Seroepidemiological survey of morbillivirus infection in Kuril harbor seals (Phoca vitulina stejnegeri) of Hokkaido, Japan

Serological analysis was performed to detect morbillivirus infection in Kuril harbor seals in Hokkaido, Japan. Serum samples were collected from the seals at Nosappu (231 sera), Akkeshi (16), and Erimo (75) between 1998 and 2005. Antibodies to phocine distemper virus (PDV) were detected by ELISA in seals from Nosappu and Erimo. Antibodies to PDV were found in 56% (5/9) of the sampled seals from Nosappu in 1998, versus only 5% (3/66) for 2003, 1% (1/79) for 2004, and 1% (1/77) for 2005. These suggest epidemic caused by the virus in or before 1998. As antibody-positive seals included juvenile seals in 2003 and 2005, sporadic infections of the virus are thought to have occurred in recent years. In Erimo, antibodies to PDV were found in 50% (14/28) of sampled seals in 2004, versus only 13% (1/8) for 1999, 7% (1/15) for 2003, and 0% (0/24) for 2005. These suggest sporadic infection by the virus before 2003 and the epizootic between after autumn in 2003, when samples of 2003 were collected, and 2004. Since antibodies to canine distemper virus (CDV) were detected in one adult seal from Nosappu in each year from 2003 to 2005, sporadic infections of the virus were suggested. There were no difference in incidence of seals with antibodies to the viruses between males and females and between juveniles and adults.     

天津地区猪流感血清学调查

2002年-2005年期间,采用血凝抑制试验对天津市10个区县44个养猪场进行了猪流感病毒抗体检测,结果75%的被调查猪场抗体检测结果有阳性,检测的648份血清样品中,69.6%为阳性。流感病毒抗体亚型调查结果显示该地区流行的猪流感病毒主要为H1和H3亚型,抗体阳性率分别为55.4%和39.4%。部分猪群中存在抗H9亚型流感病毒抗体,阳性率为5.4%,但未发现H5亚型流感病毒抗体。此外,部分猪群中同时存在2种或3种亚型(H1,H3和H9)流感病毒的抗体,表明这些猪曾经同时被2种或3种不同亚型的流感病毒感染。     

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.     

Seroepidemiology of Toxoplasma gondii and Neospora caninum in seals around Hokkaido, Japan

Serological analysis was performed to detect Toxoplasma gondii and Neospora caninum infection in seals in Hokkaido. Serum samples were collected from 322 Kuril harbor seals (Phoca vitulina stejnegeri) at Nosappu, Akkeshi and Erimo, from 46 spotted seals (P. largha) at Nosappu, Erimo, Yagishiri Island, Hamamasu and Syakotan, and from 4 ribbon seals (P. fasciata) and a bearded seal (Erignathus barbatus) at Nosappu between 1998 and 2006. Recombinant surface antigen of T. gondii (SAG2t) and N. caninum (NcSAG1t) were used as antigens for ELISA to detect antibodies. Antibodies against SAG2t were detected from 4% of 77 Kuril harbor seals at Nosappu in 2005. Antibodies against NcSAG1t were detected from 2% (1/66) in 2003, 5% (4/79) in 2004 and 10% (8/77) in 2005 of Kuril harbor seals and 11% of 9 spotted seals in 2004 sampled at Nosappu. Eight percent of 12 Kuril harbor seals from Akkeshi and 25% of 4 spotted seals from Erimo in 2005 also contained antibodies against NcSAG1t. These suggest sporadic infection of T. gondii and N. caninum in Kuril harbor seals and spotted seals in Hokkaido. Of the ELISA-positive seals, 2 seals having anti-SAG2t antibodies and 3 seals having anti-NcSAG1t antibodies in 2005 were judged to be juveniles that have no maternal antibodies. These suggest that the protozoan infections have occurred in recent years. Infection of terrestrial protozoa such as T. gondii and N. caninum in seals indicates that the sea environment has been contaminated with protozoa.     

Australian surveillance for avian influenza viruses in wild birds between July 2005 and June 2007

Objective   To identify and gain an understanding of the influenza viruses circulating in wild birds in Australia.
Design   A total of 16,303 swabs and 3782 blood samples were collected and analysed for avian influenza (AI) viruses from 16,420 wild birds in Australia between July 2005 and June 2007. Anseriformes and Charadriiformes were primarily targeted.
Procedures   Cloacal, oropharyngeal and faecal (environmental) swabs were tested using polymerase chain reaction (PCR) for the AI type A matrix gene. Positive samples underwent virus culture and subtyping. Serum samples were analysed using a blocking enzyme-linked immunosorbent assay for influenza A virus nucleoprotein.
Results   No highly pathogenic AI viruses were identified. However, 164 PCR tests were positive for the AI type A matrix gene, 46 of which were identified to subtype. A total of five viruses were isolated, three of which had a corresponding positive PCR and subtype identification (H3N8, H4N6, H7N6). Low pathogenic AI H5 and/or H7 was present in wild birds in New South Wales, Tasmania, Victoria and Western Australia. Antibodies to influenza A were also detected in 15.0% of the birds sampled.
Conclusions   Although low pathogenic AI virus subtypes are currently circulating in Australia, their prevalence is low (1.0% positive PCR). Surveillance activities for AI in wild birds should be continued to provide further epidemiological information about circulating viruses and to identify any changes in subtype prevalence.     

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.     

An immunoperoxidase monolayer assay for the detection of antibodies against swine influenza virus.

An immunoperoxidase monolayer assay (IPMA) has been developed to detect antibodies against swine influenza A virus (SIV) in pig sera. The test was evaluated by using sequential sera from pigs experimentally infected with H1N1 subtype of SIV. Two hundred field serum samples that had been examined by the hemagglutination inhibition (HI) test were also tested. Antibodies specific to SIV were detected as early as 3 days postinoculation (dpi) in the IPMA test as compared with 7 dpi by the HI test. Unlike HI, no serum treatment was required in the IPMA test. Regardless of the virus used in the test, IPMA detected antibodies to both H1N1 and H3N2 subtypes of SIV whereas HI detects antibodies against either H1N1 or H3N2, depending upon the virus used in the test. Results of this study indicate that IPMA is a useful test for screening of pig sera for SIV antibodies.     

Seroprevalence and genetic characteristics of five subtypes of influenza A viruses in the Chinese pig population: A pooled data analysis

A literature review and pooled data analysis were carried out to examine the prevalence of antibodies against five influenza virus subtypes in pigs in China over a 10-year period (1999–2009). The average seropositive frequencies of subtypes H1, H3, H5, H7 and H9 were 3478/11,168 (31.1%), 2900/10,139 (28.6%), 77/5945 (1.3%), 0/1440 (0%) and 86/3619 (2.4%), respectively. There was a geographical variation in the seroprevalence of subtype H1, with the highest seroprevalence in pigs in South and East China. BLAST analysis of genetic sequences revealed that genome segments with moderate homology to the 2009 pandemic influenza A (H1N1) virus were present among swine influenza viruses isolated in China, especially in South and East China. It was concluded from both serological and genetic studies that subtypes H1, H3, H5 and H9 are currently co-circulating in pigs in China, with the H1 subtype most commonly detected, followed by H3.     

Evidence of the concurrent circulation of H1N2, H1N1 and H3N2 influenza A viruses in densely populated pig areas in Spain

This paper reports on a serological and virological survey for swine influenza virus (SIV) in densely populated pig areas in Spain. The survey was undertaken to examine whether the H1N2 SIV subtype circulates in pigs in these areas, as in other European regions. Six hundred sow sera from 100 unvaccinated breeding herds across Northern and Eastern Spain were examined using haemagglutination inhibition (HI) tests against H1N1, H3N2 and H1N2 SIV subtypes. Additionally, 225 lung samples from pigs with respiratory problems were examined for the presence of SIV by virus isolation in embryonated chicken eggs and by a commercial membrane immunoassay. The virus isolates were further identified by HI and RT-PCR followed by partial cDNA sequencing. The HI test on sera revealed the presence of antibodies against at least one of the SIV subtypes in 83% of the herds and in 76.3% of the animals studied. Of the 600 sow sera tested, 109 (18.2%), 60 (10%) and 41 (6.8%) had SIV antibodies to subtype H1N2 alone, H3N2 alone and H1N1 alone, respectively. Twelve H3N2 viruses, 9 H1N1 viruses and 1 H1N2 virus were isolated from the lungs of pigs with respiratory problems. The analysis of a 436 nucleotide sequence of the neuraminidase gene from the H1N2 strain isolated further confirmed its identity. Demonstrably, swine influenza is still endemic in the studied swine population and a new subtype, the H1N2, may be becoming established and involved in clinical outbreaks of the disease in Spain.     

上海地区野鸟中禽流感的流行病学监测

为了评估野鸟在禽流感流行病学中的作用,于2004年4月-2005年6月间对上海地区捕捉到的63个品种的1 010只野鸟进行了血样和喉肛棉拭样品的采集.采用病毒分离试验和荧光RT-PCR试验对喉肛棉拭样品进行了病原学检测,结果均为阴性;采用HA和HI试验进行了禽流感血清抗体的检测,结果在15种野鸟的587份血样中检出了44份AIV阳性抗体,其中H1阳性数3份,阳性率为0.51%;H3阳性数4份,阳性率为0.68%;H5阳性数11份,阳性率为1.87%;H9阳性数26份,阳性率为4.43%.     

H6亚型禽流感病毒血凝素蛋白线性表位的预测与鉴定

应用生物信息学方法预测H6亚型禽流感病毒血凝素蛋白(HA)线性抗原表位,并对所获表位的免疫原性进行初步鉴定,为流感病毒表位疫苗研制和H6亚型特异性ELISA检测方法奠定基础。依据近年流感病毒流行趋势,从GenBank下载具有代表性的H6、H5、H7和H9亚型禽流感病毒血凝素蛋白的氨基酸序列。利用DNA Star软件进行H6同亚型间氨基酸序列保守性分析,再将H6与H5、H7、H9不同亚型之间氨基酸同源性进行比较,然后借助在线服务器ExPASy和IEDB对序列进行抗原性、亲水性、柔韧性、二级结构和表面可及性预测。最后去除H6与H5、H7、H9亚型氨基酸同源区域,选择H6亚型氨基酸相对保守区域并且抗原表位综合预测结果较优的几个片段,所选择表位长度为15个氨基酸。合成所获得的优势线性表位,并用间接ELISA方法对所获表位的免疫原性进行鉴定。预测后获得4个线性抗原表位,经鉴定免疫原性最好的为表位A,最差的为表位B。     

部分猪场H1和H3亚型猪流感的血清学调查

为了解中国部分省市规模化猪场H1和H3亚型猪流感病毒的流行情况,采用血凝抑制试验对采集于广东、湖南、河南省12个市县28个规模化猪场的799份血清进行H1和H3亚型猪流感病毒的抗体检测。结果表明,H1亚型抗体阳性率在0~83.33%之间,猪抗体总阳性率为46.18%(369/799),猪场阳性率为89.29%(25/28)。H3亚型抗体阳性率在0~100.00%之间,猪抗体总阳性率为61.33%(490/799),猪场阳性率为85.71%(24/28)。广东、湖南和河南地区H1亚型抗体阳性率分别为48.91%、40.26%和50.67%,H3亚型抗体阳性率分别为58.55%、70.78%和78.67%。在被调查的上述3个地区的猪群中,H1和H3亚型猪流感病毒的感染较为普遍,其中H3亚型感染率高于H1亚型,且各地区猪流感病毒的流行情况存在地域性差异。     

两株H6N2亚型鸡源禽流感病毒的分离鉴定

本研究利用血凝抑制试验(HI)、反转录-聚合酶链式反应(RT-PCR)、基因测序等方法,对广东省某活禽交易市场进行流行病学调查时获得的两株非H5、H9亚型禽流感病毒65株和C7株进行了亚型鉴定。结果表明这两株病毒均具有血凝活性,且能被抗H6亚型禽流感病毒标准阳性血清特异性抑制。用针对禽流感病毒的M基因、H6亚型禽流感病毒HA基因、N2亚型禽流感病毒NA基因特异性鉴定引物对65株和C7株进行RT-PCR扩增,分别获得特异性目的片段。测序及BLAST分析表明两株分离株与H6N2亚型禽流感广东分离株的HA基因和NA基因核苷酸序列相似性均高达95%以上。将该两分离株鉴定为H6N2亚型禽流感病毒,并命名为A/Chicken/Guangdong/65/2009、A/Chicken/Guangdong/C7/2009。     

抗H3N2亚型猪流感病毒单克隆抗体的制备与鉴定

采用纯化的H3N2亚型猪流感病毒(SIV)尿囊液作为免疫原免疫6～8周龄Balb/c小鼠,取免疫小鼠脾细胞与骨髓瘤细胞(SP2/0)融合,用间接ELISA方法筛选分泌抗SIV-H3N2的阳性细胞株,经克隆获得7株亲和力较高的杂交瘤细胞株,分别命名为1C9、2C5、2F10、3D3、4E8、5C7、5D12,用其制备的腹水ELISA效价可达1×106。通过抗体亚型测定,间接免疫荧光试验及免疫印迹试验分析鉴定,该7株单抗均为抗H3N2亚型SIV的特异性单克隆抗体,而且与其他亚型猪流感病毒、猪细小病毒、猪繁殖与呼吸综合征病毒、猪圆环病毒和猪瘟病毒等均无交叉反应,为H3N2亚型SIV的鉴别诊断奠定了基础。     

Epidemiological survey of swine influenza A virus in selected wild boar populations in Germany

The aim of this study was to evaluate the epidemiological situation of swine influenza virus (SIV) infections in different wild boar populations in Germany based on a serological surveillance in some Bundeslaender (federal states) in connection with virological investigations in wild boar shot in Northern Germany (Mecklenburg-Western Pomerania, district of Nordvorpommern). Altogether, 1245 sera from wild boar were tested using the hemagglutination inhibition test. The established seroprevalence rate was low (on average 5.2%). Antibodies were only detected against the subtypes H1N1 and H3N2 showing differences between wild boar populations and age classes. The virological investigation of samples derived from lungs of wild boar shot in Mecklenburg-Western Pomerania, district of Nordvorpommern (n=242), revealed that the virus prevalence (two virologically positive animals, 0.8%) was very low. Based on serological typing, the isolated SIV was identified as subtype H3N2. Molecular biological investigations of the hemagglutinin (HA) and neuraminidase (NA) genes confirmed this result. This study suggests that SIV infections in wild boar seem to be no serious threat for domestic pigs.     

A PCR based method for the identification of equine influenza virus from clinical samples.

In this paper we describe the development of a nested RT-PCR assay for the rapid diagnosis and characterisation of influenza virus directly from clinical specimens. Viral RNA is extracted from nasal swabs by the guanidine thiocyanate extraction method, and subsequently reverse transcribed. The complementary DNA is then used as template in a nested PCR reaction. Primers designed for use in this assay are specific for three templates; (1) the nucleoprotein (NP) gene, (2) the haemagglutinin gene of the H7N7 equine influenza virus (A1), and (3) the haemagglutinin gene of the H3N8 equine influenza virus (A2). We show that the assays are specific for the target genes chosen, and display sensitivity similar to virus isolation. The NP assay detects a variety of different influenza subtypes, whereas A1 and A2 assays are specific for influenza subtypes H7N7 and H3N8, respectively. Sequencing of amplicons obtained in the A2 assay yields information on antigenic regions of the haemagglutinin molecule, and use of this procedure in the routine surveillance of equine influenza will enable tentative characterisation of circulating viruses despite difficulties in isolating field strains of the H3N8 subtype. The A1 assay will be useful in ascertaining whether viruses of the H7N7 subtype still circulate amongst horses, or whether these are extinct.     

广西玉林市规模化禽场禽流感病毒检测

为了解广西玉林市2020年规模禽场禽流感病毒感染状况,采用荧光RT-PCR方法,对广西玉林市7个县(市、区)42个规模化禽场采集的1260份禽喉/泄殖腔棉拭子样品进行了通用型禽流感病毒核酸检测(荧光PCR),并对检测为阳性的样本进行H5、H7亚型(双重荧光PCR)和H9亚型(荧光PCR)分型鉴定.结果显示:在42个规模...     

H7N9亚型禽流感病毒三重PCR检测方法的建立

In order to develop a rapid and simultaneous assay for H7N9 subtype avian influenza virus （AIV）, three pairs of specific primers were designed according to the conserved sequences of the hemagglutinin (HA) gene of H7 subtype AIV, the neuramidinase (NA) gene of N9 subtype AIV, and the matix (M) gene of all subtypes AIV. The reaction conditions were optimized, and the specificity and sensitivity of this method were evaluated to develop a triplex PCR assay. It was shown that H7N9 subtype AIV could be amplified into three specific bands by this triplex PCR, the lengths of these bands were 330 (H7 AIV), 207 (N9 AIV) and 632 bp (all AIV), respectively. Samples containing H7 or N9 subtype AIV could be amplified into two specific bands, which were 330 and 632, 207 and 632 bp, respectively. Samples containing other subtypes AIV could be amplified into a 632 bp specific band. No specific band was amplified from other avian pathogenic virus. Sensitivity test results showed that as low as 10³ copies/μL H7N9 subtype AIV could be detected. This triplex PCR could simultaneously diagnose H7N9 subtype AIV, single H7 subtype AIV, single N9 subtype AIV and other subtype AIV in one tube. This assay was a rapid, specific and sensitive method for the detection of H7N9 subtype AIV. It could be applied in rapid diagnosis for clinical samples, and also provided a technical support to prevent and control H7N9 subtype AIV.     

一株鸡源H6N1亚型禽流感病毒全基因的分子特征

2008年国家禽流感参考实验室在我国禽流感流行病学调查期间分离到1株鸡源H6N1亚型禽流感病毒(AIV)A/Chicken/ZheJiang/80/2008(H6N1)(简称为CK/ZJ/80/08),为了弄清该病毒的分子特征,我们对其8个基因片段分别进行扩增和序列测定,对每个基因进行BLAST分析,找出同源性最高的毒株。利用DNAStar中的Megalign功能进行进化分析。结果表明CK/ZJ/80/08的HA裂解位点附近的氨基酸序列为QIETR↓GLF,推测可能为一株低致病力AIV。其HA基因与日本北海道的A/duck/Hokkaido/228/2003(H6N8)和黑龙江的A/mallard/Heilongjiang/131/2006(H6N2)以及香港早期分离株A/chicken/HongKong/17/77(H6N1)等处于同一分支;NA基因在颈部没有缺失,与A/duck/Tsukuba/718/2005(H1N1)、A/goose/Guangdong/1/96(H5N1)等处于同一分支;M基因与A/duck/Hokkaido/W90/2007(H10N7)高度同源(同源性为99%);NS基因与A/duck/Denmark/65047/04(H5N2)和A/goose/Guangdong/1/96(H5N1)处于同一分支。NP、PA、PB1、PB2分别与贵州和江西分离的H5N2亚型AIV的相应基因关系密切,同源性分别为98%、97%、97%、97%。由此推测CK/ZJ/80/08可能是由H6N2、H1N1、H10N7、H5N2等多个亚型病毒重组而成。     

H6N1亚型禽流感病毒二重荧光RT-PCR检测方法的建立

根据GenBank中H6、N1亚型禽流感病毒(AIV)的HA、NA基因序列,设计2对特异性引物和2条用不同荧光基团标记的TaqMan探针.经反应条件优化,本试验建立了检测H6N1亚型AIV的二重荧光RT-PCR方法.该法特异性强,只对H6亚型和N1亚型AIV进行特异性扩增,对其他H亚型AIV、N亚型AIV及新城疫病毒、传染性支气管炎病毒等病原体的检测均为阴性;该法敏感性好,对H6N1亚型AIV的检测限为100拷贝/μL.本试验建立的H6N1亚型AIV的二重荧光RT-PCR方法,具有快速、敏感、特异的优点,为H6N1亚型AIV的防控提供技术支撑.