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.     

Multiple lines of evidence on the genetic relatedness of the parthenogenetic and bisexual Haemaphysalis longicornis (Acari: Ixodidae)

As an obligate hematophagous ectoparasite, the hard tick Haemaphysalis longicornis exhibits two reproductive strategies, bisexual reproduction, and obligate parthenogenesis, which have attracted a widespread attention. However, the speciation of parthenogenetic population remained ambiguous due to its similarity in morphology but the remarkable differences in cytogenetics as compared with those of the bisexual ones. In the present study, we explored several new lines of genetic evidence to resolve this controversial issue. The number of the chromosomes in two lineages was checked by classical methods and their total DNA levels were determined utilizing flowcytometry. In addition, the sequences of 12S rDNA, 16S rDNA, cytochrome c oxidase I and II (COI, COII) and internal transcribed spacer-2 (ITS-2) genes were used to assess their phylogenetic relationship. We observed that the chromosome ploidy of bisexual and parthenogenetic H. longicornis collected by our laboratory was diploid and triploid, respectively. Flowcytometry analysis indicated a ratio close to 2:3 in the DNA contents of bisexual to parthenogenetic H. longicornis. Although the chromosome ploidy is different, their gene sequences are extremely similar. Analogous to the intra-species genetic difference of other invertebrates, sequence differences of all loci examined are below 2%. Phylogenetic trees constructed from 12S rDNA, 16S rDNA, COI, and ITS-2 genes revealed that they were all in the same monophyletic clade instead of splitting independently into evolutional branches. Moreover, according to 4× Rule, the K/θ ratio of two reproductive populations calculated based on COI was much smaller than four, strongly supporting that they belong to the same species. Therefore, we conclude that the evolutionary process just disturbs the chromosome ploidy and the sexual determination of parthenogenetic population and that it would be better to consider parthenogenetic H. longicornis as a metapopulation rather than a cryptic species.     

A rare G1P[6] super-short human rotavirus strain carrying an H2 genotype on the genetic background of a porcine rotavirus

Rotavirus strains with a rearranged 11th genome segment may show super-short RNA electropherotypes. Examples from human strains were limited to seven strains, 69M, 57M, B37, Mc345, AU19, B4106 and BE2001, which have a variety of G and P genotypes. AU19 is a rare G1P[6] human rotavirus strain detected in a Japanese infant with severe acute gastroenteritis. This study was undertaken to better understand the origin of AU19 by determining the genotype constellation of AU19. Upon nearly-full genome sequencing, AU19 had a G1-P[6]-I5-R1-C1-M1-A8-N1-T1-E1-H2 genotype constellation. Possession of I5 and A8 genotypes is indicative of its porcine rotavirus origin, whereas possession of H2 genotype is indicative of its DS-1 like human rotavirus origin. At the phylogenetic lineage level for the genome segments that share the genotype between porcine and human rotaviruses, the VP1-4, VP7, NSP3-4 genes were most closely related to those of porcine rotaviruses, but the origin of the NSP2 gene was inconclusive. As to the NSP5 gene, the lineage containing AU19 and the other three super-short human strains, 69M, 57M and B37, carrying the H2 genotype (H2b) clustered with the lineage to which DS-1- like short strains belonged (H2a) albeit with an insignificant bootstrap support. Taken all these observations together, AU19 was likely to emerge as a consequence of interspecies transmission of a porcine rotavirus to a child coupled with the acquisition of a rare H2b genotype by genetic reassortment probably from a co-circulating human strain. The addition of the AU19 NSP5 sequence to much homogeneous H2b genotypes shared by previous super-short rotavirus strains made the genetic diversity of H2b genotypes as diverse as that of the H2a genotype, lending support to the hypothesis that super-short strains carrying H2b genotype have long been circulating unnoticed in the human population.     

Discovery of novel [Arg49]phospholipase A2 isozymes from Protobothrops elegans venom and regional evolution of Crotalinae snake venom phospholipase A2 isozymes in the southwestern islands of Japan and Taiwan.

Protobothrops (formerly Trimeresurus) elegans, a Crotalinae snake, inhabits Ishigaki and Iriomote islands of the Sakishima Islands of Japan which are located between Okinawa island of Japan and Taiwan. Two phospholipase A(2) (PLA(2)) isozymes were purified to homogeneity from P. elegans venom and sequenced. This led to a discovery of novel PLA(2) isozymes with Arg at position 49, that is, [Arg(49)]PLA(2) forms, named PeBP(R)-I and PeBP(R)-II. They are polymorphic at position 3, Val for PeBP(R)-I and Ile for PeBP(R)-II. The cDNAs encoding PeBP(R)-I and PeBP(R)-II were cloned. The cDNA encoding an [Asp(49)]PLA(2) named PePLA(2) was also obtained. In contrast to PLA(2) isozymes from Protobothrops genus with 122 amino acid residues, PeBP(R)-I and PeBP(R)-II are composed of 121 amino acid residues due to lack of Pro at position 90. They exhibited necrotic and edema-inducing activities but no hemorrhagic activity was detected. A phylogenetic tree constructed for venom PLA(2) isozymes of Protobothrops genus and of related genera in the southwestern islands of Japan and Taiwan revealed that PeBP(R)-I and PeBP(R)-II of P. elegans are evolutionarily much closer to PmK49PLA(2), a [Lys(49)]PLA(2), from P. mucrosquamatus (Taiwan) than BPI and BPII, both [Lys(49)]PLA(2) forms, from P. flavoviridis (Amami-Oshima and Tokunoshima islands of Japan). Such evolutionary relationships are also seen in neutral [Asp(49)]PLA(2) isozymes from the three Protobothrops species. Thus, P. elegans is the species much closer to P. mucrosquamatus than P. flavoviridis. Their evolutionary distances seem to be well related to geological history of the islands where they have lived. In addition, it was clearly noted that Ovophis okinavensis (Amami-Oshima), which had formerly belonged to the Trimeresurus genus, and Trimeresurus stejnegeri (Taiwan) are the species fairly distant from Protobothrops genus.     

Characterization of antimicrobial peptides from the skin secretions of the Malaysian frogs, Odorrana hosii and Hylarana picturata (Anura:Ranidae)

Peptidomic analysis of norepinephrine-stimulated skin secretions from Hose's rock frog Odorrana hosii (Boulenger, 1891) led to the isolation of 8 peptides with differential antibacterial activities. Structural characterization demonstrated that the peptides belonged to the esculentin-1 (1 peptide), esculentin-2 (1 peptide), brevinin-1 (2 peptides), brevinin-2 (2 peptides), and nigrocin-2 (2 peptides) families of antimicrobial peptides. Similar analysis of skin secretions from the Malaysian fire frog Hylarana picturata (Boulenger, 1920) led to the isolation and characterization of peptides belonging to the brevinin-1 (2 peptides), brevinin-2 (5 peptides), and temporin (1 peptide) families. The differences in antimicrobial activities of paralogous peptides provide insight into structure-activity relationships, emphasizing the importance of cationicity in determining potency. The substitution Lys(11)-->Gln in brevinin-1HSa (FLPAVLRVAAKIVPTVFCAISKKC) from O. hosii abolishes growth inhibitory activity against Escherichia coli but has no effect on the high potency (MIC=8mug/ml) against Staphylococcus aureus. In contrast, the substitution (Gly(4)-->Asp) in brevinin-2PTb (GFKGAFKNVMFGIAKSAGKSALNALACKIDKSC) from H. picturata reduces activity against both E. coli and S. aureus. Cladistic analysis based upon the amino acid sequences of the brevinin-2 peptides from Asian frogs provides evidence for sister taxon relationships between O. hosii and O. livida and between H. picturata and H. güntheri.     

Genetic characterization of three Cuban Trichomonas vaginalis virus. Phylogeny of Totiviridae family

Trichomonas vaginalis can be infected with double stranded RNA (dsRNA) viruses known as T. vaginalis virus (TVV). This viral infection may have important implications for trichomonal virulence and disease pathogenesis. In this study we identified and genetic characterized three strains of TVVs isolated from T. vaginalis in Cuba. The three new predicted sequences of capsid protein and RNA-dependent RNA polymerase amounted to the previously determined 20 TVV sequences and other 21 viruses of Totiviridae family were used for a phylogenetic analysis. Four distinct monophyletic clades are shown in a phylogenetic tree. One corresponds with TVVs, other with Victorivirus, Leishmaniavirus and Eimeria brunetti virus and, other with viruses of the genus Totivirus and the last with Giardiavirus. The E. brunetti virus is identified in the phylogenetic tree as independent taxon between Leishmaniavirus and Victorivirus isolates, most closely related to Victorivirus. TVV constitute a monophyletic cluster distinguishable from all other viruses in Totiviridae family. This result suggested that TVV may be grouped in a separated genus and not inside of Giardiavirus. TVVs appear to be more closely related to protozoan viruses in the genus Leishmaniavirus and to fungal viruses in the genus Victorivirus than to other protozoan and fungal viruses in Giardiavirus and Totivirus. Among TVVs, four main groups can be recognized within Trichomonasvirus cluster, which correspond with the previous species classification proposed. Further studies, with more TVV strains, especially TVV3 and 4 strains, are needed in order to determine the phylogenetic relationship among Trichomonasvirus genus and specifically if TVV2 and 3 each also constitute a well-delimited group.     

刺果甘草全基因组Survey及叶绿体基因组特征分析＊

目的 基于基因组Survey分析对刺果甘草Glycyrrhiza pallidiflora Maxim.基因组大小和杂合率进行估计，并通过叶绿体基因组序列特征对其在甘草属Glycyrrhiza L.中的系统发育位置进行研究。方法 使用二代测序技术对刺果甘草进行测序，采用K-mer方法对测序reads进行分析，估算刺果甘草基因组大小和杂合率，使用生物信息学方法进行叶绿体基因组组装、注释和系统发育分析。结果 Survey分析结果显示其基因组大小约为577.82 Mb，杂合度约为0.31%，重复序列比例约为53.72%。叶绿体基因组长度为127，267 bp，不具有典型的四分体结构，总GC含量为34.32%，包含110个基因，其中76个蛋白质编码基因，30个tRNA基因和4个rRNA基因。系统发育分析表明，刺果甘草与圆果甘草G. squamulosa Franch.亲缘较接近。结论 刺果甘草存在低杂合和重复序列较多的特点，为了更好地对全基因组进行序列拼接和组装，可尝试采用三代测序结合二代测序的分析策略进行基因组组装；刺果甘草叶绿体全基因组比对和系统发育分析，为后续开展甘草属遗传多样性研究和分子鉴定标记筛选提供了重要依据。     

2010年安徽省蚊虫标本分离到基因Ⅰ型流行性乙型脑炎病毒

目的在安徽省不同地区开展流行性乙型脑炎病毒(JEV)病原学调查,了解当地JEV的基因型别及分子特征。方法 2010年8月在安徽省阜阳、淮南、安庆市的3个标本采集点使用诱蚊灯采集蚊虫标本,通过组织培养法分离病毒,并对病毒分离物进行血清学和分子生物学鉴定;利用生物信息学技术对新分离病毒的序列进行分析,完成同源性和系统发生分析。结果采集到3属3种共计7651只蚊虫标本,通过组织培养技术分离得到11株病毒分离物,鉴定结果显示均为基因Ⅰ型JEV。安徽省JEV新分离株与基因Ⅰ型JEV的同源性最高,核苷酸同源性为96.8%～99.5%,氨基酸同源性为97.8%～100%。结论在安徽省首次分离到基因Ⅰ型JEV,与我国上海市及浙江省JEV分离株进化关系较近。     

Genetic characterization and molecular clock analyses of the Crimean-Congo hemorrhagic fever virus from human and ticks in India, 2010–2011

A nosocomial outbreak of Crimean Congo hemorrhagic fever (CCHF) was reported among humans in Ahmadabad district, Gujarat, India during January, 2011. In the present study we provide the complete genomic sequences of four CCHFV isolates derived from two human patients and two pools of Hyalomma anatolicum ticks during the period of this outbreak and the complete S segment sequence of two retrospective human serum samples, positive for CCHFV in 2010. Sequence-based molecular characterization of the Indian CCHFV showed that they possessed the functional motifs known to occur in the S, M and L gene segment products as in other CCHF viruses. The S segment of the six Indian CCHF viruses showed 99.8% nucleotide identity. Notably both tick isolates shared 100% nucleotide identity with one of the Indian human isolates of 2011. Phylogenetic analysis based on the S segment demonstrated that the Indian CCHFV isolates formed a distinct cluster in the Asian-Middle East group IV of CCHF viruses. The S segment was closest to a Tajikistan strain TADJ/HU8966 of 1990 (98.5% nucleotide identity) and was of South-Asia 2 type while the M segment was of type M2. Both M and L segments were closest to an Afghanistan strain Afg09-2990 of 2009 (93% and 98% nucleotide identity, respectively). The Indian isolates were thus identified as a South-Asia 2/M2 far-east virus combination and the differing parental origin in the S and L/M segments is suggestive that it may be an intra-genotypic reassortant. Molecular clock studies further revealed that the ancestry of the viruses was not very recent and dated back to about 33 years on the basis of the S segment while it was about 15 years based on the M segment. Thus though the 2011 outbreak may not have resulted from a very recent introduction, considering that so far there is no evidence of multiple circulating strains in the country, the possibility of a recent re-introduction of the virus from any of the neighboring countries cannot be ruled out. The study thus warrants the need for continued surveillance and increased sampling of CCHFV in different parts of the country.