Isolation and characterization of two H5N1 influenza viruses from swine in Jiangsu Province of China

Pigs are susceptible to infection with both human and avian influenza A viruses and are considered intermediate hosts that facilitate virus reassortment. Although H5N1 virus has spread to a wide range of avian and mammalian species, data about swine H5N1 isolates are scarce. To determine whether Asian H5N1 influenza viruses had been transmitted to pigs, a total of 1,107 nasal swab samples from healthy swine were collected from 2008 to 2009 in Jiangsu province of eastern China. In this survey, two H5N1 viruses A/swine/Jiangsu/1/2008 (JS/08) and A/swine/Jiangsu/2/2009 (JS/09) were isolated and identified. Phylogenetic analysis showed that JS/08 and JS/09 belonged to clade 7 and clade 2.3.4, respectively, and shared over 99.0 % sequence identity with poultry H5N1 isolates of the same clade in China. Receptor specificity analysis also showed that both of the swine H5N1 isolates bound preferentially to avian-type receptors. However, experiments in mammals indicated that JS/09 was moderately pathogenic to mice without prior adaption, whereas JS/08 had limited ability to replicate. Our findings suggest that pigs are naturally infected with avian H5N1 virus and highlight the potential threat to public health due to adaption or reassortment of H5N1 virus in this species.     

Isolation and phylogenetic analysis of H1N1 swine influenza virus isolated in Korea

A swine influenza H1N1 virus was isolated from a pig during a severe outbreak of respiratory disease in Korea. All genes of the H1N1 isolate, including hemagglutinin (HA), neuraminidase (NA), matrix (M), nucleoprotein (NP), non-structural (NS), PA, PB1 and PB2, were of swine origin. Also, all these genes showed a close phylogenic relationship with those of H1N1 viruses previously isolated from pigs in the United States. These results suggest that North American swine influenza virus has actually been transmitted to pigs in Korea.     

Isolation of two H1N2 influenza viruses from swine in France

Summary Samples collected in 1987 and 1988 in Brittany from influenzainfected swine made it possible to isolate and antigenically characterize two H1N2 recombinant viruses (Sw/France/5027/87 and Sw/France/5550/88). The former virus was cloned and reinoculated to swine to allow reproduction of the disease and reisolation of a strain similar to the original one. The serodiagnostic tests carried out on both the original sera and those from the experimentally infected animals confirmed that the virus was actually type Sw/H1N2.     

Serological profiles after consecutive experimental infections of pigs with European H1N1, H3N2, and H1N2 swine influenza viruses

Swine influenza viruses (SIVs) of H1N1, H3N2, and H1N2 subtypes, with antigenically different hemagglutinins, are currently cocirculating in pigs in Europe. This study aimed to determine whether the hemagglutination inhibition (HI) test, which is the primary serological test for SIV, is sufficiently specific to discriminate between infections with the three subtypes. In experiment 1, pigs were consecutively inoculated with European H1N1, H3N2, and H1N2 SIVs by the intranasal route, or with the respective subtypes only. In a second experiment, a commercial, inactivated H1N1- and H3N2- based SIV vaccine was administered once to pigs previously infected with one to three SIV subtypes or to influenza-naive pigs. Sequential serum samples were examined in HI and virus-neutralizing (VN) tests to the three strains used for pig inoculations. Of the 160 sera collected after infection with one or two SIV subtypes, only 8 showed cross-reactive antibodies to the remaining subtype(s) in the HI test, and 11 in the VN test. Consecutive inoculations with H1N1 and H1N2 or vice versa were followed by a significant rise in preexisting antibody titers to the first subtype after the second inoculation. When dually infection-immune pigs were inoculated with the third, remaining SIV subtype, nasal virus excretion was undetectable or reduced and the serological response was absent to moderate. A single vaccination of infection-immune pigs resulted in a dramatic rise in HI and VN antibody titers to any of the previously encountered subtypes, whereas SIV-naive pigs barely seroconverted. Most important, pigs previously infected with H1N1 but not with H1N2 developed crossreactive antibodies to H1N2 after the vaccination. In conclusion, the HI test remains adequate for the differential diagnosis of infections with H1N1, H3N2, and H1N2 in European swine populations if it is properly used and if the SIV vaccination status is taken into account.     

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

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

Short communication: isolation and phylogenetic analysis of an avian-origin H3N2 canine influenza virus in dog shelter, China

A H3N2 canine influenza virus, A/canine/Guangdong/3/2011 (H3N2), was isolated from roaming dogs in rural China. Sequence and phylogenetic analysis of eight gene segments revealed that the A/canine/Guangdong/3/2011 (H3N2) was most similar to a recent H3N2 canine influenza virus isolated in cats from South Korea, which originated from an avian strain. To our knowledge, this is the first report of an avian-origin H3N2 CIV which was isolated from roaming dogs in China. The epidemiologic information provided herein suggests that continued study is required to determine if this virus could be established in the roaming dog population in rural China and pose potential threats to public health.     

Antigenic variation of H1N1, H1N2 and H3N2 swine influenza viruses in Japan and Vietnam

The antigenicity of the influenza A virus hemagglutinin is responsible for vaccine efficacy in protecting pigs against swine influenza virus (SIV) infection. However, the antigenicity of SIV strains currently circulating in Japan and Vietnam has not been well characterized. We examined the antigenicity of classical H1 SIVs, pandemic A(H1N1)2009 (A(H1N1)pdm09) viruses, and seasonal human-lineage SIVs isolated in Japan and Vietnam. A hemagglutination inhibition (HI) assay was used to determine antigenic differences that differentiate the recent Japanese H1N2 and H3N2 SIVs from the H1N1 and H3N2 domestic vaccine strains. Minor antigenic variation between pig A(H1N1)pdm09 viruses was evident by HI assay using 13 mAbs raised against homologous virus. A Vietnamese H1N2 SIV, whose H1 gene originated from a human strain in the mid-2000s, reacted poorly with post-infection ferret serum against human vaccine strains from 2000-2010. These results provide useful information for selection of optimal strains for SIV vaccine production.     

Two genotypes of H1N2 swine influenza viruses appeared among pigs in China

BackgroundH1N2 is one of the main subtypes of influenza, which circulates in swine all over the world.ObjectivesTo investigate the prevalence and genetic of H1N2 in swine of China.Study designTwo H1N2 swine influenza viruses were isolated from Tianjin and Guangdong province of China in 2004 and 2006, respectively. The molecular evolution of eight gene segments was analyzed.ResultA/Swine/Tianjin/1/2004 has low identity with A/Swine/Guangdong/2006; in the phylogenetic tree of PA gene, A/Swine/Guangdong/1/2006 and A/Swine/Guangxi/1/2006 along with the H1N2 swine isolates of North America formed a cluster; and A/Swine/Tianjin/2004 and A/Swine/Zhejiang/2004, along with the classical H1N1 swine isolates formed another cluster; except that NA gene of A/Swine/Tianjin/1/2004 fell into the cluster of the H3N2 human influenza virus, indicating the reassortment between H3N2 human and H1N1 swine influenza viruses.ConclusionTwo different genotypes of H1N2 appeared among pigs in China. A/swine/Guangdong/1/06 was probably from H1N2 swine influenza viruses of North America; while A/swine/Tianjin/1/04 maybe come from reassortments of classical H1N1 swine and H3N2 human viruses prevalent in North America.     

Pathogenesis and inflammatory responses of swine H1N2 influenza viruses in pigs

Swine influenza viruses are an important pathogen in pig industry. In this study, we wanted to know whether swine H1N2 influenza viruses circulating in Korean pigs would cause clinical signs in pigs when experimentally infected. When pigs were infected with swine H1N2 viruses isolated from Korean pigs, pigs suffered from severe clinical signs of coughing, nasal discharge, labored breathing, facial edema, anorexia, and diarrhea. When the level of cytokine induction was measured using lung tissues, pro-inflammatory cytokines such as TNF-alpha, IL-1, and IL-8 were induced higher in lungs of infected pigs than in lungs of uninfected pigs. However, no increased induction of the anti-inflammatory cytokines such as IL-4 and IL-10 was observed in lungs of infected pigs. These results suggest that the pathogenesis induced in pigs by H1N2 influenza viruses may be induced by pro-inflammatory cytokines instead of anti-inflammatory cytokines.     

Isolation and characterization of novel H3N1 swine influenza viruses from pigs with respiratory diseases in Korea

Pigs can play an important role in the genetic reassortment of influenza viruses and as a reservoir for another lineage of influenza viruses that have the ability to reassort and be transmitted between species. In March and April 2006, novel H3N1 influenza A viruses were isolated from pigs with respiratory diseases at two different commercial swine farms in Korea. Genetic and phylogenetic analyses of the sequences of all eight viral RNA segments showed that the novel H3N1 swine influenza viruses were reassortants that acquired the hemagglutinin gene from an H3 human-like virus and other genes from swine influenza viruses that are currently circulating in Korea. Serologic and virologic tests in the infected farms suggested that pig-to-pig and farm-to-farm transmissions occurred. Clinical signs in pigs and experimentally infected mice suggest the potential to transmit the virus between swine and other mammalian hosts. To our knowledge, this is the first report of the isolation of the swine H3N1 subtype from domestic pigs under field conditions in Korea. Further surveillance will be needed to determine whether this novel subtype will continue to circulate in the swine population.     

Multiplex RT-PCR assay for differentiating European swine influenza virus subtypes H1N1, H1N2 and H3N2

In Europe, three major swine influenza viral (SIV) subtypes (H1N1, H1N2 and H3N2) have been isolated in pigs. Developing a test that is able to detect and identify the subtype of the circulating strain rapidly during an outbreak of respiratory disease in the pig population is of essential importance. This study describes two multiplex RT-PCRs which distinguish the haemagglutinin (HA) gene and the neuraminidase (NA) gene of the three major subtypes of SIV circulating in Europe. The HA PCR was able to identify the lineage (avian or human) of the HA of H1 subtypes. The analytical sensitivity of the test, considered to be unique, was assessed using three reference viruses. The detection limit corresponded to 1×10(-1) TCID(50)/200μl for avian-like H1N1, 1×10(0) TCID(50)/200μl for human-like H1N2 and 1×10(1) TCID(50)/200μl for H3N2 SIV. The multiplex RT-PCR was first carried out on a collection of 70 isolated viruses showing 100% specificity and then on clinical samples, from which viruses had previously been isolated, resulting in an 89% positive specificity of the viral subtype. Finally, the test was able to identify the viral subtype correctly in 56% of influenza A positive samples, from which SIV had not been isolated previously. It was also possible to identify mixed viral infections and the circulation of a reassortant strain before performing genomic studies.     

猪型(H1N1)流感病毒血凝素和神经氨酸酶基因来源的研究

目的 研究2002年我国内地从猪群中分离的猪型(H1N1)毒株HA和NA基因来源。及其使猪致病的原因。方法 用PCR扩增目的基因，用P^GEM-T Easy Vector，4℃过夜连接，重组质粒转入DH-10B细菌，筛选阳性菌落，酶切鉴定，送六合通公司自动测序，并作进化树分析。结果 3株猪型(H1N1)病毒的HA和NA基因属猪型(H1N1)流感病毒，而不同于其他禽或人的H1N1亚型流感病毒。2002年猪型毒株由1991年猪型毒株演变而来。近来我国内地猪群中猪型毒株活动增强，其对猪能致病是由于病毒粒HA和NA蛋白抗原性发生变异所造成。结论 3株猪型病毒的HA和NA基因来源于猪型(H1N1)毒株。近来猪型毒株对猪具有致病性和活动增强是由于其HA和NA蛋白分子上氨基酸序列发生替换所造成。     

Molecular characterization and phylogenetic analysis of H1N1 and H3N2 human influenza A viruses among infants and children in Thailand

The annual influenza outbreaks can cause a high mortality rate among infants and children. In the tropics, influenza shows no clear dependence on seasons. In the present study, we performed molecular and phylogenetic analysis of H1N1 and H3N2 influenza virus isolated from infants and children diagnosed with respiratory tract illness between February 2006 and February 2007. A total of 33 samples (10.92%) were found positive for human influenza virus infection. Characterization of the hemagglutinin gene revealed conserved sequences at the receptor-binding site as well as variations due to amino acid substitutions at the antigenic site, potentially resulting in an N-linked glycosylation site. As for the neuraminidase gene, amino acid substitutions were found in N1 and N2 but not directly at the catalytic or framework sites of this enzyme. Based on the phylogenetic tree, the hemagglutinin 1 (HA1) region and the neuraminidase (NA) gene of both H1N1 and H3N2 isolated subtypes clustered with the current vaccine strain for the Northern Hemisphere 2007-2008. This finding contributes to understanding the evolution of influenza A viruses in humans and is useful for surveillance and vaccine strain selection.     

Sequence and phylogenetic analysis of H7N3 avian influenza viruses isolated from poultry in China in 2011

Four H7N3 avian influenza viruses (AIVs) were isolated from domestic ducks in live-poultry markets in Zhejiang Province, Eastern China, in 2011. All viruses were characterized by whole-genome sequencing with subsequent phylogenetic analysis and genetic comparison. Phylogenetic analysis of all eight viral genes showed that the viruses clustered in the Eurasian lineage of influenza viruses. The hemagglutinin cleavage site of all viruses indicated that the four strains were low-pathogenic avian influenza viruses.     

The first isolation of swine H1N1 influenza viruses from pigs in Thailand

Summary Two influenza A viruses were isolated from pigs in Thailand in January 1988 during the early febrile stage of an influenza-like illness. The isolates contained hemagglutinin and neuraminidase antigens related to those of swine H1N1 influenza virus. This result based on the virus isolation is compatible with the epizootiological evidence that, unlike the human influenza with peak activity in summer (May–July), swine influenza virus is prevalent in the winter season (November–January) in Thailand. The proportion of sera with hemagglutination-inhibiting antibody was higher to A/NJ/8/76 than to A/sw/Iowa/15/30. Likewise, hemagglutination-inhibition tests with monoclonal antibodies indicated that hemagglutinin antigen of the isolates was very similar to that of A/NJ/8/76 virus. In agreement with the serological survey and antigenic characteristic, genetic relatedness between the isolates from Thailand and A/NJ/8/76 virus was also demonstrated by the oligonucleotide mapping of RNA, suggesting that they may be of the same origin.     

Comparative study of the differential susceptibility of different cell lines to pandemic H1N1v influenza viruses and avian influenza, swine influenza, and human influenza viruses

The proliferation characteristics of influenza viruses of different origin were tested in various human and animal cell cultures. Pandemic H1N1v influenza and swine influenza viruses were shown to have a low infectious activity in virtually all the test lines. In spite of this, the replication of this group of viruses may be detected by de novo NP synthesis. These viruses are able to activate programmed cell death. Moreover, a low inoculative virus dose exerts a stimulating effect on cell proliferation in both suspension and monolayer cell lines.     

Classical swine H1N1 influenza viruses confer cross protection from swine-origin 2009 pandemic H1N1 influenza virus infection in mice and ferrets

The hemagglutinin of the 2009 pandemic H1N1 influenza virus is a derivative of and is antigenically related to classical swine but not to seasonal human H1N1 viruses. We compared the A/California/7/2009 (CA/7/09) virus recommended by the WHO as the reference virus for vaccine development, with two classical swine influenza viruses A/swine/Iowa/31 (sw/IA/31) and A/New Jersey/8/1976 (NJ/76) to establish the extent of immunologic cross-reactivity and cross-protection in animal models. Primary infection with 2009 pandemic or NJ/76 viruses elicited antibodies against the CA/7/09 virus and provided complete protection from challenge with this virus in ferrets; the response in mice was variable and conferred partial protection. Although ferrets infected with sw/IA/31 virus developed low titers of cross-neutralizing antibody, they were protected from pulmonary replication of the CA/7/09 virus. The data suggest that prior exposure to antigenically related H1N1 viruses of swine-origin provide some protective immunity against the 2009 pandemic H1N1 virus.     

Identification and genetic characterization of avian-origin H3N2 canine influenza viruses isolated from the Liaoning province of China in 2012

A total of 158 serum samples and 510 nasal swab specimens were collected between September 2010 and May 2012, from dogs exhibiting respiratory symptoms, in order to investigate the epidemiology of H3N2 canine influenza viruses (CIVs) in the Liaoning province of China. Serological surveillance demonstrated that 10.8 % (17/158) of serum samples were positive for H3N2 canine influenza. Two H3N2 influenza viruses, A/canine/Liaoning/27/2012 and A/canine/Liaoning/H6/2012, were isolated from pet dogs in 2012. Phylogenetic analysis indicated that the genes from these two viruses were closely related to those of avian-origin, H3N2 subtype CIVs from China and Thailand. Genetic analysis of eight genes revealed that these two H3N2 canine influenza isolates were highly similar (99.2–99.8 %) to the current common strains in Asia. Analysis of the genotype demonstrated that each gene of the two strains in this study had the same genotype (K, G, E, 3B, F, 2D, F, 1E) as those prevalent in H3N2 CIVs. Our findings further confirm that avian-origin H3N2 canine influenza has become established in China. Conducting extensive serological and epidemiological surveillance is necessary to develop an effective vaccine against this disease.