Influenza A viruses: epidemiologic study in fatteners in Spain (1987-89).

2,979 sera were collected from slaughtered swine in two geographic areas of Spain from 1987 to 1989. They were tested for antibodies against an H1N1- and H3N2-influenza virus by haemagglutination-inhibition tests (HI). The percentage of positive sera was higher in area I (78%-69.2%) than in area II (63.1%-60.4%) for both viruses respectively. The coexistence of high titres to both H1N1- and H3N2-influenza virus became apparent in cold months simultaneously in each area, although influenza viruses circulated in the Spanish swine population for two years. Also this study suggests the possible circulation of A/Texas/1/77-like strains in Spain, results which have not been reported before.     

供港猪群流感病毒的分离鉴定及流行病学调查

为了对供港猪群中的猪流感流行情况进行分析,从华南地区供港猪群中用无菌棉拭子采集鼻腔粘液样品,采用鸡胚接种方法,从供港猪群中分离出了2株不同亚型的猪流感病毒株,经国家流感中心鉴定分别为H1N1和H3N2亚型。本研究设计了猪流感常见亚型的HA和NA分型特异性引物,建立了猪流感型特异性RT-PCR检测方法;对分离鉴定的2株猪流感病毒和禽流感H5N1 HI检测抗原进行了RT-PCR检测,并对其部分HA和NA基因进行克隆测序分析。对供港猪群的血清检测结果表明:供港猪群中H1N1和H3N2亚型抗体阳性率分别为26.87%、38.26%,禽流感H5N1和H9N2亚型抗体阳性率均为0%。     

上海地区猪群中H1N1甲型流感抗体检测

对2009年H1N1甲型流感流行前后的上海地区养殖场户410份猪血清样品,分别采用血凝抑制试验（hemagglutination inhibition,HI）和酶联免疫吸附试验（enzyme-linked immunosorbent assay,ELISA）进行检测H1N1甲型流感病毒和猪流感病毒（Swine in？uenza virus,SIV）。检测结果表明,除2007年外,2008~2010年猪血清中均存在不同水平的HI抗体,阳性率呈显著上升趋势,且抗体水平与猪群饲养周期及饲养密度正相关,而与猪流感病毒的流行无相关性。     

Influenza surveillance in birds in Italian wetlands (1992-1998): is there a host restricted circulation of influenza viruses in sympatric ducks and coots?

We report the results of a 6-year serological and virological monitoring performed in ducks and coots in Italy, in order to assess the degree of influenza A virus circulation in these birds during wintering. A total of 1039 sera collected from 1992 to 1998 was screened by a double antibody sandwich blocking ELISA (NP-ELISA): seroprevalence of antibodies to influenza A viruses was significantly higher in ducks compared to coots (52.2% vs. 7.1%, respectively). The hemagglutination-inhibition (HI) assay, performed on NP-ELISA positive sera, showed that 16.9% of these duck sera and 33.3% of these coot sera had antibodies to at least one influenza virus HA subtype: ducks showed HI antibodies against most of the HA subtypes, except for the H3, H4, H7, and H12; coots were seropositive to the H3 and H10 subtypes, only. From 1993 to 1998, 22 virus strains were obtained from 802 cloacal swabs, with an overall virus isolation frequency of 2.7%. Viruses belonging to the H1N1 subtype were by far the most commonly circulating strains (18/22) and were isolated mainly from ducks (17/18). The remaining viruses were representative of the H10N8, H5N2 and H3N8 subtypes. Our data indicate some differences between influenza A virus circulation in sympatric ducks and coots and a significant antigenic diversity between some reference strains and viruses recently isolated in Italy.     

Seroepidemiological evidence of avian H4, H5, and H9 influenza A virus transmission to pigs in southeastern China

Pig serum samples collected in southeastern China were examined for antibodies to influenza A viruses. Since the hemagglutination inhibition (HI) test does not accurately detect antibodies to the hemagglutinins (HAs) of "avian" influenza viruses, we utilized the neutralization (NT) test to detect subtype-specific antibodies to the HA of avian viruses in pig sera. Neutralizing antibodies to H1, H3, H4, and H5 influenza viruses were detected in the serum samples collected in 1977-1982 and 1998, suggesting that pigs in China have been sporadically infected with avian H4 and H5 viruses in addition to swine and human H1 and H3 viruses. Antibodies to H9 virus, on the other hand, were found only in the sera collected in 1998, not in those collected in 1977-1982, correlating with the recent spread in poultry and subsequent isolation of H9N2 viruses from pigs and humans in 1998. The present results indicate that avian influenza viruses have been transmitted to pig populations in southeastern China.     

猪流感病毒的分离及NA基因的遗传进化分析

通过鸡胚接种分离到3株猪流感病毒,经HI、RT-PCR鉴定,其中GXHZ株为H1N2亚型毒株,GXLZ、GXXY株为H3N2亚型毒株,并对分离株进行EID50测定及GXHZ株的猪体攻毒试验。所扩增分离到3株SIV病毒NA基因与A/Sw/Hainan/1/2005(H1N2)的核苷酸和氨基酸序列同源性最高。3株SIV分离株的NA基因在氨基端胞浆尾区均由K6→R6,在非极性跨膜区均由V20→M20。系统发育分析表明本试验所分离的3个SIV毒株与H1N2亚型毒株亲缘性最相近,且来源于猪源H1N2亚型流感病毒。从时间上也发现,3个分离株的NA基因与1996年以后获得的H3N2毒株亲缘关系很近,都隶属于一个亚分支。     

Experimental infection with H1N1 European swine influenza virus protects pigs from an infection with the 2009 pandemic H1N1 human influenza virus

The recent pandemic caused by human influenza virus A(H1N1) 2009 contains ancestral gene segments from North American and Eurasian swine lineages as well as from avian and human influenza lineages. The emergence of this A(H1N1) 2009 poses a potential global threat for human health and the fact that it can infect other species, like pigs, favours a possible encounter with other influenza viruses circulating in swine herds. In Europe, H1N1, H1N2 and H3N2 subtypes of swine influenza virus currently have a high prevalence in commercial farms. To better assess the risk posed by the A(H1N1) 2009 in the actual situation of swine farms, we sought to analyze whether a previous infection with a circulating European avian-like swine A/Swine/Spain/53207/2004 (H1N1) influenza virus (hereafter referred to as SwH1N1) generated or not cross-protective immunity against a subsequent infection with the new human pandemic A/Catalonia/63/2009 (H1N1) influenza virus (hereafter referred to as pH1N1) 21 days apart. Pigs infected only with pH1N1 had mild to moderate pathological findings, consisting on broncho-interstitial pneumonia. However, pigs inoculated with SwH1N1 virus and subsequently infected with pH1N1 had very mild lung lesions, apparently attributed to the remaining lesions caused by SwH1N1 infection. These later pigs also exhibited boosted levels of specific antibodies. Finally, animals firstly infected with SwH1N1 virus and latter infected with pH1N1 exhibited undetectable viral RNA load in nasal swabs and lungs after challenge with pH1N1, indicating a cross-protective effect between both strains.     

Seroprevalence of porcine and human influenza A virus antibodies in pigs between 1986 and 1988 in Hassia

1,268 sera collected from slaughtered pigs in Hassia (FRG) from 1986 to 1988 were tested for antibodies against porcine and human influenza A virus strains using the single radial haemolysis test (SRHT). Antibodies against the porcine strains (subtype H1N1) A/Swine/Arnsberg/1/81, A/Swine/Iowa/15/30 and A/New Jersey/7/76 were detected in 411 (32.4%), 318 (25.1%) and 304 (24.0%) of sera, respectively. Up to 1988 a slight increase (10%) in the seroprevalence to A/Swine/Arnsberg/1/81 was noticed, whereas the results obtained with the other strains showed little variation. Antibodies against the human H1N1 strain A/Singapore/6/86 were only found in sera collected 1987 and 1988 in rates of 1.6% and 3.0%. Serological indication of infections with the human H3N2 strains A/Victoria/1/75, A/Hong Kong/1/68 and A/Philippines/2/82 could be shown in 286 (22.6%), 178 (14.4%) and 135 (10.6%) of the serum samples. Within the three year period the rate of sera positive for antibodies against A/Philippines/2/82 increased from 6.5% to 23.0%, whereas no variation in the rates were found using the other H3N2 strains. Antibodies simultaneously against porcine (H1N1) and human (H3N2) virus strains were detected in 9.9% of all sera tested.     

Serological evidence of H3N8 canine influenza-like virus circulation in USA dogs prior to 2004

H3N8 canine influenza virus (H3N8 CIV) was first reported as a novel canine respiratory pathogen in racing greyhounds and shelter dogs in the U.S.A. in 2004. Phylogenetic analyses determined that this host-adapted pathogen originated from interspecies transmission of an equine influenza virus (EIV), but it is unknown when the transmission occurred prior to discovery in 2004. The objective of this study was to determine if racing greyhound and shelter dog sera collected from 1984 to 2004 had serological evidence of exposure to H3N8 CIV or EIV. Archived sera from 702 racing greyhounds and 1568 shelter dogs were tested for H3 antibodies to the original 2004 CIV isolate, as well as EIV isolates from 1991 to 1999. None of the racing greyhounds from 1984 and 1985 had detectable H3 antibodies. One of the shelter dogs, which entered a north Florida shelter in 2004, was seropositive. For racing greyhounds sampled from 1999 to 2004, 133/520 (26%) dogs had antibodies to both CIV and EIV H3 proteins. The annual seroprevalence was 27% in 1999, 28% in 2000, 10% in 2001, 1% in 2002, 41% in 2003, and 28% in 2004. The odds of H3 seropositivity were greater among dogs that raced > or =6 months, raced on > or =2 tracks, and raced in 1998, 2002, and 2003. Many of the seropositive dogs raced at tracks that were involved in 'kennel cough' epidemics in 1998-1999 and 2002-2003. Based on serological evidence, a H3N8 canine influenza-like virus was circulating in racing greyhounds in the U.S.A. as early as 1999.     

Phylogenetic analysis of swine influenza viruses isolated in Poland

Swine influenza virus (SIV) of H1N1 and H3N2 subtypes are dominated in European pigs population. "Classical swine" H1N1 subtype was replaced by "avian-like" H1N1 subtype. It co-circulates with H3N2 reassortant possessing "avian" genes. In the present study, 41 SIV strains isolated from pigs with pneumonia, raised in 20 Polish farms, were identified and characterised. Since it was evidenced that isolates from the same geographic district and the same year of isolation are in 100% similar, 15 strains representing different district and different year of isolation were chosen to construct phylogenetic trees. Two genes, conservative matrix 1 (M1) and the most variable, haemagglutynin (HA), were sequenced and subjected into phylogenetic analysis. The results of the analysis confirmed that "avian-like" swine H1N1 strains evolved faster than classical SIV strains. HA gene of these isolates have been derived from contemporary strains of "avian-like" SIV. In contrast, the M1 gene segment may have originated from avian influenza viruses. H3N2 strain is located in swine cluster, in the main prevalent European group of H3N2 isolates called A/Port Chalmers/1/73-like Eurasian swine H3N2 lineage, which has evolved separately from the human H3N2 virus lineage around 1973.     

新型A型H1N1猪流感疫情动态及防控措施

2009年3月18日由墨西哥发端的人类大流行性甲型(A型)H1N1流感疫情迅速在全球蔓延,并已扩散到中国。新型A型H1N1流感突破种间屏障由人传染给猪、禽,并相继在北美、南美、大洋洲出现疫情。2009年4月21日,新型A型H1N1猪流感疫情首次在北美的加拿大发生;2009年6月24日,在南美的阿根廷发生猪流感疫情;2009年7月24日,远在大洋洲的澳大利亚也出现猪流感疫情;更糟糕的是在2009年7月23日,在南美紧邻阿根廷的智利的禽类(火鸡)首次发生A型流感疫情。此次新型A型H1N1流感疫情呈现大流行病学显著变化:①突破种间屏障由人传染给猪、禽;②突破地理屏障,在三大洲先后暴发规模不等、强度不一的多起疫情。因此,我国卫生主管部门、兽医主管部门和出入境检验检疫部门应高度戒备,密切关注,采取严格的生物安全措施,严防疫情疫病跨越国境,特别是防止新型A型H1N1流感疫情在人群、猪群、禽群之间相互传播,避免疫情在人类和动物进一步扩散。     

Serological and virological surveillance of swine H1N1 and H3N2 influenza virus infection in two farms located in Hubei province, central China

Swine influenza viruses H1N1 and H3N2 have been reported in the swine population worldwide. From June 2008 to June 2009, we carried out serological and virological surveillance of swine influenza in the Hubei province in central China. The serological results indicated that antibodies to H1N1 swine influenza virus in the swine population were high with a 42.5% (204/480) positive rate, whereas antibodies to H3N2 swine influenza virus were low with a 7.9% (38/480) positive rate. Virological surveillance showed that only one sample from weanling pigs was positive by RT-PCR. Phylogenetic analysis of the hemagglutinin and neuraminidase genes revealed that the A/Sw/HB/S1/2009 isolate was closely related to avian-like H1N1 viruses and seemed to be derived from the European swine H1N1 viruses. In conclusion, H1N1 influenza viruses were more dominant in the pig population than H3N2 influenza viruses in central China, and infection with avian-like H1N1 viruses persistently emerged in the swine population in the area.     

猪流感与公共卫生

猪流感(Swine influenza,SI)是目前危害全世界养猪业的重要呼吸道传染病之一.导致猪发病的致病毒株主要有H1N1、H1N2、H3N1、H3N2、H2N3、H5N1和H9N2等亚型流感病毒,特别是从猪体分离H5N1和H9N2亚型流感病毒对禽流感的控制及人类公共卫生有重要意义.针对目前流行的甲型H1N1疫情,对猪流感病毒的分子生物学、临床症状、病理变化及公共卫生意义等方面进行了综述,以期对其有一个较为全面的了解.     

猪流感病毒H1N1、H1N2和H3N2亚型多重RT-PCR诊断方法的建立

对我国分离到的猪流感病毒和GenBank数据库中已有的猪流感病毒H1N1、H1N2和H3N2亚型毒株的HA、NA基因核苷酸序列进行分析,分别选出各个病毒亚型HA和NA基因中高度保守且特异的核苷酸区域,设计扩增猪流感病毒H1和H3、N1和N2亚型的2套多重PCR特异性引物,建立了猪流感H1N1、H1N2和H3N2亚型病毒多重RT-PCR诊断方法。采用该方法对H1N1、H1N2、H3N2亚型猪流感病毒标准参考株进行RT-PCR检测,结果均呈阳性,对扩增得到的片段进行序列测定和BLAST比较,表明为目的基因片段。其它几种常见猪病病毒和其它亚型猪流感病毒的RT-PCR扩增结果都呈阴性。对107EID50/0.1mL病毒进行稀释,提取RNA进行敏感性试验,RT-PCR最少可检测到102EID50的病毒量核酸。对40份阳性临床样品的检测结果是H1N1、H1N2和H3N2亚型分别为16份、1份和20份,其它3份样品同时含有H1N1和H3N2亚型猪流感病毒,和鸡胚分离病毒结果100%一致。试验证明建立的猪流感病毒H1N1、H1N2和H3N2亚型多重RT-PCR诊断方法是一种特异敏感的诊断方法,可用于临床样品的早期快速诊断和分型。     

Induction of a cross-reactive antibody response to influenza virus M2 antigen in pigs by using a Sendai virus vector

Protecting pigs from simultaneous infection with avian, swine, and human influenza viruses would be an effective strategy to prevent the emergence of reassortants with pandemic potential. M2 protein is a candidate antigen for so-called 'universal vaccines,' which confer cross-protection to different influenza viruses in a strain- and subtype-independent manner. We tested whether a recombinant F gene-deleted Sendai virus vector that contained an M2 gene derived from an H5N1 avian influenza virus (SeV/ΔF/H5N1M2) could induce a cross-reactive antibody response to the extracellular domain of M2 protein (M2e) in pigs. SeV/ΔF/H5N1M2 induced an antibody response to M2e when the vector was inoculated intramuscularly. The antibodies induced by SeV/ΔF/H5N1M2 cross-reacted with M2e derived from different avian, swine, and human influenza viruses. In mice, however, SeV/ΔF/H5N1M2 did not confer cross-protection to challenge with a heterologous H3N2 influenza virus. Our results confirm those of other groups indicating that antibodies to M2e do not mediate protection to influenza viruses in pigs.     

Genetic diversity of H9N2 influenza viruses from pigs in China: a potential threat to human health?

Pandemic strains of influenza A virus might arise by genetic reassortment between viruses from different hosts. Pigs are susceptible to both human and avian influenza viruses and have been proposed to be intermediate hosts or mixing vessels, for the generation of pandemic influenza viruses through reassortment or adaptation to the mammalian host. In this study, we summarize and report for the first time the coexistence of 10 (A-J) genotypes in pigs in China by analyzing the eight genes of 28 swine H9N2 viruses isolated in China from 1998 to 2007. Swine H9N2 viruses in genotype A and B were completely derived from Y280-like and Shanghai/F/98-like viruses, respectively, which indicated avian-to-pig interspecies transmission of H9N2 viruses did exist in China. The other eight genotype (C-J) viruses might be double-reassortant viruses, in which six genotype (E-J) viruses possessed 1-4 H5-like gene segments indicating they were reassortants of H9 and H5 viruses. In conclusion, genetic diversity of H9N2 influenza viruses from pigs in China provides further evidence that avian to pig interspecies transmission of H9N2 viruses did occur and might result in the generation of new reassortant viruses by genetic reassortment with swine H1N1, H1N2 and H3N2 influenza viruses, therefore, these swine H9N2 influenza viruses might be a potential threat to human health and continuing to carry out swine influenza virus surveillance in China is of great significance.     

四株分离自同一猪场的H3N2亚型猪流感病毒的进化分析

猪作为流感病毒异源毒株间发生基因重组的"混合容器",其呼吸道上皮细胞上同时存在着能够感染人（SA α-2,6-Gal）和禽（SA α-2,3-Gal）两种流感病毒的受体,具备产生新型流感病毒的潜力。在我们的前期研究中,连续两年（2013年和2014年）从南宁地区某个规模化养猪场当中分离获得了2株新型甲型流感病毒重配的H3N2亚型猪流感病毒（swine influenza viruses,SIVs）。为了解SIVs在同一地方的遗传进化规律,我们在2018年至2019年间对该猪场进行了持续的监测,并于2019年再次成功分离获得了2株H3N2亚型的三源重组毒株,命名为A/swine/Guangxi/JG13/2019（简称JG13/2019）和A/swine/Guangxi/JG20/2019（简称JG20/2019）。遗传进化分析表明其基因重配形式与2013年分离株A/swine/Guangxi/JGB4/2013（简称JGB4/2013）和2014年分离株A/swine/Guangxi/JG1/2014（简称JG1/2014）相同,表面基因HA和NA来源于类人H3N2谱系,内部基因NP、M、PA、PB1和PB2来源于2009年甲型H1N1大流感谱系（pdm/09H1N1）,NS基因来源于古典型H1N1谱系。此外,新分离株JG13/2019和JG20/2019同早期分离株JGB4/2013和JG1/2014 HA和NA基因的核苷酸相似性分别为95.3%~97.4%和93.9%~97.0%,内部基因（NP、M、PA、PB1和PB2）的核苷酸相似性为96.2%~98.1%,NS基因的核苷酸相似性为97.1%~97.6%。通过分析比较这些年代不同毒株之间的关键氨基酸位点差异,结果发现JG20/2019和JG13/2019的HA蛋白仍旧保持了与人型受体结合的分子特征（190D、226I和228S）,却也出现了V223I或P227S的新变化,JG13/2019的PA蛋白（R356K）和PB2蛋白（I588T）也与之前的毒株有所不同。这些位点上的氨基酸改变是否影响到病毒的致病能力、复制能力以及跨种间传播能力,有待今后进一步研究。历经6年,携带有pdm/09 H1N1多种内部基因片段（PB2、PB1、PA、M、NP）和类人表面基因（HA和NA）的H3N2亚型SIVs依旧在同一个猪场的猪群中流行,虽然其关键的功能区域出现了基因突变,但是仍然保持着能够感染人的受体结合特性。因此,加强对SIVs流行情况的监测,将为今后防控人类流感大暴发提供预警。     

The epidemiology and evolution of influenza viruses in pigs

Pigs serve as major reservoirs of H1N1 and H3N2 influenza viruses which are endemic in pig populations world-wide and are responsible for one of the most prevalent respiratory diseases in pigs. The maintenance of these viruses in pigs and the frequent exchange of viruses between pigs and other species is facilitated directly by swine husbandry practices, which provide for a continual supply of susceptible pigs and regular contact with other species, particularly humans. The pig has been a contender for the role of intermediate host for reassortment of influenza A viruses of avian and human origin since it is the only domesticated mammalian species which is reared in abundance and is susceptible to, and allows productive replication, of avian and human influenza viruses. This can lead to the generation of new strains of influenza, some of which may be transmitted to other species including humans. This concept is supported by the detection of human-avian reassortant viruses in European pigs with some evidence for subsequent transmission to the human population. Following interspecies transmission to pigs, some influenza viruses may be extremely unstable genetically, giving rise to variants which could be conducive to the species barrier being breached a second time. Eventually, a stable lineage derived from the dominant variant may become established in pigs. Genetic drift occurs particularly in the genes encoding the external glycoproteins, but does not usually result in the same antigenic variability that occurs in the prevailing strains in the human population. Adaptation of a 'newly' transmitted influenza virus to pigs can take many years. Both human H3N2 and avian H1N1 were detected in pigs many years before they acquired the ability to spread rapidly and become associated with disease epidemics in pigs.     

Serological Evidence of Pandemic H1N1 Influenza Virus Infections in Greek Swine

The introduction of the 2009 pandemic H1N1 (pH1N1) influenza virus in pigs changed the epidemiology of influenza A viruses (IAVs) in swine in Europe and the rest of the world. Previously, three IAV subtypes were found in the European pig population: an avian‐like H1N1 and two reassortant H1N2 and H3N2 viruses with human‐origin haemagglutinin (HA) and neuraminidase proteins and internal genes of avian decent. These viruses pose antigenically distinct HAs, which allow the retrospective diagnosis of infection in serological investigations. However, cross‐reactions between the HA of pH1N1 and the HAs of the other circulating H1 IAVs complicate serological diagnosis. The prevalence of IAVs in Greek swine has been poorly investigated. In this study, we examined and compared haemagglutination inhibition (HI) antibody titres against previously established IAVs and pH1N1 in 908 swine sera from 88 herds, collected before and after the 2009 pandemic. While we confirmed the historic presence of the three IAVs established in European swine, we also found that 4% of the pig sera examined after 2009 had HI antibodies only against the pH1N1 virus. Our results indicate that pH1N1 is circulating in Greek pigs and stress out the importance of a vigorous virological surveillance programme.     

Exposure of domestic swine to influenza A viruses in Ghana suggests unidirectional,reverse zoonotic transmission at the human–animal interface

Influenza A viruses (IAVs) have both zoonotic and anthroponotic potential and are of public and veterinary importance. Swine are intermediate hosts and ‘mixing vessels’ for generating reassortants, progenies of which may harbour pandemic propensity. Swine handlers are at the highest risk of becoming infected with IAVs from swine but there is little information on the ecology of IAVs at the human–animal interface in Africa. We analysed and characterized nasal and throat swabs from swine and farmers respectively, for IAVs using RT‐qPCR, from swine farms in the Ashanti region, Ghana. Sera were also analysed for IAVs antibodies and serotyped using ELISA and HI assays. IAV was detected in 1.4% (n = 17/1,200) and 2.0% (n = 2/99) of swine and farmers samples, respectively. Viral subtypes H3N2 and H1N1pdm09 were found in human samples. All virus‐positive swine samples were subtyped as H1N1pdm09 phylogenetically clustering closely with H1N1pdm09 that circulated among humans during the study period. Phenotypic markers that confer sensitivity to Oseltamivir were found. Serological prevalence of IAVs in swine and farmers by ELISA was 3.2% (n = 38/1,200) and 18.2% (n = 18/99), respectively. Human H1N1pdm09 and H3N2 antibodies were found in both swine and farmers sera. Indigenous swine influenza A viruses and/or antibodies were not detected in swine or farmers samples. Majority (98%, n = 147/150) of farmers reported of not wearing surgical mask and few (4%, n = 6) reported to wear gloves when working. Most (n = 74, 87.7%) farmers reported of working on the farm when experiencing influenza‐like illness. Poor husbandry and biosafety practices of farmers could facilitate virus transmission across the human–swine interface. Farmers should be educated on the importance of good farm practices to mitigate influenza transmission at the human–animal interface.