Correlation of population indices of female Culex tritaeniorhynchus with Japanese encephalitis viral activity in Kapuk, Indonesia

Nineteen consecutive monthly light trap collections of mosquitoes were made between October 1978 and April 1980 in Kapuk, Indonesia. Kapuk is a small suburb of Jakarta where pigs are raised in close proximity to rice paddies which are breeding sites for Culex tritaeniorhynchus. Japanese encephalitis (JE) virus is believed to be endemic and has been recovered from mosquitoes and pigs in the area on several occasions. A total of 18,435 female Cx. tritaeniorhynchus were allocated to 359 pools of approximately 50 per pool. Virus isolations were attempted in both Vero and BHK-21 cells and agents producing cytopathic effect were identified in a micro-neutralization test. Nineteen strains of JE were recovered from the 359 pools of Cx. tritaeniorhynchus tested. The light trap index of female Cx. tritaeniorhynchus (X) and the relative frequency of pools positive for JE (Y) for each month of the study were plotted and correlation coefficients (r) calculated after transforming the mosquito population data logarithmically and the relative frequencies of isolation by arcsine square root. The close fit of the data (p less than 0.001) to an inverse linear model (1/y = a + b log10X) suggests a close dependence of JE viral activity on the population dynamics of Cx. tritaeniorhynchus. Three additional strains of JE were recovered from other Culex spp. at the same study site. One strain each was isolated from individual pools of Cx. gelidus, Cx. vishnui and Cx. fuscocephala. Cx. tritaeniorhynchus was more frequently infected with JE than the other species tested.     

Observations on the D/V ratio of male genitalia of Culex pipiens complex in Amphur Muang Chiang Mai, Northern Thailand

Mosquitoes belonging to the Culex pipiens complex were collected from various locations in Amphur Muang Chiang Mai, Northern Thailand, and identified by using D/V ratios of male genitalia. Most of the mosquitoes were Cx. pipiens quinquefasciatus, having D/V ratios of 0.46 +/- 0.09. Among these mosquitoes, 1.23% (6/490) had D/V ratios between 0.8-0.9 These forms were considered as Cx. p. quinquefasciatus which showed extreme variations from the generally acceptable criterion.     

Arbovirus investigations in Argentina, 1977-1980. II. Arthropod collections and virus isolations from Argentine mosquitoes

Prospective surveys for arboviruses were carried out in Santa Fe, Corrientes, and Chaco provinces, Argentina, aperiodically during 1977-1980. A total of 313,233 mosquitoes and 598 biting flies other than mosquitoes were collected and tested for virus in 5,197 and 45 pools, respectively. Forty virus strains were isolated, all from mosquitoes, as follows: Santa Fe Province: 4 Gamboa group viruses from Aedeomyia squamipennis, 1 strain each of St. Louis encephalitis virus from Culex pipiens quinquefasciatus and Culex (Culex) spp.; Corrientes Province: a single strain of a newly discovered Anopheles A serogroup virus, Las Maloyas, from Anopheles albitarsis; and Chaco Province: 4 Gamboa group viruses from Ad. squamipennis, 6 strains of new Bunyaviridae (1 Antequera, 1 Barranqueras, and 4 Resistencia) from Culex (Melanoconion) delpontei, 3 strains of a new subtype of western equine encephalitis virus and 1 strain of Para virus from the Cx. (Mel.) ocossa group, 12 strains of a newly discovered subtype (VI) of the Venezuelan equine encephalitis complex from Cx. (Mel.) delpontei, and 1 strain each from Ad. squamipennis, Aedes scapularis, Ae. spp., Cx. (Cux.) spp., Cx. (Mel.) ocossa group, Mansonia spp., and Psorophora spp. Bloodmeals from 265 engorged mosquitoes were identified by precipitin test. These data, coupled with data on engorgement rates for 25,995 mosquitoes from bait collections, provide information on the host feeding patterns of several mosquito species. This information is discussed, along with data on relative abundance of mosquito species, within the context of the vector relationships of the species from which viruses were isolated. The association of Cx. (Mel.) delpontei with 18 strains of 4 different viruses in Chaco Province, plus its catholic feeding habits, clearly indicate for the first time the importance of this species as an arbovirus vector.     

Replication of dengue type 2 virus in Culex quinquefasciatus (Diptera: Culicidae)

We were able to infect Culex quinquefasciatus by the parenteral route with dengue virus type 2. The percentage of mosquitoes infected was dose dependent and we obtained a rate of 45.6% infected Cx. quinquefasciatus when a 10(5.9) MID50 (mosquito infectious dose for 50% of the individuals as measured in Aedes aegypti) of dengue virus type 2 per mosquito was used. Infection was detected by an immunofluorescent assay performed on mosquito head squashes 14 days after infection. The replication of dengue virus in Cx. quinquefasciatus was either at a very low level of magnitude or generated a large number of noninfectious particles since the triturated bodies of infected Cx. quinquefasciatus did not infect Ae. aegypti mosquitoes when inoculated parenterally. We were unable to infect Cx. quinquefasciatus females orally with an artificial meal that infected 100% of Ae. aegypti females. These findings lead us to agree with the consensus that Cx. quinquefasciatus should not be considered a biological vector of dengue viruses.     

Geographic variation in vector competence for West Nile virus in the Culex pipiens (Diptera: Culicidae) complex in California

We evaluated the susceptibility to infection and transmission of West Nile virus (WNV) in seven populations of Culex pipiens pipiens (L.), Cx. p. quinquefasciatus Say, and from populations containing Cx. pipiens/quinquefasciatus hybrids in a north-south transect of California. Samples were identified to species or as hybrid forms based on morphology of male terminalia. After 7 and 14 days of extrinsic incubation, few females were infected and none transmitted WNV from samples of Cx. p. pipiens from northern Shasta County and of Cx. p. quinquefasciatus from southern Los Angeles County. Seven days after infective feeding, 13%-36% of mosquitoes from the counties of Merced, Fresno, Kern, and San Bernardino were infected, and 12%-40% of infected mosquitoes expressed WNV in salivary expectorate. Fourteen days after infective feeding, 18%-43% of mosquitoes from these counties were infected, and 50%-69% of infected mosquitoes transmitted WNV in salivary expectorate. A sample of Cx. p. quinquefasciatus from Riverside County did not transmit WNV after 7 days, but 71% transmitted 14 days after infective feeding. These results reveal extensive geographic variation in vector competence for WNV in the Culex pipiens complex in California.     

Vectors of Japanese encephalitis virus (JEV): species complexes of the vectors

The vectors of JEV are Cx. tritaeniorhynchus, Cx. vishnui, Cx. pseudovishnui, Cx. gelidus, Cx. fuscocephala, Cx. quinquefasciatus, Cx. pipiens pallens, Cx. bitaeniorhynchus, Cx. annulirostris, Aedes togoi, Ae. japonicus, Ae. vexans nipponii, Anopheles annularis and An. vagus. Cx. tritaeniorhynchus is in the tritaeniorhynchus complex, breeds in rice fields, ground pools in vast areas. Two types of mating behavior, eurygamy and moderate stenogamy were detected. In the case of the eurygamy type, the mosquitoes were from Southern Thailand and hilly areas near Kanchanaburi, Thailand. Female mosquitoes are usually dark in color, the cibarial armature has rod teeth and the posterior end of the cibarial armature is bowl shaped with a typical rim. The rim of the bowl is everted. The moderate stenogamy type were mosquitoes from the plain areas such as Bangkok, Ayutthaya, Suphan Buri and Saraburi. The posterior end of the cibarial armature is bowl shaped with a stout rim. The larvae were characteristic in their siphon index, antennal index, hair O of prothoracic segment, and comb scale number and arrangement. Cx. tritaeniorhynchus summorosus from Japan, Los Banos and Luzon, Philippines, differed from Cx. tritaeniorhynchus in that on the lateral plate of the phallosome tritaeniorhynchus teeth are somewhat weakly developed and only gently curved whereas in tritaeniorhynchus summorosus they are strongly developed, considerably longer, and sharply recurved. The siphons of larvae are short, the sides parallel and the apex truncate in tritaeniorhynchus whereas in tritaeniorhynchus summorosus they are long and slender. Cx. tritaeniorhynchus var. siamensis is possibly present. Colonies have been maintained in the Department of Medical Entomology for 31 generations. The characteristics are in hair O (short, less than 20 branches, and without secondary branching and the larval siphon (short and broad where the others are long). Cx. vishnui and Cx. pseudovishnui are in the vishnui complex. Cx. quinquefasciatus and Cx. pipiens pallens are in the Cx. pipiens complex comprising: (1) Cx. pipiens; (2) Cx. quinquefasciatus Say; (3) Cx. molestus Forskal; (4) Cx. pipiens pallens; (5) Cx. australicus; (6) Cx. globocoxitus. Anopheles annularis is a species complex evidenced by two types of polytene chromosomes.     

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     

Flaviviruses isolated from mosquitoes collected during the first recorded outbreak of Japanese encephalitis virus on Cape York Peninsula, Australia.

In response to an outbreak of Japanese encephalitis (JE) virus on Cape York Peninsula, Australia, in 1998, mosquitoes were collected using CO2 and octenol-baited Centers for Disease Control and Prevention light traps. A total of 35,235 adult mosquitoes, comprising 31 species, were processed for virus isolation. No isolates of JE virus were recovered from these mosquitoes. However, 18 isolates of Kokobera virus, another flavivirus were obtained from Culex annulirostris. Twelve isolates were from western Cape York (minimum infection rate (MIR) of 0.61: 1,000 mosquitoes) and 6 were from the Northern Peninsula Area (MIR of 1.0:1,000). Potential explanations for the failure to detect JE virus in mosquitoes collected from Cape York Peninsula include the timing of collections, the presence of alternative bloodmeal hosts, differences in pig husbandry, asynchronous porcine seroconversion, and the presence of other flaviviruses.     

Vector competence of Aedes aegypti (L.) and Culex quinquefasciatus (Say) for Dirofilaria immitis (Leidy)

This study was performed to examine the vector competence of Aedes aegypti and Culex quinquefasciatus for Dirofilaria immitis. Eleven individual experiments were conducted in this study. Nonthaburi and Udon Thani strains of Ae. aegypti were allowed to feed on infected dogs that had 5,750 and 4,600 microfilariae (mW) per ml of blood, respectively. Three groups of Bangkok-strain Cx. quinquefasciatus were allowed to feed on dogs that had 4,800, 5,200, and 5,850 mf per ml of blood. Six groups of Liverpool-strain Ae. aegypti were allowed to feed on dogs with 1,650, 1,950, 3,350, 9,000, 9,250, and 11,550 mf per ml of blood. Three to 4% of Nonthaburi-strain, and 0-6% of Udon Thani-strain Ae. aegypti became infected and had infective-stage larvae (L3) of D. immitis in their probosces. Zero to 1 and 7% of Bangkok-strain Cx. quinquefasciatus had L3 in their probosces after taking blood meals with 4,800 and 5,850 mf per ml of blood, respectively. The percent-infected Liverpool-strain Ae. aegypti with L3 in their probosces were 3-12, 0-12, 10, 16, 7-19, and 0-21 after taking blood meals with 1,650, 1,950, 3,350, 9,000, 9,250, and 11,550 mf per ml of blood, respectively, when tested at different post-blood-feeding days. This study showed both Ae. aegypti and Cx. quinquefasciatus from Thailand can become vectors for D. immitis; however, Liverpool-strain Ae. aegypti are more likely to be competent vectors for D. immitis than Ae. aegypti and Cx. quinquefasciatus from Thailand. The percent infection rates of Ae. aegypti and Cx. quinquefasciatus with D. immitis in the field in Thailand need to be investigated, to confirm the role of these mosquitoes in the life cycle of D. immitis in nature.     

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。     

Isolation of Japanese encephalitis virus from mosquitoes (Diptera: Culicidae) collected in the Western Province of Papua New Guinea, 1997-1998

After Japanese encephalitis (JE) virus emerged in the Torres Strait in Australia in 1995, investigations were initiated into the origin of the incursion. New Guinea was considered the most likely source, given its proximity to islands of the Torres Strait. Almost 400,000 adult mosquitoes were processed for virus isolation from 26 locations in the Western Province of Papua New Guinea (PNG) between February 1996 and February 1998, yielding three isolates of JE virus. Two isolates of Murray Valley encephalitis, 17 isolates of Sindbis, and 1 each of Sepik and Ross River viruses were also obtained. Nucleic acid sequences of the PNG JE isolates were determined in the prM region, and in a region overlapping a part of the fifth nonstructural protein and the 3' untranslated region. The PNG isolates belonged to genotype II, and shared > 99.2% identity with isolates from humans and mosquitoes from the Torres Strait, suggesting that PNG is the source of incursions of JE virus into Australia.     

Reexamination of Culex pipiens hybridization zone in the Eastern United States by ribosomal DNA-based single nucleotide polymorphism markers

Mosquitoes in the Culex pipiens complex are important vectors of several disease-causing pathogens, including West Nile virus. In North America, the complex consists of Cx. pipiens pipiens form pipiens, Cx. pipiens pipiens form molestus, Cx. pipiens quinquefasciatus, and their hybrids that exhibit substantial diversity in physiology, behavior, and geographic range. Hybridization among these mosquitoes is of concern because of potential implications for disease transmission. Currently, several morphological and molecular markers exist for differentiating members of the Cx. pipiens complex; however, these markers have specific limitations. We report here two highly reliable ribosomal DNA-based single nucleotide polymorphism (SNP) markers, CxpG2T and CxpA2d, for detecting Cx. pipiens complex mosquitoes containing Cx. p. quinquefasciatus alleles. Both CxpG2T and CxpA2d contain one allele that is present in all members of the Cx. pipiens complex, and the other allele is specific to Cx. p. quinquefasciatus. Testing of field populations from the eastern United States showed that these two SNP markers are capable of identifying a south to north gradient of Cx. p. quinquefasciatus and hybrids. The northern limit of detection of Cx. p. quinquefasciatus alleles in this study was in Fort Totten, NY (40.79°N), whereas the southern boundary was determined between Atlanta, GA (33.81°N) and Gainesville, FL (29.64°N). CxpG2T and CxpA2d were more accurate than the ACE-2 marker, and they may conceivably provide comparable resolution with microsatellite markers for detecting Cx. p. quinquefasciatus alleles.     

Behavioural biting preference of Culex quinquefasciatus in human host in Lagos metropolis Nigeria

BACKGROUND & OBJECTIVES: Olfactory cues play an important role in the attraction of major disease vectors towards their host. Hence we intend to investigate the contribution of selected parts of the human host on the behavioural biting preference of Culex quinquefasciatus. METHODS: Two volunteers were trained to collect host seeking female mosquitoes landing to feed on different parts of the lower limbs. The Cx. quinquefasciatus were collected in paper cups with the aid of a flash-light and aspirator. Each paper cup was labeled to represent the selected parts where the mosquitoes were collected. RESULTS: The composition of Cx. quinquefasciatus from the total mosquitoes collected showed that Cx. quinquefasciatus was more predominant (90%) over other species present. The average minimum and maximum atmospheric temperature recorded during the night catches were 27 and 29.5 degrees C while the average relative humidity range was 78-81.6%. The behavioural biting preference results obtained showed that the density of Cx. quinquefasciatus mosquitoes preferring the foot region (298) was significantly higher (p > 0.05) when compared with other different parts of the human host such as the ankle, calf and the thigh. INTERPRETATION & CONCLUSION: The study indicates that the foot region of the human host has a stronger influence in orientating mosquitoes towards the human host. Hence, the exploitation of the characteristic human odour will add to the existing vector control strategies.     

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.     

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.     

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     

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.     

Detection and stability of Japanese encephalitis virus RNA and virus viability in dead infected mosquitoes under different storage conditions

A semi-nested polymerase chain reaction (PCR) was evaluated for detection of Japanese encephalitis (JE) virus in infected mosquitoes stored under simulated northern Australian summer conditions. The effect of silica gel, thymol, and a combination of the two on RNA stability and virus viability in dead mosquitoes were also examined. While JE virus RNA was relatively stable in mosquitoes held for up to 14 days after death, viable virus was not detected after day 1. Thymol vapor inhibited fungal contamination. Detection of single mosquitoes infected with JE virus in large pools of mosquitoes was also investigated. Single laboratory-infected mosquitoes were detected in pools of < or = 200 mosquitoes and in pools diluted to 0.2/100 and 0.1/100 mosquitoes, using the semi-nested PCR. However, the ability to detect live virus decreased as pool size increased. The semi-nested PCR proved more expensive than virus isolation for pools of 100 mosquitoes. However, the semi-nested PCR was faster and more economical using larger pools. Results indicate that surveillance of JE virus in mosquitoes using the semi-nested PCR is an alternative to monitoring seroconversions in sentinel pigs.