Encephalitis Virus In Mosquitoes Research Articles

Development and Validation of a Colorimetric Reverse Transcriptase Loop-Mediated Isothermal Amplification Assay for Detection of Eastern Equine Encephalitis Virus in Mosquitoes.

Eastern equine encephalitis virus (EEEV) is a highly pathogenic alphavirus that causes periodic outbreaks in the eastern USA. Mosquito abatement programs are faced with various challenges with surveillance and control of EEEV and other mosquito-borne illnesses. Environmental sampling of mosquito populations can be technically complex. Here we report the identification of biomarkers, development and validation of a colorimetric reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay for the detection of EEEV. Positive samples are easily visualized by a color change from pink to yellow. The assay was validated using EEEV from viral culture, experimentally spiked mosquito pools, and previously tested mosquito pools. The RT-LAMP assay detected viral titers down to approximately 10% of what would be present in a single infectious mosquito, based upon EEEV viral titers determined by previous competency studies. The RT-LAMP assay efficiently detected EEEV in combined aliquots from previously homogenized pools of mosquitoes, allowing up to 250 individual mosquitoes to be tested in a single reaction. No false positive results were obtained from RNA prepared from negative mosquito pools acquired from known and potential EEEV vectors. The colorimetric RT-LAMP assay is highly accurate, technically simple, and does not require sophisticated equipment, making it a cost-effective alternative to real time reverse transcriptase-polymerase chain reaction (RT-PCR) for vector surveillance.

Detection of Japanese encephalitis virus in mosquitoes from Xinjiang during next-generation sequencing arboviral surveillance.

A total of 548 mosquitoes were collected from different animal farms located near to highly populated cities in Xinjiang and were subjected to metagenomic next-generation sequencing (mNGS). The mNGS data demonstrated that 18,842 (XJ1 strain) and 1,077 (XJ2 strain) of Japanese encephalitis virus (JEV)-related reads were detected in XJ1 and XJ2 mosquito samples collected from Wushi and Wensu counties of Aksu area, which accounted for 0.032% and 0.006% of the total clean reads generated from XJ1 and XJ2 samples, respectively. The Bayesian molecular phylogenetic analysis suggested that XJ1 and XJ2 strains belonged to JEV genotype III and were clustered with JEV strains isolated in China. Notably, Bayesian molecular time line phylogeny revealed that XJ1 strain shared its MRCA with JEV GSS strain about 67 YA, suggesting that XJ1 strain likely originated from linages closely related to GSS strain and spread to Xinjiang later. Overall, these findings suggest that Xinjiang was probably not free from JEV, and thus, a further surveillance of JEV is required in Xinjiang.

Twenty years of surveillance for Eastern equine encephalitis virus in mosquitoes in New York State from 1993 to 2012

BackgroundThe year 1971 was the first time in New York State (NYS) that Eastern equine encephalitis virus (EEEV) was identified in mosquitoes, in Culiseta melanura and Culiseta morsitans. At that time, state and county health departments began surveillance for EEEV in mosquitoes.MethodsFrom 1993 to 2012, county health departments continued voluntary participation with the state health department in mosquito and arbovirus surveillance. Adult female mosquitoes were trapped, identified, and pooled. Mosquito pools were tested for EEEV by Vero cell culture each of the twenty years. Beginning in 2000, mosquito extracts and cell culture supernatant were tested by reverse transcriptase-polymerase chain reaction (RT-PCR).ResultsDuring the years 1993 to 2012, EEEV was identified in: Culiseta melanura, Culiseta morsitans, Coquillettidia perturbans, Aedes canadensis (Ochlerotatus canadensis), Aedes vexans, Anopheles punctipennis, Anopheles quadrimaculatus, Psorophora ferox, Culex salinarius, and Culex pipiens-restuans group. EEEV was detected in 427 adult mosquito pools of 107,156 pools tested totaling 3.96 million mosquitoes. Detections of EEEV occurred in three geographical regions of NYS: Sullivan County, Suffolk County, and the contiguous counties of Madison, Oneida, Onondaga and Oswego. Detections of EEEV in mosquitoes occurred every year from 2003 to 2012, inclusive. EEEV was not detected in 1995, and 1998 to 2002, inclusive.ConclusionsThis was the first time in NYS that EEEV was detected in Cx. salinarius, Ps. ferox and An. punctipennis. The detection of EEEV in mosquitoes every year for 10 years was the longest time span since surveillance began in 1971. The calendar date of the earliest annual appearance of EEEV in mosquitoes did not change during surveillance spanning 42 years.

Molecular Epidemiology of Japanese Encephalitis Virus in Mosquitoes during 2013- 2014

Japanese encephalitis (JE) is a mosquito-borne zoonotic disease caused by the Japanese encephalitis virus (JEV). Japanese encephalitis is widespread in Asia and endemic in Taiwan. Virological surveillance in mosquitoes showed that the genotype III (GIII) strains of JEV were the predominant epidemic strains circulating in Asia before 1990; however, the genotype I (GI) strains have been introduced into China, Japan, Vietnam, South Korea, and Thailand, and cocirculated with or replaced the GIII strains in Asia. Previous studies showed that in Taiwan, all of the JEV strains obtained during 2005- 2007 belonged to GIII of JEV, but the predominant genotype has shifted from GIII to GI during 2008-2012. This study continued to report the genotype distribution, genetic variation, and mosquito species potentially involved in the transmission of JEV during 2013-2014. Overall, 37,637 mosquitoes were collected; 96 pools were tested JEV positive by RT-PCR, including Culex tritaeniorhynchus (94 pools) and Cx. annulus (2 pools). Phylogenetic analysis of envelope gene sequences of JEV isolated from mosquitoes demostrated that GI was the predominant genotype of JEV between 2013 and 2014, whilst only a few GIII strains of JEV were found in Eastern Taiwan. We will continue the monitoring of endemic and newly introduced JEV in Taiwan.

Molecular detection of Eastern Equine Encephalitis virus in mosquitoes from La Pintada (Antioquia)

ABSTRACT Objective. The detection of emerging and re-emerging arboviruses in mosquitoes from urban and rural areas, is fundamental for predict possible epidemic outbreaks in human populations. The Municipality of La Pintada (Antioquia), is characterized by the presence of dry tropical forest relicts, fishing, tourism, farms and mining. An entomological research was performed for explore the possible circulation of arboviruses of public health importance. Materials and methods. Mosquitoes were captured in urban and rural sites in February-April of 2012. The specimens were stored in liquid nitrogens tanks and were grouped using taxonomic keys for genera. RNA extraction from pools and generic/nested RT-PCR was performed for Flavivirus, Alphavirus, Orthobunyavirus (Group Bunyamwera) and Phlebovirus. Results. 1274 mosquitoes were collected, mainly belonging to Culex and Aedes genera. RNA extracts of 64 pools were tested by RT-PCR and one pool was positive for Alphavirus. Sequencing of the RT- PCR product and the analysis with sequences storage in GenBank designate the presence of Eastern equine encephalitis virus (EEEV). Conclusions. This is the first record of natural infection from EEEV in mosquitoes from La Pintada (Antioquia), an area with ecological elements that favor the emergence of emerging and re-emerging arboviruses of medical and veterinarian importance.

Molecular epidemiology of Japanese encephalitis virus in mosquitoes in Taiwan during 2005-2012.

Japanese encephalitis (JE) is a mosquito-borne zoonotic disease caused by the Japanese encephalitis virus (JEV). Pigs and water birds are the main amplifying and maintenance hosts of the virus. In this study, we conducted a JEV survey in mosquitoes captured in pig farms and water bird wetland habitats in Taiwan during 2005 to 2012. A total of 102,633 mosquitoes were collected. Culex tritaeniorhynchus was the most common mosquito species found in the pig farms and wetlands. Among the 26 mosquito species collected, 11 tested positive for JEV by RT-PCR, including Cx. tritaeniorhynchus, Cx. annulus, Anopheles sinensis, Armigeres subalbatus, and Cx. fuscocephala. Among those testing positive, Cx. tritaeniorhynchus was the predominant vector species for the transmission of JEV genotypes I and III in Taiwan. The JEV infection rate was significantly higher in the mosquitoes from the pig farms than those from the wetlands. A phylogenetic analysis of the JEV envelope gene sequences isolated from the captured mosquitoes demonstrated that the predominant JEV genotype has shifted from genotype III to genotype I (GI), providing evidence for transmission cycle maintenance and multiple introductions of the GI strains in Taiwan during 2008 to 2012. This study demonstrates the intense JEV transmission activity in Taiwan, highlights the importance of JE vaccination for controlling the epidemic, and provides valuable information for the assessment of the vaccine's efficacy.

Molecular epidemiology of Japanese encephalitis virus in mosquitoes during an outbreak in China, 2013.

Japanese encephalitis virus (JEV) can cause serious encephalitis and Culex mosquitoes are the primary vector. In 2013, a JE outbreak occurred in Shandong Province, China with 407 confirmed cases, including 11 deaths. An investigation on JEV in mosquitoes during the outbreak was conducted. A total of 14,719 mosquitoes were collected at 3 sites. For the 12,695 Culex tritaeniorhynchus mosquitoes, 88/201 pooled samples were positive by RT-PCR for the presence of the pre-membrane or envelope protein coding genes. The maximum likelihood estimates of JEV positive individuals per 1,000 vectors were 12.0, 7.2, and 6.0 in the 3 sites respectively with an overall estimate of 9.1. Phylogenetic analysis on these pre-membrane (n = 72) and envelope (n = 26) sequences with those of reference strains revealed they belonged to genotype I. This study describes the molecular epidemiology of JEV and suggests the high infection rate in mosquitoes is an important factor for the outbreak.

Molecular Detection and Genotyping of Japanese Encephalitis Virus in Mosquitoes during a 2010 Outbreak in the Republic of Korea

Japanese encephalitis virus (JEV), a mosquito-borne zoonotic pathogen, is one of the major causes of viral encephalitis. To reduce the impact of Japanese encephalitis among children in the Republic of Korea (ROK), the government established a mandatory vaccination program in 1967. Through the efforts of this program only 0–7 (mean 2.1) cases of Japanese encephalitis were reported annually in the ROK during the period of 1984–2009. However, in 2010 there was an outbreak of 26 confirmed cases of Japanese encephalitis, including 7 deaths. This represented a >12-fold increase in the number of confirmed cases of Japanese encephalitis in the ROK as compared to the mean number reported over the last 26 years and a 3.7-fold increase over the highest annual number of cases during this same period (7 cases). Surveillance of adult mosquitoes was conducted during the 2010 outbreak of Japanese encephalitis in the ROK. A total of 6,328 culicine mosquitoes belonging to 12 species from 5 genera were collected at 6 survey sites from June through October 2010 and assayed by reverse-transcription polymerase chain reaction (RT-PCR) for the presence of JEV. A total of 34/371 pooled samples tested positive for JEV (29/121 Culex tritaeniorhynchus, 4/64 Cx. pipiens, and 1/26 Cx. bitaeniorhynchus) as confirmed by sequencing of the pre-membrane and envelope protein coding genes. The maximum likelihood estimates of JEV positive individuals per 1,000 culicine vectors for Cx. tritaeniorhynchus, Cx. pipiens, and Cx. bitaeniorhynchus were 11.8, 5.6, and 2.8, respectively. Sequences of the JEV pre-membrane and envelope protein coding genes amplified from the culicine mosquitoes by RT-PCR were compared with those of JEV genotypes I-V. Phylogenetic analyses support the detection of a single genotype (I) among samples collected from the ROK in 2010.

Japanese Encephalitis Virus in Mosquitoes and Swine in Yunnan Province, China 2009–2010

The residential regions of Yunnan province, canton of Jing Hong, in China were surveyed for Japanese encephalitis virus (JEV) infection in mosquito and swine vectors to determine the frequency of JEV-carrying zoonotic vectors in 2009-2010. A total of 21,500 mosquitoes were collected and divided by species, and brain tissue was collected from 108 stillborn piglets. The infection rates for the different JEV species were 13.2% for Culex tritaeniorhynchus, 2.7% for Anopheles sinensis, 0.7% for Armigeres subalbatus, and 18.5% for stillborn piglets. The complete genomes of two JEV samples that were collected in different seasons and different regions, Yunnan 0901 and Yunnan 0902, were sequenced from a pool of Culex mosquitoes and stillborn piglets that had been collected randomly from several piggeries. Multiple sequence alignment with 24 fully-sequenced genes and 93 complete sequences of the JEV-encoded E gene revealed nucleotide homologies ranging from 97.2-99.6% and 94.5-99.7% in mosquitoes and piglets, respectively, and deduced amino acid homologies ranging from 97.4-98.1% and 96.0-98.2%, respectively. Phylogenetic analyses of the Yunnan 0901 and Yunnan 0902 strains' full-length genomes and E gene sequences indicated that these strains are most closely related to six Chinese SA14-derived viruses, and distantly related to the Australian FU, vellore P20778, and Japanese Ishikawa strains, and the previously isolated YN86-B8639 strains. The phylogenetic relationships based on the full-length genome were similar to those found for the E gene, indicating that phylogenetic analysis of the E gene will be a useful approach for genotyping of JEV, but not to better understand the potential changes in the biological characteristics and genetic relationship of JEV isolates.

Detection and Differentiation of Genotype I and III Japanese Encephalitis Virus in Mosquitoes by Multiplex Reverse Transcriptase-Polymerase Chain Reaction

Japanese encephalitis (JE) is a disease that threatens both human and animal populations in Asian countries, and the causative agent of JE, Japanese encephalitis virus (JEV), has recently changed from genotype III (GIII) to genotype I (GI). However, a test for the rapid differentiation of GI and GIII JEV is still unavailable, especially one that can be used for mosquito-based surveillance. We have designed GI- and GIII-specific primer sets for the rapid detection and differentiation of GI and GIII JEV by multiplex reverse transcriptase-polymerase chain reaction (multiplex RT-PCR). The GI-specific and GIII-specific primer sets were able to specifically amplify the target gene from GI and GIII JEV, respectively. The limitations of detection were 0.00225 and 0.225 pfu for the GI-specific and GIII-specific primers, respectively. Using a mixture of GI-specific and GIII-specific primers, the multiplex RT-PCR was able to specifically detect and differentiate GI and GIII JEV. The multiplex RT-PCR was able to successfully differentiate GI and GIII virus in JEV-infected mosquitoes. Thus, a sensitive and specific multiplex RT-PCR system for the rapid detection and differentiation of GI and GIII JEV has been developed, and this test is likely to be valuable when carrying out mosquito-based JEV surveillance.

Eastern Equine Encephalitis Virus in Mosquitoes and Their Role as Bridge Vectors

Eastern equine encephalitis virus (EEEV) is maintained in an enzootic cycle involving Culiseta melanura mosquitoes and avian hosts. Other mosquito species that feed opportunistically on mammals have been incriminated as bridge vectors to humans and horses. To evaluate the capacity of these mosquitoes to acquire, replicate, and potentially transmit EEEV, we estimated the infection prevalence and virus titers in mosquitoes collected in Connecticut, USA, by cell culture, plaque titration, and quantitative reverse transcription-PCR. Cs. melanura mosquitoes were the predominant source of EEEV (83 [68%] of 122 virus isolations) and the only species to support consistently high virus titers required for efficient transmission. Our findings suggest that Cs. melanura mosquitoes are primary enzootic and epidemic vectors of EEEV in this region, which may explain the relative paucity of human cases. This study emphasizes the need for evaluating virus titers from field-collected mosquitoes to help assess their role as vectors.

Development and field evaluation of a nested RT-PCR kit for detecting Japanese encephalitis virus in mosquitoes

A novel nested reverse transcription-polymerase chain reaction (RT-PCR)-based kit is described for detecting Japanese encephalitis virus (JEV), especially for genotype 1 and 3 strains. The assay consists of a first round RT-PCR and a subsequent nested PCR amplification. It has unique features such as the use of a premix system in which all reagents are lyophilized in reaction tubes and the inclusion of control RNA in each reaction to monitor false negative results. In addition, an automatic tissue homogenizer and a RNA extraction system are used concurrently for assay standardization and increasing throughput. The assay using the kit proved specific for JEV with no amplification of other JEV-related flaviviruses. The detection limits were approximately 0.1 PFU/ml and 1 PFU/ml for JEV genotypes 1 and 3, respectively. The assay protocol has been validated in large-scale field trials in South Korea during the 2008–2009 surveillance seasons. Nineteen of 1136 pools of mosquitoes (54,583 mosquitoes total) were identified as JEV positive. This nested RT-PCR kit combined with control RNA and an automatic RNA extraction system should be suitable for routine JEV surveillance programs.

PooledInfRate Software

PooledInfRate Software

Trajectory analysis of winds and eastern equine encephalitis in USA, 1980–5

Backward trajectories of winds were determined to identify possible sources of eastern equine encephalitis virus associated with isolation of virus from mosquitoes or birds or outbreaks in horses between 1980 and 1985 in Maryland, New Jersey, New York and Michigan, USA. The results of the trajectory analyses suggested that eastern equine encephalitis virus could have been carried by infected mosquitoes on surface winds at temperatures 13 degrees C or higher from North Carolina north-eastwards along the Atlantic Coast to Maryland and New Jersey and thence to upstate New York and from western Kentucky to Michigan. Landing of mosquitoes was associated with the presence of a cold front and rain leading to variations in the location and timing of outbreaks from year to year. The mosquito responsible was most likely to have been Culiseta melanura, but Coquillettidia perturbans and Aedes sollicitans could also have been involved. There may be a continual cycle of eastern equine encephalitis virus in mosquitoes and birds in south-eastern USA, from where the virus could be distributed by infected mosquitoes on the wind along the Gulf and Atlantic Coasts and up the Mississippi Valley.

An orbivirus of mosquitoes which induces CO2 sensitivity in mosquitoes and is lethal for rabbits.

An orbivirus, JKT-7400, isolated from Culex mosquitoes in Indonesia, replicated to a high titer and induced cytopathic effects in Aedes albopictus cell cultures. The virus produced lethal sensitivity to carbon dioxide in Culex and Aedes mosquitoes as well as in Drosophila melanogaster fruit flies but was not the agent of the hereditary sensitivity to carbon dioxide previously described for Culex quinquefasciatus. When injected intravenously in high doses, JKT-7400 virus was lethal for rabbits, apparently without replicating to a significant extent. It was not pathogenic for adult mice inoculated intravenously or for adult or suckling mice inoculated intracerebrally and intraperitoneally. Unlike an orbivirus isolated from Culex mosquitoes in China, JKT-7400 did not interfere with the replication of Japanese encephalitis virus in mosquitoes.

Effect of some environmental factors on survival of overwintering mosquitoes, Culex pipiens pallens.

In connection with the possible overwintering of Japanese B encephalitis virus in mosquitoes, some experiments were carried out to examine the effect of temperature, day-length and blood feeding on the survival of the overwintering population of the mosquito, Culex pipiens pallens. No significant difference was observed in the survival rate between the blood-fed and unfed groups under a higher temperature (24 degrees C) and longer day-length (L:D 14:10). Under a lower temperature (16 degrees C) and shorter day-length (L:D 10:14), blood-fed mosquitoes survived twice as long as those under a higher temperature and longer day-length condition. Based on the analysis of wing-length distribution, it was clarified that the blood-fed mosquitoes were a uniform longer-winged group and that most of the shorter-winged mosquitoes were included in the unfed group. Judging from such lower blood-feeding activity of the shorter-winged mosquitoes, their survival rate is suggested to be low during or after overwintering.

Effect of rearing temperature on transovarial transmission of St. Louis encephalitis virus in mosquitoes.

Female Aedes albopictus and Aedes epactius infected by inoculation with St. Louis encephalitis (SLE) virus transmitted virus transovarially to their F1 progeny. With Ae. epactius, minimal infection rates (MIR) in F1 larvae were similar for progeny reared at 18 (+/- 1) degree C or 27 (+/- 1) degree C, whereas, MIR's were significantly higher in F1 adults reared at 18 degrees C, than they were in F1 adults reared at 27 degrees C. Use of different strains of SLE virus appeared to influence transovarial transmission of virus by Ae. albopictus, but not by Ae. epacticus. Transovarial transmission of SLE virus was not demonstrated with colonized strains of Aedes dorsalis, Ades sierrensis, Ades triseriatus, Ades vexans, Culex pipiens, Culex quinquefasciatus and Culex tarsalis, and field strains of Ae. vexans.

St. Louis encephalitis virus in mosquitoes.

Journal Article ST. LOUIS ENCEPHALITIS VIRUS IN MOSQUITOES Get access R. W. CHAMBERLAIN, R. W. CHAMBERLAIN Search for other works by this author on: Oxford Academic PubMed Google Scholar W. D. SUDIA, W. D. SUDIA Search for other works by this author on: Oxford Academic PubMed Google Scholar J. D. GILLETT J. D. GILLETT Search for other works by this author on: Oxford Academic PubMed Google Scholar American Journal of Epidemiology, Volume 70, Issue 3, November 1959, Pages 221–236, https://doi.org/10.1093/oxfordjournals.aje.a120072 Published: 01 November 1959 Article history Received: 22 April 1959 Published: 01 November 1959

Multiplication of eastern equine encephalitis virus in mosquitoes following intrathoracic inoculation.

Multiplication of eastern equine encephalitis virus in mosquitoes following intrathoracic inoculation.

ON SUSCEPTIBILITY OF JAPANESE WILD BIRDS FOR JAPANESE B. ENCEPHALITIS VIRUS

We have previously reported on the studies of the pattern of Japanese B encephalitis virus in mosquitoes (Culex pipiens var, pallens and Culex tryteniorhyncus), and various relationships between these species of mosquitoes and the virus, and concluded that Japanese B encephalitis virus which was sucked by mosquitoes reappered after a certain period of external incubation, and that the final concentration of the virus in mosquito bodies depends on the concentration of the virus in the first feeding to the mosquitoes. It was further concluded that the experimental transmission of the virus to mice through bite of these virus-sucking mosquitoes are only possible (10%) when the concentration of virus in the mosquito is high. We have considered, therefore, that there must be certain species of animals in nature, whose blood contained Japanese B encephalitis virus in high concentration as sources of the virus to these mosquitoes, if the latter had transmitted the virus in nature. In the same year (1941), W. McD. Hammon and W. C. Reeves in America, demonstrated the St. Louis virus and the western equine encephalomyelitis virus in the mosquitoes (Culex tarsalis), which were captured in Yakima Valley, Washington, and neutralizing antibodies in the blood of chickens in the same area, and proposed the hypothesis of infection cycle for these viruses (mosquito-chicken-mosquito) .These authors later concluded however, that the chickens were i not true reservoirs or latent carriers, though the possibility had not been ruled out of an occasional fowl having an unusual course of infection, because the domestic birds such as chicken, ducks and doves could not maintain these viruses in the blood for a long period, and they could not domenstrate the viruses in hibernated mosquitoes or transovalian infection of these viruses and mosquitoes.From the results of our foundamental experiments on the relationships between mosquitoes and J apanese B encephalitis virus and of the studies carried out by Hammon et al. in America, we thought that it was necessary to search for the reservoir of Japanese B encephalitis virus in Japan, from the standpoint of epidemiology and of prophylaxis against this disease. In the summer of 1947, Dr. Hammon, Dr. Casals and Dr. Meiklejohn came to Japan and, after a conference with these investigaters, and with the Virus and Rickettsia Committee of G. H. Q., staff of 406th Gen. Med. Lab. and N. I. H, it was decided to carry out the studies herein reported. We are indepted to Dr. W. McD. Hammon, Dr. J: Casals, Dr. G. Meiklejohn, Mr. Austine, Dr. H. Hamlin and Dr. Uchida for many valuable suggestions and assistance in our studies.