Novel H1N2 swine influenza reassortant strain in pigs derived from the pandemic H1N1/2009 virus

Swine influenza monitoring programs have been in place in Italy since the 1990 s and from 2009 testing for the pandemic H1N1/2009 virus (H1N1pdm) was also performed on all the swine samples positive for type A influenza. This paper reports the isolation and genomic characterization of a novel H1N2 swine influenza reassortant strain from pigs in Italy that was derived from the H1N1pdm virus. In May 2010, mild respiratory symptoms were observed in around 10% of the pigs raised on a fattening farm in Italy. Lung homogenate taken from one pig showing respiratory distress was tested for influenza type A and H1N1pdm by two real time RT-PCR assays. Virus isolation was achieved by inoculation of lung homogenate into specific pathogen free chicken embryonated eggs (SPF CEE) and applied onto Caco-2 cells and then the complete genome sequencing and phylogenetic analysis was performed from the CEE isolate. The lung homogenate proved to be positive for both influenza type A (gene M) and H1N1pdm real time RT-PCRs. Virus isolation (A/Sw/It/116114/2010) was obtained from both SPF CEE and Caco-2 cells. Phylogenetic analysis showed that all of the genes of A/Sw/It/116114/2010, with the exception of neuraminidase (NA), belonged to the H1N1pdm cluster. The NA was closely related to two H1N2 double reassortant swine influenza viruses (SIVs), previously isolated in Sweden and Italy. NA sequences for these three strains were clustering with H3N2 SIVs. The emergence of a novel reassortant H1N2 strain derived from H1N1pdm in swine in Italy raises further concerns about whether these viruses will become established in pigs. The new reassortant not only represents a pandemic (zoonotic) threat but also has unknown livestock implications for the European swine industry.     

1株猪源2009/H1N1流感病毒反向遗传系统的建立

为建立1株猪源2009/H1N1流感病毒A/swine/Heilongjiang/44/2009(HLJ44)的反向遗传系统,利用双向表达质粒p HW2000,分别构建了该病毒株8个基因节段的重组质粒,将其共转染于293T和MDCK混合培养的细胞,拯救出重组病毒R-HLJ44。测序结果表明,R-HLJ44与亲本病毒HLJ44的核苷酸序列完全一致;二者在细胞上具有相似的增殖特性;抗原性未发生变化;分别以106TCID50的剂量鼻腔感染BALB/c小鼠,结果显示R-HLJ44与亲本HLJ44在小鼠脏器中的复制滴度也基本一致。以上结果表明拯救的重组病毒保持了与亲本病毒一致的生物学特性。该病毒反向遗传系统的建立,为进一步研究H1N1亚型流感病毒的致病分子机制及新型疫苗研制等奠定了基础。     

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.     

Reassortant H1N1 influenza virus vaccines protect pigs against pandemic H1N1 influenza virus and H1N2 swine influenza virus challenge

Influenza A (H1N1) virus has caused human influenza outbreaks in a worldwide pandemic since April 2009. Pigs have been found to be susceptible to this influenza virus under experimental and natural conditions, raising concern about their potential role in the pandemic spread of the virus. In this study, we generated a high-growth reassortant virus (SC/PR8) that contains the hemagglutinin (HA) and neuraminidase (NA) genes from a novel H1N1 isolate, A/Sichuan/1/2009 (SC/09), and six internal genes from A/Puerto Rico/8/34 (PR8) virus, by genetic reassortment. The immunogenicity and protective efficacy of this reassortant virus were evaluated at different doses in a challenge model using a homologous SC/09 or heterologous A/Swine/Guangdong/1/06(H1N2) virus (GD/06). Two doses of SC/PR8 virus vaccine elicited high-titer serum hemagglutination inhibiting (HI) antibodies specific for the 2009 H1N1 virus and conferred complete protection against challenge with either SC/09 or GD/06 virus, with reduced lung lesions and viral shedding in vaccine-inoculated animals compared with non-vaccinated control animals. These results indicated for the first time that a high-growth SC/PR8 reassortant H1N1 virus exhibits properties that are desirable to be a promising vaccine candidate for use in swine in the event of a pandemic H1N1 influenza.     

Detection and isolation of 2009 pandemic influenza A/H1N1 virus in commercial piggery,Lagos Nigeria

WHO declared pandemic of A/H1N1influenza in 2009 following global spread of the newly emerged strain of the virus from swine. Presently there is a dearth of data on the ecology of pandemic influenza H1N1 required for planning of intervention measures in sub Saharan Africa. Herein we report isolation of 2009 pandemic influenza A/H1N1 in an intensive mega piggery farms operation in South West Nigeria.     

Efficacy of a pandemic (H1N1) 2009 virus vaccine in pigs against the pandemic influenza virus is superior to commercially available swine influenza vaccines

In April 2009 a new influenza A/H1N1 strain, currently named "pandemic (H1N1) influenza 2009" (H1N1v), started the first official pandemic in humans since 1968. Several incursions of this virus in pig herds have also been reported from all over the world. Vaccination of pigs may be an option to reduce exposure of human contacts with infected pigs, thereby preventing cross-species transfer, but also to protect pigs themselves, should this virus cause damage in the pig population. Three swine influenza vaccines, two of them commercially available and one experimental, were therefore tested and compared for their efficacy against an H1N1v challenge. One of the commercial vaccines is based on an American classical H1N1 influenza strain, the other is based on a European avian H1N1 influenza strain. The experimental vaccine is based on reassortant virus NYMC X179A (containing the hemagglutinin (HA) and neuraminidase (NA) genes of A/California/7/2009 (H1N1v) and the internal genes of A/Puerto Rico/8/34 (H1N1)). Excretion of infectious virus was reduced by 0.5-3 log(10) by the commercial vaccines, depending on vaccine and sample type. Both vaccines were able to reduce virus replication especially in the lower respiratory tract, with less pathological lesions in vaccinated and subsequently challenged pigs than in unvaccinated controls. In pigs vaccinated with the experimental vaccine, excretion levels of infectious virus in nasal and oropharyngeal swabs, were at or below 1 log(10)TCID(50) per swab and lasted for only 1 or 2 days. An inactivated vaccine containing the HA and NA of an H1N1v is able to protect pigs from an infection with H1N1v, whereas swine influenza vaccines that are currently available are of limited efficaciousness. Whether vaccination of pigs against H1N1v will become opportune remains to be seen and will depend on future evolution of this strain in the pig population. Close monitoring of the pig population, focussing on presence and evolution of influenza strains on a cross-border level would therefore be advisable.     

The 2009 pandemic (H1N1) viruses isolated from pigs show enhanced pathogenicity in mice

Since the emergence of the 2009 pandemic (H1N1) virus (2009/H1N1) in April 2009, cases of transmission from humans to pigs have been reported frequently. In our previous studies, four 2009/H1N1 variants were isolated from pigs. To better understand the phenotypic differences of the pig isolates compared with the human isolate, in this study mice were inoculated intranasally with different 2009/H1N1 viruses, and monitored for morbidity, mortality, and viral replication, cytokine production and pathological changes in the lungs. The results show that all isolates show effective replication in lungs, but varying in their ability to cause morbidity. In particular, the strains of A/swine/Nanchang/3/2010 (H1N1) and A/swine/Nanchang/F9/2010 (H1N1) show the greatest virulence with a persisting replication in lungs and high lethality for mice, compared with the human isolate A/Liaoning /14/2009 (H1N1), which shows low virulence in mice. Furthermore, the lethal strains could induce more severe lung pathological changes and higher production of cytokines than that of other strains at an early stage. Amino acid sequence analysis illustrates prominent differences in viral surface glycoproteins and polymerase subunits between pig isolates and human strains that might correlate with their phenotypic differences. These studies demonstrate that the 2009/H1N1 pig isolates exhibit heterogeneous infectivity and pathogencity in mice, and some strains possess an enhanced pathogenicity compared with the human isolate.     

Phylogenetic evolution of swine-origin human influenza virus: a pandemic H1N1 2009

The knowledge of the genome constellation in pandemic influenza A virus H1N1 2009 from different countries and different hosts is valuable for monitoring and understanding of the evolution and migration of these strains. The complete genome sequences of selected worldwide distributed influenza A viruses are publicly available and there have been few longitudinal genome studies of human, avian and swine influenza A viruses. All possible to download SIV sequences of influenza A viruses available at GISAID Platform (Global Initiative on Sharing Avian Influenza Data) were analyzed firstly through the web servers of the Influenza Virus Resource in NCBI. Phylogenetic study of circulating human pandemic H1N1 virus indicated that the new variant possesses a distinctive evolutionary trait. There is no one way the pandemic H1N1 have acquired new genes from other distinguishable viruses circulating recently in local human, pig or domestic poultry populations from various geographic regions. The extensive genetic diversity among whole segments present in pandemic H1N1 genome suggests that multiple introduction of virus have taken place during the period 1999-2009. The initial interspecies transmission could have occurred in the long-range past and after it the reassortants steps lead to three lineages: classical SIV prevalent in the North America, avian-like SIV in Europe and avian-like related SIV in Asia. This analysis contributes to the evidence that pigs are not the only hosts playing the role of "mixing vessel", as it was suggested for many years.     

Fatal pandemic (H1N1) 2009 influenza A virus infection in a Pennsylvania domestic cat

We report the earliest recognized fatality associated with laboratory-confirmed pandemic H1N1 (pH1N1) influenza in a domestic cat in the United States. The 12-year old, indoor cat died on 6 November 2009 after exposure to multiple family members who had been ill with influenza-like illness during the peak period of the fall wave of pH1N1 in Pennsylvania during late October 2009. The clinical presentation, history, radiographic, laboratory and necropsy findings are presented to assist veterinary care providers in understanding the features of this disease in cats and the potential for transmission of infection to pets from infected humans.     

Descriptive clinical and epidemiological characteristics of influenza A H1N1 2009 virus infections in pigs in England

Infection of pigs with influenza A H1N1 2009 virus (A(H1N1)pdm09) was first detected in England in November 2009 following global spread of the virus in the human population. This paper describes clinical and epidemiological findings in the first English pig farms in which A(H1N1)pdm09 influenza virus was detected. These farms showed differences in disease presentation, spread and duration of infection. The factors likely to influence these features are described and relate to whether pigs were housed or outdoors, the age of the pigs, inter-current disease and the management system of the unit. Infection could be mild or clinically inapparent in breeding pigs with more typical respiratory disease being identified later in their progeny. Mortality was low where disease was uncomplicated by environmental stresses or concurrent infections. Where deaths occurred in pigs infected with A(H1N1)pdm09 influenza, they were mainly due to other infections, including streptococcal disease due to Streptococcus suis infection. This paper demonstrates the ease with which A(H1N1)pdm09 virus was transmitted horizontally and maintained in a pig population.     

Comparative study of pandemic (H1N1) 2009, swine H1N1, and avian H3N2 influenza viral infections in quails

Quail has been proposed to be an intermediate host of influenza A viruses. However, information on the susceptibility and pathogenicity of pandemic H1N1 2009 (pH1N1) and swine influenza viruses in quails is limited. In this study, the pathogenicity, virus shedding, and transmission characteristics of pH1N1, swine H1N1 (swH1N1), and avian H3N2 (dkH3N2) influenza viruses in quails was examined. Three groups of 15 quails were inoculated with each virus and evaluated for clinical signs, virus shedding and transmission, pathological changes, and serological responses. None of the 75 inoculated (n = 45), contact exposed (n = 15), or negative control (n = 15) quails developed any clinical signs. In contrast to the low virus shedding titers observed from the swH1N1-inoculated quails, birds inoculated with dkH3N2 and pH1N1 shed relatively high titers of virus predominantly from the respiratory tract until 5 and 7 DPI, respectively, that were rarely transmitted to the contact quails. Gross and histopathological lesions were observed in the respiratory and intestinal tracts of quail inoculated with either pH1N1 or dkH3N2, indicating that these viruses were more pathogenic than swH1N1. Sero-conversions were detected 7 DPI in two out of five pH1N1-inoculated quails, three out of five quails inoculated with swH1N1, and four out of five swH1N1-infected contact birds. Taken together, this study demonstrated that quails were more susceptible to infection with pH1N1 and dkH3N2 than swH1N1.     

Evaluation of transmission of swine influenza type A subtype H1N2 virus in seropositive pigs

OBJECTIVE: To examine clinical signs, virus infection and shedding, and transmission of swine influenza virus (SIV) subtype H1N2 among seropositive pigs. ANIMALS: Eighteen 3-week-old pigs with maternal antibodies against SIV subtypes H1N1, H3N2, and H1N2. PROCEDURE: Ten pigs (principal) were inoculated intranasally with subtype H1N2 and 2 groups of contact pigs (n = 4) each were mixed with principal pigs on day 7 (group 1) or 28 (group 2). Two principal pigs each were necropsied on days 4, 14, 21, 28, and 42 days after inoculation. Four pigs in each contact group were necropsied 35 and 14 days after contact. Virus excretion was evaluated after inoculation or contact. Lung lesions and the presence of SIV in various tissues were examined. RESULTS: Mild coughing and increased rectal temperature were observed in principal pigs but not in contact pigs. Nasal virus shedding was detected in all principal pigs from day 2 for 3 to 5 days, in group 1 pigs from day 2 for 4 to 9 days after contact, and in group 2 pigs from day 4 for 2 to 6 days after contact. Trachea, lung, and lymph node specimens from infected pigs contained virus. Antibody titers against all 3 subtypes in all pigs gradually decreased. CONCLUSIONS AND CLINICAL RELEVANCE: Protection from viral infection and shedding was not observed in pigs with maternal antibodies, but clinical disease did not develop. Vaccination programs and good management practices should be considered for control of SIV subtype H1N2 infection on swine farms.     

甲型H1N1(2009)流感病毒实时荧光RT-PCR检测方法的建立与评价

自甲型H1N1(2009)流感病毒于2009年在世界多个国家暴发流行后,许多国家的猪群中检测到该病毒的存在,因此建立快速准确的甲型H1N1流感病毒的检测方法成为迫切需求。本研究针对甲型H1N1(2009)流感病毒NA基因设计特异性引物和探针,建立甲型H1N1(2009)流感病毒特异性实时荧光RT-PCR快速检测方法。并将该方法与WHO推荐美国CDC建立的检测方法和美国农业部推荐的检测方法进行比较。通过田间试验验证该方法在实际应用中的效果。结果表明:本研究建立的方法对检测甲型H1N1流感病毒裂解疫苗的灵敏度达到10-6,与WHO推荐的A型流感病毒实时荧光PCR检测方法的灵敏度一致,并且高于美国农业部推荐方法的检测灵敏度(10-5);该方法特异性强,与经典型H1N1和其他亚型流感病毒株无任何交叉反应。与香港兽医化验所进行了检测比对,检测灵敏度和特异性完全一致。近3 800份的田间试验表明,所建立的方法准确可靠。本研究所建立检测方法快速灵敏,检测结果准确可靠,适合用于甲型H1N1(2009)流感病毒的分子生物学检测和监测。     

Pandemic A/H1N1/2009 influenza virus in swine, Cameroon, 2010

Although swine origin A/H1N1/2009 influenza virus (hereafter "pH1N1″) has been detected in swine in 20 countries, there has been no published surveillance of the virus in African livestock. The objective of this study was to assess the circulation of influenza A viruses, including pH1N1 in swine in Cameroon, Central Africa. We collected 108 nasal swabs and 98 sera samples from domestic pigs randomly sampled at 11 herds in villages and farms in Cameroon. pH1N1 was isolated from two swine sampled in northern Cameroon in January 2010. Sera from 28% of these herds were positive for influenza A by competitive ELISA and 92.6% of these swine showed cross reactivity with pandemic A/H1N1/2009 influenza virus isolated from humans. These results provide the first evidence of this virus in the animal population in Africa. In light of the significant role of swine in the ecology of influenza viruses, our results call for greater monitoring and study in Central Africa.     

中国猪源HSN1和H9N2亚型流感病毒的分离鉴定

猪是禽流感病毒"禽-猪-人"传播链中重要的中间宿主,了解猪流感的疫情动态将为动物流感及人流感的疾病预测及防制提供重要依据.1999～2001年间进行的血清学和病毒学监测发现我国猪群存在大范围的H1和H3亚型猪流感感染(李海燕等,2002).2002～2003年,我们进一步对来自全国14个省市的1936份血清进行了H9亚型猪流感的检测,同时在广东、福建等省进行了H5亚型猪流感的检测.2002年辽宁、广东、山东及重庆猪血清中出现H9亚型流感抗体,阳性率分别为7.3%、6.8%、5.1%和1.6%.2003年采集的猪血清H9亚型流感抗体均为阴性,同时发现广东、福建两省2003年出现H5亚型流感阳性猪群,阳性率分别为4.7%和8.2%.从2001～2003年收集和送检的样品中分离鉴定了6株H9N2亚型和2株H5N1亚型猪流感病毒,部分序列分析发现H9和H5亚型猪流感病毒均与我国分离的禽流感病毒高度同源.本研究进一步确证了我国猪群中存在H9N2亚型流感病毒,并且首次发现我国猪群已出现H5N1亚型流感病毒,为人类流感及动物流感的防制敲响了警钟.对这两个亚型流感病毒所具有的公共卫生和兽医公共卫生危害性应予以高度重视.     

A serological survey of canine H3N2, pandemic H1N1/09 and human seasonal H3N2 influenza viruses in dogs in China

Influenza viruses have been isolated from dogs in China; however, the extent of influenza infection among dogs is not yet clear. Here, we examined the seroprevalence of avian-origin canine H3N2, pandemic H1N1/09 and human seasonal H3N2 influenza viruses in pet dogs in China during January 2012 to June 2013. The seropositivity rate of canine H3N2, H1N1/09 and human H3N2 were 3.5%, 1.5%, and 1.2%, respectively. Dogs aged 2–5 years were most commonly seropositive to canine H3N2 virus. It is worth noting that two serum samples were positive against both canine H3N2 and H1N1/09 viruses, suggesting the possibility of coinfection with both viruses. Our findings emphasize the necessity for continued surveillance of influenza viruses in dogs in China.