吉林部分地区TTSuVs与PCV2混合感染情况调查及其与PMWS相关性分析

为了解吉林地区猪细环病毒(TTSuVs)与II型猪圆环病毒(PCV2)混合感染情况,分析TTSuVs感染与仔猪多系统衰竭征(PMWS)的相关性。试验通过PCR技术对2019年收集到的吉林部分地区的10家规模化猪场130份血清样本进行检测;并应用本实验已建立的PCV2、TTSuVs实时荧光定量PCR检测方法,对PCV2阳性样品进行病毒载量的分析,区分PMWS病猪与PCV2亚临床感染猪,比较PMWS病猪与PCV2亚临床感染猪中TTSuVs的载量。结果显示:TTSuV1感染率为38.46%;TTSuV2感染率为57.69%;PCV2感染率为46.15%;TTSuV1与TTSuV2的混合感染率为31.54%;TTSuV1与PCV2的混合感染率为32.30%;TTSuV2与PCV2的混合感染率为44.62%;三种病毒的混合感染率为22.30%。且PMWS病猪血清中TTSuV2载量明显高于PCV2亚临床感染猪(P<0.01)。结果表明,TTSuV2载量与PMWS病的发生存在一定程度的相关性。     

广东猪输血传播病毒流行病学调查及其主要抗原编码区进化分析

为了解广东省猪群中猪输血传播病毒(TTSuVs)的感染情况,采用Nest-PCR方法对广东省7个不同地区规模化猪场抽检采集的159份育肥猪血清进行检测.结果表明,TTSuVs的2个种——TTSuV1和TTSuV2的阳性检出率分别为54.72％和35.22％,二者的共感染率为29.56％,说明TTSuVs在广东猪群中有较为广泛的传播.挑选Nest-PCR检测TTSuV1和TTSuV2为阳性的样品,分别在病毒保守区设计引物,扩增病毒基因,测序验证后,与其它已发表的不同属种TTVs ORF1序列进行进化树分析.结果表明猪TTVs与人、犬、猫、猴的TTVs分别处在进化树的不同分枝,具有很大差异 ;本次研究获得的TTSuV1 ORF1序列与国内多株已发表的TTSuV1 ORF1序列相似性为72％～75％;获得的TTSuV2ORF1序列与现有测序的多株TTSuV2的ORF1序列相似性为88％～97％.此研究为进一步研究TTSuVs流行状况、血清学诊断方法和临床防治技术奠定基础.     

四川地区猪细环病毒1型的流行病学调查和分子特征研究

为了解猪细环病毒1型(TTSuV1)在四川地区猪群中的流行情况及其遗传进化关系,采用PCR对四川省不同地区的5个规模化猪场采集的140份临床健康猪血清进行检测,并根据NCBI已发表的TTSuV1的全基因组序列,对TTSuV1中的2个亚型TTSuV1a和TTSuV1b进行全基因的扩增。结果显示,在140份临床健康猪血清样本中TTSuV1a和TTSuV1b的阳性检出率分别为49.29%(95%CI:40.7%~57.9%)和39.29%(95%CI:31.1%~47.9%)。通过克隆转化,序列测定后拼接获得TTSuV1a的近似全基因组长为2 497bp,TTSuV1b为2 693bp,对测得序列进行同源性和遗传进化分析。结果表明,扩增得到TTSuV1a的ORF1和ORF2序列,与国内外已发表的参考毒株间的核苷酸同源性分别为79.3%~97.5%和92.7%~98.2%,TTSuV1b的ORF1、ORF2序列与其他参考毒株间的核苷酸同源性分别为100%和97.1%~98.6%,分析表明其差异较小,亲缘关系较近。研究结果为四川省部分地区猪群中TTSuV1的流行情况提供数据参考,为进一步研究TTSuV的遗传进化提供一定的理论依据。     

两广地区3株猪非典型瘟病毒的流行及遗传演化分析

为了解我国两广地区猪非典型瘟病毒(APPV)的流行及变异情况,本研究于2017年～2018年从该地区的两个规模化养殖场采集了10份患先天性震颤仔猪的各组织病料样品,利用RT-PCR进行APPV检测,挑选3份阳性样品进行该病毒的开放阅读框(ORF)基因序列的RT-PCR扩增、测序,并对ORF基因、该基因包含的E2和P7基因进行核苷酸和氨基酸序列同源性分析及该3种基因的遗传进化分析。结果显示,10份病料样品均为APPV阳性,采用RT-PCR分段扩增获得3株APPV的ORF基因全长序列。ORF基因序列分析显示,3株APPV之间的核苷酸同源性为81.6%～99.8%,与41株中国株的同源性为80.8%～99.3%,与19株国外参考株的同源性为81.3%～91.1%;E2和P7基因的同源性分析结果与ORF基因同源性分析结果一致。ORF、E2和P7基因的遗传进化树显示,广西猪场分离的GX-GL1株和GX-GL67株属于基因1型,而广东猪场分离的GD-CT4株属于基因3型。上述结果表明3株APPV与中国株的亲缘关系更近,两广地区的APPV基因型呈现地域差异性,本研究有助于了解APPV在中国的流行情况。     

福建省羊传染性脓疱病毒F1L、B2L和VIR基因的遗传变异分析

为探明2014年-2015年在福建省流行的羊传染性脓疱病毒(ORFV)遗传变异情况,对10株ORFV流行毒株的F1L、B2L和VIR基因进行克隆、测序及分析。结果表明,10株ORFV F1L基因之间的核苷酸序列同源性为97.6%~100%,与国内株的核苷酸序列同源性为96.8%~99.7%,与NZ2参考株的核苷酸序列同源性为96.3%~97.1%;同NZ2参考株比较,FJ-YT2014缺失2个氨基酸;10株ORFV B2L基因之间核苷酸序列同源性为97.5%~99.9%,与国内株的核苷酸序列同源性为96.7%~99.5%,与NZ2参考株的核苷酸序列同源性为96.7%~97.7%;10株ORFV VIR基因之间的核苷酸序列同源性为95.8%~99.5%,与国内株的核苷酸序列同源性为94.6%~99.6%,与NZ2参考株的核苷酸序列同源性为94.6%~96.4%。基于基因核苷酸序列的遗传进化分析表明,10株ORFV F1L基因与福建省分离株、山西株和新疆株亲缘关系较近;10株ORFV B2L基因与新疆、山西、德国毒株亲缘关系较近;10株ORFV VIR基因与台湾、新疆株亲缘关系较近。结果提示,当前福建省流行的ORFV F1L、B2L和VIR基因尚未出现明显变异,但是其F1L、B2L和VIR基因核苷酸序列之间普遍存在异质性。     

吉林地区部分规模化猪场猪圆环病毒流行情况调查

为了解猪圆环病毒(Porcine circovirus, PCV)在吉林部分规模化猪场的流行情况,对吉林部分地区的10家规模化猪场的704份样品进行PCR检测,分析猪场PCV流行情况、感染季节及感染地区情况。结果表明,2017-2019年吉林地区部分规模化猪场PCV2的阳性率为42.0%(296/704),PCV3阳性率为34.2%(241/704),PCV2与PCV3混合感染率为22.7%(160/704);PCV2在仔猪中感染率较高,为49.7%(96/193),且秋季为PCV主要感染季节。研究结果表明,2017-2019年间吉林地区部分规模化猪场PCV感染普遍发生,且PCV2更易在仔猪群中流行。     

2008年我国部分地区新城疫病毒分离株的分子生物学特征分析

从中国部分地区分离到9株新城疫病毒(NDV),采用RT-PCR方法扩增NDV F基因(535 bp),参照国内外已经发表的F基因构建系统进化树。结果:6株吉林分离株和2株山东分离株属于VIId基因型,氨基酸裂解位点序列为112R-R-Q-K-R-F117,为典型的强毒株特征。吉林分离株之间的核苷酸同源性为93.5%～99.1%,山东分离株之间的核苷酸同源性为99.3%,吉林和山东分离株之间的核苷酸同源性较高,约为93.5%～96.3%。1株安徽分离株属于Ⅰ基因型,氨基酸裂解位点序列为112G-K-Q-G-R-L117,为典型的弱毒株特征,与V4毒株核苷酸同源性为95.7%,与吉林、山东分离株存在一定差异。表明我国新城疫病毒主要以VIId基因型为主,也存在其他基因型的流行。     

2013年-2014年江西地区PRRSV分离株NSP2及ORF5基因的遗传变异分析

为研究江西地区猪繁殖与呼吸综合征病毒(PRRSV) ORF5基因的变异情况及NSP2基因的结构特征,采用RT-PCR方法扩增了12份江西地区猪场疑似患PRRS的猪肺脏样品中的ORF5全序列和NSP2部分序列,应用DNAStar和Mega 6.0等软件对所得序列进行同源性比对及遗传变异分析。12株PRRSV ORF5核苷酸同源性为83.7%~99.8%,氨基酸同源性为82.1%~99.5%;与参考毒株JXA1、VR-2332和LV的核苷酸同源性分别为84.9%~99.7%、85.2%~91.0%和62.4%~64.8%。对阳性病料进行了NSP2基因部分序列的扩增,测序结果显示12株PRRSV均属于美洲型毒株,12株PRRSV的NSP2部分序列均存在30个氨基酸的不连续缺失,与高致病性PRRSV有相同的缺失特征。12株PRRSV的ORF5遗传进化树分析显示,10株与高致病性PRRSV处在同一进化分支,进一步说明高致病性PRRSV已成为江西地区的优势流行毒株。     

2013年-2014年江西地区PRRSV分离株NSP2及ORF5基因的遗传变异分析

为研究江西地区猪繁殖与呼吸综合征病毒(PRRSV)ORF5基因的变异情况及NSP2基因的结构特征,采用RT-PCR方法扩增了12份江西地区猪场疑似患PRRS的猪肺脏样品中的ORF5全序列和NSP2部分序列,应用DNAStar和Mega 6.0等软件对所得序列进行同源性比对及遗传变异分析。12株PRRSV ORF5核苷酸同源性为83.7%~99.8%,氨基酸同源性为82.1%~99.5%;与参考毒株JXA1、VR-2332和LV的核苷酸同源性分别为84.9%~99.7%、85.2%~91.0%和62.4%~64.8%。对阳性病料进行了NSP2基因部分序列的扩增,测序结果显示12株PRRSV均属于美洲型毒株,12株PRRSV的NSP2部分序列均存在30个氨基酸的不连续缺失,与高致病性PRRSV有相同的缺失特征。12株PRRSV的ORF5遗传进化树分析显示,10株与高致病性PRRSV处在同一进化分支,进一步说明高致病性PRRSV已成为江西地区的优势流行毒株。     

水貂肠炎病毒LN-10分离株的分离鉴定

为了解我国水貂肠炎病毒(MEV)的流行情况,本研究采用F81细胞从疑似患有肠炎的水貂粪便样品中分离出一株病毒,经形态学、血清学、动物回归试验和分子生物学鉴定,分离的病毒为MEV,命名为LN-10。对该病毒主要结构蛋白VP2基因进行克隆测序和基因进化分析表明,LN-10分离株VP2基因与GenBank中的其他18株MEV株核苷酸和氨基酸均有较高的同源性,分别为99.3%～100%和99%～100%,其中核苷酸同源性与ZYL-1株为100%,而氨基酸同源性与ZYL-1株和Manzhouli株均为100%。本研究为MEV分子流行病学调查和疫苗的研究奠定了基础。     

猪流产胎儿中猪圆环病毒3型的检测及其遗传演化分析

旨在分析猪流产胎儿中猪圆环病毒3型(porcine circovirus type 3,PCV3)的感染及其遗传进化情况。对2015—2017年采集的湖南省680份猪流产胎儿病料样品进行PCV3检测、扩增和测序,并利用生物信息学软件对获得的序列进行遗传进化分析。结果显示,猪流产胎儿样品中PCV3阳性率为24.4%(166/680),其中,2015—2017年阳性率分别为18.2%(26/143)、22.7%(54/238)和28.8%(86/299)。从PCV3阳性样本中获得了1株PCV3基因组全长序列,命名为PCV3/CN/Hunan-57(GenBank序列登录号为MZ934695),与国内外毒株基因组序列相比,相似性为97.8%~99.2%;获得了5株PCV3 ORF2基因序列,它们的核苷酸和氨基酸相似性为97.5%~98.3%和96.7%~99.7%,与国内外参考毒株ORF2基因序列相比,其核苷酸和氨基酸相似性为96.3%~99.2%和96.8%~100%。基于ORF2基因序列构建遗传进化树和进行多重序列比对,发现获得的PCV3阳性序列分别属于PCV3a(1株)和PCV3c(4株),并鉴定出T100A、G113S和A184S突变位点。综上表明,猪流产胎儿中PCV3感染率较高,且存在不同类型的PCV3毒株感染,为后续深入研究PCV3遗传变异及其相关致病性提供了重要参考信息。     

Type I restriction-modification system and its resistance in electroporation efficiency in Flavobacterium columnare

The role of swine torque teno sus viruses (TTSuVs) as co-factors in disease syndromes involving porcine circovirus strain 2 (PCV2) and porcine reproductive and respiratory disease syndrome virus (PRRSV) has been a debatable subject. In this study, the prevalence of TTSuVs in Iowa, the leading pork producing state in the U.S., was estimated by a duplex PCR. The PCR is capable of simultaneously detecting both teno sus viruses 1 and 2 (TTSuV1 and 2). Based on an analysis of 300 random samples representing six major geographical regions of the state, the overall prevalence rates for TTSuV1 and 2 were 47.34% and 24.67% respectively while the combined prevalence rate was 52.33%. The epidemiological association of TTSuV1 and 2 with the common etiological agents of the porcine respiratory disease complex (PRDC) namely porcine PRRSV, PCV2, Mycoplasma hyopneumoniae and swine influenza virus (SIV) was estimated in lung tissue derived from 45 pigs showing clinical signs of PRDC. Notably, 86.67% of the PRDC-suspect samples were positive for TTSuV1 in comparison to the baseline population prevalence rate of 47.34%. However, the prevalence of TTSuV2 (26.67%) was not significantly different. TTSuV1 was detected in 80.00%, 81.81%, 75.00% and 77.78% of the PRRSV, SIV, M. hyopneumoniae and PCV2 positive PRDC-suspect samples respectively. Our results indicate that TTSuV1 is strongly associated with clinical PRDC and support the hypothesis that TTSuVs might function as co-factors in PRDC. Further studies to define their possible role in the pathogenesis of swine respiratory diseases are warranted.     

北京市猪场猪圆环病毒3型感染状况调查及其ORF2基因遗传进化分析

为了解猪圆环病毒3型(PCV3)在北京市不同区县猪场中的流行情况,研究建立PCR检测方法,对来自北京市8个区县56个养殖场的1177份临床样品进行检测,并对获得的部分ORF2全基因序列进行遗传进化分析。结果显示,PCV3总体阳性率为1.0%(12/1177),猪场阳性率为8.9%(5/56)。对PCV3阳性样本进行ORF2基因测序及同源性比对,共测得12株PCV3 ORF2全长基因序列,其中包括6株不同的ORF2全长基因序列。结果显示,该6株序列之间的核苷酸相似性为97.7%～99.5%,推导氨基酸序列的相似性为97.2%～100%;与参考毒株之间的核苷酸同源性为96.0%～99.5%,推导氨基酸同源性为92.1%～100.0%;进化树显示北京毒株属于PCV3b基因型。试验表明,PCV3在北京多个猪场呈现一定的流行趋势,流行毒株以PCV3b基因型为主。     

Seroprevalence of Torque teno sus virus types 1 and 2 in postweaning multisystemic wasting syndrome-suspected pigs and porcine circovirus type 2-vaccinated normal pigs in southern Japan

We investigated the seroprevalence rate of Torque teno sus virus types 1 (TTSuV1) and 2 (TTSuV2) in the sera of 38 post weaning multisystemic wasting syndrome (PMWS)-suspected pigs and 43 porcine circovirus type 2 (PCV2)-vaccinated normal pigs on 3 commercial pig farms in southern Japan by using nested polymerase chain reaction. High seroprevalence rate of TTSuVs was observed in both PMWS-suspected pigs (100%) and PCV2-vaccinated normal pigs (90.7%). The seroprevalence rate of TTSuV2 was significantly higher in the PMWS-suspected pigs than in PCV2-vaccinated normal pigs (97.4% versus 81.4%, P<0.05), whereas no such difference was observed for TTSuV1 between the 2 groups of pigs. In both pig groups, the seroprevalence rate of TTSuV2 was significantly higher than that of TTSuV1 (P<0.01-0.05). These results show that TTSuVs are highly seroprevalent in both PMWS-suspected pigs and PCV2-vaccinated normal pigs with TTSuV2 being more seroprevalent in the former than in the latter.     

Infection dynamics of Torque teno sus virus types 1 and 2 in serum and peripheral blood mononuclear cells

This study was carried out to investigate the presence of Torque teno sus virus types 1 (TTSuV1) and 2 (TTSuV2) in a longitudinally (14 to 150 days of age) collected paired pooled sera (pSE) and peripheral blood mononuclear cells (pPBMCs) using nested polymerase chain reaction. The detection rate of TTSuV1 in pSE increased from 14 to 90 days of age, but a progressive decline was observed from 120 to 150 days of age, while in pPBMC, a high value was maintained till the end of growing-finishing period. On the contrary, except in PBMCs at 30 days of age, high detection rates of TTSuV2 were found in both pSE and pPBMCs in all sampling ages. The detection rate of TTSuVs between pSE and pPBMCs was positively correlated at all sampling ages except for TTSuV1 at 150 days of age. This is the first study showing the presence of TTSuVs in PBMCs from pigs and describing the in vivo infection dynamics of TTSuV in paired sera and PBMCs during the entire growing and finishing periods of pigs reared in conventional farms.     

Detection of Torque teno sus virus types 1 and 2 by nested polymerase chain reaction in sera of sows at parturition and of their newborn piglets immediately after birth without suckling colostrum and at 24 hr after suckling colostrum

This study was performed to clarify the sow-to-fetus transmission pathway of Torque teno sus virus (TTSuV) types 1 (TTSuV1) and 2 (TTSuV2). For this purpose, detection of TTSuV1 and TTSuV2 (TTSuVs) in sera of 6 sows (Sows 1-6) at parturition and in sera of their newborn piglets immediately after birth without suckling colostrum was performed by nested polymerase chain reaction (nPCR). These sows were bred using semen that had tested negative for TTSuVs. In a TTSuV1- and TTSuV2-positive sow (Sow 1), TTSuV1 and TTSuV2 were detected in 4 and 5 of 12 newborn littermates, respectively. In a TTSuV1-positive sow (Sow 2), TTSuV1 was detected in 1 of 8 newborn littermates. In 4 TTSuV1- and TTSuV2-negative sows (Sows 3-6), TTSuV1 was detected in 6 out of the 25 newborn piglets of 3 sows (Sows 3-5), while TTSuVs were not detected in all 13 piglets of 1 sow (Sow 6). In addition, to investigate the possibility of a sow-to-piglet transmission pathway of TTSuV via colostrum, TTSuV1 and TTSuV2 in sera of 12 newborn piglets from Sows 1-3 were examined by nPCR. Immediately after birth without suckling colostrum, TTSuV1 and TTSuV2 were not detected in 10 and 8 of 12 newborn piglets, respectively; however, at 24 hr after suckling colostrum, TTSuV1 was detected in 6 piglets, while TTSuV2 was not detected in any piglets. These results confirmed the existence of a sow-to-fetus transmission pathway of TTSuV during normal pregnancy and suggested a possibility of sow-to-piglet transmission of TTSuV via colostrum.     

吉林地区1株小鹅瘟病毒NS1和VP1基因的克隆与序列分析

为了解吉林地区小鹅瘟病毒（Gosling plague virus,GPV）的基因特征,及其与黑龙江及中国其他省份和国外流行毒株的相关性,本研究采用PCR方法鉴定2018年吉林某养鹅场送检的病死雏鹅的肠组织,同时对GPV的非结构蛋白NS1基因及结构蛋白VP1基因进行了克隆和测序,并与国内外16株GPV参考毒株的相应序列进行分析。结果表明,病死雏鹅为GPV与减蛋综合征病毒（Egg drop syndrome virus,EDSV）混合感染;吉林地区GPV的NS1基因长为1 884 bp,编码627个氨基酸,与参考毒株核苷酸序列同源性为93.8%~99.8%,氨基酸序列同源性为97.1%~99.7%;VP1基因长为2 199 bp,编码732个氨基酸,与参考毒株核苷酸序列同源性为93.4%~99.9%,氨基酸序列同源性为96.4%~99.9%。NS1及VP1基因的系统进化树分析均表明,吉林地区GPV与哈尔滨分离株98E属于同一进化分支,亲缘关系最近,与国外分离株、中国台湾和安徽分离株亲缘关系均较远。吉林地区GPV与鹅源GPV具有较近的亲源关系,同源性明显高于其他水禽来源的GPV。该研究为明确中国东北地区GPV空间的流行规律提供基础数据,为东北地区GPV的诊断与治疗提供参考依据。     

Gene Analysis of Penton Protein of 12 Serotypes of Fowl Adenovirus

In order to understand the genetic evolution of Penton protein of fowl adenovirus(FAdV),the specific primers of Penton were designed on the basis of published sequences of different genotypes on GenBank.Then Penton gene of 12 serotypes were amplified by PCR and constructed into pEASY-Blunt Simple Cloning Vector for sequencing.The nucleotide sequences of Penton protein of 12 serotypes were analyzed and compared with the nucleotide sequences of standard strains published on NCBI by ClustalW method to draw a genetic phylogenetic tree.The results showed that the nucleotide sequences of Penton protein of 12 serotypes had more than 99.2% homology with their respective standard strain,among which the homology of FAdV-1,FAdV-2,FAdV-3,FAdV-4,FAdV-6,FAdV-7,FAdV-8a,FAdV-8b,FAdV-10 and FAdV-11 were as high as 100%.This further confirmed the consistency between the laboratory preserved strains and the world standard strains.However,although Penton protein was highly conserved,there were still significant differences among different species.The nucleotide homology between FAdV-A1 and other species was 70.6%-73.3%,while that of FAdV-B5 was 70.6%-77.9%.The nucleotide homology between FAdV-C strains of different serotypes was 99.6%,while homology between FAdV-C4 and other species was 71.2%-73.4%.The nucleotide homology between FAdV-D strains of different serotypes was 95.6%-98.7%,while the highest homology between FAdV-D and other species was 85.3%.The nucleotide homology between FAdV-E strains of different serotypes was 98.2%-99.4%,while the highest homology between FAdV-E and other species was 85.3%.In conclusion,the homology difference between strains of the same species was small,and that of different species was significant.FAdV could be divided into A,B,C,D and E species according to nucleotide sequence of Penton protein.This study successfully cloned 12 serotypes Penton gene of FAdV and performed genetic evolution analysis,which laid the foundation for the diagnosis,monitoring and identification of FAdV in the future.     

鸡腺病毒12个血清型代表毒株Penton蛋白基因分析

为了解鸡腺病毒（fowl adenovirus,FAdV）Penton基因的遗传进化规律,试验根据GenBank中已公布的各基因型序列设计特异性引物,通过PCR技术分别扩增12个血清型毒株的Penton基因,连接至pEASY-Blunt Simple Cloning Vector上进行序列测定,并将12个血清型毒株Penton蛋白的核苷酸序列与NCBI上已公布的标准毒株的核苷酸序列使用ClustalW法进行分析比对,绘制遗传进化树。结果显示,12种血清型Penton蛋白核苷酸序列与各血清型标准毒株同源性均在99.2%以上,其中FAdV-1、FAdV-2、FAdV-3、FAdV-4、FAdV-6、FAdV-7、FAdV-8a、FAdV-8b、FAdV-10、FAdV-11与对应标准株同源性高达100%,这进一步证实了实验室保存毒株与世界相应标准毒株的一致性。Penton蛋白虽然保守性较高,但不同种之间仍存在明显差异。FAdV-A1与其他种毒株同源性为70.6%~73.3%;FAdV-B5与其他种毒株同源性为70.6%~77.9%;FAdV-C同种不同血清型之间核苷酸同源性为99.6%,FAdV-C4与其他种之间同源性71.2%~73.4%;FAdV-D同种不同血清型之间核苷酸同源性为95.6%~98.7%,与其他种之间最高为85.3%;FAdV-E同种不同血清型之间核苷酸同源性为98.2%~99.4%,不同种间最高为85.3%。即相同种毒株之间差异较小,不同种毒株之间差异较大。依据Penton蛋白的核苷酸序列同样可以将FAdV分为A、B、C、D、E 5个种。本研究通过成功克隆FAdV 12种血清型Penton基因,并进行遗传进化分析,为今后对FAdV诊断、监测及毒株鉴定提供了参考。