Molecular characterization of human isolates of Giardia duodenalis from Ethiopia

Giardia duodenalis, a flagellated protozoan, represents a common cause of gastroenteritis in Ethiopia, however very little information is available on the epidemiology and transmission routes of this pathogen, and a genetic characterization of the parasite has never been attempted in this country. The aim of this study was the genetic analysis of human isolates of G. duodenalis collected in different localities across the country, both from urban and rural areas. A fragment of the beta-giardin gene was amplified by nested PCR and analyzed by restriction and sequence analyses. Of the 59 isolates examined, 31 (52%) were typed as assemblage A and 13 (22%) as assemblage B. A strong correlation between the presence of symptoms and infection with assemblage B was observed. The remaining 15 (25%) isolates were typed as mixed infections by PCR-RFLP, specifically, A+F (in seven isolates) and A+B (in eight isolates). Sequencing of the A+F products confirmed the presence of assemblage F in three isolates, whereas the remaining four were identified as assemblage A. The detection of assemblage F, a cat-specific assemblage that to date has not been associated with human infections, was not able to be confirmed by the analysis of two commonly used markers (small subunit ribosomal RNA and triosephosphate isomerase). The analysis of the one isolate that was successfully amplified with the glutamate dehydrogenase primers unambiguously identified it as G. duodenalis, yet it was distinct from the established A and F sequences; thus the exact genetic identity of these isolates remains unclear.     

Isolation and characterization of 115 street rabies virus isolates from Ethiopia by using monoclonal antibodies: identification of 2 isolates as Mokola and Lagos bat viruses.

There were 115 isolates of rabies viruses recovered by tissue culture technique from 119 animal brains collected in Ethiopia. By using 17 selected antinucleocapsid monoclonal antibodies (MAbs), 113 isolates were classic street rabies viruses (serotype 1). An isolate of feline origin (Eth-16) was a Mokola virus (serotype 3) and another isolate (Eth-58, obtained from a rabid dog) was serotype 2 (Lagos bat virus). None of the 16 antiglycoprotein MAbs used neutralized the Eth-16 isolate, whereas Eth-58 was neutralized by 1 (TERA543). Antirabies vaccines prepared from Pitman-Moore and Pasteur virus strains protected mice against homologous challenge, but neither was protective against the 2 rabies-related virus isolates. The isolation of Mokola and Lagos bat viruses from domestic animals in eastern Africa is of public and veterinary concern mainly due to lack of effective vaccines against these agents and the difficulty of proper diagnosis.     

Molecular analysis of hepatitis B virus isolates in Mexico： Predominant circulation of hepatitis B virus genotype H

INTRODUCTIONHepatitis B virus(HBV)is an important cause of morbidity and mortality worldwide.It is estimated that2billion people are infected with HBV and350million individuals suffer from chronic HBV infection in the world[1,2].Chronic HBV infection may …     

Molecular analysis of hepatitis B virus isolates in Mexico: Predominant circulation of hepatitis B virus genotype H

AIM:To determine the genotypes in Mexican hepatitis B virus (HBV) isolates and characterize their precore and core promoter mutations.METHODS: Forty-nine HBV isolates of Mexico obtained from sera of 15 hepatitis patients, 6 hemodialysis patients, 20 men seeking HIV testing, and 8 AIDS patients were analyzed. HBV isolates were amplified by PCR,and genotyped by line probe assay (INNO-LiPA HBV Genotyping; INNOGENETICS N V, Ghent, Belgium).HBV genotype confirmation was performed by DNA sequencing part of the sAg region. Precore and core promoter mutation characterization was performed by line probe assay (INNO-LiPA HBV PreCore; INNOGENETICS N.V.; Ghent, Belgium).RESULTS: Overall, HBV genotype H was found in 37(75.5%) out of the 49 isolates studied. HBV genotypes G, A, and D were found in 5 (10.2%), 4 (8.2%), and 3(6.1%) isolates, respectively. HBV genotype H was predominant in isolates from hemodialysis patients (100%),hepatitis patients (80%), and men seeking HIV testing (75%), and accounted for half of infections in AIDS patients (50%). Six (12.2%) out of the 49 HBV isolates showed both wild type and mutant populations at precore codon 28. These mixed wild type and precore murant populations were observed in one HBV genotype A isolate and in all HBV genotype G isolates. A dual variant core promoter mutation was observed in 1 (2%) of the isolates, which was genotype H.CONCLUSION: HBV genotype H is highly predominant in HBV isolates of Mexico followed by genotypes G, A and D. A low frequency of precore and core promoter mutations is observed in HBV Mexican isolates.     

Molecular characterization of field isolates of human pathogenic trypanosomes

The accurate identification of each of the three subspecies of Trypanosoma brucei remains a challenging problem in the epidemiology of sleeping sickness. Advances in molecular characterization have revealed a much greater degree of heterogeneity within the species than previously supposed. Only group 1 T. b. gambiense stands out as a separate entity, defined by several molecular markers. T. b. rhodesiense is generally too similar to sympatric T. b. brucei strains to be distinguished from them by any particular molecular markers. Nevertheless, characterization of trypanosome isolates from humans and other animals has allowed the identification of potential reservoir hosts of T. b. rhodesiense. The recent discovery of a gene for human serum resistance may provide a useful marker for T. b. rhodesiense in the future. There have been few attempts to find associations between genetic markers and other biological characters, except human infectivity. However, virulence or fly transmissibility have been correlated with molecular markers in some instances.     

Heterosexual HIV transmission dynamics: implications for prevention and control

Understanding the epidemiologic definition of epidemic versus non-epidemic spread of an infectious disease agent and the different patterns of heterosexual HIV transmission are needed to fully understand the low potential for heterosexual HIV epidemics in most heterosexual populations. Epidemic sexual HIV transmission can occur only in populations where there are large numbers of persons who have unprotected sex with multiple and concurrent sex partners. How high HIV prevalence may reach in these populations depends on the size and overlap of sex networks, and the prevalence of facilitating and protective factors that can greatly increase or limit the amount of infected blood and sexual fluids exchanged during intercourse. The wide difference in potentials for heterosexual HIV epidemics that exists within and between countries must be recognized, accepted and monitored in order to design and focus prevention strategies where they are most needed and most effective.     

Molecular characterization of vancomycin-resistant Enterococcus spp. clinical isolates from Hungary and Serbia

The objectives of this work were to collect and characterize vancomycin-resistant Enterococcus faecium (VREF) clinical isolates from Hungary and Serbia and to analyse their genetic relatedness. VREF isolates were initially typed by PFGE. A selection of VREF isolates representing all participating hospitals was further examined by multiple-locus variable-number tandem repeat analysis (MLVA) and multilocus sequence typing (MLST). VanB VREF isolates (n=18) recovered from blood, urine and faecal cultures at a Budapest hospital between August 2003 and December 2004 were molecularly characterized. Macrorestriction analysis of the isolates revealed their monoclonal relatedness. A cluster of infections caused by 2 distinct VanA VREF clones recovered from 6 departments was identified in a Belgrade hospital in Serbia. The vanA resistance determinant was transferable by in vitro conjugation experiments. We also identified 2 vanA-positive E. gallinarum blood culture isolates in this Belgrade hospital. Molecular typing of representative VREF isolates from Hungary and Serbia by MLVA and MLST revealed that all tested isolates belonged to MLST complex CC17 and the corresponding MLVA cluster 1. Our results extend the documented occurrence of CC17 to a new region in Europe.     

Transmission dynamics of rabies virus in Thailand: Implications for disease control

Background  

In Thailand, rabies remains a neglected disease with authorities continuing to rely on human death statistics while ignoring the financial burden resulting from an enormous increase in post-exposure prophylaxis. Past attempts to conduct a mass dog vaccination and sterilization program have been limited to Bangkok city and have not been successful. We have used molecular epidemiology to define geographic localization of rabies virus phylogroups and their pattern of spread in Thailand.     

Molecular characterization of clinical Burkholderia pseudomallei isolates from India

Multilocus sequence typing of seven isolates of Burkholderia pseudomallei from India showed considerable diversity, with six different sequence types. Possible dissemination of melioidosis by historical trading routes is supported by links to strains from Southeast Asia, China, and Africa and the presence of the Burkholderia mallei allele of the bimA gene.     

狂犬病病毒HEP-Flury-mEG株的拯救与鉴定

目的拯救狂犬病毒HEP-Flury-mEG株,为疫苗制备奠定基础。方法将ERA株G蛋白333位毒力位点修饰为谷氨酸后替换至HEP-Flury株基因组。重组感染性克隆质粒和4个辅助质粒,共转染BHK-21细胞,拯救重组病毒。结果 IFA鉴定成功拯救出了HEP-Flury-mEG株狂犬病病毒。重组病毒G基因经XholⅠ酶切,片段大小为1 071bp和520bp,与预期结果相符。重组病毒在BHK-21细胞中传代4次,滴度维持在1×107.5 FFU/ml。重组病毒经常规负染后在电镜下为弹状粒子,长度和直径与亲本株一致。结论获得了重组狂犬病毒HEP-Flury-mEG株。该病毒滴度高,形态完整,传代稳定,为进一步研究狂犬病毒病毒的生物学特性和新型基因工程疫苗奠定了基础。     

Molecular determinants of human T-lymphotropic virus type 1 transmission and spread

Human T-lymphotrophic virus type-1 (HTLV-1) infects approximately 15 to 20 million people worldwide, with endemic areas in Japan, the Caribbean, and Africa. The virus is spread through contact with bodily fluids containing infected cells, most often from mother to child through breast milk or via blood transfusion. After prolonged latency periods, approximately 3 to 5% of HTLV-1 infected individuals will develop either adult T-cell leukemia/lymphoma (ATL), or other lymphocyte-mediated disorders such as HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). The genome of this complex retrovirus contains typical gag, pol, and env genes, but also unique nonstructural proteins encoded from the pX region. These nonstructural genes encode the Tax and Rex regulatory proteins, as well as novel proteins essential for viral spread in vivo such as, p30, p12, p13 and the antisense encoded HBZ. While progress has been made in the understanding of viral determinants of cell transformation and host immune responses, host and viral determinants of HTLV-1 transmission and spread during the early phases of infection are unclear. Improvements in the molecular tools to test these viral determinants in cellular and animal models have provided new insights into the early events of HTLV-1 infection. This review will focus on studies that test HTLV-1 determinants in context to full length infectious clones of the virus providing insights into the mechanisms of transmission and spread of HTLV-1.     

Reduced avian virulence and viremia of West Nile virus isolates from Mexico and Texas

A West Nile virus (WNV) isolate from Mexico (TM171-03) and BIRD1153, a unique genotype from Texas, have exhibited reduced murine neuroinvasive phenotypes. To determine if murine neuroinvasive capacity equates to avian virulence potential, American crow (Corvus brachyrhynchos) and house sparrows (Passer domesticus) were experimentally inoculated with representative murine neuroinvasive/non-neuroinvasive strains. In both avian species, a plaque variant from Mexico that was E-glycosylation competent produced higher viremias than an E-glycosylation-incompetent variant, indicating the potential importance of E-glycosylation for avian replication. The murine non-neuroinvasive BIRD1153 strain was significantly attenuated in American crows but not house sparrows when compared with the murine neuroinvasive Texas strain. Despite the loss of murine neuroinvasive properties of nonglycosylated variants from Mexico, our data indicate avian replication potential of these strains and that unique WNV virulence characteristics exist between murine and avian models. The implications of reduced avian replication of variants from Mexico for restricted WNV transmission in Latin America is discussed.     

Immunopathogenesis of human immunodeficiency virus: implications for immune-based therapies.

Human immunodeficiency virus (HIV) infection leads to a state of CD4 lymphopenia and generalized immune activation with subsequent development of opportunistic infections and neoplasms. The use of highly active antiretroviral treatment has dramatically improved the clinical outcome for HIV-infected patients, but the associated cost and toxicity and the eventual development of drug resistance have underscored the need for additional therapeutic strategies. Immune-based therapies, such as treatment with cytokines or immunosuppressants, adoptive immunotherapy, and therapeutic immunizations, are being intensely investigated as potential supplements to antiretroviral therapy. Although much data have been generated as a result of these efforts, to date there has been little evidence of the clinical efficacy of these strategies. Randomized clinical studies remain critical in evaluating the clinical significance and the role of immune-based therapies in the therapeutic armamentarium against HIV.     

广西狂犬病野毒株N基因的测序与分析

目的 对广西狂犬病野毒株N基因进行测序和分析，了解其遗传特点和进化规律。方法 2003至2004年从广西各地采集595份动物脑材料，经RT-PCR法确定16份为阳性。对此16株狂犬病野毒株N基因全序列进行RT-PCR扩增、克隆和测序分析。结果 广西狂犬病病毒株属于I型，可分为Ⅰ、Ⅱ和Ⅲ群。GX01、GX08、GX09、GX014、GX091、GX195、GX260、GXLA、GXHX和GXSL株的同源性在97．6％～99．8％，为Ⅰ群，它们与其他毒株的同源性为89．0％～89．7％；GX219、GX074、GX304、GXBM和GXPX株的核苷酸同源性在98．4％～99．9％，为Ⅱ群，它们与其他各株的同源性为88．6％～89．7％；GXN119株为Ⅲ群，与泰国的8738THA株核苷酸同源性为95．3％。遗传进化分析表明，389位磷酸化位点、主要的4个抗原区及Th细胞表位区非常保守。N蛋白上的氨基酸变异主要集中在42、90、110、128、135、332、379和397位氨基酸上，这些氨基酸的变异与同源性分群结果一致。结论 根据基因同源性、遗传进化树及氨基酸变异分析，广西狂犬病野毒株分3群，呈地区性分布。     

Treatment of chronic hepatitis C: lessons from human immunodeficiency virus dynamics.

Treatment of chronic hepatitis C is a major problem. A sustained viral response to interferon alpha monotherapy occurs in <20% of patients. Using a combination therapy of interferon alpha and ribavirin. the sustained response rate in naive hepatitis C patients has increased to 31%-47%. The success of therapy for chronic hepatitis C depends on both virus- and host-related factors, such as age, histology, duration of hepatitis C virus (HCV) carriage and biochemical parameters. During the last 5 years, insight into the dynamics of human immunodeficency virus (HIV) has been obtained by analysing the changes in viral load after starting antiviral treatment. By using a mathematical model of HIV kinetics as an example, an exponentially rapid decline in serum HCV RNA level was seen after the first dose of interferon alpha, followed by a slower exponential decline: a so-called biphasic pattern. The estimated virion half-life varies between 2.7 and 16.8 h. The high virion turnover allows the generation of a heterogeneous quasi-species population of HCVs. It is therefore supposed that initial aggressive treatment can be helpful to prevent the development of mutations that make the virus more defensible for the interferon alpha treatment. Various trials are now being conducted based on this principle of high induction antiviral therapy.     

Pathogenicity of different rabies virus isolates and protection test in vaccinated mice

This study was aimed to evaluate and compare the pathogenicity of rabies virus isolated from bats and dogs, and to verify the efficacy of a commercial rabies vaccine against these isolates. For evaluation of pathogenicity, mice were inoculated by the intramuscular route (IM) with 500MICLD??/0.03 mL of the viruses. The cross-protection test was performed by vaccinating groups of mice by the subcutaneous route and challenged through the intracerebral (IC) route. Isolates were fully pathogenic when inoculated by the IC route. When inoculated intramuscularly, the pathogenicity observed showed different death rates: 60.0% for the Desmodus rotundus isolate; 50.0% for dog and Nyctinomops laticaudatus isolates; 40.0% for Artibeus lituratus isolate; 9.5% Molossus molossus isolate; and 5.2% for the Eptesicus furinalis isolate. Mice receiving two doses of the vaccine and challenged by the IC route with the isolates were fully protected. Mice receiving only one dose of vaccine were partially protected against the dog isolate. The isolates from bats were pathogenic by the IC route in mice. However, when inoculated through the intramuscular route, the same isolates were found with different degrees of pathogenicity. The results of this work suggest that a commercial vaccine protects mice from infection with bat rabies virus isolates, in addition to a canine rabies virus isolate.