Genetic diversity and phylogenetic analysis of glycoprotein 5 of PRRSV isolates in mainland China from 1996 to 2006: coexistence of two NA-subgenotypes with great diversity

GP5, the most important neutralizing antigen of porcine reproductive and respiratory syndrome virus (PRRSV), has the highest genetic diversity among isolates. To more fully understand the extent of genetic diversity of PRRSV in China, we analyzed and compared the GP5 sequences of 42 PRRSV isolated from 1996 to 2006 in mainland China. We found that all of the Chinese isolates examined belong to the North American (NA) type. Among them two highly diverse subgroups were clearly demarcated on the NA-genotype phylogenetic tree. All the subgroup 1 isolates were found to be high variable in the primary neutralizing epitope and the viruses were geographically restricted to regions in southeast China. The subgroup 2 isolates shared a high identity with MLV vaccine and its parent virus VR-2332. These results may contribute to the knowledge of PRRSV epidemiology in China, and may help to explain the low efficiency of MLV or killed CH-1a vaccine to protect the subgroup 1 virus infected pigs, and the great genetic diversity should be taken into consideration for control and preventive measures.     

Genetic diversity and phylogenetic analysis of Feline Coronavirus sequences from Portugal

The distribution of FCoV Types I and II in a Portuguese cat population was studied by a RT-PCR assay targeting the 3′-end of the viral RNA. For a period of 3 years, 120 samples were collected and 57 were found positive for FCoV RNA. Within the positive samples the presence of FCoV Type I was found in 79%. Type II was only detected in 3.5% in animals with Feline Infectious Peritonitis. The remaining 17.5% could not be differentiated. These viral sequences, comprising a region within gene S were further subjected to a heteroduplex mobility assay (HMA) detecting the presence of viral quasispecies in 17% of the samples. Phylogenetic analysis for FCoV Type I revealed high genetic diversity between the Portuguese sequences and other previously characterized strains, while Type II tree showed a higher genetic homogeneity. This study confirmed the presence of FCoV Types I and II circulating in Portugal and detected high genetic diversity between circulating strains suggesting that the virus persists within the host as mixed viral populations.     

高致病性猪繁殖与呼吸综合征病毒变异株主要囊膜糖蛋白GP5的遗传变异分析

为了探究高致病性猪蓝耳病病毒（HP-PRRSV）的遗传变异特征,本研究对GenBank中235株HP—PRRSV GP5序列的遗传进化、主要氨基酸基序、抗原性以及N-糖基化位点数量和位置的变异进行了分析。结果表明HP-PRRSV之间的同源性较高,与其他参考毒株相比,这些病毒处于一个相对独立的分支中,而且与经典疫苗株的亲缘关系较远。这有助于解释经典疫苗株对于HP—PRRSV为何起不到理想的免疫保护效果。在病毒的中和表位序列中存在着规律的点突变,同时抗原性比较显示HP-PRRSV与经典毒株之间存在着一定的差异。绝大多数的HP-PRRSV的N-糖基化位点在数量上多一个,而且位置要向羧基端平移2个氨基酸,从而使得中和表位两侧直接与糖链相连,可能会造成中和表位被糖侧链所遮掩,本研究由此推测减弱中和抗体对HP-PRRSV的有效识别,促进病毒逃避机体体液免疫。     

Genetic and phylogenetic analysis of glycoprotein of rabies virus isolated from several species in Brazil

Genetic and phylogenetic analyses of the region containing the glycoprotein (G) gene, which is related to pathogenicity and antigenicity, and the G-L intergenic region were carried out in 14 Brazilian rabies virus isolates. The isolates were classified as dog-related rabies virus (DRRV) or vampire bat-related rabies virus (VRRV), by nucleoprotein (N) analysis. The nucleotide and amino acid (AA) homologies of the area containing the G protein gene and G-L intergenic region were generally lower than those of the ectodomain. In both regions, nucleotide and deduced AA homologies were lower among VRRVs than among DRRVs. There were AA differences between DRRV and VRRV at 3 antigenic sites and epitopes (IIa, WB+ and III), suggesting that DRRV and VRRV can be distinguished by differences of antigenicity. In a comparison of phylogenetic trees between the ectodomain and the area containing the G protein gene and G-L intergenic region, the branching patterns of the chiropteran and carnivoran rabies virus groups differed, whereas there were clear similarities in patterns within the DRRV and VRRV groups. Additionally, the VRRV isolates were more closely related to chiropteran strains isolated from Latin America than to Brazilian DRRV. These results indicate that Brazilian rabies virus isolates can be classified as DRRV or VRRV by analysis of the G gene and the G-L intergenic region, as well as by N gene analysis.     

Genetic diversity and phylogenetic relationships between Leishmania infantum from dogs,humans and wildlife in south‐east Spain

Leishmania infantum causes human and canine leishmaniosis. The parasite, transmitted by phlebotomine sand flies, infects species other than dogs and people, including wildlife, although their role as reservoirs of infection remains unknown for most species. Molecular typing of parasites to investigate genetic variability and evolutionary proximity can help understand transmission cycles and designing control strategies. We investigated Leishmania DNA variability in kinetoplast (kDNA) and internal transcribed spacer 2 (ITS2) sequences in asymptomatically infected wildlife (n = 58) and symptomatically and asymptomatically infected humans (n = 38) and dogs (n = 15) from south‐east Spain, using single nucleotide polymorphisms (SNPs) and in silico restriction fragment length polymorphism (RFLP) analyses. All ITS2 sequences (n = 76) displayed a 99%–100% nucleotide identity with a L. infantum reference sequence, except one with a 98% identity to a reference Leishmania panamensis sequence, from an Ecuadorian patient. No heterogeneity was recorded in the 73 L. infantum ITS2 sequences except for one SNP in a human parasite sequence. In contrast, kDNA analysis of 44 L. infantum sequences revealed 11 SNP genotypes (nucleotide variability up to 4.3%) and four RFLP genotypes including B, F and newly described S and T genotypes. Genotype frequency was significantly greater in symptomatic compared to asymptomatic individuals. Both methods similarly grouped parasites as predominantly or exclusively found in humans, in dogs, in wildlife or in all three of them. Accordingly, the phylogenetic analysis of kDNA sequences revealed three main clusters, two as a paraphyletic human parasites clade and a third including dogs, people and wildlife parasites. Results suggest that Leishmania infantum genetics is complex even in small geographical areas and that, probably, several independent transmission cycles take place simultaneously including some connecting animals and humans. Investigating these transmission networks may be useful in understanding the transmission dynamics, infection risk and therefore in planning L. infantum control strategies.     

Genetic diversity and relatedness of Fasciola spp. isolates from different hosts and geographic regions revealed by analysis of mitochondrial DNA sequences

The present study examined sequence variability in a portion of the mitochondrial cytochrome c oxidase subunit 1 (pcox1) and NADH dehydrogenase subunits 4 and 5 (pnad4 and pnad5) among 39 isolates of Fasciola spp., from different hosts from China, Niger, France, the United States of America, and Spain; and their phylogenetic relationships were re-constructed. Intra-species sequence variations were 0.0-1.1% for pcox1, 0.0-2.7% for pnad4, and 0.0-3.3% for pnad5 for Fasciola hepatica; 0.0-1.8% for pcox1, 0.0-2.5% for pnad4, and 0.0-4.2% for pnad5 for Fasciola gigantica, and 0.0-0.9% for pcox1, 0.0-0.2% for pnad4, and 0.0-1.1% for pnad5 for the intermediate Fasciola form. Whereas, nucleotide differences were 2.1-2.7% for pcox1, 3.1-3.3% for pnad4, and 4.2-4.8% for pnad5 between F. hepatica and F. gigantica; were 1.3-1.5% for pcox1, 2.1-2.9% for pnad4, 3.1-3.4% for pnad5 between F. hepatica and the intermediate form; and were 0.9-1.1% for pcox1, 1.4-1.8% for pnad4, 2.2-2.4% for pnad5 between F. gigantica and the intermediate form. Phylogenetic analysis based on the combined sequences of pcox1, pnad4 and pnad5 revealed distinct groupings of isolates of F. hepatica, F. gigantica, or the intermediate Fasciola form irrespective of their origin, demonstrating the usefulness of the mtDNA sequences for the delineation of Fasciola species, and reinforcing the genetic evidence for the existence of the intermediate Fasciola form.     

Genetic diversity of the envelope glycoprotein E2 of classical swine fever virus: recent isolates branched away from historical and vaccine strains

The envelope glycoprotein E2 of classical swine fever virus (CSFV) plays multiple roles in the viral life cycle, interaction with host and pathogenesis. We sequenced the E2 gene of 34 CSFV isolates from southeastern China for analysis of genetic diversity in this particular region. Phylogenetic analysis revealed that genotype 2.1b viruses became predominant in southeastern China with 33 isolates clustered in 2.1b and only 1 isolate belonged to 2.2. Pairwise comparisons demonstrated isolates in this study showed a homology of 78.4-82.6% to Chinese C-strain in the 190nt of the E2 fragment. The minimum similarity within the 2.1b isolates was 91.1%. Variability of the full length E2 amino acid sequences of 45 CSFV isolates representative of three main groups of CSFV including 19 from southeastern China during 2004--2007 and 26 from other parts of China and other countries was analyzed by the differences between non-synonymous (dN) and synonymous (dS) rates and the entropy values. Two variable and three conserved regions as well as some specific positions under positive selection were defined. Our results suggest that recent isolates from southeastern China have shifted away from historical CSFV isolates including the vaccine strains probably as a result of their adaptive abilities to the selection forces within the host.     

Molecular characterization and phylogenetic analysis of new Newcastle disease virus isolates from the mainland of China

Seventy-nine velogenic Newcastle disease virus (NDV) isolates were obtained from infected chicken flocks during the outbreaks of Newcastle disease (ND) in various regions of the mainland of China in 2006. The F gene fragment (535 bp, from nt 47 to 581 of the F gene) which codes the main functional region of the F protein was obtained by RT-PCR and sequenced. All sequences obtained in this study have been submitted to GenBank. All the isolates have the motif ¹¹²R-R-Q/R-K/R-R-F¹¹⁷ at the cleavage site of the fusion protein, which is typical of velogenic NDV isolates. For genotyping, a phylogenetic tree based on nucleotides 47–435 of the F gene was constructed, and the 79 isolates could be divided into two genotypes, namely VIId and III. Most of the isolates proved to be of genotype VIId; only two isolates were of genotype III. Genotype VIId NDV has been the predominant pathogen responsible for most Newcastle disease outbreaks in China. The proportion of isolates of genotype VIId NDV shows an increasing trend, according to studies on the molecular epidemiology of NDV in China from 2002 to 2006.     

Molecular characterization and phylogenetic analysis of new Newcastle disease virus isolates from the mainland of China

Seventy-nine velogenic Newcastle disease virus (NDV) isolates were obtained from infected chicken flocks during the outbreaks of Newcastle disease (ND) in various regions of the mainland of China in 2006. The F gene fragment (535 bp, from nt 47 to 581 of the F gene) which codes the main functional region of the F protein was obtained by RT-PCR and sequenced. All sequences obtained in this study have been submitted to GenBank. All the isolates have the motif ¹¹²R-R-Q/R-K/R-R-F¹¹⁷ at the cleavage site of the fusion protein, which is typical of velogenic NDV isolates. For genotyping, a phylogenetic tree based on nucleotides 47–435 of the F gene was constructed, and the 79 isolates could be divided into two genotypes, namely VIId and III. Most of the isolates proved to be of genotype VIId; only two isolates were of genotype III. Genotype VIId NDV has been the predominant pathogen responsible for most Newcastle disease outbreaks in China. The proportion of isolates of genotype VIId NDV shows an increasing trend, according to studies on the molecular epidemiology of NDV in China from 2002 to 2006.     

Genetic and phylogenetic analysis of South Korean sacbrood virus isolates from infected honey bees (Apis cerana)

Sacbrood virus (SBV) is one of the most destructive honey bee viruses. The virus causes failure to pupate and death in both larvae and adult bees. Genetic analysis of SBV infected honey bees (Apis cerana) from five different provinces was carried out based on three nucleotide sequences; one partial structural protein coding sequence and two non-structural protein coding sequences. Sequences amplified by three specific primer pairs were aligned and compared with reference sequences deposited in the GenBank database. Sequence alignments revealed a low level of sequence variation among Korean isolates (≥ 98.6% nucleotide identity), regardless of the genome regions studied or the geographic origins of the strains. Multiple sequence comparisons indicated that Korean SBV isolates are genetically closely related to Chinese and other Asian strains. Interestingly, the Korean SBV isolates showed a number of unique nucleotides and amino acids that had not been observed in other published strains. Korean and other Asian isolates from the host A. cerana and the UK, European and Japanese strains from the host Apis mellifera showed differences in nucleotide and deduced amino acid identities. This suggests that host-specificity exists among SBV strains isolated from different species. Phylogenetic relatedness between compared sequences was analyzed by MEGA 4.1 software using the neighbor-joining (NJ) method with a boot-strap value of 1000 replicates. Obtained topologies were in agreement with previous studies, in which a distinct group of SBV was formed by UK and European genotypes and another group was comprised of Asian genotypes including strains that originated from China, Japan (japonica), India and Nepal. However, phylogeny based on a partial protein structural coding sequence grouped all Korean SBV isolates identified in A. cerana as a separate cluster. Our findings suggest that further study, including Korean SBV isolated from A. mellifera, is needed.     

Genetic diversity of avian infectious bronchitis virus isolates in Korea between 2003 and 2006

Thirty-three field isolates of avian infectious bronchitis virus (IBV) were recovered from commercial chicken flocks in Korea between 2003 and 2006 and were characterized phylogenetically by nucleotide sequence analysis of the IBV S1 gene hyper-variable region. Our phylogenetic analysis revealed that recent field isolates of IBV formed at least three distinct phylogenetic types, including K-I, K-II, and K-III. K-I type IBV consisted of indigenous, 13 IBV isolates which evolved from the Kr-EJ/95 strain and then separated into the lineages of type K-Ia and type K-Ib. K-II type IBV isolates (n = 19) were closely related to nephropathogenic IBV variants from China and Japan. The K-III type isolate (Kr/D064/05), first identified by this study, was closely related to enteric IBV variants from the Chinese strains that cause proventriculitis. Sequence comparisons showed amino acid differences of >27.5% between IBV types. The molecular epidemiologic characteristics of IBV field isolates are briefly discussed.     

猪繁殖与呼吸综合征病毒乌鲁木齐分离株GP5基因遗传变异分析

为对乌鲁木齐市猪繁殖与呼吸综合征病毒(PRRSV)的分子遗传变异情况进行研究与分析,应用RT-PCR方法对疑似感染PRRSV猪的组织脏器进行PRRSV GP5基因的扩增,将阳性样品PCR产物进行克隆和测序,并应用分子生物学软件对测序结果进行比对和遗传进化分析。结果显示,5株病毒分离株GP5基因全长均为603 bp,与JXA1株亲缘关系较近,同属于美洲株的同一基因亚群。本试验结果为掌握乌鲁木齐市PRRSV的分子遗传变异情况提供了依据。     

西藏部分山羊群体线粒体DNA D-loop区遗传多样性及系统进化分析

为了探究西藏不同山羊群体的遗传多样性和亲缘关系,提取4个山羊群体DNA,扩增其mtDNA D-loop区,并测序。结果显示:西藏山羊群体mtDNA D-loop区长度在1 200～1 212 bp,各群体山羊D-loop区富含A、T碱基,共发现106个多态位点,分离出21个单倍型。西藏山羊群体单倍型多样性(Hd)和核苷酸多样性(Pi)分别为0.085 7～1.000 0,0.007 04～0.019 14,4个群体山羊碱基突变率高,表明西藏山羊的遗传多样性非常丰富。核苷酸歧义度、NJ系统进化树表明西藏山羊群体间有共同母系血源,部分支系母系起源于镰刀型角野山羊(Capra aegagrus)和捻角山羊(Capra falconeri),同时存在其他的母系起源,支持山羊品种内的多起源说。     

2012年~2014年我国东南地区HP-PRRSV GP5基因的遗传进化与变异分析

为分析高致病性猪繁殖与呼吸综合征病毒(HP-PRRSV)的遗传变异情况,本研究对2012年~2014年东南地区8个省市21份HP-PRRSV阳性样品的GP5基因进行了RT-PCR扩增、克隆和测序,并将其进行序列比对和遗传进化分析。结果表明:本研究检测的21株HP-PRRSV GP5基因的核苷酸和氨基酸同源性分别为81.1%~99.8%和75.1%~100%。其中有13个病毒株的GP5基因与JXA1株同属于2.2亚群,其余8个病毒株的GP5基因与台湾2007年、2013年分离株属于2.3亚群。遗传进化树分析表明,2.3亚群是2014年流行的一个新兴亚群,它的流行可能与两地猪群贸易交流频繁有关,其GP5基因在抗原表位和糖基化位点发生了一定的变异。本研究结果为HP-PRRSV遗传进化分析和疫病防制提供了新的参考。     

2014年华南地区PRRSV ORF5基因的遗传变异分析

为了研究华南地区PRRSV的遗传变异情况,本研究对2014年华南地区16份PRRSV阳性样品的ORF5基因进行了RT-PCR扩增、克隆和测序,并将其进行序列比对和遗传进化分析。结果表明:本研究检测的16株PRRSV ORF5基因的核苷酸同源性为82.8%~100%,与JXA1,CH-1a以及VR2332核苷酸同源性分别为84.4%~99.3%,85.9%~95.0%和83.6%~89.2%。遗传进化树分析显示,所获得16株毒株序列中有13株属于以JXA1为代表的变异株亚群,另外3株属于以GM2,QYYZ为代表的新分支亚群。GP5氨基酸位点分析发现新分支亚群存在较为广泛的变异,尤其在信号肽区,诱导表位,中和表位及高变区。     

西南地区白玉米地方种质资源分布及遗传多样性

通过表型性状结合的主成分分析,SSR标记分析和群体结构分析,对50个西南地区白玉米地方品种的种质资源分布及遗传多样性进行了研究。结果表明,白玉米地方品种农艺性状表现出很大差异,主成分聚类将其分为7个类群,大部分品种聚在一个群内,但也有少数品种单独成类;利用筛选出来的51对扩增条带清晰,具有明显多态性的SSR引物,共检测到 515个多态性位点,SSR位点的平均多态性位点数为10.1个,每个位点的等位基因数为 5~19个,平均为12个,多态信息量(PIC)分布范围为0.764～0.958,平均为0.885,遗传相似系数变幅在0.574～0.840,平均为0.684,标记索引指数(MI)分布范围为3.820～18.208,平均值为9.775。SSR分析将所选品种分为8类,且84%的组合集中在前三大类。从地方品种来源地分析来看,地理位置相似或接近的地方品种大多两两成类,遗传相似系数很高,而群体结构分析显示,白玉米地方品种的群体结构受其地理环境气候的影响。少数地方品种较独立,可以进一步从种质资源方面研究利用。     

Genetic diversity analysis of the ORF5 gene in porcine reproductive and respiratory syndrome virus samples from South China

To understand the genetic diversity of porcine reproductive and respiratory syndrome virus (PRRSV) in South China, we collected 231 clinical samples from pigs with suspected PRRSV infection in Guangdong between 2007 and 2009. We found that 74 of 231 samples were positive by RT-PCR. The PCR products of the ORF5 gene of 35 isolates from different farms were sequenced and their DNA sequences were compared to 23 other PRRSV isolates in the GenBank. We found that the nucleotide similarity among all South China isolates ranged from 87.6% to 100%, and all belonged to the North American genotype. Most of them were classified into subgenotype I, but the rest mapped to subgenotypes III, V or VI. Those in subgenotypes I and III were found to be highly variable in the primary neutralising epitope (PNE) with a specific amino acid mutation (F39/L39→I39), and a few isolates in subgenotypes I and III isolates also had a mutation at L41 (L41→S41). PRRSV isolates in subgenotypes III, V and VI had less potential glycosylation sites than those in subgenotype I. Our data contribute to the understanding of molecular variation of PRRSV in South China.     

Detection and phylogenetic analysis of Mycoplasma hyopneumoniae from Tibetan pigs in western China

Tropical Animal Health and Production - Enzootic pneumonia (EP), often caused by Mycoplasma hyopneumoniae, occurs in Tibetan pigs between October and December in Western China. The aim of this...     

2013-2014年我国东北地区猪繁殖与呼吸综合征病毒GP5和NSP2基因遗传变异分析

为了解2013-2014年我国东北地区猪繁殖与呼吸综合征病毒(PRRSV)的遗传变异情况,研究对东北地区3个省份29个地区采集的123份样品进行了检测,并对20份阳性样品的GP5和部分NSP2基因进行了RT-PCR扩增、测序、遗传进化及序列比对分析。结果表明:我国东北地区PRRSV阳性率为16%,与国内外已发表的10株PRRSV参考株相比,GP5基因的核苷酸同源性为84.4%~100%。GP5氨基酸序列分析表明,大多数毒株在氨基酸高变区、中和表位和诱导表位发生了变异,主要变异为:C24→Y24,F25→L25,V27→A27,L28→I28,N34→G34/S34/D34。LN283株GP5和NSP2基因遗传进化树的不同结果表明,LN283株可能涉及PRRSV基因重组。HLJ13株NSP2基因PCR扩增产物有2条相差90 bp的目的条带,且NSP2基因遗传进化树显示两条序列分别属于CH-1a亚群和HuN4亚群,推断HLJ13株可能为经典和高致病性混合感染株。GP5和NSP2遗传进化及氨基酸序列分析结果表明,所有病毒株均属于北美洲型,且以HP-PRRSV为主,经典株与变异株共存。本研究结果可为我国东北地区PRRSV遗传进化研究及疫病防控提供参考。     

猪繁殖与呼吸综合征病毒ORF5基因的遗传变异和系统进化分析

猪繁殖与呼吸综合征病毒(PRRSV)毒株间变异广泛,而且这种变异能够筛选出强毒株。在结构蛋白中GP5的变异率最高,其变异能够导致病毒毒力的变化。为了确定PRRSV在我国的进化趋势和毒力增强的原因,对从2003年以来分离到14株PRRSV GP5蛋白的核苷酸序列和推导的氨基酸序列进行分析并构建了系统进化树。结果显示2006年以来从国内分离的毒株其糖基化位点发生了明显改变,并且在系统进化树上形成了新的分支,进化分析显示它们可能来源于1995年的中国分离株CH-1 a。