重组GII型诺如病毒长沙株全基因组序列的测定与分析

目的 对2016年长沙地区一起由GII型诺如病毒引起的急性胃肠炎暴发疫情致病原进行全基因组序列测定,掌握其基因类型、分子进化特征和抗原重组情况。方法 提取疫情中患者粪便标本的总RNA,反转录成cDNA,PCR扩增病毒全基因组并采用Sanger法测序,比对拼接后获得病毒全基因组序列;通过BLAST比对和诺如病毒在线分型工具(typing online tool)确定其基因型别;从GenBank中下载GII型诺如病毒参考序列,采用DNA Star软件进行序列多序列比对和同源性分析,绘制系统遗传进化树,基因重组特征分析采用SimPlot软件。结果 通过一代测序获得病毒基因组序列长7491bp,有3个开放阅读框(ORF),长度分别为5100bp,1647bp,765bp。多序列比对和同源分析发现ORF1区与GII.P12型代表株同源性最高,VP1区则与GII.3型同源性最高;因此,将该毒株命名为Hu/GII.P12-GII.3/CS02/2016 /CHN。分子遗传进化分析显示其与中国其他地区如北京、上海、广东等地流行的GII.P12-GII.3重组型诺如病毒亲缘关系最为接近。抗原重组分析发现Hu/GII.P12-GII.3/CS02/2016 /CHN长沙株重组位点在5080bp,为ORF1与 ORF2重叠区的起点。结论 除了GII.P16-GII.2重组诺如病毒的广泛流行外,长沙地区仍存在GII.P12/GII.3重组诺如病毒的散发流行,需加其强监测。     

Viral hit and run-oncogenesis: Genetic and epigenetic scenarios

It is well documented that viral genomes either inserted into the cellular DNA or coreplicating with it in episomal form can be lost from neoplastic cells. Therefore, “hit and run”-mechanisms have been a topic of longstanding interest in tumor virology. The basic idea is that the transient acquisition of a complete or incomplete viral genome may be sufficient to induce malignant conversion of host cells in vivo, resulting in neoplastic development. After eliciting a heritable change in the gene expression pattern of the host cell (initiation), the genomes of tumor viruses may be completely lost, i.e. in a hit and run-scenario they are not necessary for the maintenance of the malignant state. The expression of viral oncoproteins and RNAs may interfere not only with regulators of cell proliferation, but also with DNA repair mechanisms. DNA recombinogenic activities induced by tumor viruses or activated by other mechanisms may contribute to the secondary loss of viral genomes from neoplastic cells. Viral oncoproteins can also cause epigenetic dysregulation, thereby reprogramming cellular gene expression in a heritable manner. Thus, we expect that epigenetic scenarios of viral hit and run-tumorigenesis may facilitate new, innovative experiments and clinical studies in spite of the fact that the regular presence of a suspected human tumor virus in an early phase of neoplastic development and its subsequent regular loss have not been demonstrated yet. We propose that virus-specific “epigenetic signatures”, i.e. alterations of the host cell epigenome, especially altered DNA methylation patterns, may help to identify viral hit and run-oncogenic events, even after the complete loss of tumor viruses from neoplastic cells.     

EGF-PE4O重组毒素对喉鳞癌细胞株Hep-2的毒性作用

观察EGF－PE40重组毒素对喉鳞癌细胞的毒性作用,为寻找新型的治疗头颈鳞癌的生物制剂奠定基础。应用MTT比色法测定EGF－PE40重组毒素的生物学活性。并在光镜下观察Hep－2细胞形态变化。确定MTT比色法中Hep－2细胞浓度为2×104／well。选择10％SDS－0．01mol／LHCI作为MTT结晶的溶解液,当EGF－PE40重组毒素为0．53ng／ml时,50％的Hep－2细胞死亡,在一定范围内,随重组毒素增加,细胞损伤加重。     

Determination of ionisation chamber collection efficiency in a swept electron beam by means of thermoluminescent detectors and the "two-voltage" method

Two methods for determining the collection efficiency of a 0.6 cm3 thimble ionisation chamber exposed to the swept electron beam of a linear accelerator Therac 20 Saturne (CGR MeV) have been compared. In one method the chamber signal has been compared to that of simultaneously exposed thermoluminescent LiF dosemeters (TLD), in the other the "two-voltage" method of Boag, adapted for swept beams, has been used. By variation of the electron energy between 20 and 13 MeV, of the focus-skin-distance (FSD) between 200 and 100 cm and of the monitor rate between 400 monitor units (m.u.) and 100 m.u. per minute, different values could be produced for the peak charge density M. The collection efficiency of the chamber, operating at a standard voltage of 250 V, decreases from 0.99 to 0.84 for a charge density increasing from 0.3 X 10(-4) C/m3 to 7.5 X 10(-4) C/m3, respectively. The maximum deviation observed between the TLD and the "two-voltage" method adopted for similar M is never more than 2% and mostly smaller than 1%. It can be concluded that, under the present experimental conditions, the calculated ionisation chamber collection efficiency is confirmed by the experimental method using TL dosimetry.     

Mixed vaginal infections of Balb/c mice with low virulent herpes simplex type 1 strains result in restoration of virulence properties: vaginitis/vulvitis and neuroinvasiveness

Vaginal infections of BALB/c Ann mice with herpes simplex virus type 1 (HSV-1) were studied. Mice were inoculated with virulent strains ANG path and 17 syn⁺ or low-virulent recombinant strains 27/III and 17-syn3 that differ from parental strains in their glycoprotein B (gB) gene sequences. When low-virulent strains were inoculated separately, no vaginitis/vulvitis was produced despite replication in the vagina. In contrast, after coinfection of mice with the two low-virulent strains, vaginitis/vulvitis was produced and virus could be recovered from the central nervous system (CNS). Two of the CNS isolates produced vaginitis/vulvitis, neuroinvasiveness and death of mice after vaginal infection. Restriction fragment analysis and sequencing were used to assess recombination events in the gB gene sequence of the CNS isolates. After mixed vaginal infection recombination between non-virulent HSV strains occurs, resulting in vaginitis/vulvitis and neuroinvasiveness. No correlation was detected between the syncytial phenotype and local vaginal virulence. Virulence of HSV is not solely dependent on gB function; it seems to be more probable that several genes act in concert to induce virulence and neuroivasiveness. Received: 28 May 1996     

Cloning the REC1 gene of Ustilago maydis

Summary The REC1 gene of Ustilago maydis plays a key role in homologous recombination and the repair of damaged DNA. In order to understand the nature and functions of the gene product, the gene has been cloned by functional complementation. A 3.8 kb cloned fragment complements the pleiotropic mitotic phenotype of different rec1 alleles. It does not complement the UV sensitivity of two other sensitive mutants. Disruption of the chromosomal copy of the 1.566 kb open reading frame within this fragment reproduces the rec1 pleiotropic phenotype. Furthermore, in diploids this disrupted reading frame is unable to complement previously characterised rec1 alleles.     

Superinfection and recombination of infectious laryngotracheitis virus vaccines in the natural host

Infectious laryngotracheitis virus (ILTV, Gallid alphaherpesvirus 1) causes severe respiratory disease in chickens and has a major impact on the poultry industry worldwide. Live attenuated vaccines are widely available and are administered early in the life of commercial birds, often followed by one or more rounds of revaccination, generating conditions that can favour recombination between vaccines. Better understanding of the factors that contribute to the generation of recombinant ILTVs will inform the safer use of live attenuated herpesvirus vaccines. This study aimed to examine the parameters of infection that allow superinfection and may enable the generation of recombinant progeny in the natural host. In this study, 120 specific-pathogen free (SPF) chickens in 8 groups were inoculated with two genetically distinct live-attenuated ILTV vaccine strains with 1–4 days interval between the first and second vaccinations. After inoculation, viral genomes were detected in tracheal swabs in all groups, with lowest copies detected in swabs collected from the groups where the interval between inoculations was 4 days. Superinfection of the host was defined as the detection of the virus that was inoculated last, and this was detected in tracheal swabs from all groups. Virus could be isolated from swabs at a limited number of timepoints, and these further illustrated superinfection of the birds as recombinant viruses were detected among the progeny. This study has demonstrated superinfection at host level and shows recombination events occur under a very broad range of infection conditions. The occurrence of superinfection after unsynchronised infection with multiple viruses, and subsequent genomic recombination, highlight the importance of using only one type of vaccine per flock as the most effective way to limit recombination.     

Sequence types diversity of Legionella pneumophila isolates from environmental water sources in Guangzhou and Jiangmen,China

In this study, 159 Legionella pneumophila strains isolated from various natural and artificial water sources in Guangzhou and Jiangmen, China, were subjected to genotyping by the sequence-based typing (SBT) scheme. These isolates were assigned into 53 sequence types (STs) (50 STs with seven loci data and three unidentified STs with incomplete loci profiles) with ST1 as the dominant one (14.5%), and the index of diversity (IOD) was 0.950. Eight new alleles and 34 new STs were reported here. Notably, most of the newly identified STs with seven loci data (24/34) contained no new allele, implying frequent recombination events in L. pneumophila. Five intragenic recombination events were identified in the concatenated sequences of seven loci. The diversity of STs in natural environmental isolates (41 STs, IOD = 0.956) is higher than that of artificial environmental ones (17 STs, IOD = 0.824). The ST patterns varied in isolates from these two sources: the most common STs from artificial water sources, ST1 and ST752 (39.2% and 13.7%), were only occasionally isolated from natural water sources (2.9% and 3.8%, respectively); while the predominant STs from natural water sources, ST1048, ST739 and ST1267 (15.2%, 6.7% and 6.7%), were less frequently seen in artificial environments (2.0%, 0% and 0%, respectively). We also found out that Legionnaires’ disease associated STs might be more frequently isolated in artificial environments than in natural ones. Our data revealed remarkable genetic diversity of L. pneumophila isolates from environmental water systems of Guangzhou and Jiangmen, and the different ST distribution patterns between natural water and artificial water sources as well.     

Hepatitis C virus genetic variability and evolution

Hepatitis C virus(HCV) has infected over 170 million people worldwide and creates a huge disease burden due to chronic, progressive liver disease. HCV is a singlestranded, positive sense, RNA virus, member of the Flaviviridae family. The high error rate of RNA-dependent RNA polymerase and the pressure exerted by the host immune system, has driven the evolution of HCV into 7 different genotypes and more than 67 subtypes. HCV evolves by means of different mechanisms of genetic variation. On the one hand, its high mutation rates generate the production of a large number of different but closely related viral variants during infection, usually referred to as a quasispecies. The great quasispecies variability of HCV has also therapeutic implications since the continuous generation and selection of resistant or fitter variants within the quasispecies spectrum might allow viruses to escape control by antiviral drugs. On the other hand HCV exploits recombination to ensure its survival. This enormous viral diversity together with some host factors has made it difficult to control viral dispersal. Current treatment options involve pegylated interferon-α and ribavirin as dual therapy or in combination with a direct-acting antiviral drug, depending on the country. Despite all the efforts put into antiviral therapy studies, eradication of the virus or the development of a preventive vaccine has been unsuccessful so far. This review focuses on current available data reported to date on the genetic mechanisms driving the molecular evolution of HCV populations and its relation with the antiviral therapies designed to control HCV infection.