Swine Influenza Virus Surveillance Research Articles

Swine Influenza Viruses Isolated from 2019 to 2022 in Shandong Province, China, Exemplify the Dominant Genotype.

Swine influenza viruses (SIVs) have been circulating in swine globally and are potential threats to human health. During the surveillance of SIVs in Shandong Province, China, from 2019 to 2022, 21 reassortant G4 genotype Eurasian avian-like (EA) H1N1 subtypes containing genes from the EA H1N1 (HA and NA), 2009 pandemic (pdm/09) H1N1 virus (PB2, PB1, PA, NP, and M), and classical swine (CS) H1N1 (NS) lineages were isolated. The analysis of the key functional amino acid sites in the isolated viruses showed that two mutation sites (190D and 225E) that preferentially bind to the human α2-6 sialic acid receptor were found in HA. In PB2, three mutation sites (271A, 590S, and 591R) that may increase mammalian fitness and a mutation site (431M) that increases pathogenicity in mice were found. A typical human signature marker that may promote infection in humans, 357K, was found in NP. The viruses could replicate efficiently in mouse lungs and turbinates, and one of the H1N1 isolates could replicate in mouse kidneys and brains without prior adaption, which indicates that the viruses potentially pose a threat to human health. Histopathological results showed that the isolated viruses caused typical bronchopneumonia and encephalitis in mice. The results indicate that G4 genotype H1N1 has potential transmissibility to humans, and surveillance should be enhanced, which could provide important information for assessing the pandemic potential of the viruses.

Antigenic and Genetic Characterization of Swine Influenza Viruses Identified in the European Region of Russia, 2014-2020.

Pigs have long been recognized as “mixing vessels” in which new viruses are formed by reassortment involving various influenza virus lineages (avian, animal, human). However, surveillance of swine influenza viruses only gained real significance after the 2009 pandemic. A fundamentally important point is the fact that there is still no regular surveillance of swine flu in Russia, and the role of swine viruses is underestimated since, as a rule, they do not cause serious disease in animals. Since the pig population in Russia is large, it is obvious that the lack of monitoring and insufficient study of swine influenza evolution constitutes a gap in animal influenza surveillance, not only for Russia, but globally. A 6 year joint effort enabled identification of SIV subtypes that circulate in the pig population of Russia’s European geographic region. The swine influenza viruses isolated were antigenically and genetically diverse. Some were similar to human influenza viruses of A(H1N1)pdm09 and A(H3N2) subtype, while others were reassortant A(H1pdm09N2) and A(H1avN2) and were antigenically distinct from human H1N1 and H1N1pdm09 strains. Analysis of swine serum samples collected throughout the seasons showed that the number of sera positive for influenza viruses has increased in recent years. This indicates that swine populations are highly susceptible to infection with human influenza viruses. It also stresses the need for regular SIV surveillance, monitoring of viral evolution, and strengthening of pandemic preparedness.

Molecular epidemiology and glycomics of swine influenza viruses circulating in commercial swine farms in the southeastern and midwest United States

Tracking the genetic diversity and spread of swine influenza viruses (SIVs) in commercial swine farms is central for control and to reduce the potential emergence of SIV reassortants. We analyzed the diversity of SIVs in nasal washes or oral fluids from commercial swine farms in North Carolina using influenza M qRT-PCR and hemagglutinin (HA) and neuraminidase (NA) subtyping. We found a predominance of H1 HAs and N2 NAs in the samples examined. The majority of the H1 HAs could be further classified into gamma and delta subclusters. We also identified HAs of the H1 alpha cluster, and those of human novel pandemic origin. Glycan binding profiles from a representative subset of these viruses revealed broad α2,6 sialylated glycan recognition, though some strains exhibited the ability to bind to α2,3 sialic acid. These data show that SIV surveillance can aid our understanding of viral transmission dynamics and help uncover the diversity at the human-swine interface.

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.

Swine influenza viruses in Northern Vietnam in 2013–2014

Swine are an important intermediate host for emergence of pandemic influenza. Vietnam is the largest swine producer in South East Asia. Systematic virological and serological surveillance of swine influenza viruses was carried out in Northern Vietnam from May 2013 to June 2014 with monthly sampling of pigs in local and large collective slaughterhouses and in a live pig market. Influenza A seroprevalence in the local slaughterhouses and in the large collective slaughterhouse was 48.7% and 29.1%, respectively. Seventy-seven influenza A viruses were isolated, all from the large collective slaughterhouse. Genetic analysis revealed six virus genotypes including H1N1 2009 pandemic (H1N1pdm09) viruses, H1N2 with H1 of human origin, H3N2 and H1N1pdm09 reassortants, and triple-reassortant H3N2 viruses. Phylogenetic analysis of swine and human H1N1pdm09 viruses showed evidence of repeated spill-over from humans to swine rather than the establishment of H1N1pdm09 as long-term distinct lineage in swine. Surveillance at the large collective slaughterhouse proved to be the most efficient, cost-effective, and sustainable method of surveillance for swine influenza viruses in Vietnam.

Subtyping of Swine Influenza Viruses Using a High-Throughput Real-Time PCR Platform.

Influenza A viruses (IAVs) are important human and animal pathogens with high impact on human and animal health. In Denmark, a passive surveillance program for IAV in pigs has been performed since 2011, where screening tests and subsequent subtyping are performed by reverse transcription quantitative real-time PCR (RT-qPCR). A disadvantage of the current subtyping system is that several assays are needed to cover the wide range of circulating subtypes, which makes the system expensive and time-consuming. Therefore, the aim of the present study was to develop a high-throughput method, which could improve surveillance of swine influenza viruses (swIAVs) and lower the costs of virus subtyping. Twelve qPCR assays specific for various hemagglutinin and neuraminidase gene lineages relevant for swIAV and six assays specific for the internal genes of IAV were developed and optimized for the high-throughput qPCR platform BioMark (Fluidigm). The qPCR assays were validated and optimized to run under the same reaction conditions using a 48.48 dynamic array (48.48DA). The sensitivity and specificity was assessed by testing virus isolates and field samples with known subtypes. The results revealed a performance of the swIAV 48.48DA similar to conventional real-time analysis, and furthermore, the specificity of swIAV 48.48DA was very high and without cross reactions between the assays. This high-throughput system provides a cost-effective alternative for subtyping of swIAVs.

Distribution of gene segments of the pandemic A(H1N1) 2009 virus lineage in pig populations.

Swine influenza viruses (SIVs) are important not only for pig farming, but also for public health. In fact, pandemic A(H1N1) 2009 viruses [A(H1N1)pdm09] were derived from SIVs. Therefore, timely characterization of locally circulating SIVs is necessary for understanding the global status of SIVs. To genetically characterize SIVs circulating in Japanese pig populations, we isolated 24 SIVs of three subtypes (17 H1N1, four H1N2 and three H3N2 strains) from 14 pig farms in Japan from 2013 to 2016. Genetic analyses revealed that the haemagglutinin (HA) and neuraminidase (NA) genes of the 17 H1N1 and the HA gene of one H1N2, A/swine/Aichi/02/2016 (H1N2), SIVs belonged to the A(H1N1)pdm09 lineage. More importantly, all of the remaining six gene segments (i.e., PB1, PB1, PA, NP, M and NS) of the 24 SIVs, regardless of the HA and NA subtype, were also classified as belonging to the A(H1N1)pdm09 lineage. These results indicate that gene segments of A(H1N1)pdm09 lineage are widely distributed in SIVs circulating in Japanese pig populations In addition, the NA gene of A/swine/Aichi/02/2016 (H1N2) shared less than 88.5% nucleotide identity with that of the closest relative A/swine/Miyagi/5/2003 (H1N2), which was isolated in Japan in 2003. These results indicate the sustained circulation of classical H1N2-derived SIVs with remarkable diversity in the NA genes in Japanese pig populations. These findings highlight the necessity of both intensive biosecurity systems and active SIV surveillance in pig populations worldwide for both animal and public health.

Molecular evolution of H1N1 swine influenza in Guangdong, China, 2016–2017

Swine are the main host of the H1N1 swine influenza virus (SIV), however, H1N1 can also infect humans and occasionally cause serious respiratory disease. To trace the evolution of the SIV in Guangdong, China, we performed an epidemic investigation during the period of 2016–2017. Nine H1N1 influenza viruses were isolated from swine nasal swabs. Antigenic analysis revealed that these viruses belonged to two distinct antigenic groups, represented by A/Swine/Guangdong/101/2016 and A/Swine/Guangdong/52/2017. Additionally, three genotypes, known as GD52/17-like, GD493/17-like and GD101/16-like, were identified by phylogenetic analysis. Importantly, the genotypes including a minimum of 4 pdm/09-origin internal genes have become prevalent in China in recent years. A total of 2966 swine serum samples were used to perform hemagglutination inhibition (HI) tests, and the results showed that the seroprevalence values of SW/GD/101/16 (32.2% in 2016, 32.1% in 2017) were significantly higher than the seroprevalence values of SW/GD/52/17 (18.0% in 2016, 16.7% in 2017). Our study showed that the three reassortant genotypes of H1N1 SIV currently circulating in China are stable, but H1N1pdm09 poses challenges to human health by the introduction of internal genes into these reassortant genotypes. Strengthening SIV surveillance is therefore critical for SIV control and minimizing its potential threat to public health.

Rapid detection and subtyping of European swine influenza viruses in porcine clinical samples by haemagglutinin- and neuraminidase-specific tetra- and triplex real-time RT-PCRs.

BackgroundA diversifying pool of mammalian‐adapted influenza A viruses (IAV) with largely unknown zoonotic potential is maintained in domestic swine populations worldwide. The most recent human influenza pandemic in 2009 was caused by a virus with genes originating from IAV isolated from swine. Swine influenza viruses (SIV) are widespread in European domestic pig populations and evolve dynamically. Knowledge regarding occurrence, spread and evolution of potentially zoonotic SIV in Europe is poorly understood.ObjectivesEfficient SIV surveillance programmes depend on sensitive and specific diagnostic methods which allow for cost‐effective large‐scale analysis.MethodsNew SIV haemagglutinin (HA) and neuraminidase (NA) subtype‐ and lineage‐specific multiplex real‐time RT‐PCRs (RT‐qPCR) have been developed and validated with reference virus isolates and clinical samples.ResultsA diagnostic algorithm is proposed for the combined detection in clinical samples and subtyping of SIV strains currently circulating in Europe that is based on a generic, M‐gene‐specific influenza A virus RT‐qPCR. In a second step, positive samples are examined by tetraplex HA‐ and triplex NA‐specific RT‐qPCRs to differentiate the porcine subtypes H1, H3, N1 and N2. Within the HA subtype H1, lineages “av” (European avian‐derived), “hu” (European human‐derived) and “pdm” (human pandemic A/H1N1, 2009) are distinguished by RT‐qPCRs, and within the NA subtype N1, lineage “pdm” is differentiated. An RT‐PCR amplicon Sanger sequencing method of small fragments of the HA and NA genes is also proposed to safeguard against failure of multiplex RT‐qPCR subtyping.ConclusionsThese new multiplex RT‐qPCR assays provide adequate tools for sustained SIV monitoring programmes in Europe.

A Novel H1N2 Influenza Virus Related to the Classical and Human Influenza Viruses from Pigs in Southern China.

Southern China has long been considered to be an epicenter of pandemic influenza viruses. The special environment, breeding mode, and lifestyle in southern China provides more chances for wild aquatic birds, domestic poultry, pigs, and humans to be in contact. This creates the opportunity for interspecies transmission and generation of new influenza viruses. In this study, we reported a novel reassortant H1N2 influenza virus from pigs in southern China. According to the phylogenetic trees and homology of the nucleotide sequence, the virus was confirmed to be a novel triple-reassortant H1N2 virus containing genes from classical swine (PB2, PB1, HA, NP, and NS genes), triple-reassortant swine (PA and M genes), and recent human (NA gene) lineages. It indicated that the novel reassortment virus among human and swine influenza viruses occurred in pigs in southern China. The isolation of the novel reassortant H1N2 influenza viruses provides further evidence that pigs are “mixing vessels,” and swine influenza virus surveillance in southern China will provide important information about genetic evaluation and antigenic variation of swine influenza virus to formulate the prevention and control measures for the viruses.

Determination of current reference viruses for serological study of swine influenza viruses after the introduction of pandemic 2009 H1N1 (pdmH1N1) in Thailand

Since the introduction of pandemic H1N1 2009 virus (pdmH1N1) in pigs, the status of Thai swine influenza virus (SIV) has changed. The pdmH1N1 and its reassortant viruses have become the predominant strain circulating in the Thai swine population based on the surveillance data from 2012 to 2014. For this reason, the reference viruses for serological assays using the hemagglutination inhibition (HI) test needed to be updated. Six anti-sera against reference viruses from 2006 to 2009 (enH1N1-06, enH1N1-09, enH1N2-09, pdmH1N1-09, enH3N2-07 and enH3N2-09) were used for the HI test with four contemporary viruses (enH1N1-10, pdmH1N1-10, rH1N2 and rH3N2) and the selected reference viruses were tested with sera collected from the field to determine the current SIV status. The results showed that anti-sera of swH1N1-06 had the highest titers against enH1N1-10. Anti-sera of pdmH1N1-09 had the highest titers against pdmH1N1-10 and rH1N2, whereas, anti-sera of enH3N2-09 had the highest titers against rH3N2. The results demonstrated that enH1N1-06, pdmH1N1-09 and enH3N2-09 should be selected as reference viruses for contemporary serological studies and HI tests. The seroprevalence results from 410 samples revealed enH1N1 (37.79%), pdmH1N1 (37.32%) and H3N2 (35.86%), respectively. The present study indicated that pdmH1N1 was widespread and commonly found in the Thai pig population increasing the risk of novel reassortant viruses and should be added as a reference virus for HI test. SIV surveillance program and serological studies should be conducted for the benefits of SIV control and prevention as well as monitoring for zoonotic potential.

Novel triple-reassortant H1N1 swine influenza viruses in pigs in Tianjin, Northern China

Pigs are susceptible to both human and avian influenza viruses and therefore have been proposed to be mixing vessels for the generation of pandemic influenza viruses through reassortment. In this study, for the first time, we report the isolation and genetic analyses of three novel triple-reassortant H1N1 swine influenza viruses from pigs in Tianjin, Northern China. Phylogenetic analysis showed that these novel viruses contained genes from the 2009 pandemic H1N1 (PB2, PB1, PA and NP), Eurasian swine (HA, NA and M) and triple-reassortant swine (NS) lineages. This indicated that the reassortment among the 2009 pandemic H1N1, Eurasian swine and triple-reassortant swine influenza viruses had taken place in pigs in Tianjin and resulted in the generation of new viruses. Furthermore, three human-like H1N1, two classical swine H1N1 and two Eurasian swine H1N1 viruses were also isolated during the swine influenza virus surveillance from 2009 to 2013, which indicated that multiple genetic lineages of swine H1N1 viruses were co-circulating in the swine population in Tianjin, China. The emergence of novel triple-reassortant H1N1 swine influenza viruses may be a potential threat to human health and emphasizes the importance of further continuous surveillance.

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.

European Surveillance Network for Influenza in Pigs: Surveillance Programs, Diagnostic Tools and Swine Influenza Virus Subtypes Identified in 14 European Countries from 2010 to 2013

Swine influenza causes concern for global veterinary and public health officials. In continuing two previous networks that initiated the surveillance of swine influenza viruses (SIVs) circulating in European pigs between 2001 and 2008, a third European Surveillance Network for Influenza in Pigs (ESNIP3, 2010–2013) aimed to expand widely the knowledge of the epidemiology of European SIVs. ESNIP3 stimulated programs of harmonized SIV surveillance in European countries and supported the coordination of appropriate diagnostic tools and subtyping methods. Thus, an extensive virological monitoring, mainly conducted through passive surveillance programs, resulted in the examination of more than 9 000 herds in 17 countries. Influenza A viruses were detected in 31% of herds examined from which 1887 viruses were preliminary characterized. The dominating subtypes were the three European enzootic SIVs: avian-like swine H1N1 (53.6%), human-like reassortant swine H1N2 (13%) and human-like reassortant swine H3N2 (9.1%), as well as pandemic A/H1N1 2009 (H1N1pdm) virus (10.3%). Viruses from these four lineages co-circulated in several countries but with very different relative levels of incidence. For instance, the H3N2 subtype was not detected at all in some geographic areas whereas it was still prevalent in other parts of Europe. Interestingly, H3N2-free areas were those that exhibited highest frequencies of circulating H1N2 viruses. H1N1pdm viruses were isolated at an increasing incidence in some countries from 2010 to 2013, indicating that this subtype has become established in the European pig population. Finally, 13.9% of the viruses represented reassortants between these four lineages, especially between previous enzootic SIVs and H1N1pdm. These novel viruses were detected at the same time in several countries, with increasing prevalence. Some of them might become established in pig herds, causing implications for zoonotic infections.

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.

Editorial Commentary: Variant Influenza A(H3N2) Virus: Looking Through a Glass, Darkly

First detected in July 2011 [1], the influenza A(H3N2) variant virus (H3N2v), a swine-like progeny virus from the 2009 pandemic H1N1 virus, has caused considerable public health alarm. Most children <10 years of age are thought to be susceptible [2], and evidence of limited H3N2v person-to-person transmission has been reported [1]. Soon after its discovery, public health officials became quite concerned regarding this novel viral threat. In November 2011, the World Health Organization released a virus construct A/Minnesota/11/2010 for vaccine development. In August 2012, the US Centers for Disease Control and Prevention released special H3N2v diagnostic and agricultural fair guidance. In this issue of Clinical Infectious Diseases, Jhung et al [3] summarize the largest human H3N2v outbreak to date (306 cases in 10 states), clearly associating it with pig exposure at agricultural fairs. The authors’ case identification methods and exposure assessments were sound and a number of their findings were quite remarkable: Among the infected persons, 82% were ≤11 years of age, 95% had recent swine exposure, the estimated incubation period was 2.9 days after pig exposure, 16 patients were hospitalized (1 patient died), and 15 likely acquired H3N2v from another human. Although agricultural fairs have been previously shown to be a risk factor for swine influenza virus (SIV) infections in humans [4–8], never before have the epidemiological data been so strong and geographically widespread. The legion of authors in this paper demonstrates the huge interagency effort that was required to gain such a comprehensive view. The authors are to be commended for this important work. However, it seems rather tragic that after the 2009 H1N1 pandemic and this large outbreak, we still do not have a clear view of the dynamic ecology of SIV-like viruses that are freely circulating between swine and human populations. Instead we must often wait for humans to serve as sentinels through their infection with such novel viruses. Although novel SIV viruses are reportable when detected in humans or in pigs, there is sparse testing of healthy pigs for novel SIV; when influenza testing does occur, swine farmers’ participation in the US Department of Agriculture’s influenza surveillance programs is for the most part voluntary. Why should the swine industry pay for SIV surveillance and virus characterization among pigs, when pigs often carry influenza A viruses without clinical signs [8] and the viruses are not transmitted to humans via meat products? Unless a novel virus emerges to cause significant morbidity in pigs, and that virus is characterized, the novel virus is not likely to be discovered and reported by veterinary professionals. Lack of SIV ecologic data is also due in part to concerns that research findings might cause economic damage to the pork industries. Why should pork farmers permit researchers to study SIV transmission in their farms when such research could lead to discoveries that would hamper the sale of a farmer’s pigs or require expensive renovations to swine barns? Hence, although we clearly see that novel SIV human infections can be numerous and widespread and that these crossspecies influenza A virus jumps can occur at agriculture fairs or among individuals occupationally exposed to pigs [9–11], we still do not yet know how to effectively mitigate this risk. It seems to these authors that what we must do is engage in multidisciplinary research to prospectively study the large commercial and small hobby farms that Received 12 September 2013; accepted 15 September 2013; electronically published 24 September 2013. Correspondence: Gregory C. Gray, MD, MPH, FIDSA, Department of Environmental and Global Health, College of Public Health and Health Professions, University of Florida, Box 100188, Gainesville, FL 32610 (gcgray@phhp.ufl.edu). Clinical Infectious Diseases 2013;57(12):1713–4 © The Author 2013. Published by Oxford University Press on behalf of the Infectious Diseases Society of America. All rights reserved. For Permissions, please e-mail: journals. permissions@oup.com. DOI: 10.1093/cid/cit650

Serological surveillance of H5 and H9 avian influenza A viral infections among pigs in southern China

Pigs are susceptible to both human and avian influenza viruses (AIV). Moreover, they are suspected of being the intermediate hosts or mixing vessels of pandemic influenza viruses. Researchers suspect that the influenza viruses are able to undergo reassortment or to adapt to various mammalian hosts while they incubate in pigs. For the present report, we conducted a serological surveillance of pigs in southern China from 2008 to 2012 to establish the prevalence of antibodies against H5N1 and H9N2 AIV. A total of one hundred pig farms from the Guangdong, Zhejiang, Fujian, and Yunnan Provinces were sampled, yielding a total of 3960 serum specimens. The haemagglutination inhibition (HI) tests revealed no evidence of H5 infection when the Clade 2.3.2 virus was used as the antigen, but a 4.6% positive rate of H9 infection was observed when using the Beijing/1/94-like virus as the antigen. The positive sera for H9 infection were further verified with neutralization tests, which confirmed a 3.7% rate of positive sera of H9 infection. In summary, the results imply that the swine populations in southern China had not been affected greatly by the H5N1 avian influenza virus. Nevertheless, these swine H9N2 influenza viruses might pose a threat to human health, and so researchers should continue to carry out swine influenza virus surveillance in China.

Imported pigs may have introduced the first classical swine influenza viruses into Mainland China

ObjectivesThe first classical swine influenza A H1N1 viruses were isolated in Mainland China in 1991. To aid surveillance of swine influenza viruses as part of pandemic preparedness, we sought to identify their origin. MethodsWe sequenced and phylogenically analyzed 19 swine influenza viruses isolated in 1991 and 1992 in China and compared them with viruses isolated from other regions during the same period. ResultsAll 19 swine influenza viruses analyzed in our study shared the highest similarity with the classical swine influenza virus A/Swine/Maryland/23239/1991 (H1N1). Phylogenetic trees of eight segmented genes exhibited similar topology, with all segments in the cluster of classical swine influenza viruses. In addition, antigenic analysis also indicated that the tested isolated were related to classical swine influenza isolates. ConclusionsClassical swine H1N1 influenza viruses were predominant in Beijing pig herds during this period. Since both antibody and virus detections did not indicate the presence of CS H1N1 before 1991 in Mainland China, we combined with the data on pigs imported to and exported from China and concluded that these viruses might spread to China via pigs imported from North America and that they could affect the genetic evolution and transmission dynamics of swine influenza viruses in Hong Kong.

Swine influenza virus infection in different age groups of pigs in farrow-to-finish farms in Thailand

BackgroundUnderstanding swine influenza virus (SIV) ecology has become more and more important from both the pig industry and public health points of views. However, the mechanism whereby SIV occurs in pig farms is not well understood. The purpose of this study was to develop a proper strategy for SIV surveillance.FindingsWe conducted longitudinal monitoring in 6 farrow-to-finish farms in the central region of Thailand from 2008 to 2009. Nasal swabs and serum samples were collected periodically from clinically healthy pigs consisting of sows, fattening pigs, weaned piglets and pigs transferred from other farms. A total of 731 nasal swabs were subjected to virus isolation and 641 serum samples were subjected to detection of SIV antibodies against H1 and H3 subtypes using the hemagglutination inhibition test and ELISA. Twelve SIVs were isolated in this study and eleven were from piglets aged 4 and 8 weeks. Phylogenetical analysis revealed that SIVs isolated from different farms shared a common ancestor. Antibodies against SIVs were detected in fattening pigs on farms with no SIV isolation in the respective periods studied. These observations suggested that piglets aged 8 weeks or younger could be a main target for SIV isolation. Farm-to-farm transmission was suggested for farms where pigs from other farms are introduced periodically. In addition, antibodies against SIVs detected in fattening pigs could be a marker for SIV infection in a farm.ConclusionsThe present study provided important information on SIV surveillance that will enable better understanding of SIV ecology in farrow-to-finish farms.

Pandemic (H1N1) 2009 Virus in Swine Herds, China

To the Editor: During March and early April 2009, a new swine-origin influenza A (H1N1) virus emerged in Mexico and the United States; this virus subsequently spread across the globe by human-to-human transmission at an unprecedented rate. Pandemic (H1N1) 2009 virus also affected pigs. On May 2, 2009, the Canadian Food Inspection Agency notified the World Organisation for Animal Health that the novel influenza A virus had been confirmed on a pig farm in Alberta, Canada. Infection of pigs with pandemic (H1N1) 2009 virus has been observed in multiple countries (1). In this study, we report transmission of pandemic (H1N1) 2009 virus from humans to pigs in the People’s Republic of China. During August 2009–April 2010, swine influenza virus surveillance was conducted in the provinces of central and eastern China, including Henan, Hubei, Hunan, Jiangxi, and Anhui. A total of 1,021 samples, comprising tracheal mucus swabs and lungs, from pigs on 30 farms, were collected, and dozens of swine influenza viruses, H1N1, H1N2, H3N8, H9, and H10 subtypes, were isolated. Eight isolates were subtype H1N1, including 4 novel pandemic (H1N1) 2009 viruses: A/swine/Nanchang/3/2010 (H1N1) (GenBank accession nos. {type:entrez-nucleotide,attrs:{text:JF275917,term_id:321496099,term_text:JF275917}}JF275917–24), A/swine/Nanchang/5/2010 (H1N1) (GenBank accession nos. {type:entrez-nucleotide,attrs:{text:JF275933,term_id:321496135,term_text:JF275933}}JF275933–40) and A/swine/Nanchang/6/2010 (H1N1) (GenBank accession nos. {type:entrez-nucleotide,attrs:{text:JF275941,term_id:321496153,term_text:JF275941}}JF275941–48), which were isolated from tracheal mucus, and A/swine/Nanchang/F9/2010 (H1N1) (GenBank accession nos. {type:entrez-nucleotide,attrs:{text:JF275925,term_id:321496117,term_text:JF275925}}JF275925–32), isolated from the lung. Pigs from which the novel viruses were isolated showed mild respiratory signs, including depression, cough, and transient increase in body temperature. Compared with the sequence of A/California/04/2009 (H1N1), genomic sequencing of the 4 pandemic viruses showed 15 common point mutations, such as polymerase basic protein 2, T588I; polymerase acidic protein, A70V, P224S, D547E; hemagglutinin, P100S, D103E, S145P, T214A, S220T, I338V; nucleocapsid protein, V100I, H289Y; neuraminidase, V106I, N248D; and nonstructural protein, I123V. Recent studies have shown that the novel pandemic (H1N1) 2009 human influenza viruses were almost avirulent for mice (50% mouse lethal dose >106 PFU for A/CA/04/09 [2,3]). In this study, mice were anesthetized with ketamine/xylazine, as described (4) and 50 μL of phosphate-buffered saline containing the indicated doses (105 50% egg infectious dose) of the 4 viruses were instilled into anesthetized mice through nostrils. Interestingly, the 4 pandemic (H1N1) 2009 viruses isolated from pigs could cause systemic infection on mice. All mice had extensive loss of body weight, and some of the infected mice died within 2 weeks postinfection (data not shown). To detect whether the 4 isolated pandemic (H1N1) 2009 viruses could cause clinical diseases in pigs and what kinds of pathologic damage could be induced in infected pigs, pigs were intratracheally challenged with the 4 pandemic (H1N1) 2009 viruses at a dose of 107 50% egg infectious dose and monitored for clinical signs and pathologic changes. Clinical signs in pigs were mild, and no deaths occurred. Except for slightly labored breathing, no infected pigs showed dominant signs, such as cough, nasal discharge, facial edema, and dyspnea. Body temperatures of infected pigs started to increase at 2 days postinfection (dpi), peaked ≈3 dpi at 41.5°C, and returned to the initial temperature by 5 dpi. We observed necrosis of the tracheal wall and pneumonia foci in the 4 pandemic (H1N1) 2009 virus–infected pigs. Hematoxylin and eosin–stained sections of the trachea from the infected pigs showed mild necrotizing tracheitis. The necrotic epithelial cells were present in the lumen, and a mixed inflammatory cell infiltrate was present throughout (Figure). In the lung, we also observed alveolar septal edema and interstitial inflammatory cell infiltrates, as well as histologic changes, including alveolar epithelial hyperplasia, a mixed inflammatory infiltrate, and interstitium broadening (Figure). Additionally, immunohistochemical analysis for the distribution of viral antigens showed positive staining in bronchial epithelial cells and alveolar pneumocytes. Figure Hematoxylin and eosin–stained trachea and lung controls and samples from pigs infected with pandemic (H1N1) 2009 virus. A) Control trachea sample; B) mixed inflammatory cell infiltrate present throughout trachea sample from infected pig. C) Control ... Previous studies showed that pandemic (H1N1) 2009 may have become established in swine populations in Canada, Norway, and Hong Kong (1,5–8). The human-to-pig transmission of pandemic (H1N1) 2009 may substantially affect virus evolution and subsequent epidemiology. Although the pandemic was mild, the virus could develop further reassortment in swine and gain virulence. On the other hand, subtype H5N1 and H9N2 viruses have become established in pigs, so the introduction of pandemic (H1N1) 2009 virus to pigs has provided the possibility for the incorporation of avian virus genes into mammalian-adapted viruses. That transmission could occur from humans to pigs and vice versa is especially troublesome. Given the possible production of novel viruses of potential threat to public health, we should emphasize influenza surveillance in pigs and establishment of the genetic basis of the viral genome for rapidly identifying such reassortment events.