Serologic evidence for widespread infection with La Crosse and St. Louis encephalitis viruses in the Indiana human population

The vast under-reporting of La Crosse virus and St. Louis encephalitis virus infections in Indiana residents was evident when numerous inapparent infections were detected retrospectively using serum dilution neutralization analyses of serum obtained in November 1978-April 1979 from 10,208 persons (0.2% of the state's population). An antibody prevalence rate of 3.6% to St. Louis encephalitis virus was detected in the sample population as a whole, with rates as high as 13.2% for residents of individual counties. The estimated average annual rate of infection for the whole population was 0.32%. The antibody prevalence to La Crosse virus in the sample population as a whole was 2.3%, with rates ranging up to 12.5% for residents of individual counties. The estimated average annual rate of infection for the whole population was 0.29%. The epidemiologic behavior of the two viruses was quite different. Age-specific antibody prevalence for St. Louis encephalitis virus indicated a pattern of endemic infection existed in the population as a whole; antibody prevalence rose as the population aged. However, many other infections apparently occurred during the 1975 and earlier epidemics. Age-specific antibody prevalence for La Crosse virus indicated a typical pattern of endemic infection was present. The antibody prevalence to La Crosse virus was best described by the Poisson distribution and that of St. Louis encephalitis virus by the negative binomial distribution. These data support the hypothesis that St. Louis encephalitis virus primarily produces intermittent epidemics in the Midwest while La Crosse virus produces continuous seasonal endemic infections. However, evidence suggestive of a low level of interepidemic St. Louis encephalitis virus infection in the population was also obtained. Computer-drawn synagraphic mapping view "maps" of regional antibody prevalence rates demonstrated the existence of distinct foci of infection for each virus in the human population.     

微流芯片检测流感病毒多重逆转录聚合酶链反应产物的实验研究

[目的 ]　建立应用微流芯片检测甲 1型、甲 3型、乙型流感病毒多重逆转录聚合酶链反应 (mRT -PCR)产物的方法 ,在mRT -PCR扩增核酸的基础上自动化灵敏的定量检测扩增产物 ,快速检测甲亚型、乙型流感病毒 ,帮助临床快速诊断和鉴别诊断其他呼吸道病毒感染 ,以及明确流感病毒在人群中感染、流行情况。　 [方法 ]　采用经MDCK细胞分离培养的甲 1型、甲 3型、乙型流感病毒毒株病毒液 ,使用 3组特异引物经mRT -PCR扩增核酸 ,扩增产物分别采用毛细管电泳技术经Caliper10 0 0微流芯片分析仪自动化检测和经 2 %琼脂糖凝胶电泳法检测。　[结果 ]　设计三组引物对相应甲 1型、甲 3型、乙型流感病毒靶基因的mRT -PCR扩增产物片段分别为 43 0bp、2 10bp、3 91bp ,本实验mRT -PCR扩增产物经 2 %琼脂糖凝胶电泳 ,与Marker比照 ,DNA条带基本在此位点附近 ;产物采用毛细管电泳法经Caliper10 0 0微流芯片分析仪后 ,分别于 413bp、2 0 3bp、3 79bp处出现陡峭的峰 ,如图所示。　[结论 ]　应用微流芯片采用毛细管电泳法可对流感病毒mRT -PCR产物进行定位及相对定量 ,有助于快速诊断流感病毒感染 ,有助于对流感的监测。     

Detection of St. Louis encephalitis virus in mosquitoes by use of the polymerase chain reaction.

We recently developed an assay using the polymerase chain reaction (PCR) for the specific detection of St. Louis encephalitis (SLE) virus RNA. This assay was tested in a blind study on 7 samples of pooled mosquitoes (50 mosquitoes/pool) which were also characterized for SLE virus by plaque assay in Vero cell culture. One sample was positive for the SLE virus as determined by both the PCR assay and a combination of the plaque assay and the indirect fluorescent antibody assay. The remaining 6 samples were negative for the presence of SLE virus as determined by both methods. These data indicate that this PCR assay can be used to monitor for the presence of SLE virus in pools of homogenized mosquitoes. This approach could provide early data on which to base disease control decisions.     

Wild terrestrial rainforest mammals as potential reservoirs for flaviviruses (yellow fever, dengue 2 and St Louis encephalitis viruses) in French Guiana

A serological survey for yellow fever virus (YFV), dengue 2 virus (DENV-2), and St Louis encephalitis virus (SLEV) was undertaken using a seroneutralization technique in 27 wild forest mammal species (574 individuals) in French Guiana. Evidence of yellow fever infection was observed in 10 species, with high prevalence recorded in howler monkey (18%) and agouti (20%). Antibodies against DENV-2 and SLEV were found sporadically in various species. This potential host diversity and the range of potential vectors might explain the behaviour of the viruses in epidemic outbreaks and the emergence of periurban loci.     

Quantification ofCoxiella burnetii by polymerase chain reaction (PCR) and a colorimetric microtiter plate hybridization assay (CMHA)

A colorimetric microtiter plate hybridization assay (CMHA) for the quantitative determination ofCoxiella burnetii DNA after amplification by externally controlled polymerase chain reaction (PCR) is described. The quantification assay is based on an enzyme linked immunosorbent assay (ELISA) format. Cloned DNA, representing a sequence complementary to an internal part of the diagnostic amplicon, was noncovalently attached to the wells of a microtiter plate. Biotinylated PCR product was hybridized to the immobilized capture probe. Bound product was detected via streptavidin horseradish peroxidase. The devised nonisotopic technique allows specific, rapid, and convenient quantification ofC. burnetii DNA. Additionally, it is compatible with standard laboratory ELISA equipment, making this assay amenable to automation and permitting processing of large sample numbers.     

Characterization of vitellogenin gene expression in round goby (Neogobius melanostomus) using a quantitative polymerase chain reaction assay

A growing concern over endocrine disruption in aquatic species has prompted the development of molecular assays to monitor environmental impacts. This study describes the development of quantitative polymerase chain reaction (qPCR) assays to characterize the expression of two vitellogenin (Vtg) genes in the invasive round goby (Neogobius melanostomus). Fragments from the 18SrRNA (housekeeping gene), Vtg II, and Vtg III genes were cloned and sequenced. The qPCR assays were developed to detect hepatic Vtg expression in goby. The assays detected induction of both Vtg genes in nonreproductive males following a two-week laboratory exposure to 17β-estradiol (≥1 mg/kg i.p. injection). The assays were applied to goby from Hamilton Harbour, Lake Ontario (Canada), including those from sites where feminization and intersex of goby has been documented. Both Vtg genes had significantly higher expression in females compared to males. Male reproductive goby adopt either parental or sneaker tactics; Vtg II expression was higher in sneaker than in parental males but parental and nonreproductive males did not differ from each other. The Vtg III expression was significantly higher in sneaker males followed by parental males and nonreproductive males, respectively. The Vtg II and III expression in nonreproductive males was elevated in the contaminated site with documented intersex. This assay provides an important tool for the use of an invasive species in monitoring endocrine disruption in the Great Lakes region.     

Use of polymerase chain reaction (PCR) for the detection of vaccine contamination by infectious laryngotracheitis virus.

Quality control of biologicals for veterinary use includes certification of freedom from extraneous agents. Contamination of vaccines may originate from various materials used for production and during manufacturing process. Requirements for avian virus vaccines to demonstrate freedom of adventitious agents are stated in the European Pharmacopoeia and include monitoring for infectious laryngotracheitis virus (ILTV). ILTV is an avian herpesvirus belonging to the alphaherpesvirus subfamily causing acute respiratory disease. To date the methods to detect ILTV contaminating biologicals consist of demonstration of antibody induction in chicken after immunization or virus cultivation in embryonated eggs. These methods are time consuming and laborious. Therefore, a specific, simple and sensitive in vitro polymerase chain reaction (PCR) for the detection of ILTV contamination in avian virus vaccines was developed. Primers were designed to amplify part of the p32 gene. Four different ILTV vaccine strains could be unequivocally detected. The identity of the amplified fragment was confirmed by restriction endonuclease analysis.     

Detection of dengue viruses in field-caught Aedes aegypti (Diptera: Culicidae) in Maracay, Aragua state, Venezuela by type-specific polymerase chain reaction.

Virological surveillance of dengue viruses in Aedes aegypti populations constitutes a powerful tool for early prediction of dengue outbreaks. We have standardized a protocol for viral RNA extraction from individual and pools of mosquitoes that permits a sensitive detection of dengue virus without RNA degradation or PCR inhibition when we apply a semi-nested RT-PCR. The limit of detection for each dengue serotype was 0.1 PFU. In a prospective field study conducted from November 2000 to December 2001, adult female A. aegypti mosquitoes from several municipalities with high dengue transmission in Maracay, Aragua State, Venezuela were collected and screened for dengue viruses using RT-PCR. We analyzed a total of 296 A. aegypti pools (1,632 mosquitoes); of these, 154 pools (469 mosquitoes) were collected from houses with persons with clinical diagnosis of dengue (dengue houses), and 142 pools (1,163 mosquitoes) from adjacent residences (neighbour houses). From the dengue houses, eight mosquito pools (5.2%) were positive for DENV-1 (0.7%), DENV-3 (3.2%) and DENV-4 (1.3%) viruses. From the neighbour houses, 18 mosquito pools (12.7%) were positive for DENV-3 (12%) and DENV-4 (0.7%) viruses. From these 26 RT-PCR positive mosquito pools (containing 1-25 mosquitoes each), 22 pools (84.6%) were positive for DENV-3. The most prevalent serotype in the 2001 dengue outbreak was also DENV-3. The minimum infection rate in both A. aegypti collections, from dengue houses and neighbour houses was 17 and 15 per 1,000, respectively. The relevance of these results for dengue surveillance is discussed.     

Detection of bovine herpesvirus 1 sequences in yaks (Bos grunniens) with keratoconjunctivitis, using a highly sensitive nested polymerase chain reaction

Thirty-seven yaks (Bos grunniens) with keratoconjunctivitis and 22 healthy yaks were used to investigate the role of bovine herpesvirus 1 (BoHV-1) in keratoconjunctivitis in yaks. Nucleic acid sequences of BoHV-1 glycoproteins B and E were detected in conjunctival swabs from all yaks with keratoconjunctivitis using a nested polymerase chain reaction (PCR). In 21 yaks, BoHV-1 sequences were detected along with Moraxella bovis (M. bovis) and Neisseria spp. The amplified BoHV-1 sequences were identical, and no nucleotide variation was observed when compared with a BoHV-1 reference strain using single-strand conformation polymorphism analysis of the amplified DNA sequences. Interestingly, BoHV-1 sequences could not be detected in samples from healthy yaks. However, conjunctival swabs from two healthy yaks (9.09%) yielded M. bovis and Neisseria spp. Samples from 35 yaks with keratoconjunctivitis showed positive reactions in an avidin biotin enzyme-linked immunosorbent assay for BoHV-1 antibodies; all the healthy yaks were seronegative. This is the first report of a possible association of BoHV-1 with keratoconjunctivitis in yaks.     

利用TaqMan-MGB探针检测A(H3N2)亚型流行性感冒病毒E119V耐药突变

目的 建立快速检测A(H3N2)亚型流行性感冒病毒(简称流感病毒)神经氨酸酶(M)基冈E119V奥司他韦耐药突变位点的双重实时荧光定量逆转录PCR(rRT-PCR)方法.方法 由GenBank获取2000-2012年A(H3N2)亚型流感病毒NA基因序列26条,据此设计特异性靶向NA E119V突变位点的TaqMan-MGB探针进行双重rRT-PCR反应,并利用耐药参考株、临床分离株进行敏感性、特异性和重复性评价.结果 建立了快速检测NA基因E119V位点的双重rRT-PCR检测方法.本方法在A(H3N2)病毒(HA=8)稀释度至10-5仍可检测到荧光信号,病毒滴度的对数与Ct值之间呈线性相关,具有较高的灵敏度;与无耐药突变的A(H3N2)流感病毒及其他呼吸道病毒均无交叉反应,两条TaqMan-MGB探针之问不存在交叉反应,具有很高的特异性,并能够检测耐药株和敏感株混合病毒中的耐药突变,在较高浓度的混合病毒中对耐药突变的检测限是5％,在低浓度混合病毒中的检测限是50％.重复性试验得到H3N2-119E和H3N2-119V两组探针批内平均变异系数(CV)值分别为2.32％和0.57％,批间CV值分别为1.77％和0.97％,具有较好的重复性.对其中20株病毒的NA基因序列进行分析,证实这些毒株的NA基因119位点均为谷氨酸(E),表明本研究设计的方法与序列测定结果一致.结论 建立了基于TaqMan-MGB探针检测A(H3N2)亚型流感病毒E119V耐药突变的双重rRT-PCR方法,该方法灵敏、特异、重复性好,实验过程和结果判读简单易操作.     

Comparison of influenza vaccine effectiveness using different methods of case detection: clinician-ordered rapid antigen tests vs. active surveillance and testing with real-time reverse-transcriptase polymerase chain reaction (rRT-PCR)

Annual evaluation of influenza vaccine effectiveness (VE) is needed to assess ongoing impact of immunization efforts in the setting of antigenic drift and periodic vaccine reformulation. Optimal methodology for determining VE remains unclear. We compared influenza VE generated from prospective enrollment and rRT-PCR testing (active surveillance group) with VE based on clinician-ordered diagnostic tests (clinical testing group) in a defined population over four seasons. VE was calculated as (1 - adjusted OR) for vaccination in cases vs. test-negative controls. VE based on clinical testing underestimated VE based on active surveillance and testing with rRT-PCR by 5-33% depending on season.