Novel flaviviruses detected in different species of mosquitoes in Spain

We report the characterization of three novel flaviviruses isolated in Spain. Marisma Mosquito virus, a novel mosquito borne virus, was isolated from Ochlerotatus caspius mosquitoes; Spanish Ochlerotatus flavivirus and Spanish Culex flavivirus, two novel insect flaviviruses, were isolated from Oc. caspius and Culex pipiens, respectively. During this investigation, we designed a sensitive RT-nested polymerase chain reaction method that amplifies a 1019bp fragment of the flavivirus NS5 gene and could be directly used in clinical or environmental samples for flavivirus characterization and surveillance. Analysis of the sequence generated from that amplicon contains enough phylogenetic information for proper taxonomic studies. Moreover, the use of this tool allowed the detection of additional flavivirus DNA forms in Culex, Culiseta, and Ochlerotatus mosquitoes.     

Evaluation of a Field-Portable DNA Microarray Platform and Nucleic Acid Amplification Strategies for the Detection of Arboviruses,Arthropods, and Bloodmeals

Highly multiplexed assays, such as microarrays, can benefit arbovirus surveillance by allowing researchers to screen for hundreds of targets at once. We evaluated amplification strategies and the practicality of a portable DNA microarray platform to analyze virus-infected mosquitoes. The prototype microarray design used here targeted the non-structural protein 5, ribosomal RNA, and cytochrome b genes for the detection of flaviviruses, mosquitoes, and bloodmeals, respectively. We identified 13 of 14 flaviviruses from virus inoculated mosquitoes and cultured cells. Additionally, we differentiated between four mosquito genera and eight whole blood samples. The microarray platform was field evaluated in Thailand and successfully identified flaviviruses (Culex flavivirus, dengue-3, and Japanese encephalitis viruses), differentiated between mosquito genera (Aedes, Armigeres, Culex, and Mansonia), and detected mammalian bloodmeals (human and dog). We showed that the microarray platform and amplification strategies described here can be used to discern specific information on a wide variety of viruses and their vectors.     

The Development of a Loop-Mediated Isothermal Amplification Method (LAMP) for Echinococcus granulosis Coprodetection

We have previously developed a polymerase chain reaction (PCR) assay for detection of Echinococcus granulosus infection, which proved very sensitive and specific for identification of infected dogs. We have now developed a loop-mediated isothermal amplification (LAMP) assay, which amplifies the same genomic repeated sequences of E. granulosus for coprodetection. This assay enabled detection of a single egg in fecal samples and showed high species specificity for E. granulosus with no cross-amplification of DNA from closely related helminths, including Echinococcus multilocularis. Because the method does not require thermocycling for DNA amplification, or electrophoresis for amplicon detection, it can potentially be used for premortem identification of E. granulosus-infected dogs to enable large-scale surveys in endemic countries where highly specialized equipment to undertake PCR analysis is rare.     

Dogs as Sentinels for Flavivirus Exposure in Urban,Peri-Urban and Rural Hanoi,Vietnam

Diseases caused by flaviviruses, including dengue fever and Japanese encephalitis, are major health problems in Vietnam. This cross-sectional study explored the feasibility of domestic dogs as sentinels to better understand risks of mosquito-borne diseases in Hanoi city. A total of 475 dogs serum samples from 221 households in six districts of Hanoi were analyzed by a competitive enzyme-linked immunosorbent assay (cELISA) for antibodies to the pr-E protein of West Nile virus and other flaviviruses due to cross-reactivity. The overall flavivirus seroprevalence in the dog population was 70.7% (95% CI = 66.4–74.8%). At the animal level, significant associations between seropositive dogs and district location, age, breed and keeping practice were determined. At the household level, the major risk factors were rural and peri-urban locations, presence of pigs, coil burning and households without mosquito-borne disease experience (p < 0.05). Mosquito control by using larvicides or electric traps could lower seropositivity, but other measures did not contribute to significant risk mitigation of flavivirus exposure in dogs. These results will support better control of mosquito-borne diseases in Hanoi, and they indicate that dogs can be used as sentinels for flavivirus exposure.     

West Nile virus serosurveillance in Iowa white-tailed deer (1999-2003)

Sera from white-tailed deer (Odocoileus virginianus) were collected in Iowa during the winter months (1999-2003), 2 years before and after West Nile virus (WNV) was first reported in Iowa (2001), and were analyzed for antibodies to WNV. Samples from 1999 to 2001 were antibody negative by blocking enzyme-linked immunosorbent assay (bELISA) and plaque reduction neutralization test (PRNT(90)). Prevalence derived from bELISA (2002, 12.7%; 2003, 11.2%) and WNV PRNT(90) (2002, 7.9%; 2003, 8.5%) assays were similar. All samples were negative for antibodies against St. Louis encephalitis virus as determined by PRNT(90). Antibodies to flaviviruses were detected by indirect enzyme-linked immunosorbent assay (iELISA) prior to the first WNV cases reported in Iowa (1999-2001) with prevalence ranging from 2.2% to 3.2%, suggesting the circulation of an additional undescribed flavivirus prior to the introduction of WNV into the area. Flavivirus prevalence as determined by iELISA increased in 2002 and 2003 (23.3% and 31.9%, respectively). The increase in prevalence exceeded estimates of WNV prevalence, suggesting that conditions favored general flavivirus transmission (including WNV) during the 2002-2003 epizootic. These data indicate that serologic analysis of deer sera collected from hunter harvests may prove useful for surveillance and evidence of local transmission of WNV and other pathogens and identify white-tailed deer as a species for further studies for host competency.     

The laboratory as a tool to qualify tuberculosis diagnosis

OBJECTIVES: To evaluate the performance of laboratory diagnosis of tuberculosis, clinical samples underwent culture, species identification and drug susceptibility testing (DST). METHODS: A total of 554 samples from 269 patients were tested for smear microscopy using Kinyoun stain. Culture was performed in Ogawa-Kudoh medium and species identification was performed using the IS6110 amplified region. DST for rifampicin, isoniazid (INH) and streptomycin were carried out using the Resazurin assay. RESULTS: Cultures augmented the number of cases diagnosed by 22.1%, IS6110 amplification identified all Mycobacterium tuberculosis strains thus isolated and DST detected three strains resistant to INH and one multidrug-resistant strain. CONCLUSION: Simultaneous use of different techniques enhanced culture yield, species identification and detection of drug resistance even in a laboratory with limited facilities.     

Do wild ungulates allow improved monitoring of flavivirus circulation in Spain?

As a response to the need for improved and cost-efficient West Nile virus (WNV) and other flavivirus surveillance tools, we tested 887 juvenile free-living red deer, 742 free-living juvenile wild boar, and 327 farmed deer, to detect temporal variability in exposure to these viruses. Thirty of 742 wild boar samples (4%; 95% CI 2.8,5.7) yielded a positive ELISA result. Antibody-positive individuals had been sampled between 2003 and 2011 in localities from central and southern Spain. No wild boar from the northern half of Spain (n=120) tested positive. Regarding juvenile wild red deer, only two out of 887 samples yielded a positive ELISA result (0.2%; 95% CI 0.1,0.8). These two samples came from the same site and sampling year. The likelihood of detecting contact with WNV or cross-reacting flaviviruses was 18 times higher among juvenile wild boar than among juvenile red deer. ELISA positivity among farmed deer increased 10-fold after local flavivirus outbreaks recorded in the summer and autumn of 2010. This survey demonstrated the potential usefulness of juvenile wild ungulates, particularly wild boar, as suitable flavivirus sentinels in southwestern Europe, and that systematic serum banking of samples from hunter-harvested wildlife or from individual farmed ungulates provides valuable material for retrospective epidemiological surveys and future disease monitoring.     

Detection of Schistosoma mansoni cercariae in plankton samples by PCR

A PCR assay on the basis of a tandemly repeated DNA sequence was employed for the detection of Schistosoma mansoni in artificial plankton samples. It was highly specific, since as few as 1fg DNA from this species were sufficient to obtain a clear signal, while 10pg DNA of Schistosoma rodhaini were required and no PCR products were obtained with even 10ng DNA of planktonic organisms and any other trematode species tested. In areas with transmission of different Schistosoma species 10pg DNA should be used for amplification, which would allow detection of 20 S. mansoni cercariae in 0.05g plankton without interference caused by DNA of other Schistosoma species. In other areas 10ng DNA from plankton samples can be amplified, detecting less than one S. mansoni cercaria specifically in 0.05g plankton. This assay might help to identify S. mansoni in samples from field studies, where a multitude of different organisms hinder a correct species identification.     

Enhancement of dengue virus infection in monocytes by flavivirus antisera

Enhanced dengue 2 virus (D2V) infection in suspension cultures of human peripheral blood mononuclear phagocytes (PBL) produced by subneutralizing concentrations of dengue antisera has been described previously. In this study, the enhancement phenomenon was found to be a general property of representative flavivirus antisera. All except one of 24 antisera, which had been raised by 1-3 injections of flaviviruses in rabbits, enhanced the growth of dengue 2 virus in human PBL. Flavivirus antisera showing the greatest level of cross-reactivity against a battery of 42 flavivirus antigens in the hemagglutination-inhibition test were most potent in enhancing dengue replication in PBL cultures. Cross-neutralizing reactivity did not relate to enhanced D2V infection. However nearly one-half of studied flavivirus antisera neutralized D2V at dilutions of 1:10 or 1:20. Heterotypic D2V neutralizing antibody could serve as a "brake" on infection enhancement in vivo. Observations should be made in the field to look for possible enhancement of dengue infection in heterotypic flavivirus immunes.     

Overview of flavivirus molecular biology and future vaccine development via recombinant DNA

Studies in many laboratories over the last several years have elucidated the structures of several different flavivirus genomes. Conserved features include the production of at least 10 different virus encoded proteins from a single long open reading frame by a combination of host and virus-encoded proteases. The established gene order is 5'-C- prM(M)-E-NS1-NS2A-NS2B-NS3-NS4A-NS4B-NS 5-3' and these proteins exhibit varying degrees of homology in comparisons among flaviviruses. Conserved RNA sequences and structures have also been identified for the mosquito-borne flaviviruses but are absent in sequenced tick-bone viruses. Relevant to the development of efficacious flavivirus vaccines, studies aimed at defining the antigenic determinants necessary for eliciting protective immunity have focused primarily on the structural proteins, in particular the E protein, as well as the nonstructural secreted glycoprotein, NS1. Other work, which has led to the derivation of live-attenuated flavivirus strains, should eventually allow the genetic determinants of flavivirus attenuation and pathogenesis to be understood at the molecular level. The successful recovery infectious flaviviruses from cloned cDNA raises the possibility of manipulating these viral genomes as cDNA to construct or propagate candidate live-attenuated vaccine strains. Several applications of this technology are discussed.     

St. Louis encephalitis virus: first isolation from a human in São Paulo State, Brazil

This paper reports the isolation of St. Louis encephalitis virus (SLEV) from a febrile human case suspected to be dengue, in S?o Pedro, S?o Paulo State. A MAC-ELISA done on the patient's acute and convalescent sera was inconclusive and hemagglutination inhibition test detected IgG antibody for flaviviruses. An indirect immunofluorescent assay done on the C6/36 cell culture inoculated with the acute serum was positive for flaviviruses but negative when tested with dengue monoclonal antibodies. RNA extracted from the infected cell culture supernatant was amplified by RT-PCR in the presence of NS5 universal flavivirus primers and directly sequenced. Results of BLAST search indicated that this sequence shares 93% nucleotide similarity with the sequence of SLEV (strain-MSI.7), confirmed by RT-PCR performed with SLEV specific primers. Since SLEV was identified as the cause of human disease, it is necessary to improve surveillance in order to achieve early detection of this agent in the state of S?o Paulo and in Brazil. This finding is also an alert to health professionals about the need for more complete clinical and epidemiological investigations of febrile illnesses as in the reported case. SLEV infections can be unrecognized or confused with other ones caused by an arbovirus, such as dengue.     

Chikungunya and dengue fever among hospitalized febrile patients in northern Tanzania

Consecutive febrile admissions were enrolled at two hospitals in Moshi, Tanzania. Confirmed acute Chikungunya virus (CHIKV), Dengue virus (DENV), and flavivirus infection were defined as a positive polymerase chain reaction (PCR) result. Presumptive acute DENV infection was defined as a positive anti-DENV immunoglobulin M (IgM) enzyme-linked immunsorbent assay (ELISA) result, and prior flavivirus exposure was defined as a positive anti-DENV IgG ELISA result. Among 870 participants, PCR testing was performed on 700 (80.5%). Of these, 55 (7.9%) had confirmed acute CHIKV infection, whereas no participants had confirmed acute DENV or flavivirus infection. Anti-DENV IgM serologic testing was performed for 747 (85.9%) participants, and of these 71 (9.5%) had presumptive acute DENV infection. Anti-DENV IgG serologic testing was performed for 751 (86.3%) participants, and of these 80 (10.7%) had prior flavivirus exposure. CHIKV infection was more common among infants and children than adults and adolescents (odds ratio [OR] 1.9, P = 0.026) and among HIV-infected patients with severe immunosuppression (OR 10.5, P = 0.007). CHIKV infection is an important but unrecognized cause of febrile illness in northern Tanzania. DENV or other closely related flaviviruses are likely also circulating.     

荧光定量PCR快速检测鼠疫耶尔森氏菌的实验研究

目的　利用LightCycler建立一种简便、特异的荧光定量PCR检测方法 ,用于鼠疫耶尔森氏菌的快速检测。 方法　采用SYBRGreenI随机掺入法 ,以Roche试剂盒为标准 ,比较两公司的DNA聚合酶 ,用鼠疫菌 310 0 4建立荧光PCR反应体系 ;针对鼠疫耶尔森氏菌特异的染色体标识序列和质粒上F1抗原基因设计引物 ,检测其灵敏度和特异性 ,以盲测试验进行验证 ;在此基础上鉴定 2 75株鼠疫耶尔森氏菌 ,并测试该法检测脏器污染模拟标本的灵敏度。结果　建立以一个公司试剂为主较低成本的PCR反应体系 ;EV76的检测灵敏度可达每反应体系 1.6个菌 ;检测我国 18个生态型共计 2 75株鼠疫耶尔森氏菌及 2 8株非鼠疫菌株的PCR扩增结果表明 ,鼠疫菌株均出现特异的扩增结果 ,2 8株对照菌株均为阴性 ;肝、脾、肺模拟标本检测灵敏度可达每反应体系 4 0 0 0个菌。结论　该方法对于检测鼠疫耶尔森氏菌具有简便、快捷、高敏感性和特异性的特点 ,适用于鼠疫紧急疫情时的快速诊断和疫源地监测     

Rapid identification of Leishmania complexes by a real-time PCR assay

A real-time PCR assay for the detection of four Leishmania complexes (L. Viannia, L. mexicana, L. donovani/infantum, and L. major) was developed and evaluated. The assay was developed to detect the glucosephosphate isomerase gene and capitalizes on DNA sequence variability within that gene for Leishmania complex identification. Primer/probe sets were created and tested against a panel of 21 known negative controls and on DNA extracted from cultured promastigotes or from tissue biopsies from patients with cutaneous leishmaniasis. The assay was highly specific, as no amplification products were detected in the negative control samples while simultaneously retaining a high degree of complex-specific diagnostic accuracy for cultured organisms and patient clinical samples. Real-time PCR offers rapid (within hours) identification of Leishmania to the complex level and provides a useful molecular tool to assist both epidemiologists and clinicians.     

多重PCR种特异性检测恶性疟原虫和间日疟原虫方法的建立

目的建立恶性疟原虫和间日疟原虫种特异性检测的多蕈PCR方法,用于疟疾的检测和诊断.方法根据疟原虫18S核糖体小亚基ssRNA的基因序列设计合成8对11条引物,通过对恶性疟、间日疟患者及健康对照者血样的DNA进行扩增,选择出敏感性和特异性最佳的引物用于建立多重PCR方法,并用梯度变化的方法分别对引物浓度、复性温度、延伸温度和循环次数等反应参数进行比较分析,优化PCR反应条件.利用优化后的多重PCR埘采自云南和上海的139份疟疾患者血样和32份非疟疾患者血样进行检测,以镜检方法为金标准,分析多重PCR方法检测患者血样的敏感性和特异性.结果从8对11条引物中优选出2对共3条引物用于建立多重PCR.利用这3条引物进行多重PCR,一次反应即可完成对恶性疟原虫和间日疟原虫的种特异性鉴定.对疟疾和非疟疾患者血样检测结果显示,该方法检测患者血样的敏感性为97.8%,特异性为100%.结论多重PCR方法敏感、特异、可进行批量检测,适用于对人群的疟疾监测和疑似疟疾病例的诊断,并能鉴定恶性疟原虫和间日疟原虫虫种.