First isolation of Japanese encephalitis from Culex quinquefasciatus in Thailand

Isolation of Japanese encephalitis (JE) virus using C6/36 cell and immunofluorescence virus antigen detection techniques was attempted from female mosquitoes collected with CDC gravid traps in Samut Songkhram Province in the central region and in Phuket Province in southern Thailand, in 2003. One thousand and eighty female mosquitoes including 6 species of the Culicidae family (Culex quinquefasciatus, Cx. gelidus, Cx. tritaeniorhynchus, Cx. whitmorei, Cx. vishnui complex, Cx. s.g. culiciomyia) (pooled by specific specimen), were processed for virus isolation. Two pools of Cx. quinquefasciatus yielded a JE virus isolation. This represents the first report of JE virus isolation from Cx. quinquefasciatus in Thailand.     

中国云南湄公河上游乙型脑炎传播媒介生物学及行为的研究

目的　探讨乙型脑炎病毒媒介在人房的季节消长、嗜血习性和叮人行为,以及其幼虫相关的生物学习性, 从而制定出有效的媒介控制措施。　方法　2001 年 6~12 月,在云南南部湄公河上游,选取一个种植场,采用 CDC 诱蚊灯和人工诱捕的方法捕蚊;蚊胃血源环状沉淀法鉴定其嗜血习性以及孳生地调查幼虫孳生习性。　结果　共捕获乙脑病毒媒介蚊虫5属、11种、5 726只。无论是诱蚊灯还是人工诱蚊法,三带喙库蚊、伪杂鳞库蚊、棕头库蚊和中华按蚊的季节密度高峰都出现于 6~8 月的雨季;3 种库蚊在村内和村外有整夜叮咬活动,但中华按蚊的叮人高峰出现在 21:00 之前。人房室内诱蚊灯捕蚊与室外人诱观察发现,室内捕捉乙脑病媒数量远高于室外。蚊胃血检测结果发现它们的人血指数都比较高;幼虫具有明显的特征性分布。　结论　湄公河上游地区人房优势蚊种三带喙库蚊、伪杂鳞库蚊、棕头库蚊和中华按蚊的生物学及行为与以往畜圈调查的结果存在着差异。     

Mosquito blood feeding patterns as a factor in the epidemiology of Japanese encephalitis in southern India.

Determinations were made of the source of 16,330 bloodmeals from 10 species of Culex mosquitoes, including recognized vectors of Japanese encephalitis (JE) virus, in two epidemiologically distinct areas in southern India. In Madurai, where cases occurred sporadically and pigs were reared only in some villages, Cx. tritaeniorhynchus, Cx. pseudovishnui, and Cx. vishnui had fed predominantly on cattle (89.2-91%), but less frequently on humans (2.1-6.2%) and on pigs and ardeid birds (0-0.1%). In Nallur, which was endemic for JE and had a large pig population, 4.4-5.4% of the feedings were on these hosts. Cattle feedings accounted for 84.6-88% of the total feedings, human feedings for 2.4-6.2%, but there were no ardeid-positive feedings. Culex tritaeniorhynchus and Cx. vishnui showed a marked increase in the proportion of human feedings during the hot season, due to increased availability of humans sleeping outdoors to mainly exophagic mosquitoes. Feeding indices were corrected for spatial and temporal concurrence of hosts in each season, but these factors were found to require further elucidation. Discrepancies in the relative abundance of vectors as monitored by two different methods are discussed in the light of these observations.     

Longitudinal studies in South Indian villages on Japanese encephalitis virus infection in mosquitoes and seroconversion in goats

Japanese encephalitis (JE) is endemic in Cuddalore district, Tamil Nadu, where Culex tritaeniorhynchus Giles was the major vector. We screened 45 100 adult female Cx. tritaeniorhynchus (902 pools) by enzyme-linked immunosorbent assay and isolated and confirmed JE virus (JEV) by using an insect bioassay system. We had 69 isolates of which 62 (90%) were identified as JEV. The average vector abundance per man hour for Cx. tritaeniorhynchus was 324.5 per month for the period June 1998-May 2000. The average minimum infection rate (MIR) per month in Cx. tritaeniorhynchus was 1.4 (range 0.0-5.6). Every year, a new batch of goats, 20 in the first year and 31 in the second year, born during the non-JE transmission period (January-June), aged <6 months and negative for haemagglutination inhibition (HI) antibodies were procured and placed in the villages as sentinels. Fortnightly, blood specimens were collected from these goats and tested for JE antibodies by HI test. Seroconversions (SCs) were recorded in 14 goats (70%) in the first year and 23 goats (74%) in the second year. JE HI antibody titres in goats were low (1:10-1:80) and these levels declined to undetectable levels in about 4 weeks following SCs. The time sequence of events indicated that four of five peaks of MIR in mosquitoes were followed 1-3 months later by peaks in the proportion of seroconverted goats. We suggest the screening of goats and cattle as a more feasible tool to stratify areas according to JE infection risk to the human population through the regular health system rather than screening mosquitoes using monoclonal antibodies, which is possible only in specialized laboratories.     

云南省景洪市虫媒病毒调查分析

近１０多年来，先后从云南省景洪市各乡镇的当地病人、猪、蝙蝠和蚊虫体内分离出乙型脑炎病毒２２株，从白纹伊蚊中分离到登革４型病毒１株，从蝙蝠和蚊体内分离到基孔肯雅病毒５株，从患急性期血液中分离到辛德毕斯病毒１株，从发热病人血液、脑炎病人脑脊液、猪血清和牛血清中分离到新环状病毒４７株，从黄胸鼠肺脏中检查出流行性出血热病毒抗原阳性６份；人群、猪恒河猴或鼠类血清中亦检出上述６种病毒的抗体，表明景洪市存在乙     

Impact of weather variables on mosquitoes infected with Japanese encephalitis virus in Kurnool district, Andhra Pradesh

ObjectiveTo assess the virus infection in mosquitoes during different seasons and correlated with various climatic factors.MethodsThe field collected vectors were screened for Japanese encephalitis (JE) virus after dessication using ELISA method. Most of the positive pools were recorded from Culex tritaeniorhynchus (Cx. tritaeniorhynchus) and Culex. gelidus (Cx. gelidus) during JE transmission season (winter) and some positive pools were also reported during non JE transmission periods (i.e. summer and rainy seasons).ResultsThe minimum infection rates (MIR) of 1.75 from Cx. tritaeniorhynchus and 0.17 from Cx. gelidus has been reported in the year 2002 at the beginning of the study and the values were found nil at the end of the study (2006) from the study areas of Kurnool district.ConclusionsFrom this study it is noted that MIR of Cx. gelidus and Cx. tritaeniorhynchus were modulated by various meteorological parameters. The mosquito vector abundance increases after the monsoon period (winter) and lowest in dry season (summer). Similarly, MIR fluctuated between seasons with higher MIR recorded after monsoon period and lower in the rest of season. Impact of these metrological parameters in JE virus infected mosquitoes is discussed in this paper.     

Short report: observations on the multiple feeding behavior of Culex tritaeniorhynchus (Diptera: culicidae), the vector of Japanese encephalitis in Kerala in southern India

A study was undertaken in villages endemic for Japanese encephalitis (JE) in Kerala in southern India during the period 1998-2001 to determine the host-feeding pattern of Culex tritaeniorhynchus, the major vector of JE in southeast Asia. A total of 3,067 blood-engorged Cx. tritaeniorhynchus were tested and 2,553 (82.2%) of the samples could be identified. Culex tritaeniorhynchus had fed mainly (56.6%) on cattle. Pig feeding accounted 6.3% of the total samples. Some samples (n = 980, 38.3%) were of serologic mixed origin. Of 980 mixed blood-fed mosquitoes, 975 (99.5%) had imbibed blood from two distinct hosts and 5 (0.5%) imbibed blood from three distinct hosts. Mixed blood meals were mostly (96.7%) from cattle and goats. The epidemiologic implications of multiple feeding of Cx. tritaeniorhynchus on dampening (dead-end) hosts such as cattle and goats in the transmission of JE virus is discussed.     

Japanese encephalitis virus infection in mosquitoes reared from field-collected immatures and in wild-caught males

Immature stages of mosquitoes collected in JE endemic areas of Karnataka, India between 1985 and 1987 were reared to adults and processed for the detection and isolation of Japanese encephalitis (JE) virus in an attempt to find naturally occurring vertical transmission of the virus. Males collected during 1985-1986 were also processed. A total of 15,785 adults reared from immatures and divided into 445 pools and 1,756 wild-collected males divided into 128 pools were processed using mosquito inoculation and immunofluorescence techniques. JE virus antigen was detected in 9 pools, 4 of which yielded JE virus. These were 2 pools of males and 1 pool of female Culex tritaeniorhynchus and 1 pool of male C. pseudovishnui, suggesting vertical transmission of JE virus in the mosquitoes.     

云南省德宏州蚊虫分布特点及乙型脑炎病毒分离

１９８３年５月以及１９８４年和１９８９年７￣８月，在云南省德宠州路西，瑞丽和盈江三县，市捕获成年雌性蚊虫９属４４种１９０８３只。三带喙库蚊，霜背库蚊，棕头库蚊和中华按蚊是农村畜圈的主要蚊种。伪白纹伊蚊和白纹伊蚊是野外竹林区的优势蚊种。对所获蚊虫用Ｃ６／３６细胞和乳鼠方法分离病毒，结果从三带喙库蚊中分离到乙型脑炎病毒２株，从霜背库蚊，白纹伊蚊和窄翅伊蚊中各分离出１株乙型脑炎病毒。分析认为，三带喙库蚊     

三带喙库蚊和致倦库蚊经口感染乙型脑炎病毒疫苗SA14-14-2株的研究

目的通过实验，确定被SA14—14—2乙脑减毒活疫苗免疫的宿主动物和人在被媒介蚊虫叮咬后，是否存在感染和传播的可能。方法建立三带喙库蚊和致倦库蚊的实验室种群，用乙型脑炎SA14—14—2疫苗株病毒和乙脑野毒株经口感染两种库蚊，感染后不同时间取一定数量的蚊，研磨制成悬液，应用空斑试验方法检测感染后不同时间蚊体内的病毒存在情况和病毒滴度。结果在用6．0610gPFU／ml SA14～14—2病毒经口感染的两种库蚊中，没有检测到病毒空斑，即没发现蚊虫的感染；在用较高的6．1810gPFU／ml病毒经口感染的三带喙库蚊和致倦库蚊中，分别有一组蚊虫出现低滴度感染，空斑形成单位分别是1．24和1．11log10PFU／ml；在用野毒株经口感染的两种库蚊，共计19组蚊虫中，有14组发生感染，空斑形成单位在3．18～4．7910g10PFU／ml。结论作为乙脑主要传播媒介的三带喙库蚊和致倦库蚊对SA14—14—2疫苗株病毒的经口感染和病毒在体内的复制严重受限，当叮咬接种疫苗的人后，不具备发生乙脑病毒感染和传播的能力。     

云南省西双版纳州蚊虫分布特点及与虫媒病毒的关系

１９８１、１９８２、１９８３、１９８６、１９８７和１９８８年在西双版纳州景洪、勐海和勐腊县（市）捕获成年雌性蚊虫８属３４种３４５０８只。夜晚在农村畜圈及其周围采获蚊虫２１种,优势蚊种为棕头库蚊和三带喙库蚊;白天在野外竹林采获蚊虫２５种,优势蚊种为贺斑伊蚊和白蚊伊蚊。从三带喙库蚊（８株）、霜背库蚊（４株）、伪杂鳞库蚊（３株）、环带库蚊（１株）、棕头库蚊（１株）、中华按蚊（３株）、刺扰伊蚊（２株）、白     

云南省乙型脑炎几个重要流行区的传播媒介调查

目的了解云南省乙型脑炎重要流行区传播媒介种群密度和地区分布。方法现场CDC诱蚊灯诱捕成蚊;吸蚊管人工捕蚊2种方法调查。结果CDC诱蚊灯诱捕成蚊法调查了5个县,捕获蚊虫6属37种8023只,其中思茅6属34种6786只,三带喙库蚊占49.01%;麻栗坡、马关、砚山、邱北、捕获蚊虫6属21种1237只,其中三带喙库蚊占71.06%;人工捕成蚊法调查了7个地区,捕获蚊虫5属21种11301只,其中思茅翠云区调查4个乡(镇)捕获蚊虫5属21种10482只,三带喙库蚊占64.44%,其它6个地区捕获蚊虫4属16种819只,乙型脑炎主要传播媒介三带喙库蚊景谷占49.13%,孟连占46.15%,陇川占35.59%;结论调查结果显示:这些地区乙型脑炎疾病传播媒介品种较多,特别是三带喙库蚊在乙型脑炎流行区种群大密度较高,地区分布较广,大量危险因素存在。     

A longitudinal study of the prevalence of Japanese encephalitis virus in adult and larval Culex tritaeniorhynchus mosquitoes in northern Taiwan

Adult females and larvae of Culex tritaeniorhynchus were collected year-round for 3 1/2 years at a site near Taipei, Taiwan. One hundred sixty-four isolates of Japanese encephalitis (JE) virus were obtained from approximately 142,000 adult females and 1 isolate of the virus was obtained from approximately 382,000 larvae. Virus was recovered from adult females every year, except the first, beginning in May. The single larval isolate was from specimens collected in June. The vertical transmission of JE virus in Cx. tritaeniorhynchus as a possible inter-epidemic viral survival mechanism is examined.     

Continuity and change of Japanese encephalitis virus in Toyama Prefecture, Japan

To determine the mechanisms of maintenance and evolution of Japanese encephalitis virus (JEV) in a temperate zone, we attempted to isolate JEV from mosquitoes and pigs in Toyama Prefecture, Japan. A total of 87 JEVs were isolated from female Culex tritaeniorhynchus mosquitoes and pigs during 2005-2009. The prevalence of JEV in Toyama Prefecture was seasonally late in comparison with that of the virus during 1966-1972. Furthermore, JEVs were isolated after the peak in the number of female Cx. tritaeniorhynchus. Among JEV strains isolated in this study, two distinct groups were observed within genotype I of the phylogeny generated from nucleotide sequence information derived from the envelope and capsid/premembrane genes: strains belonging to the major type were isolated during 2005-2009, and strains from the minor type were isolated only in 2007. The major type has exhibited gradual change in its envelope and capsid/premembrane genes, and all isolates obtained in 2008 and 2009 had a novel deletion of seven nucleotides in the variable region of the 3'-untranslated region.     

Distribution of mosquitoes and mosquito-borne viruses along the China-Myanmar border in Yunnan Province

A total of 54,673 mosquitoes were collected at 11 sites located near the China-Myanmar border in the western part of Yunnan Province during July and August 2007. There were 29 species in 4 genera identified from the collections, including 12 species of Culex, 12 species of Anopheles, 3 species of Aedes, and 2 species of Armigeres. Culex tritaeniorhynchus Giles (67.9%, 37,119/54,673) and Anopheles sinensis Wiedemann (25.9%, 14,170/54,673) were the most abundant species in this investigation. Virus was isolated using BHK-21 and C6/36 cells from 22 of 510 mosquito pools. Isolates included Japanese encephalitis virus (JEV) and Getah virus (GETV), which were identified by serological and molecular methods. Twenty JEV strains were isolated from Cx. tritaeniorhynchus (15 isolates), An. sinensis (3 isolates), and Armigeres subalbatus Coquillett (2 isolates), and 2 GETV strains were isolated from Culex pseudovishnui Colless and Cx. tritaeniorhynchus. This study suggests that Ar. subalbatus is a potentially important local vector because of the high JEV infection ratio found in this species. Enzootic JEV transmission persists in this area and therefore, surveillance for human disease caused by JEV and GETV should be conducted in the region.     

Growth characteristics of the chimeric Japanese encephalitis virus vaccine candidate, ChimeriVax-JE (YF/JE SA14--14--2), in Culex tritaeniorhynchus, Aedes albopictus, and Aedes aegypti mosquitoes

The Japanese encephalitis (JE) virus vaccine candidate, ChimeriVax-JE, which consists of a yellow fever (YF) 17D virus backbone containing the prM and E genes from the JE vaccine strain JE SA14--14--2, exhibits restricted replication in non-human primates, producing only a low-level viremia following peripheral inoculation. Although this reduces the likelihood that hematophagous insects could become infected by feeding on a vaccinated host, it is prudent to investigate the replication kinetics of the vaccine virus in mosquito species that are known to vector the viruses from which the chimera is derived. In this study ChimeriVax-JE virus was compared to its parent viruses, as well as to wild-type JE virus, for its ability to replicate in Culex tritaeniorhynchus, Aedes albopictus, and Aedes aegypti mosquitoes. Individual mosquitoes were exposed to the viruses by oral ingestion of a virus-laden blood meal or by intrathoracic (IT) virus inoculation. ChimeriVax-JE virus did not replicate following ingestion by any of the three mosquito species. Additionally, replication was not detected after IT inoculation of ChimeriVax-JE in the primary JE virus vector, Cx. tritaeniorhynchus. ChimeriVax-JE exhibited moderate growth following IT inoculation into Ae. aegypti and Ae. albopictus, reaching titers of 3.6-5.0 log(10) PFU/mosquito. There was no change in the virus genotype associated with replication in mosquitoes. Similar results were observed in mosquitoes of all three species that were IT inoculated or had orally ingested the YF 17D vaccine virus. In contrast, all mosquitoes either IT inoculated with or orally fed wild-type and vaccine JE viruses became infected, reaching maximum titers of 5.4-7.3 log(10) PFU/mosquito. These results indicate that ChimeriVax-JE virus is restricted in its ability to infect and replicate in these mosquito vectors. The low viremia caused by ChimeriVax-JE in primates and poor infectivity for mosquitoes are safeguards against secondary spread of the vaccine virus.     

Blood meal identification by using Microdot ELISA in vector mosquitoes

The blood meals of five mosquito species which are vectors for malaria, J.E. and Filaria were determined by using Microdot ELISA technique, which is a rapid and sensitive method. A total of 4354 freshly fed mosquitoes comprising of Anopheles culicifacies, An stephensi, Culex quinquefasciatus, Cx. vishnui gp. and Cx. tritaeniorhynchus were tested. Results of the study reveal that highest proportion of An. culicifacies between 76-100% fed on bovine host followed by 0.3 to 24.2% on human. Cx. vishnui showed 1.9% feeding on human, 49-87.7% on bovine and 7.4-12.3% on the pigs. It has been observed that malaria vectors namely An. culicifacies and An. stephensi are still zoophilic in nature. Similarly, the vector of J.E. namely Culex vishnui gp. and Cx. tritaeniorhynchus also fed on cattle but some samples have indicated feeding of blood meal from pigs.     

Outdoor resting preference of Culex tritaeniorhynchus, the vector of Japanese encephalitis in Warangal and Karim Nagar districts, Andhra Pradesh

BACKGROUND & OBJECTIVES: Entomological investigations were carried out in Warangal and Karim Nagar districts of Andhra Pradesh during viral encephalitis outbreak in July 2003. METHODS: Adult mosquito collections in outdoor were done using sweep cage method and in indoor with the help of aspirator tube and flash light. Larval collections were done by dipping method. JE virus positivity was tested by IgM capture ELISA test. RESULTS: Outdoor collections revealed presence of Culex tritaeniorhynchus, Cx. bitaeniorhynchus and Cx. gelidus and in indoor collections--Cx. quinquefasciatus, Cx. tritaeniorhynchus, An. vagus and An. subpictus. In the outdoor collections Cx. tritaeniorhynchus was predominant (96.3% of total collection). Three samples out of 55 serum samples from human cases and five from contacts showed the presence of antibodies against JE virus. INTERPRETATION & CONCLUSION: Cx. tritaeniorhynchus, a known vector of JE is predominant in outdoors and playing a main role in JE transmission in this area. Vector control aimed at the outdoor resting population might limit virus circulation in the mosquito vertebrate host cycle and prevent human infection.     

Mosquito larvicidal properties of essential oil of an indigenous plant, Ipomoea cairica Linn

Laboratory bioassay of the essential oil extracted from an indigenous plant, Ipomoea cairica, commonly known as 'Railway creeper', was carried out against the larvae of four vector species of mosquitoes in order to evaluate its mosquito larvicidal effect. Bioassay test revealed that the essential oil of the plant possess remarkable larvicidal properties as it could induce 100% mortality in the larvae of Culex tritaeniorhynchus (100 ppm), Aedes aegypti (120 ppm), Anopheles stephensi (120 ppm), and Culex quinquefasciatus (170 ppm) mosquitoes at concentrations rangeing from 100 to 170 ppm. The LC(50) and LC(90) values estimated for Cx. tritaeniorhynchus, Ae. aegypti, An. stephensi, and Cx. quinquefasciatus were 14.8 and 78.3, 22.3 and 92.7, 14.9 and 109.9, and 58.9 and 161.6 ppm, respectively. The essential oil was found to be most highly toxic to the larvae of Cx. tritaeniorhynchus followed by Ae. aegypti, An. stephensi, and Cx. quinquefasciatus mosquitoes.