Cytochrome c oxidase subunit III (COX3) gene,an informative marker for phylogenetic analysis and differentiation of Babesia species in China

In this study a 552-bp region of the cytochrome c oxidase subunit III (COX3) was amplified by polymerase chain reaction (PCR) and sequenced from individual Babesia species. Sequence variation between Babesia species from China ranged between 0 and 32.4%. We analyzed the phylogenetic performance of the partial sequence of the COX3 gene to resolve Babesia relationships as compared to the nuclear 18S rRNA and the mitochondrial cytochrome b (COB) gene, These data indicate that the COX3 gene seems to be superior to the COB gene and the 18S rRNA in recognizing close lineages among some Babesia species. Our work indicates that the COX3 gene does complement and corroborate the phylogenetic inferences observed with the nuclear 18S rRNA and the COB gene previously reported. The combined phylogenetic analysis based on the nuclear 18S rRNA and the COX3 gene significantly improved (bootstrap) intraspecies support of the phylogenetic relationship. The presence of additional variable sites in the COX3 gene allowed an improved interspecies differentiation of Babesia species in this study. The data could be applicable for the survey of parasite dynamics, epidemiological studies as well as prevention and control of the disease.     

Phylogenetic analysis of the Trypanosoma genus based on the heat-shock protein 70 gene

Trypanosome evolution was so far essentially studied on the basis of phylogenetic analyses of small subunit ribosomal RNA (SSU-rRNA) and glycosomal glyceraldehyde-3-phosphate dehydrogenase (gGAPDH) genes. We used for the first time the 70 kDa heat-shock protein gene (hsp70) to investigate the phylogenetic relationships among 11 Trypanosoma species on the basis of 1380 nucleotides from 76 sequences corresponding to 65 strains. We also constructed a phylogeny based on combined datasets of SSU-rDNA, gGAPDH and hsp70 sequences. The obtained clusters can be correlated with the sections and subgenus classifications of mammal-infecting trypanosomes except for Trypanosoma theileri and Trypanosoma rangeli. Our analysis supports the classification of Trypanosoma species into clades rather than in sections and subgenera, some of which being polyphyletic. Nine clades were recognized: Trypanosoma carassi, Trypanosoma congolense, Trypanosoma cruzi, Trypanosoma grayi, Trypanosoma lewisi, T. rangeli, T. theileri, Trypanosoma vivax and Trypanozoon. These results are consistent with existing knowledge of the genus' phylogeny. Within the T. cruzi clade, three groups of T. cruzi discrete typing units could be clearly distinguished, corresponding to TcI, TcIII, and TcII + V + VI, while support for TcIV was lacking. Phylogenetic analyses based on hsp70 demonstrated that this molecular marker can be applied for discriminating most of the Trypanosoma species and clades.     

Phylogenetic analysis of the genus Plasmodium based on the gene encoding adenylosuccinate lyase.

Phylogenetic studies of the genus Plasmodium have been performed using sequences of the nuclear, mitochondrial and plastid genes. Here we have analyzed the adenylosuccinate lyase (ASL) gene, which encodes an enzyme involved in the salvage of host purines needed by malaria parasites for DNA synthesis. The ASL gene is present in several eukaryotic as well as prokaryotic organisms and does not have repeat regions, which facilitates the accuracy of the alignment. Furthermore, it has been shown that ASL is not subject to positive natural selection. We have sequenced the ASL gene of several different Plasmodium species infecting humans, rodents, monkeys and birds and used the obtained sequences along with the previously known P. falciparum ASL sequence, for structural and phylogenetic analysis of the genus Plasmodium. The genetic divergence of ASL is comparable with that observed in other nuclear genes such as cysteine proteinase, although ASL cannot be considered conserved when compared to aldolase or superoxide dismutase, which exhibit a slower rate of evolution. Nevertheless, a protein like ASL has a rate of evolution that provides enough information for elucidating evolutionary relationships. We modeled 3D structures of the ASL protein based on sequences used in the phylogenetic analysis and obtained a consistent structure for four different species despite the divergence observed. Such models would facilitate alignment in further studies with a greater number of plasmodial species or other Apicomplexa.     

Molecular survey of bovine Babesia species in Bactrian camels (Camelus bactrianus) in Mongolia

Bovine babesiosis, which is caused by species of genus Babesia, is a leading cause of considerable economic losses to the cattle industry each year. Bovine Babesia species have frequently been detected in non-cattle hosts, such as water buffalo (Bubalus bubalis), from which the parasites can be transmitted by ticks to cattle. Therefore, Babesia infections should be minimized not only in cattle but also in non-cattle carriers. In the present study, we surveyed the Bactrian camels (Camelus bactrianus) in Mongolia for three clinically significant bovine Babesia species, including Babesia bovis, B. bigemina, and Babesia sp. Mymensingh, which had been detected previously in Mongolian cattle. We screened blood DNA samples from 305 Bactrian camels in six Mongolian provinces for these species, using parasite-specific PCR assays. Our findings showed that the Bactrian camels in Mongolia were infected with all three Babesia species surveyed. The overall positive rates of B. bovis, B. bigemina, and Babesia sp. Mymensingh were 32.1%, 21.6%, and 24.3%, respectively, whereas 52.5% of the surveyed animals were infected with at least one parasite species. We also found that the female Bactrian camels and the Mongolian native camel breed had significantly higher Babesia positive rates than the male Bactrian camels and the Hos Zogdort breed. In Mongolia, cattle and Bactrian camels usually share common pasture lands for grazing; furthermore, tick species infesting cattle also infest Bactrian camels. Our findings, together with these observations, suggest that the tick transmission of bovine Babesia species might be possible between cattle and Bactrian camels. Therefore, strategies for the control of bovine babesiosis in Mongolia should include methods to minimize bovine Babesia species infections in Bactrian camels.     

Phylogenetic analysis of nonstructural protein 5 (NSP5) gene sequences in porcine rotavirus B strains

Porcine rotavirus B (RVB) has frequently been detected in the diarrhea of suckling and weaned pigs. Because it is difficult to propagate RVBs serially in cell culture, little genetic information about RNA segments other than VP7, NSP1 and NSP2 is available for porcine RVBs. We conducted a phylogenetic analysis focusing on nonstructural protein 5 (NSP5) using 22 porcine RVB strains, which were identified in fecal samples collected around Japan. Sequence analysis showed that NSP5 genes of RVBs contain one ORF, in contrast to the corresponding gene of RVAs that have two ORFs. Comparison of NSP5 amino acid sequences from porcine RVBs with other RVBs revealed that eight serine and serine/threonine residues at the N- and C-terminal regions are highly conserved among RVBs. Phylogenetic analysis also indicated the presence of six clusters (H1–H6) including rat, human, porcine and bovine plus ovine clusters with cut-off values of 78% at the nucleotide level. Moreover, the NSP5 genes of porcine RVBs were divided to three clusters. The data presented here demonstrates that several porcine RVBs with distinctive genotypes are circulating among farms throughout Japan. Our findings provide important new insights into the evolutionary dynamics of RVBs.     

中国柯萨奇病毒A组2型的遗传进化分析

目的  探讨中国柯萨奇病毒A组2型(coxsackievirus A2, CV-A2)毒株的遗传进化特征。方法  收集中国CV-A2毒株的VP1区及全基因序列,采用MEGA 10.2.4软件构建系统进化树并开展氨基酸位点突变分析,采用SimPlot 3.5.1软件进行基因重组分析。结果  共收集到118条VP1序列和26条全基因序列。CV-A2毒株主要来源于广东省,毒株数量自2011年开始迅速增多。基于VP1序列构建的进化树有7个分支,原型毒株为独立A组,多数CV-A2毒株聚集于G组。具有全基因组序列的CV-A2代表株的基因重组分析显示毒株间存在基因重组。另外,VP1氨基酸序列共有22个突变位点。结论  中国流行的CV-A2毒株已发生较明显的基因重组和VP1区域氨基酸位点突变,监测CV-A2的演化对于手足口病的防控具有重要意义。     

Phylogenetic analysis and geographical distribution of Theileria equi and Babesia caballi sequences from horses residing in Spain

The intraerythrocytic protozoans Theileria equi and Babesia caballi are the causative agents of equine piroplasmosis (EP), one of the most important equine tick-borne diseases due to its significant impact on global international horse trade. Although EP is known to be endemic in Spain, previous phylogenetic studies have only been conducted for limited geographical regions. Therefore, the objective of this study was to evaluate the genetic diversity and distribution of these parasite species nationwide. This was performed by amplification of the 18S small subunit (SSU) rRNA gene from 100 EP positive equine blood samples using a nested PCR protocol, and sequencing the obtained amplicons. Seventy-seven T. equi and six B. caballi isolates were successfully sequenced and phylogenetic analysis revealed that the T. equi isolates grouped into the previously described clades A (n = 21/77), D (n = 1/77) and E (n = 55/77), while B. caballi isolates were placed into clades A (n = 5/6) and B (n = 1/6). Isolates from T. equi clade D and B. caballi clade B have not previously been reported in Spain. A greater intra-clade diversity (97.3–98.3 % identity) was observed between T. equi clade E isolates compared to those within clade A (99.7–100 % identity). Additionally, a multivariable logistic regression model was used to analyse associations between the clade of T. equi infection and available epidemiological data. Horses residing in Spanish northern regions were statistically more likely to be infected with T. equi clade E (p = 0.01). We conclude that while extensive sequence variation of equine piroplasms exists in Spanish infected horses, a requirement for increased equine movement controls between Spain and EP-endemic countries should be considered.     

福州市2010年甲型H1N1流感病毒分离株血凝素基因分子进化分析

目的 了解福州市2010年甲型H1N1流感病毒HA基因的突变和分子进化.方法 采用RT-PCR技术扩增流感病毒A/Fuzhou/2/2010(H1N1)血凝素HA基因,测定核苷酸序列,用Clus-talX 1.83软件进行序列比对,用Bi0edit软件的ClustaW程序进行序列同源性分析,用Mega 5.0软件以邻位...     

Strong conservation of rhoptry-associated-protein-1 (RAP-1) locus organization and sequence among Babesia isolates infecting sheep from China (Babesia motasi-like phylogenetic group)

Rhoptry-associated-protein 1 (RAP-1) is considered as a potential vaccine candidate due to its involvement in red blood cell invasion by parasites in the genus Babesia. We examined its value as a vaccine candidate by studying RAP-1 conservation in isolates of Babesia sp. BQ1 Ningxian, Babesia sp. Tianzhu and Babesia sp. Hebei, responsible for ovine babesiosis in different regions of China. The rap-1 locus in these isolates has very similar features to those described for Babesia sp. BQ1 Lintan, another Chinese isolate also in the B. motasi-like phylogenetic group, namely the presence of three types of rap-1 genes (rap-1a, rap-1b and rap-1c), multiple conserved rap-1b copies (5) interspaced with more or less variable rap-1a copies (6), and the 3′ localization of one rap-1c. The isolates Babesia sp. Tianzhu, Babesia sp. BQ1 Lintan and Ningxian were almost identical (average nucleotide identity of 99.9%) over a putative locus of about 31 Kb, including the intergenic regions. Babesia sp. Hebei showed a similar locus organization but differed in the rap-1 locus sequence, for each gene and intergenic region, with an average nucleotide identity of 78%. Our results are in agreement with 18S rDNA phylogenetic studies performed on these isolates. However, in extremely closely related isolates the rap-1 locus seems more conserved (99.9%) than the 18S rDNA (98.7%), whereas in still closely related isolates the identities are much lower (78%) compared with the 18S rDNA (97.7%). The particularities of the rap-1 locus in terms of evolution, phylogeny, diagnosis and vaccine development are discussed.     

南宁市37例登革病毒1型E基因系统进化分析

目的 对南宁市37例登革病毒1型(DENV1)流行株E基因进行序列测定,分析其分子生物学特征,探讨其可能来源.方法 选取2014年12份、2019年25份,共37份经确证为DENV1感染者阳性血浆样本,RT-PCR扩增E基因片段,测序后进行同源性比较、系统进化分析.结果 测序后共获得37条长度为1 437 bp的E基因...     

H1N1流感病毒的遗传进化研究

2009年3月底,墨西哥流感样疾病（ILI）病例异常增加,2009年4月17～28日期间,共报道1551例严重肺炎流感疑似病例（其中7例确诊死亡病例）,随后与之毗邻的美国也相继出现类似病例。2009年4月25日,世界卫生组织（WHO）宣布本次疫情为国际关注的突发公共卫生事件（PHEIC）,并于4月30日将流感大流行警戒级别从4级提高到5级,     

Phylogenetic analysis using E2 gene of classical swine fever virus reveals a new subgenotype in China

Outbreaks of classical swine fever (CSF) have caused serious economic consequences in China. Phylogenetic analysis based on full-length E2 gene sequences showed that five classical swine fever virus (CSFV) isolates collected from Hunan province in 2011 and 2012, together with seven other isolates from neighboring provinces, Guangdong (5) and Guangxi (2), could be classified as a new subgenotype 2.1c, which may have been endemic in the south of China for at least fourteen years. Subgenotype 2.1c isolates share 90.2–94.9% and 89.9–93.8% nucleotide sequence similarity separately with those of subgenotype 2.1a and 2.1b in E2 gene, which are lower than the nucleotide identities between subgenotype 2.1a and 2.1b (91.1–95.7%). Further analysis based on a partial E2 gene sequence (216 nt) indicated that subgenotype 2.1c isolates are also circulating in Thailand. Alignment of E2 amino acid sequences showed that subgenotype 2.1c isolates exhibit a SPA → TPV substitution at positions 777 and 779 compared with subgenotypes 2.1a and 2.1b.     

Phylogenetic diversity of bat trypanosomes of subgenus Schizotrypanum based on multilocus enzyme electrophoresis, random amplified polymorphic DNA, and cytochrome b nucleotide sequence analyses.

Trypanosome stocks isolated from bats (Chiroptera) and belonging to the subgenus Schizotrypanum were analyzed by multilocus enzyme electrophoresis (MLEE) at 22 loci, random amplified polymorphic DNA (RAPD) with 14 primers and/or cytochrome b nucleotide sequence. Bat trypanosomes belonged to the species Trypanosoma cruzi marinkellei (10 stocks), Trypanosoma dionisii (four stocks) and Trypanosoma vespertilionis (three stocks). One T. rangeli stock and seven stocks of T. cruzi sensu stricto, the agent of Chagas disease, were included for comparison. The homology of several RAPD fragments shared by distinct species was verified by hybridization. The sequence of a 516-nucleotide portion of the maxicircle-encoded cytochrome b (CYb) coding region was determined in representative stocks of the species under study. Phylogenetic analysis of the data confirmed the previous taxonomic attribution of these bat trypanosomes based on biological, epidemiological and ecological features. However, a new finding was that within T. cruzi marinkellei two major subdivisions could be distinguished, T.c.m. I, found in the spear-nose bats Phyllostomus discolor and Phyllostomus hastatus, and T.c.m. II, from P. discolor. In addition, the T. c. marinkellei 'Z' stock from a short-tailed bat (Carollia perspicillata) was distantly related to these two subdivisions, and the monophyly of T. c. marinkellei is unclear based on the present data. Based on the present sample, the European species T. dionisii and T. vespertilionis appeared to be more homogeneous. RAPD and CYb data both suggested the monophyly of a group composed of T. cruzi and the two major subdivisions of T. cruzi marinkellei. This study shows that MLEE, RAPD and CYb can be used for taxonomic assignment and provide valuable phylogenetic information for strains and taxa within the subgenus Schizotrypanum. An evolutionary scenario in which the broad host-range parasite T. cruzi would be derived from a bat-restricted trypanosome ancestor is discussed.     

Phylogenetic analysis supports horizontal gene transfer of L-amino acid oxidase gene in Streptococcus oligofermentans

Phylogenetic analysis of 10 amino acid sequences from 19 Streptococcus species showed that S. oligofermentans clustered within the mitis group. However, the l-amino acid oxidase (LAAO) of S. oligofermentans showed a different clustering pattern from the other proteins analyzed implicating horizontal gene transfer (HGT) in the origin of the S. oligofermentans LAAO gene. LAAO of S. oligofermentans is known to confer ability to compete with other oral cavity bacteria, most notably S. mutans; therefore, the HGT event may have been important in extending the ecological niche occupied by this species, consistent with those of other studies suggesting that HGT can play a key role in enabling bacterial species to occupy new ecological niches.     

华东地区散发性戊型肝炎病毒系统进化分析

目的 了解华东地区散发性戊型肝炎病毒(HEV)的系统进化特征.方法 2005-2008年收集华东地区14家二级或三级医院413份散发戊型肝炎患者血清,应用巢式RT-PCR方法检测HEVRNA并测序.参照GenBank相关序列,分析各序列之间的同源性,并构建进化树.结果 413例患者的男女性别比为1.75:1,40～69岁患者居多(61.5%).413份患者血清有140份分离出HEV RNA,阳性分离率为34%.所有分离的140份病毒株均属于HEV Ⅳ型,与Ⅰ、Ⅱ、Ⅲ和Ⅳ型参考病毒株的核苷酸同源性分别为77.9%～88.3%、80.8%～90.6%、73.4%～85.2%和91.0%～95.4%.结论 基因Ⅳ型HEV是引起华东地区散发性戊型肝炎的优势病毒株,其起源和进化等问题仍需进一步研究.     

浙江省鼠中巴尔通体rpoB基因系统进化分析

目的初步了解巴尔通体在浙江省鼠群中携带状况及流行特征,为制定预防措施提供依据。方法 2009年8月于浙江省磐安县捕鼠101只,提取其肝、脾DNA后,选择巴尔通体属rpoB基因进行PCR扩增,并对所得产物进行测序及进一步的系统进化分析。结果 2009年在磐安县捕获的101只鼠中肝脾标本检出巴尔通体rpoB基因阳性1例,其中黄胸鼠中巴尔通体分离率为4.3%;此分离株与格雷汉巴尔通体（GenBank序列号：AB426694.1）处于同一进化分支上（自展值为96%）。结论在浙江省鼠群中存在巴尔通体感染,分离株种属为格雷汉巴尔通体。     

红色蚋细胞色素氧化酶COI基因克隆及鉴定

目的对红色蚋细胞色素氧化酶亚基I(COI)基因进行克隆、鉴定与序列分析。方法以红色蚋成虫基因组DNA为模板,设计特异性引物进行PCR扩增、纯化基因片段后连接、转化大肠杆菌DH5a,筛选阳性克隆、测序并与NCBI数据库中的核酸序列进行同源性比对分析,掌握其基因序列及其蛋白质结构特征。结果克隆序列的结果表明COI基因全长1542 bp,编码513个氨基酸,编码蛋白等电点为5.84,相对分子量为56 KDa,具有一个细胞色素氧化酶亚基I的核心保守结构;其基因碱基组成A、T、C、G分别为28.60%,36.90%,17.76%,16.74%,G+C含量为34.5%,A+T含量为65.5%。结论成功克隆出红色蚋CO I基因序列,可为进一步研究奠定基础。     

东南沿海地区汉坦病毒的遗传进化分析

目的 对浙江、福建省及上海市东南沿海地区汉坦病毒的基因进行比较.方法 收集浙江、福建省及上海市汉坦病毒部分M基因序列,应用Mega 4.0和DNAStar软件包进行系统发生分析,以邻位相连法(NJ)构建系统发生树,分析东南沿海地区浙江、福建省及上海市汉坦病毒的基因差异,分析采用1000个多序列组.结果 经对浙江、福建省及上海市若干株汉坦病毒基因的序列比较与系统进化分析,浙江省分离的同型别毒株的基因序列同源性高于福建省与上海市的分离毒株,而浙江省同地区分离株的核苷酸同源性更高,地区接近的分离株系统进化分支的分布也大部分更靠近.汉滩型病毒(HTNV)福建株的ZH53和汉城型病毒(SEOV)浙江建德株Gou3和ZJ5与本研究同型别的其他株及国际标准株的核苷酸同源性较低,分别为82.7％～86.3％和84.0％～85.3％,而且分别在HTNV和SEOV发生群中构成一独立分支.浙江省从田鼠中分离到SEOV,产生宿主“溢出”现象.结论 浙江、福建省及上海市东南沿海地区汉坦病毒的基因差异和亲缘远近关系主要表现在地区性,显示出高度的地理聚集现象,浙江省建德地区和福建省分别存在SEOV和HTNV的特殊亚型病毒.     

东莞地区诺如病毒ORF2基因克隆与进化树分析

目的探讨东莞地区婴幼儿感染的诺如病毒ORF2基因序列特性,分析东莞地区诺如病毒的基因型。方法收集2004年和2009年东莞地区婴幼儿腹泻标本66份,参考诺如病毒Farmington Hills株(AY502023)基因组序列,自行设计了引物两段扩增ORF2基因的引物,短片段作为检测标本的片段,分段扩增病毒ORF2全长基因,PCR产物克隆于T载体上,序列测定,用ClustalW/X和MEGA5.0等软件进行序列特性分析和基因型分析。结果获得了3株ORF2全长基因,全长为1623bp,ORF2编码主要结构蛋白衣壳蛋白(VP1),VP1蛋白有两个主要区域:P区(protruding)和S区(shell);将病毒株NVdgsl0902、NVdgsl0910的P2区与诺如病毒GⅡ-4基因型中a、b、c、d、e、f 6个亚型的P2区氨苷酸序列进行同源性比较,同源性为86.0%～91.0%,而与2006b新变株为97.0%,病毒株NVdgsl0412的P2区与诺如病毒GⅡ-4基因型中a、b、c、d、f5个亚型的P2区氨苷酸序列进行同源性比较,同源性为88.0%～93.0%,与e亚型的同源性为98.1%。结论 NVdgsl0412属于GⅡ-4e型,NVdgsl0902、NVdgsl0910属于GⅡ-4f亚型的2006b新变异株,2004年与2009年东莞地区的诺如病毒流行株发生了变异,目前是以2006b新变异株作为主要的流行株。     

Phylogenetic relationships of Leishmania species based on trypanosomatid barcode (SSU rDNA) and gGAPDH genes: Taxonomic revision of Leishmania (L.) infantum chagasi in South America

Phylogenetic studies on trypanosomatid barcode using V7V8 SSU rRNA and gGAPDH gene sequences have provided support for redefining some trypanosomatid species and positioning new isolates. The genus Leishmania is a slow evolving monophyletic group and including important human pathogens. The phylogenetic relationships of this genus have been determined by the natural history of its vertebrate hosts, vector specificity, clinical manifestations, geographical distribution and molecular approaches using different markers. Thus, in an attempt to better understand the phylogenetic relationships of Leishmania species, we performed phylogenetic analysis on trypanosomatid barcode using V7V8 SSU rRNA and gGAPDH gene sequences among a large number of Leishmania species and also several Brazilian visceral Leishmania infantum chagasi isolates obtained from dogs and humans. Our phylogenetic analysis strongly suggested that Leishmania hertigi and Leishmania equatoriensis should be taxonomically revised so as to include them in the genus Endotrypanum; and supported ancient divergence of Leishmania enriettii. This, together with recent data in the literature, throws light on the discussion about the evolutionary southern supercontinent hypothesis for the origin of Leishmania ssp. and validates L. infantum chagasi from Brazil, thus clearly differentiating it from L. infantum, for the first time.