and M genes of this isolate had the highest nucleotide homology with A/swing/Beijing/0301/2018 (H1N1), about 98.29%, 98.73%, 97.41%, 97.52%, and 99.08%, respectively. The phylogenetic tree showed that the isolate belonged to G4 genotype EA H1N1 SIV, with PB2, PB1, PA, NP and M genes from pdm/09 H1N1, HA and NA genes from EA H1N1, and NS gene from Triple-reassortant H1N1. The cleavage site of the HA protein was IPSIQSR↓G, which was the molecular characteristic of the low pathogenic influenza virus. No amino acid mutations associated with neuraminidase inhibitors were found in the NA protein. PB2 protein 701N mutation, PA protein P224S mutation, NP protein Q357K mutation, M protein P41A mutation, and NS protein 92D all indicated its enhanced adaptability to mammals. Conclusion: The patient is the first human infection with G4 genotype EA H1N1 SIV in Shaanxi province. The virus is low pathogenic, but its adaptability to mammals is enhanced. Therefore, it is necessary to strengthen the monitoring of such SIVs.

Isolation and identification of Eurasian avian-like H1N1 swine influenza virus and evaluation of their pathogenicity and immune protective effects in pigs

Swine influenza (SI) is a severe disease affecting pigs, with a huge economic impact on pig farmers. Currently, available SIV vaccines do not meet the requirements for Swine influenza prevention and control, indicating the need for vaccine development using predominant strains. Here, we isolated and identified the swine influenza virus in farms and slaughterhouses in nine provinces in China to determine the most prevalent strain. A total of 8383 samples were collected between 2013 and 2022, from which 87 swine influenza virus strains were isolated. Genome sequencing identified 62 strains of the H1N1 subtype, three strains of the H1N2 subtype, and 22 strains of the H3N2 subtype. The 521# strain virus possesses the viral ribonucleoprotein (vRNP) and matrix (M) genes from the pdm/09 lineage, the HA, NA from the original Eurasian avian-like (EA) H1N1 lineage, and the nonstructural (NS) gene from the triple-reassortant (TR) lineage. The 431# strain was also a TR, except its M-gene was derived from the original EA H1N1 lineage. The pathogenicity of two 431# strains and one typical 521# strain was evaluated in mice, and the 431# strain exhibited higher pathogenicity. Therefore, a new 521# strain was selected for vaccine production because it is the current circulating strain. The vaccine produced using the 521# strain and pre-evaluated adjuvants was effective against the homologous H05 strain, as evidenced by the normal body temperature of vaccinated pigs and low virus titer of nasal swabs. In contrast, infection with the H05 strain significantly increased the body temperature of unvaccinated pigs and increased the virus titer of nasal swabs. Notably, vaccination with the 521#-based vaccine conferred some level of protection against the heterologous B15 strain (H3N2 subtype), thus reducing the viral load in pigs.

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.

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

ABSTRACTThe 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.

Zoonotic Threat of G4 Genotype Eurasian Avian-Like Swine Influenza A(H1N1) Viruses, China, 2020

We investigated genetic and biologic characteristics of 2 Eurasian avian-like H1N1 swine influenza viruses from pigs in China that belong to the predominant G4 genotype. One swine isolate exhibited strikingly great homology to contemporaneous human Eurasian avian-like H1N1 isolates, preferential binding to the human-type receptor, and vigorous replication in mice without adaptation.

Natural Reassortment of Eurasian Avian-Like Swine H1N1 and Avian H9N2 Influenza Viruses in Pigs, China.

Several zoonotic influenza A viruses detected in humans contain genes derived from avian H9N2 subtypes. We uncovered a Eurasian avian-like H1N1 swine influenza virus with polymerase basic 1 and matrix gene segments derived from the H9N2 subtype, suggesting that H9N2 viruses are infecting pigs and reassorting with swine influenza viruses in China.

Prevalence, Genetics and Evolutionary Properties of Eurasian Avian-like H1N1 Swine Influenza Viruses in Liaoning.

Swine influenza virus (SIV) is an important zoonosis pathogen. The 2009 pandemic of H1N1 influenza A virus (2009/H1N1) highlighted the importance of the role of pigs as intermediate hosts. Liaoning province, located in northeastern China, has become one of the largest pig-farming areas since 2016. However, the epidemiology and evolutionary properties of SIVs in Liaoning are largely unknown. We performed systematic epidemiological and genetic dynamics surveillance of SIVs in Liaoning province during 2020. In total, 33,195 pig nasal swabs were collected, with an SIV detection rate of 2%. Our analysis revealed that multiple subtypes of SIVs are co-circulating in the pig population in Liaoning, including H1N1, H1N2 and H3N2 SIVs. Furthermore, 24 H1N1 SIVs were confirmed to belong to the EA H1N1 lineage and divided into two genotypes. The two genotypes were both triple reassortant, and the predominant one with polymerase, nucleoprotein (NP), and matrix protein (M) genes originating from 2009/H1N1; hemagglutinin (HA) and neuraminidase (NA) genes originating from EA H1N1; and the nonstructural protein (NS) gene originating from triple reassortant H1N2 (TR H1N2) was detected in Liaoning for the first time. According to our evolutionary analysis, the EA H1N1 virus in Liaoning will undergo further genome variation.

Cross-reaction of seasonal influenza vaccine immune serum against Eurasian Avian-like H1N1 swine influenza virus

Objective: To evaluate the cross-reaction of seasonal influenza vaccine immune serum against Eurasian avian-like H1N1 swine influenza virus. Methods: Nine human infected Eurasian avian-like H1N1 swine influenza virus strains were obtained from national influenza surveillance network laboratories in Jiangsu, Hebei, Shandong, Yunnan, Hunan, Fujian and Tianjin provinces, and their genetic characteristics of hemagglutinin were analyzed by deep sequencing. 30 volunteers were recruited respectively from children (2-5 years old), adults (24-57 years old) and elderly (60-84 years old) who received 2019-2020 seasonal influenza vaccine in Anning city, Yunnan Province in October 2019, and serum samples were collected before and 1 month after vaccination. The hemagglutination inhibition test was used to evaluate the cross-reaction of serum before and after immunization against 4 strains of human infection with Eurasian avian-like H1N1 swine influenza virus isolated since 2015. Results: The homology of hemagglutinin genes of 9 Eurasian avian-like H1N1 swine influenza viruses was similar, but the difference of hemagglutinin heavy chain and light chain amino acid genes with A (H1N1) pdm09 (vaccine strain) were 90-101 and 24-30 amino acids respectively. The antibody titer of vaccine strain antiserum to vaccine strain was 2 560; the antibody titers of the vaccine strain antiserum to Eurasian avian-like H1N1 swine influenza virus and the Eurasian avian-like H1N1 swine influenza virus antiserum to vaccine strain were same as 640. The proportion of children, adults and elderly vaccinated with seasonal influenza vaccine with antibody titer ≥40 against vaccine strain was 90.0%, 70.0% and 73.3%, respectively; while the proportion merely were 46.7%, 36.7% and 33.3%-43.3% to 4 strains of Eurasian avian-like H1N1 swine influenza virus, respectively. Conclusion: Seasonal influenza vaccination does not provide effective cross-protection against Eurasian avian-like H1N1 swine influenza virus.

Epidemiology and Genotypic Diversity of Eurasian Avian-Like H1N1 Swine Influenza Viruses in China.

Eurasian avian-like H1N1 (EA H1N1) swine influenza virus (SIV) outside European countries was first detected in Hong Kong Special Administrative Region (Hong Kong, SAR) of China in 2001. Afterwards, EA H1N1 SIVs have become predominant in pig population in this country. However, the epidemiology and genotypic diversity of EA H1N1 SIVs in China are still unknown. Here, we collected the EA H1N1 SIVs sequences from China between 2001 and 2018 and analyzed the epidemic and phylogenic features, and key molecular markers of these EA H1N1 SIVs. Our results showed that EA H1N1 SIVs distributed in nineteen provinces/municipalities of China. After a long-time evolution and transmission, EA H1N1 SIVs were continuously reassorted with other co-circulated influenza viruses, including 2009 pandemic H1N1 (A(H1N1)pdm09), and triple reassortment H1N2 (TR H1N2) influenza viruses, generated 11 genotypes. Genotype 3 and 5, both of which were the reassortments among EA H1N1, A(H1N1)pdm09 and TR H1N2 viruses with different origins of M genes, have become predominant in pig population. Furthermore, key molecular signatures were identified in EA H1N1 SIVs. Our study has drawn a genotypic diversity image of EA H1N1 viruses, and could help to evaluate thepotential risk of EA H1N1 for pandemic preparedness and response.

Prevalent Eurasian avian-like H1N1 swine influenza virus with 2009 pandemic viral genes facilitating human infection

Pigs are considered as important hosts or "mixing vessels" for the generation of pandemic influenza viruses. Systematic surveillance of influenza viruses in pigs is essential for early warning and preparedness for the next potential pandemic. Here, we report on an influenza virus surveillance of pigs from 2011 to 2018 in China, and identify a recently emerged genotype 4 (G4) reassortant Eurasian avian-like (EA) H1N1 virus, which bears 2009 pandemic (pdm/09) and triple-reassortant (TR)-derived internal genes and has been predominant in swine populations since 2016. Similar to pdm/09 virus, G4 viruses bind to human-type receptors, produce much higher progeny virus in human airway epithelial cells, and show efficient infectivity and aerosol transmission in ferrets. Moreover, low antigenic cross-reactivity of human influenza vaccine strains with G4 reassortant EA H1N1 virus indicates that preexisting population immunity does not provide protection against G4 viruses. Further serological surveillance among occupational exposure population showed that 10.4% (35/338) of swine workers were positive for G4 EA H1N1 virus, especially for participants 18 y to 35 y old, who had 20.5% (9/44) seropositive rates, indicating that the predominant G4 EA H1N1 virus has acquired increased human infectivity. Such infectivity greatly enhances the opportunity for virus adaptation in humans and raises concerns for the possible generation of pandemic viruses.

The R251K Substitution in Viral Protein PB2 Increases Viral Replication and Pathogenicity of Eurasian Avian-like H1N1 Swine Influenza Viruses.

The Eurasian avian-like swine (EA) H1N1 virus has affected the Chinese swine industry, and human infection cases have been reported occasionally. However, little is known about the pathogenic mechanism of EA H1N1 virus. In this study, we compared the mouse pathogenicity of A/swine/Guangdong/YJ4/2014 (YJ4) and A/swine/Guangdong/MS285/2017 (MS285) viruses, which had similar genotype to A/Hunan/42443/2015 (HuN-like). None of the mice inoculated with 106 TCID50 of YJ4 survived at 7 days post infection, while the survival rate of the MS285 group was 100%. Therefore, a series of single fragment reassortants in MS285 background and two rescued wild-type viruses were generated by using the reverse genetics method, and the pathogenicity analysis revealed that the PB2 gene contributed to the high virulence of YJ4 virus. Furthermore, there were 11 amino acid differences in PB2 between MS285 and YJ4 identified by sequence alignment, and 11 single amino acid mutant viruses were generated in the MS285 background. We found that the R251K mutation significantly increased the virulence of MS285 in mice, contributed to high polymerase activity and enhanced viral genome transcription and replication. These results indicate that PB2-R251K contributes to the virulence of the EA H1N1 virus and provide new insight into future molecular epidemiological surveillance strategies.

Protective efficacy of a high-growth reassortant H1N1 influenza virus vaccine against the European Avian-like H1N1 swine influenza virus in mice and pigs

Swine influenza A viruses (SIVs) causing outbreaks of acute, highly contagious respiratory disease in pigs also pose a potential threat to public health. European avian-like H1N1 (EA H1N1) SIVs are the predominant circulating viruses in pigs in China and also occasionally cause human infection. In this study, a high-growth reassortant virus (SH1/PR8), with HA and NA genes from a representative EA H1N1 isolate A/Swine/Shanghai/1/2014 (SH1) in China and six internal genes from the high-growth A/Puerto Rico/8/34 (PR8) virus, was generated by plasmid-based reverse genetics and tested as a candidate seed virus for the preparation of inactivated vaccine. The protective efficacy of inactivated SH1/PR8 was evaluated in mice and pigs challenged with wild-type SH1 virus. After primer and boost vaccination, the SH1/PR8 vaccine induced high-level hemagglutination inhibiting (HI) antibodies, IgG antibodies, and neutralization antibodies in mice and pigs. Mice and pigs in the vaccinated group showed less clinical phenomena and pathological changes than those in the unvaccinated group. In conclusion, the inactivated high-growth reassortant vaccine SH1/PR8 could induce high antibody levels and complete protection is expected against SH1 wild type SIV, and protection against heterologous EA H1N1 SIV needs further evaluation.

Substitution of D701N in the PB2 protein could enhance the viral replication and pathogenicity of Eurasian avian-like H1N1 swine influenza viruses

Eurasian avian-like H1N1 (EA H1N1) swine influenza viruses (SIVs) have become predominant in pig populations in China and have recently been reported to have the most potential to raise the next pandemic in humans. The mutation D701N in the PB2 protein, which accounts for 31% of H1N1 SIVs, has previously been shown to contribute to the adaptation of the highly pathogenic H5N1 or H7N7 avian influenza viruses in mammals. However, little is known of the effects of this substitution on the EA H1N1 viruses. Herein, we investigated the contributions of 701N in the PB2 protein to an EA H1N1 SIV (A/Hunan/42443/2015(H1N1), HuN EA-H1N1), which had 701D in the PB2 protein. Our results found that viral polymerase activity, viral replication, and pathogenicity in mice were indeed enhanced due to the introduction of 701N into the PB2 protein, and the increased viral growth was partly mediated by the host factor importin-α7. Thus, substantial attention should be paid to the D701N mutation in pig populations.

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.

Predicted Enhanced Human Propensity of Current Avian-Like H1N1 Swine Influenza Virus from China.

Influenza A virus (IAV) subtypes against which little or no pre-existing immunity exists in humans represent a serious threat to global public health. Monitoring of IAV in animal hosts is essential for early and rapid detection of potential pandemic IAV strains to prevent their spread. Recently, the increased pandemic potential of the avian-like swine H1N1 IAV A/swine/Guangdong/104/2013 has been suggested. The virus is infectious in humans and the general population seems to lack neutralizing antibodies against this virus. Here we present an in silico analysis that shows a strong human propensity of this swine virus further confirming its pandemic potential. We suggest mutations which would further enhance its human propensity. We also propose conserved antigenic determinants which could serve as a component of a prepandemic vaccine. The bioinformatics tool, which can be used to further monitor the evolution of swine influenza viruses towards a pandemic virus, are described here and are made publically available (http://www.vin.bg.ac.rs/180/tools/iav_mon.php; http://www.biomedprotection.com/iav_mon.php).

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.

Reassortants of the pandemic (H1N1) 2009 virus and establishment of a novel porcine H1N2 influenza virus, lineage in Germany

The incursion of pandemic (H1N1) 2009 virus (pdmH1N1) into the German pig population was investigated in a serosurvey and by virological means between June 2009 and December 2012. Analysis of 23,116 pig sera from a total of 2,666 herds revealed 224 herds that reacted with pdmH1N1 but not with the prevalent avian-like H1N1 swine influenza virus. Sixty-six pdmH1N1 strains and their reassortant derivatives (pdmH1huN2, huH3pdmN1) have been collected since November 2009. Sequencing of three pdmH1N1, 20 pdmH1huN2 and one huH3pdmN1 strains with conventional and next generation sequencing techniques and subsequent phylogenetic analyses with available sequence data revealed the emergence of five distinct reassortant genotypes in Europe. The most frequent genotype emerged at least three times independently, one of which (Papenburg lineage) established a stable infection chain and became more prevalent in pigs than pdmH1N1 in Germany.

Complete Genome Sequences of Six Avian-Like H1N1 Swine Influenza Viruses from Northwestern China

Very little is known about swine influenza in northwestern China. Here, we report the complete genomic sequences of six avian-like H1N1 swine influenza viruses (SIVs) isolated in pigs in northwestern China. Phylogenetic analyses of the sequences of eight genomic segments demonstrated that they are avian-like H1N1 SIVs.

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.

Neutralizing antibodies against pandemic, seasonal and avian-like H1N1 swine influenza virus in swine contacts and swine, Western-Europe

Neutralizing antibodies against pandemic, seasonal and avian-like H1N1 swine influenza virus in swine contacts and swine, Western-Europe