Genetic characterization and phylogenetic analysis of porcine circovirus type 2 strains present in Cuban swine herds

Porcine circovirus type 2 (PCV2) is the essential infectious agent of postweaning multisystemic wasting syndrome (PMWS) considered as one of the most important swine diseases worldwide. One of the main risk factors reported for the development of PMWS is the PCV2 genotype. The presence of PCV2 in Cuban swine herds has been reported recently. However, genetic information about these viruses is not available yet. Hence, the objectives of this study were to classify the Cuban porcine circovirus type 2 sequences as well as to investigate the genetic diversity and the putative origins of the virus circulating in Cuban swine herds. PCV2 Cuban sequences appeared to be close related when an analysis of the entire viral genome sequences was performed. The main variations on amino acid sequences of the capsid protein were found within the immunoreactive areas. All the Cuban PCV2 sequences analyzed belonged to genotype 1 and were located within the same Cluster (1A). Interestingly, five of them were clustered with high confident values with those described as the PCV2 variants associated with severe porcine circovirus diseases reported in Canada from the late 2004 to 2006. Pigs imported from this source appeared to be the most probable origin of the viruses circulating in Cuban swine herds currently. The fact that one sequence was not clustered with any other group of PCV2 within genotype 1 might suggest that different introductions of the agent in the country from unknown sources have occurred.     

First report of porcine circovirus type 2 infections in Cuba

To obtain information about the porcine circovirus type 2 (PCV2) infection status of pigs in Cuba and the probable association of PCV2 with other porcine viruses, tissue samples collected from ill pigs were evaluated using polymerase chain reaction (PCR). The PCR analysis showed that 67.7% of the samples (23/34) from seven swine herds of six different geographic regions were detected to be positive for PCV2. Ten of the 23 PCV2 positive samples (43.5%) shown a concurrent infection with porcine parvovirus (PPV) and 17 of 23 PCV2 positive samples (73.9%) exhibited a concomitant infection with classical swine fever virus (CSFV). This study is the first report of PCV2 infecting pigs with different clinical conditions in Cuban swine herds and provides evidence of PCV2 co-infection with PPV and CSFV in the field.     

Molecular detection of Torque teno sus virus in lymphoid tissues in concomitant infections with other porcine viral pathogens

In this study, 40 pigs with respiratory and wasting disorders from Cuban swine herds were screened by PCR for the presence of TTSuV1, TTSuV2, PCV-2, PPV and CSFV in spleen samples. The variability of the porcine TTSuV sequences obtained was investigated by phylogenetic analysis. This study showed for the first time that TTSuV1 and TTSuV2 were present in Cuban swine herds. The investigation revealed the following infection rates: TTSuV1 40%, TTSuV2 37.5%, PCV-2 70%, PPV 37.5% and CSFV in 52.5%. The presence of two or more of these viruses at different rates in the same spleen samples was revealed. Also, a higher genetic diversity of TTSuV2 sequences was observed regarding TTSuV1 sequences.     

Classical swine fever virus isolates from Cuba form a new subgenotype 1.4

Identification and classification of classical swine fever virus (CSFV) on the basis of nucleotide sequencing and phylogenetic analysis have become an important tool to study the epidemiology and to control CSF disease. According to phylogenetic analyses of short sequences from the 5′nontranslated region (150 nt) and the E2 (190 nt), most CSFV isolates from South and Central America have been assorted to the subgenotypes 1.1 and 1.3, while CSFV isolates from Cuba have been allocated to subgenotype 1.2. Here we demonstrate that determination and comparison of full-length E2 sequences as well as of the sequences encoding for N^pro, C, E^rns, E1 and E2 (3361 nt) do not support segregation of Cuban CSFV isolates to subgenotype 1.2. In fact, our analysis revealed that the Cuban isolates are more divergent from other so far known CSFV subgenotype 1 isolates and form a novel separate subgenotype that is proposed to be designated subgenotype 1.4.     

Postweaning multisystemic wasting syndrome produced in gnotobiotic pigs following exposure to various amounts of porcine circovirus type 2a or type 2b

In late 2005, a postweaning, high mortality syndrome spread rapidly through finishing barns in swine dense areas of the United States. Diagnostic investigations consistently detected porcine circovirus type 2 (PCV2) from diseased tissues. Subsequent genetic analysis revealed that the infectious agent was a PCV2 type termed "PCV2b". Prior to late 2004, only the PCV2a type, but not PCV2b, had been reported in North America. In this communication, we produce severe postweaning multisystemic wasting syndrome (PMWS) in gnotobiotic pigs using infectious PCV2a and PCV2b generated from DNA clones constructed from field isolates identified in the 2005 outbreak. Clinical signs exhibited by diseased pigs included anorexia, dyspnea and listlessness. Mortality was typically observed within 12h of onset of dyspnea. The most striking microscopic lesions in affected animals were severe hepatic necrosis and depletion of germinal centers in lymph nodes with associated abundant PCV2 viral antigen. Clinical signs and lesions observed in these studies were comparable to those reported in experiments with gnotobiotic pigs inoculated with a PCV2a isolate while concurrently receiving immune-stimulation or co-infection with porcine parvovirus or torque teno virus. The animals in these studies were confirmed to be free of detectable porcine parvovirus, porcine reproductive and respiratory syndrome virus, bovine viral diarrhea virus, swine hepatitis E virus, and aerobic and anaerobic bacteria. Seven out of 24 PCV2 inoculated pigs had a detectable congenital torque teno virus infection with no correlation to clinical disease. Thus, in these studies, both PCV2a and PCV2b isolates were singularly capable of inducing high mortality in the absence of any detectable infectious co-factor.     

Genomic analysis of PCV2 isolates from Danish archives and a current PMWS case-control study supports a shift in genotypes with time

Porcine circovirus type 2 (PCV2) is the primary cause of Postweaning Multisystemic Wasting Syndrome (PMWS) in pigs. PCV2, however, is found in both PMWS-affected herds and non-affected herds. The objective of this study was to clarify if PCV2 genome nucleotide sequences isolated from pigs from PMWS-affected herds and non-affected herds cluster phylogenetically in two separate groups. All isolates (45) belonged to PCV2 group 1 and shared a nucleotide sequence identity of 99.4-100% indicating a very homogeneous PCV2 population in Denmark. Phylogenetic analysis of the PCV2 isolates revealed no distinctive clustering of case- and control-herds suggesting that there is no link between PCV2 sequences and herd disease status. The appearance of only PCV2 group 1 isolates in this study (isolates from 2003/2004) led us to determine if PCV2 nucleotide sequences had changed in Denmark over time. Interestingly, all PCV2 isolates from before the first outbreak of PMWS (2001) belonged either to a new PCV2 group identified for the first time in this study and named group 3 (isolates from 1980, 1987 and 1990) or PCV2 group 2 (isolates from 1993 and 1996). The shift from PCV2 group 2 to 1 was confirmed on a more global scale by placing all full genome PCV2 sequences submitted to GenBank from 1997 to 2006 in either of the groups by phylogenetic analysis. The analysis showed that the shift happened in 2003 or even earlier. This may indicate that PCV2 group 1 is a more adapted form of PCV2 and possibly could be more pathogenic.     

First evidence of the emergence of novel putative infectious bronchitis virus genotypes in Cuba

The emergence of new infectious bronchitis virus (IBV) genotypes or serotypes along with the poor cross-protection observed among IBV serotypes have complicated the avian infectious bronchitis (IB) control programs in different geographic regions. In Cuba, the lack of genetic information regarding IBV and the increasing epidemiological importance of this virus in Cuban chicken flocks demand further characterization of IBV isolates. In the present work, studies of genetic diversity and phylogenetic relationships among recent IBV isolates from Cuban chicken flocks showing respiratory disorders were performed. Two putative genotypes genetically different to the Massachusetts genotype H120 strain used in the Cuban vaccination program were found in the flocks assessed. In addition, a potential nephropathogenic IBV isolate was found by first time in Cuba.     

猪瘟病毒的分离鉴定及其E2基因序列分析

2004~2005年上海和江苏3家猪场发生仔猪急性死亡,用ELISA鉴别试剂盒证明为猪瘟感染。用PK15细胞分离病毒,传代至第3代无细胞病变,RT-PCR扩增E2基因为阳性,扩增片段经纯化后克隆入T载体并进行序列测定。通过DNAStar软件对3株病毒的基因序列进行了比较,同时构建了系统进化树,结果表明,三株病毒分离株均扩增出269bp片段并且核苷酸序列100%同源,这3株与Alfort、Shimen株、兔化弱毒株(HCLV)和Brescia4个标准毒株核苷酸序列同源性均在90%以上,说明在江苏、上海地区分离得到的3株猪瘟病毒与流行毒株在基因序列上没有发生大的变异。     

Genetic diversity of porcine circovirus type 2 (PCV2) in the Romanian wild boar population

In this study, we have analyzed 23 PCV2 ORF2 sequences recovered from wild boar population in Romania. The PCV2 sequences were originated from different geographical regions in Romania, and collected between 2008 and 2009 during the classical swine fever virus (CSFV) surveillance campaign. Complete open reading frame 2 (ORF2) nucleotide sequences were obtained and compared with sequences mainly from European and Asian isolates. The Romanian sequences were identified as belonging to previously described clusters 2a and 2b, with high degree of heterogeneity (PCV2 ORF2 nucleotide homology ranged between 90.1% and 100%). Interestingly, for cluster 2a, the majority of the sequences (8 from a total number of 9) clustered mainly with the Asian isolates (especially China, but also India and South Korea), with three exceptions from Europe previously reported in Germany, Belgium and The Netherlands.     

Genetic diversity of porcine circovirus type 2 (PCV2) in the Romanian wild boar population

In this study, we have analyzed 23 PCV2 ORF2 sequences recovered from wild boar population in Romania. The PCV2 sequences were originated from different geographical regions in Romania, and collected between 2008 and 2009 during the classical swine fever virus (CSFV) surveillance campaign. Complete open reading frame 2 (ORF2) nucleotide sequences were obtained and compared with sequences mainly from European and Asian isolates. The Romanian sequences were identified as belonging to previously described clusters 2a and 2b, with high degree of heterogeneity (PCV2 ORF2 nucleotide homology ranged between 90.1% and 100%). Interestingly, for cluster 2a, the majority of the sequences (8 from a total number of 9) clustered mainly with the Asian isolates (especially China, but also India and South Korea), with three exceptions from Europe previously reported in Germany, Belgium and The Netherlands.     

Genetic Diversity of Mitochondrial DNA D-loop Sequences in Huili Black Goat

To explore the genetic diversity and origin for genetic resource protection of Huili Black goat, the mitochondrial DNA (mtDNA) D-loop was investigated. mtDNA D-loop sequences of 41 goats were analyzed by PCR, sequencing techniques, and biological information and the phylogenetic trees were constructed. The mtDNA sequences of the Huili Black goat ranged from 1211 to 1213 bp, and 2 sequences were 1211 bp, 29 sequences 1212 bp, and 10 sequences 1213 bp. The content of A+T (60.1%) was higher than one of G+C (39.9%). There were 9 haplotypes, and the haplotype diversity was 0.842+0.00368. The nucleotide diversity was 0.01542+0.00034. The phylogenetic analysis showed that Huili Black goat was distributed in a branch, and were closed to Jianchang Black goat, Chengdu Ma goat, Jintang Black goat, Guizhou White goat, Guizhou Black goat, but they were less related to Capra falconeri. Huili Black goats had rather abundant genetic diversity, and were greatly affected by other goat breeds in history.     

Detection and genetic characterization of porcine circovirus type 2 (PCV2) in pigs from Croatia

Porcine circovirus type 2 (PCV2) from the Circoviridae family has recently been associated with two serious diseases of swine, post-weaning multisystemic wasting syndrome (PMWS) and porcine dermatitis and nephropathy syndrome (PDNS). During 2002, several outbreaks of clinical disease in pigs with weights ranging from 10 to 70 kg occurred on four farms in different locations in Croatia. The signs were consistent with PMWS and PDNS. Apart from progressive weight loss, pneumonia and/or diarrhoea, multifocal erythematous skin lesions and dermal necrosis were also observed. The PCR results obtained from PCV2 specific oligonucleotide primers confirmed a PCV2 infection. In addition, archive samples that were classical swine fever virus positive and derived from domestic pigs during an outbreak in 1997 were included in this study and one out of the three isolates was found to be positive for PCV2. For a better epizootiological understanding, genetic typing of representative isolates was carried out and compared with available isolates reported in the GenBank databases.     

Genome Sequence Analysis of Porcine Circovirus Type 2 Strains from Shanxi Province

In order to study genetic variation of porcine circovirus type 2 (PCV2) strains in Shanxi province, the genomic sequences of four PCV2 strains which were SXXZ1, SXXZ2, SXTY and SXJZ were isolated recently from some areas of Shanxi province, they were amplified, cloned and sequenced. The amplified PCV2 genomic sequences of these four strains were analyzed and compared with that of 34 published PCV2 stains by DNAStar and drawing phylogenetic tree. The results showed that the genomic sequences of SXJZ PCV2 strain was 1 768 bp, and the others were 1 767 bp, which accounted for 25% and 75%, respectively. The homologies of nucleotide sequences of the four strains were 95.9% to 100.0%, the homologies of nucleotide sequences of the four strains with the 34 isolates from different regions of the world PCV strain were 94.5% to 99.9%, and the homologies of nucleotide sequences of the four strains with the domestic vaccine strains were 95.6% to 99.8%. The phylogenetic tree analysis showed that SXXZ1, SXXZ2 and SXTY belonged to PCV-2b genotype, and SXJZ belonged to PCV-2e genotype. The evolutionary relationship among SXXZ1, SXXZ2 and the QY strain of Guangdong were closer, we also found a recent evolutionary relationship between SXTY and AUT5 of Austria, and a recent evolutionary relationship between SXJZ and FJ of Fujian. Besides the evolutionary relationship among SXXZ1, SXXZ2, SXTY and the DBN-SX07-2 of PCV2 vaccine of Shanxi province, a recent evolutionary relationship between SXJZ and LG of PCV2 vaccine were found. Thus PCV2 strains were popular based on PCV-2b in Shanxi province, also PCV-2e was isolated and existed. It laid a foundation for the molecular epidemiology, genetic variation, prevention and control of PCV2 in Shanxi province.     

猪圆环病毒2型山西株全基因组序列分析

为了解山西省猪圆环病毒2型（PCV2）的遗传变异情况,本试验采用PCR方法对山西省分离的4株猪圆环病毒流行株（SXXZ1株、SXXZ2株、SXTY株和SXJZ株）的全基因组进行了扩增、克隆和测序,并将其全基因组序列与国内外34株主要流行毒株进行核苷酸同源性与系统进化树分析。结果显示,4株PCV2山西流行株全基因组核酸序列全长SXJZ株为1 768 bp,其余均为1 767 bp,分别占25%和75%。4株毒株核苷酸同源性为95.9%~100.0%,与国内外34株参考株同源性为94.5%~99.9%,与国产疫苗株同源性为95.6%~99.8%;PCV2全基因组序列进化分析表明,本研究分离的SXXZ1株、SXXZ2株和SXTY株属于PCV-2b基因型,SXJZ株属于PCV-2e基因型,其中SXXZ1株和SXXZ2株与广东QY株的进化关系最近,SXTY与奥地利AUT5株的进化关系最近,SXJZ与福建FJ株的进化关系最近;而SXXZ1株、SXXZ2株和SXTY株与山西PCV2疫苗DBN-SX07-2株的进化关系较近,SXJZ株与国内PCV2疫苗LG株的进化关系较近。从而证实在山西省流行的PCV2毒株以PCV-2b为主,同时还分离出了PCV-2e亚型,表明山西省存在PCV-2e亚型毒株。本试验结果为山西省PCV2的分子流行病学、遗传变异及防控奠定了基础。     

山西猪瘟野毒株E_2基因序列测定与遗传变异多样性分析

为了解山西地区猪瘟病毒（CSFV）流行毒株的遗传变异情况,采用RT-PCR方法,2013年从山西部分地区分离出5株CSFV流行毒株,并进行了E2全基因扩增、克隆与序列测定,应用DNAStar分析软件对所测定的5株毒株与国内外参考毒株的相应序列进行了同源性分析,绘制系统发育进化树。结果表明：5株CSFV流行毒株之间E2基因核苷酸序列与所推导氨基酸序列的同源性分别为81.4%~100%和87.9%~100%,与CSFV石门毒株（Shimen株）的核苷酸与氨基酸的同源性分别为82.9%~94.8%和89.0%~94.9%,与CSFV兔化弱毒株（HCLV株）的核苷酸与氨基酸的同源性分别为81.6%~99.6%和87.9%~99.5%,与17株来自各国不同地区的CSFV参考毒株的核苷酸与氨基酸的同源性分别为81.5%~99.6%和86.3%~99.7%。经系统发育关系分析,4株属于基因2群,且705、713、725、729、734和738位氨基酸发生置换,另外1株属于基因1群。本研究揭示了山西猪瘟流行毒株的遗传变异多样性现状。     

Genetic diversity of Bangladeshi native chickens based on complete sequence of mitochondrial DNA D-loop region

ABSTRACT

1. The aim of this study was to explore genetic diversity and possible origin of Bangladeshi (BD) native chickens. The complete mtDNA D-loop region was sequenced in 60 chickens representing five populations; naked neck, full feathered, Aseel, Hilly and autosomal dwarf. The 61 reference sequences representing different domestic chicken clades in China, India, Laos, Indonesia, Myanmar, and other Eurasian regions were included. The mtDNA D-loop sequence polymorphism and maternal origin of five BD populations were analysed.

2. A total of 35 polymorphic sites, and 21 haplotypes were detected in 60 mtDNA D-loop sequences. The haplotype and nucleotide diversity of the five populations were 0.921 ± 0.018 and 0.0061 ± 0.0019, respectively. Both mtDNA network and phylogenetic analysis indicated four clades (four haplogroups) in BD populations (21 haplotypes) along with 61 reference haplotypes. Clade E contained the most individuals (20) and haplotypes (11) of BD chickens, followed by clade D (17, 6), clade C (12, 2) and clade F (11, 2), respectively.

3. The higher number of unique haplotypes found in Yunnan, China, suggested that the origin of BD chickens was in this region. The haplotypes from different haplogroups were introduced in Bangladeshi chickens from India, China and Myanmar. The phylogenetic tree showed a close relationship of BD chickens with the clusters from India, China, Myanmar and Laos, and indicated the dispersion of BD chickens from these sources. The phylogenetic information revealed high genetic diversity of BD chickens because of their origin from different lineages with high genetic variation and distance, which was determined from four cluster and neighbour-joining trees.

4. In conclusion, BD populations had high genetic diversity. The mtDNA network profiles and phylogenetic trees showed multiple maternal origins of BD chickens from India, China, Myanmar and Laos.     

新疆巴州牦牛种群线粒体DNA遗传多样性及其系统发育分析

研究测定新疆巴州牦牛种群线粒体DNA细胞色素b基因全长序列,在此基础上构建系统发育树,分析种群的遗传现状.结果表明,在17头巴州牦牛个体中检测到4种Cyt b基因单倍型,单倍型多样性为0.331,核苷酸多样性为0.00102;系统发育关系显示,巴州牦牛Cyt b基因单倍型明显有两个分支,一支与家牦牛单倍型聚在一起,另一支与新疆野牦牛单倍型聚在一起,表明巴州牦牛可能有2个母系起源.     

广西猪瘟病毒E0和E2基因的克隆及序列分析

通过分析目前广西猪瘟病毒(CSFV)的E0和E2基因特征,为了解广西地区CSFV的分子流行病学、遗传变异及综合防控提供科学依据。试验采用RT-PCR方法,对阳性猪瘟样品进行CSFV的E0及E2基因的扩增,经克隆、测序后,利用DNAStar软件对序列进行比对分析,同时绘制系统遗传进化树。结果表明,从阳性猪瘟样品中成功扩增CSFV的E0及E2基因。序列比对分析发现,GX2毒株与参考毒株的E0基因核苷酸同源性在83.1%~94.1%,其推导氨基酸同源性在85.9%~99.6%;与参考毒株的E2基因核苷酸同源性为81.7%~93.7%,其推导氨基酸同源性为89.0%~97.0%;E0与E2基因均属于基因Ⅱ群。氨基酸变异位点分析表明,E0蛋白的RNase活性区域氨基酸基序位点没有发生变异;E2蛋白中15个位点上的半胱氨酸均未发生变异,但单抗识别位点S734R发生变异。遗传进化分析显示测定的GX2毒株与近年来广西CSFV流行毒株的变异趋势相似,与中国传统疫苗株HCLV、经典强毒株Shimen的同源性较低,亲缘关系较远,与广西近年来的流行毒株GXWZ02株的同源性较高,亲缘关系较近。     

猪圆环病毒2型四川分离株ORF2基因的克隆与序列分析

对临床分离毒株PCV-2 sch2010 ORF2基因全序列进行了克隆和序列分析.结果表明,PCV-2sch2010 ORF2全基因共705 bp,编码234个氨基酸,与GenBank发表的21株PCV-2的ORF2参考序列的核苷酸氨基酸序列同源性分别为88.4%～99.7%和87.2%～99.6%;与2株PCV-1 ORF2(GU371908、FJ475129)的核苷酸序列和氨基酸序列同源性分别为58.7%～59.5%和62.8%～64.5%;PCV-2 sch2010分离株与PCV-2的欧洲分支群亲缘关系较近.