Swine Influenza Virus Lineages Research Articles

Origin and Evolution of H1N1/pdm2009: A Codon Usage Perspective.

The H1N1/pdm2009 virus is a new triple-reassortant virus. While Eurasian avian-like and triple-reassortant swine influenza viruses are the direct ancestors of H1N1/pdm2009, the classic swine influenza virus facilitate the spectrum of influenza A diversity in pig population when the reassortant events occurred during 1998 to April 2009. The factors that facilitate the final formation of this gene constellation for H1N1/pdm2009 virus from this complex gene pool remain unknown. Since a novel successful virus should efficiently replicate and transmit in their hosts, in this study, we estimated the adaptability of the codon usage patterns of the pool of genes from these lineages of swine influenza viruses to the human expression system. We found that the MP and NA genes of Eurasian avian-like swine influenza viruses, and the PB2, PB1 and PA genes of triple-reassortant swine influenza viruses were best adapted to the human codon usage pattern. As these genes participated in the development of H1N1/pdm2009, they might help in viral replication and strengthen its competitiveness during its emergence. After its emergence in the human population, a gradual optimization of codon usage patterns between 2009 and 2019 to the human codon usage for the H1N1/pdm2009 genes was detected. This reveals that ongoing adaptive evolution, after its original incursion, occurred to further increase the adaptability of overall gene cassette to human expression system.

Surveillance of swine influenza viruses in sentinel familial farms in Hung Yen province in Northern Vietnam in 2013-2014.

From May 2013 to April 2014, 15 swine family-run farms (17 pig litters) in two districts in Hung Yen province, near Hanoi, were virologically and epizootiologically monitored for swine influenza viruses (SIV) monthly. No SIV was isolated from nasal swabs. Maternal antibodies were detected in 10 litters, and seroconversion against SIV was detected in six litters. There was a marked difference in patterns of SIV transmission in the two districts. Van Lam district which has low density of swine with mainly smallholder farms had low intensity of SIV, with much of the infection caused by H1N1 2009 pandemic-like viruses A(H1N1)pdm09, likely originated from humans. In contrast, Van Giang district, which has high swine density and larger farms, had high levels of typical SIV (triple reassortants H3N2 and H3N2 Binh Duong lineage viruses) circulating within swine. With one exception, the SIV lineages detected were those we concurrently isolated from studies in a large central abattoir in Hanoi. Influenza-like illness symptoms reported by farmers were poorly correlated with serological evidence of SIV infection.

Efficient isolation of Swine influenza viruses by age-targeted specimen collection.

The control of swine influenza virus (SIV) infection is paramount for increasing the productivity of pig farming and minimizing the threat of pandemic outbreaks. Thus, SIV surveillance should be conducted by region and on a regular basis. Here, we established a microneutralization assay specific for SIV seroprevalence surveillance by using reporter gene-expressing recombinant influenza viruses. Growth-based SIV seroprevalence revealed that most sows and piglets were positive for neutralizing antibodies against influenza viruses. In contrast, the 90-day-old growing pigs exhibited limited neutralizing activity in their sera, suggesting that this particular age of population is most susceptible to SIV infection and thus is an ideal age group for SIV isolation. From nasal swab specimens of healthy pigs in this age population, we were able to isolate SIVs at a higher incidence (5.3%) than those of previous reports. Nucleotide sequencing and phylogenetic analysis of the hemagglutinin (HA) genes revealed that the isolated SIVs have circulated and evolved in pigs but not have been recently introduced from humans, implying that a large number of SIV lineages may remain "undiscovered" in the global porcine populations. We propose that the 90-day-old growing pig-targeted nasal swab collection presented in this study facilitates global SIV surveillance and contributes to the detection and control of SIV infection.

Expansion of genotypic diversity and establishment of 2009 H1N1 pandemic-origin internal genes in pigs in China.

Two-way transmission of influenza viruses between humans and swine has been frequently observed, and the occurrence of the 2009 H1N1 pandemic influenza virus (pdm/09) demonstrated that swine-origin viruses could facilitate the genesis of a pandemic strain. Although multiple introductions to and reassortment in swine of the pdm/09 virus have been repeatedly reported in both Eurasia and the Americas, its long-term impact on the development of swine influenza viruses (SIVs) has not been systematically explored. Our comprehensive evolutionary studies of the complete genomes of 387 SIVs obtained from 2009 to 2012 by influenza virus surveillance in China revealed 17 reassortant genotypes with pdm/09-origin genes. Even though the entire 2009 pandemic virus and its surface genes cannot persist, its internal genes have become established and are now the predominant lineages in pigs in the region. The main persistent pdm/09-origin reassortant forms had at least five pdm/09-origin internal genes, and their surface genes were primarily of European avian-like (EA) or human H3N2-like SIV origin. These findings represent a marked change in the evolutionary patterns and ecosystem of SIVs in China. It is possible that the pdm/09-origin internal genes are in the process of replacing EA or triple-reassortant-like internal genes. These alterations in the SIV gene pool need to be continually monitored to assess changes in the potential for SIV transmission to humans. Shortly after the emergence of the 2009 pandemic H1N1 (pdm/09) influenza virus, it was transmitted from humans to pigs and this continues to occur around the world. Many reassortants between pdm/09-origin viruses and enzootic swine influenza viruses (SIVs) have been detected. However, the long-term impact of pdm/09-origin viruses on the SIV gene pool, which could lead to the generation of influenza viruses with the potential to infect humans, has not been systematically examined. From extensive surveillance of SIVs over a 38-month period in southern China, it was found that although neither complete pdm/09 viruses nor their surface genes could persist in pigs, their internal genes did persist. Over the survey period, these internal genes became predominant, potentially replacing those of the enzootic SIV lineages. The altered diversity of the SIV gene pool needs to be closely monitored for changes in the potential for SIV transmission to humans.

Influenza at the animal-human interface: a review of the literature for virological evidence of human infection with swine or avian influenza viruses other than A(H5N1).

Factors that trigger human infection with animal influenza virus progressing into a pandemic are poorly understood. Within a project developing an evidence-based risk assessment framework for influenza viruses in animals, we conducted a review of the literature for evidence of human infection with animal influenza viruses by diagnostic methods used. The review covering Medline, Embase, SciSearch and CabAbstracts yielded 6,955 articles, of which we retained 89; for influenza A(H5N1) and A(H7N9), the official case counts of t he World Health Organization were used. An additional 30 studies were included by scanning the reference lists. Here, we present the findings for confirmed infections with virological evidence. We found reports of 1,419 naturally infected human cases, of which 648 were associated with avian influenza virus (AIV) A(H5N1), 375 with other AIV subtypes, and 396 with swine influenza virus (SIV). Human cases naturally infected with AIV spanned haemagglutinin subtypes H5, H6, H7, H9 and H10. SIV cases were associated with endemic SIV of H1 and H3 subtype descending from North American and Eurasian SIV lineages and various reassortants thereof. Direct exposure to birds or swine was the most likely source of infection for the cases with available information on exposure.

Genetic ancestor of external antigens of pandemic influenza A/H1N1 virus

The aim of the present investigation was to discover the genetic relationships of 2009 pandemic novel influenza A/H1N1 virus (NIV) external antigens Hemagglutinin (HA) and Neuraminidase (NA) with other influenza viruses by performing phylogenetic, comparative and statistical analyses. Phylogenetic trees of these two antigens show that the sequences of the NIV viruses are relatively homogeneous and these were derived from several viruses circulating in swine. The phylogenetic tree of HA shows that NIV had the closest relationship with North-American pig lineages whereas NA had with European pig lineages. In both segments, NIVs had the closest genetic relationship with swine influenza virus lineages. It strongly suggests that pigs are the most possible animal reservoir. Comparative analysis shows that among clade A, NIVs had very low genetic divergence as well as high similarity and also suffered strong purifying selection whereas neighbor clade B shows moderate values when compared to those of clades C-F. It indicates that classical swine influenza viruses present in clade B might be an ancestor of NIVs external antigens. The process of re-assortment occurred in classical swine influenza viruses. The mutation sites exclusively fixed in the NIV of swine and human along with vaccine strain provide an important suggestion for disease diagnosis and vaccine research.

Reassortants of pandemic influenza A virus H1N1/2009 and endemic porcine HxN2 viruses emerge in swine populations in Germany

The incursion of the human pandemic influenza A virus H1N1 (2009) (H1N1 pdm) into pig populations and its ongoing co-circulation with endemic swine influenza viruses (SIVs) has yielded distinct human-porcine reassortant virus lineages. The haemagglutinin (HA) gene of H1N1 pdm was detected in 41 influenza virus-positive samples from seven swine herds in north-west Germany in 2011. Eight of these samples yielded virus that carried SIV-derived neuraminidase N2 of three different porcine lineages in an H1N1 pdm backbone. The HA sequences of these viruses clustered in two distinct groups and were distinguishable from human and other porcine H1 pdm by a unique set of eight non-synonymous mutations. In contrast to the human population, where H1N1 pdm replaced seasonal H1N1, this virus seems to co-circulate and interact more intensely with endemic SIV lineages, giving rise to reassortants with as-yet-unknown biological properties and undetermined risks for public health.

Tissue Tropism of Swine Influenza Viruses and Reassortants in Ex Vivo Cultures of the Human Respiratory Tract and Conjunctiva

The 2009 pandemic influenza H1N1 (H1N1pdm) virus was generated by reassortment of swine influenza viruses of different lineages. This was the first influenza pandemic to emerge in over 4 decades and the first to occur after the realization that influenza pandemics arise from influenza viruses of animals. In order to understand the biological determinants of pandemic emergence, it is relevant to compare the tropism of different lineages of swine influenza viruses and reassortants derived from them with that of 2009 pandemic H1N1 (H1N1pdm) and seasonal influenza H1N1 viruses in ex vivo cultures of the human nasopharynx, bronchus, alveoli, and conjunctiva. We hypothesized that virus which can transmit efficiently between humans replicated well in the human upper airways. As previously reported, H1N1pdm and seasonal H1N1 viruses replicated efficiently in the nasopharyngeal, bronchial, and alveolar epithelium. In contrast, representative viruses from the classical swine (CS) (H1N1) lineage could not infect human respiratory epithelium; Eurasian avian-like swine (EA) (H1N1) viruses only infected alveolar epithelium and North American triple-reassortant (TRIG) viruses only infected the bronchial epithelium albeit inefficiently. Interestingly, a naturally occurring triple-reassortant swine virus, A/SW/HK/915/04 (H1N2), with a matrix gene segment of EA swine derivation (i.e., differing from H1N1pdm only in lacking a neuraminidase [NA] gene of EA derivation) readily infected and replicated in human nasopharyngeal and bronchial epithelia but not in the lung. A recombinant sw915 with the NA from H1N1pdm retained its tropism for the bronchus and acquired additional replication competence for alveolar epithelium. In contrast to H1N1pdm, none of the swine viruses tested nor seasonal H1N1 had tropism in human conjunctiva. Recombinant viruses generated by swapping the surface proteins (hemagglutinin and NA) of H1N1pdm and seasonal H1N1 virus demonstrated that these two gene segments together are key determinants of conjunctival tropism. Overall, these findings suggest that ex vivo cultures of the human respiratory tract provide a useful biological model for assessing the human health risk of swine influenza viruses.

Molecular epidemiology of novel swine origin influenza virus (S-OIV) from Gwalior, India, 2009

BackgroundThe H1N1pandemic virus is a newly emergent human influenza A virus that is closely related to a number of currently circulating pig viruses in the 'classic North American' and 'Eurasian' swine influenza virus lineages and thus referred as S-OIV. Since the first reports of the virus in humans in April 2009, H1N1 virus has spread to 168 countries and overseas territories. India also witnessed severe H1N1 pandemic virus epidemic with considerable morbidity and mortality in different parts starting from May 2009.FindingsThe suspected swine flu outbreak from Gwalior India during October- December 2009 was confirmed through S-OIV HA gene specific RT-LAMP and real time RT-PCR. Positive samples through CDC real time and Lamp assay were further processed for isolation of the virus. Full HA gene sequencing of the H1N1 isolates of Gwalior, India revealed 99% homology with California and other circulating novel swine flu viruses. Three major changes were observed at nucleotide level, while two major amino acid shifts were observed at the position C9W and I30M corresponding to the ORF with prototype strain. The HA gene sequence phylogeny revealed the circulation of two genetically distinct lineages belonging to Clade VII and Clade I of S-OIV.ConclusionsOur findings also supported the earlier report about circulation of mixed genogroups of S-OIV in India. Therefore continuous monitoring of the genetic makeup of this newly emergent virus is essential to understand its evolution within the country.

Swine influenza virus A (H3N2) infection in human, Kansas, USA, 2009.

Swine influenza virus A (H3N2) infection in human, Kansas, USA, 2009.

Genetic correlation between current circulating H1N1 swine and human influenza viruses

Background H1N1 is the main subtype influenza A virus circulating in human and swine population, and has long been a threat to economy and public health. Objective To explore the genetic correlation between current circulating H1N1 swine and human influenza viruses. Study design Three new H1N1 swine influenza viruses (SIVs) were isolated and genomes sequencing were conducted followed by phylogenetic and molecular analysis of all swine and human H1N1 influenza viruses isolated in China in the past five years. Results Homology and phylogenetic analysis revealed that the three isolates possessed different characteristics: the genome of A/Swine/Shandong/1112/2008 was closely related to that of classical H1N1 SIV, while A/Swine/Shandong/1123/2008 was a reassortant with NS gene from the human-like H3N2 influenza virus and other genes from the classical H1N1 SIV, and A/Swine/Fujian/0325/2008 fell into a lineage of seasonal human H1N1 influenza viruses. Genetically, 2009 H1N1 influenza A viruses (2009 H1N1) in China were contiguous to the SIV lineages rather than the seasonal H1N1 human influenza virus's lineage. Furthermore, molecular analysis among human and swine influenza viruses provided more detail information for understanding their genetic correlation. Conclusions These results suggested that in China in the past five years, the classical, avian-like and human-like H1N1 SIV existed in swine herds and the reassortment between H1N1 swine and H3N2 human influenza viruses was identified. In addition, the present data showed no evidence to support a strong correlation between the 2009 H1N1 and the swine influenza virus circulating in China.

Establishment of a multiplex RT-PCR assay to detect different lineages of swine H1 and H3 influenza A viruses

Classical swine H1N1, emerging European avian-like H1N1 and human-like H3N2 lineages are co-circulating in the swine population in China. The reverse transcriptase polymerase chain reaction (RT-PCR) assay is an effective method for use in influenza surveillance. In this study, a multiplex RT-PCR method was developed for simultaneous identification of hemagglutinin (HA) genes derived from the three lineages of swine influenza viruses. Three primer sets were designed and aimed specifically at HA genes of these viral lineages. The specificity of the assay showed that the established methods could efficiently differentiate the HA genes of classical swine H1N1, European avian-like H1N1, and human-like H3N2 viruses while other viruses such as classical swine fever virus, porcine reproductive and respiratory syndrome virus, pseudorabies virus, and porcine circovirus type 2, could not be detected. The assay showed a sensitivity of 1 x 10(2.5) 50% egg infectious dose for each virus lineage. The comparison of the results with those obtained from the analysis of 300 swine tracheal swab samples by means of virus isolation showed a high level of agreement. This multiplex RT-PCR method provides a rapid and specific swine influenza diagnostic tool that also has the potential for investigating the epidemiology of different lineages of swine influenza virus prevalent currently in China.

Prevalence of antibodies to swine influenza viruses in humans with occupational exposure to pigs, Thuringia, Germany, 2008–2009

The Eurasian lineages of swine influenza viruses are different genetically from classical swine H1N1 influenza viruses and comprise avian-like H1N1 and human-like H1N2 and H3N2 subtypes. Although sporadic isolation of such viruses from human specimens has been reported, the prevalence of human infections is not known. In the present study, the seroprevalence against Eurasian swine influenza viruses was investigated. Sera were collected in Thuringia, Germany, from December 2007 to April 2009. The study group comprised 118 professionals with occupational exposure to pigs (50 pig slaughterers/meat inspectors, 46 pig farmers, 22 veterinarians caring for pig herds). The control group included 118 age- and gender-matched blood donors from Thuringia. As a result, 18 sera of the study group were identified with raised hemagglutination-inhibition titers against a panel of nine swine influenza viruses (three strains/ subtype). For 17/18 sera this finding was confirmed in the neutralization assay. For 11/18 sera the raise of titers was significant, that is, a fourfold increase of hemagglutination-inhibition titers was observed. No gender-specific bias of the high titer sera was observed. Twelve sera of the control group showed increased hemagglutination-inhibition titers against swine influenza viruses. Hemagglutination-inhibition titers of 2/12 control sera were raised fourfold but did not exhibit a significant increase of neutralization titers. All increased hemagglutination-inhibition titers of the control group may be explained by cross-reactivity with seasonal influenza virus strains, as all these sera also reacted with human strains.

First whole genome characterization of swine influenza virus subtype H3N2 in Thailand

H3N2 swine influenza viruses (SIV) were first detected in Asia shortly after the 1968 pandemic emerged in humans. Subsequently, human H3N2 viruses have sporadically reappeared in swine. In Thailand, a human-like H3N2 SIV was reported in 1978 although the genetic sequence of this virus is unknown. In this study, we undertook cross sectional syndromic surveillance in pigs in four provinces in Thailand. Seven genetically similar H3N2 viruses were isolated. A representative, A/SW/Thailand/KU5.1/04, was fully sequenced and shown to contain genes from human-like influenza viruses and North American and European SIV. The results restate that transmission of influenza A virus among human and swine populations is common and that genes from both American and Eurasian SIV lineages cocirculate in Thailand.

Absence of 2009 Pandemic H1N1 Influenza A Virus in Fresh Pork

The emergence of the pandemic 2009 H1N1 influenza A virus in humans and subsequent discovery that it was of swine influenza virus lineages raised concern over the safety of pork. Pigs experimentally infected with pandemic 2009 H1N1 influenza A virus developed respiratory disease; however, there was no evidence for systemic disease to suggest that pork from pigs infected with H1N1 influenza would contain infectious virus. These findings support the WHO recommendation that pork harvested from pandemic influenza A H1N1 infected swine is safe to consume when following standard meat hygiene practices.

Origin and future distribution of the new A (H1N1) influenza virus emerging in North America in 2009

The origin of the new A (H1N1) influenza virus recently emerging in North America is a hot controversial topic of significance in disease control and risk assessment. Some experts claimed that it was an unusually mongrelized mix of human, avian and swine influenza viruses, while some others concluded that it was totally a simple re-assortment hybrid of two lineages of swine influenza viruses. Here the phylogenetic diversity of the viral PB1, PA and PB2 gene sequences using online web servers, and the results suggest that all the 8 genetic segments of the new virus were possibly from two lineages of swine influenza viruses, and one of the lineage was a mongrelized mix of human, avian and swine influenza viruses emerging in the world approximately 10 years ago. Considering the recent epidemiological trends of the new virus, we believe it will spread more widely in the world and persist long in human populations. It also could spread among swine populations. The future wide spreading of the new virus may coincide the disappearance of a subtype of previous human influenza A virus.