Detection of Insect-Specific Flaviviruses in Mosquitoes (Diptera: Culicidae) in Northeastern Regions of South Africa

Mosquitoes in the Aedes and Culex genera are considered the main vectors of pathogenic flaviviruses worldwide. Entomological surveillance using universal flavivirus sets of primers in mosquitoes can detect not only pathogenic viruses but also insect-specific ones. It is hypothesized that insect-specific flaviviruses, which naturally infect these mosquitoes, may influence their vector competence for zoonotic arboviruses. Here, entomological surveillance was performed between January 2014 and May 2018 in five different provinces in the northeastern parts of South Africa, with the aim of identifying circulating flaviviruses. Mosquitoes were sampled using different carbon dioxide trap types. Overall, 64,603 adult mosquitoes were collected, which were screened by RT-PCR and sequencing. In total, 17 pools were found positive for insect-specific Flaviviruses in the mosquito genera Aedes (12/17, 70.59%) and Anopheles (5/17, 29.41%). No insect-specific viruses were detected in Culex species. Cell-fusing agent viruses were detected in Aedes aegypti and Aedes caballus. A range of anopheline mosquitoes, including Anopheles coustani, An. squamosus and An. maculipalpis, were positive for Culex flavivirus-like and Anopheles flaviviruses. These results confirm the presence of insect-specific flaviviruses in mosquito populations in South Africa, expands their geographical range and indicates potential mosquito species as vector species.

Dynamic Surveillance of Mosquitoes and Their Viromes in Wuhan During 2020

Objective: Mosquitoes are medically important arthropod vectors that harbor a variety of viruses. Geography and climate are known to be associated with variations in mosquito density, species and viromes. Our study investigated the dynamic changes in mosquito populations, species compositions and viromes in a regularly disinfected environment in Wuhan, China, during 2020. Methods: Traps were set in different mosquito habitats, including an urban residential area, two hospitals, a scenic area and a pig farm in a rural region between April and October of 2020. The collected mosquitoes were subjected to morphological identification, RT-qPCR and metagenomic sequencing. Results: A total of 2345 adult mosquitoes were collected. Culex mosquitoes were dominant in both urban regions (90.32%, 1538/1703) and the pig farm (54.98%, 353/642). In RT-qPCR screening, the prevalence of Banna virus was 15% and 3% in mosquitoes from the urban area and the pig farm, respectively, whereas no Japanese encephalitis virus was detected. Culex viromes showed dynamic changes during the collection period. Several mosquito-specific viruses, such as Culex flavivirus, Alphamesonivirus 1, Hubei mosquito virus 2 and Hubei mosquito virus 4, showed seasonal changes and unimodal increases or declines. Other mosquito-specific viruses, such as Wuhan mosquito virus 6, Hubei virga-like virus 2 and Zhejiang mosquito virus 3, were stable in all collected Culex and are potential members of the core viromes. Conclusion: This study improves understanding of the dynamic composition of mosquito species and the viromes that they carry, and provides useful information for guiding mosquito control and mosquito-borne disease prevention strategies.

Implementación de un sistema de detección de flavivirus en mosquitos

Los Flavivirus constituyen virus transmitidos por artrópodos, principalmente mosquitos. Pueden producir enfermedades en humanos y animales, también incluyen virus específicos de insectos que solo infectan y se replican en los insectos, no así en vertebrados. En Paraguay los virus dengue, fiebre amarilla y Zika fueron detectados en infecciones humanas, pero los estudios de flavivirus en mosquitos son aún escasos. Por ello, el objetivo del presente estudio fue implementar un sistema de detección de flavivirus en mosquitos en el IICS-UNA. Primero, se organizaron capacitaciones en colecta, preparación de pools y procesamiento por técnicas de RT-PCRs convencionales realizadas por expertos internacionales a profesionales locales (bioquímicos y biólogos). Además, se implementaron planillas de registro de datos y de control de transporte de muestras de los lugares de colectas hasta el IICS-UNA. Se prepararon en total 201 pools de 1 a 35 mosquitos cada uno agrupados por especie, localidad, entre otros criterios. Para asegurar la integridad del RNA extraído se realizó la detección de un control interno (Actina-1), siendo todos los pools positivos para el mismo, 91/201 pools fueron positivos para flavivirus. Se realizó la secuenciación de 19/91 pools positivos para flavivirus identificándose flavivirus de insectos (detectándose principalmente Culex Flavivirus, cell fusing agents Flavivirus y Kamiti river virus), evidenciando la elevada distribución de estos virus. Estos resultados demuestran que fue factible implementar el sistema de detección de flavivirus en mosquitos, lo cual podría contribuir a fortalecer la vigilancia y control de estas virosis, así como el conocimiento sobre la importancia ecológica de flavivirus de insectos.

Molecular detection and identification of Culex flavivirus in mosquito species from Jeju, Republic of Korea

Background Mosquito-borne flaviviruses are prime pathogens and have been a major hazard to humans and animals. They comprise several arthropod-borne viruses, including dengue virus, yellow fever virus, Japanese encephalitis virus, and West Nile virus. Culex flavivirus (CxFV) is a member of the insect-specific flavivirus (ISF) group belonging to the genus Flavivirus, which is widely distributed in a variety of mosquito populations. Methods Viral nucleic acid was extracted from adult mosquito pools and subjected to reverse transcriptase nested polymerase chain reaction (PCR) using target-specific primers for detecting CxFV nonstructural protein 5 (NS5). The PCR-positive samples were then sequenced, and a phylogenetic tree was constructed, including reference sequences obtained from GenBank. Results 21 pools, belonging to Culex pipiens pallens (Cx. p. pallens) were found to be positive for the CxFV RNA sequence, with a minimum infection rate of 14.5/1000 mosquitoes. The phylogenetic analysis of the NS5 protein sequences indicated that the detected sequences were closely related to strains identified in China, with 95–98% sequence similarities. Conclusion Our findings highlight the presence of CxFV in Cx. p. pallens mosquito species in Jeju province, Republic of Korea. This is the first study reporting the prevalence of CxFV in Culex Pipiens (Cx. pipiens) host in the Jeju province, which can create possible interaction with other flaviviruses causing human and animal diseases. Although, mosquito-borne disease causing viruses were not identified properly, more detailed surveillance and investigation of both the host and viruses are essential to understand the prevalence, evolutionary relationship and genetic characteristic with other species.

Dynamic surveillance of mosquitoes and their viromes in Wuhan during 2020.

Mosquitoes are medically important arthropod vectors and harbor a great variety of viruses. The population density, species and virome of mosquitoes varies according to geography and climate. To investigate the dynamic changes in the species composition and diversity of mosquitoes and their viromes in Wuhan, China, a total of 2,345 adult mosquitoes collected from different habitats including an urban residential area, two hospitals, a scenic area, and a pig farm in a rural region from April to October 2020 were subjected to morphological identification, RT-qPCR and metagenomic sequencing. The results indicated that the dominant presence of Culex mosquitoes was observed in both urban regions (90.32%, 1538/1703) and the pig farm (54.98%, 353/642). Viromes of Culex showed dynamic changes during the collection time. Several viruses, such as Culex flavivirus, Alphamesonivirus 1, Hubei mosquito virus 2 and Hubei mosquito virus 4, had seasonal changes and unimodal increases or declines. Other viruses, such as Wuhan mosquito virus 6, Hubei virga-like virus 2 and Zhejiang mosquito virus 3, were stable in all collected Culex and should be potential members of "core viromes". This study improves the understanding of the dynamic composition of mosquitoes and the viromes they carry and provides useful information for informing mosquito control and mosquito-borne disease prevention strategies.

Detection of Culex flavivirus (Flaviviridae) from a natural Culex (Culex) chidesteri Dyar, 1921 population, Caatinga Biome, Semiarid Scrubland, Brazil

The first detection of Culex flavivirus (CxFV) in mosquitoes was described in 2007 in Japan and subsequently in different areas of the Americas, Africa, and Asia. In this study, we report the identification of CxFV in Culex (Culex) chidesteri Dyar, 1921 mosquitoes collected in the Açu National Forest - FLONA/ AÇU-RN, a preserved area of Biome Caatinga, State of Rio Grande do Norte, Brazil. We performed nucleotide-sequencing analysis in order to compare with other previously identified CxFV collected from around the world. A total of 129 samples (pools of ≤30 female mosquitoes) were processed for generic reverse transcriptase PCR assay. CxFV infection was identified in only two pools from Culex chidesteri. Our phylogenetic analysis revealed that the AssuCxFV identified in this study belongs to the Africa/Caribbean/South America Subtype. Our study represents the first report of the CxFV from a natural Culex chidesteri population.

A survey of mosquito-borne and insect-specific viruses in hospitals and livestock markets in western Kenya

Aedes aegypti and Culex pipiens complex mosquitoes are prolific vectors of arboviruses that are a global threat to human and animal health. Increased globalization and ease of travel have facilitated the worldwide dissemination of these mosquitoes and the viruses they transmit. To assess disease risk, we determined the frequency of arboviruses in western Kenyan counties bordering an area of high arboviral activity. In addition to pathogenic viruses, insect-specific flaviviruses (ISFs), some of which are thought to impair the transmission of specific pathogenic arboviruses, were also evaluated. We trapped mosquitoes in the short and long rainy seasons in 2018 and 2019 at livestock markets and hospitals. Mosquitoes were screened for dengue, chikungunya and other human pathogenic arboviruses, ISFs, and their blood-meal sources as determined by high-resolution melting analysis of (RT-)PCR products. Of 6,848 mosquitoes collected, 89% were trapped during the long rainy season, with A. aegypti (59%) and Cx. pipiens sensu lato (40%) being the most abundant. Most blood-fed mosquitoes were Cx. pipiens s.l. with blood-meals from humans, chicken, and sparrow (Passer sp.). We did not detect dengue or chikungunya viruses. However, one Culex poicilipes female was positive for Sindbis virus, 30 pools of Ae. aegypti had cell fusing agent virus (CFAV; infection rate (IR) = 1.27%, 95% CI = 0.87%-1.78%); 11 pools of Ae. aegypti had Aedes flavivirus (AeFV; IR = 0.43%, 95% CI = 0.23%-0.74%); and seven pools of Cx. pipiens s.l. (IR = 0.23%, 95% CI = 0.1%-0.45%) and one pool of Culex annulioris had Culex flavivirus. Sindbis virus, which causes febrile illness in humans, can complicate the diagnosis and prognosis of patients with fever. The presence of Sindbis virus in a single mosquito from a population of mosquitoes with ISFs calls for further investigation into the role ISFs may play in blocking transmission of other arboviruses in this region.

Molecular epidemiology of mosquito-borne viruses at the China–Myanmar border: discovery of a potential epidemic focus of Japanese encephalitis

Background Mosquito-based arbovirus surveillance can serve as an early warning in evaluating the status of mosquito-borne virus prevalence and thus prevent local outbreaks. Although Tengchong County in Yunnan Province—which borders Myanmar—is abundant and diverse in mosquitoes, very few mosquito-based arbovirus investigations have been conducted in the recent decade. Herein, this study aims to evaluate the presence and the diffusion of mosquito-borne pathogens, currently prevalent in this region. Methods We collected 9486 mosquitoes, representing eight species, with Culex tritaeniorhynchus and Anopheles sinensis as the dominant species, during high mosquito activity seasons (July–October) in Tengchong, in 2018. Samples collected from 342 pools were tested using reverse-transcription PCR to determine the species, distribution, and infection rates of virus and parasite, and further analyze their genotypes, phylogenetic relationships, infection rate, and potential pathogenicity. Results Fifteen Japanese encephalitis virus (JEV) strains from Cx. tritaeniorhynchus pools were detected. Seven strains of insect-specific flaviviruses (ISFVs), including two Aedes flavivirus (AeFV) and Yunnan Culex flavivirus strains each, one Culex theileri flavivirus, Yamadai flavivirus (YDFV) and Anopheles-associated flavivirus (AAFV) strains each were detected in Aedes albopictus, Cx. tritaeniorhynchus, Cx. vagans, Cx. pseudovihnui, and An. sinensis pools, respectively. The whole-genome was successfully amplified in one strain of JEV and AeFV each. Phylogenetic analysis using the E gene placed all the newly detected JEV strains into the GI-b genotype. They showed highly nucleotide identities, and were most closely related to the strain detected in Tengchong in 2010. The comparison of the E protein of JEV strains and vaccine-derived strain, showed six amino residue differences. The bias-corrected maximum likelihood estimation values (and 95% confidence interval) for JEV in Cx. tritaeniorhynchus collected in Tengchong in 2018 were 2.4 (1.4–3.9). Conclusions A potential Japanese encephalitis epidemic focus with the abundance of host mosquitoes and high JEV infection rate was observed in Tengchong. In addition, at least five species of ISFVs co-circulate in this area. This study highlights the importance of widespread and sustained mosquito-based arbovirus surveillance in local areas to prevent the transmission of JEV, and other emerging/re-emerging mosquito-borne pathogens. Graphic Abstract

Monitoring Mosquito-Borne Arbovirus in Various Insect Regions in China in 2018

BackgroundIncreases in global travel and trade are changing arbovirus distributions worldwide. Arboviruses can be introduced by travelers, migratory birds, or vectors transported via international trade. Arbovirus surveillance in field-collected mosquitoes may provide early evidence for mosquito-borne disease transmission.MethodsDuring the seasons of high mosquito activity of 2018, 29,285 mosquitoes were sampled from seven sentinel sites in various insect regions. The mosquitoes were analyzed by RT-PCR for alphaviruses, flaviviruses, and orthobunyaviruses.ResultsWe detected three strains of Japanese encephalitis virus (JEV), five strains of Getah virus (GETV), and 45 strains of insect-specific flaviviruses including Aedes flavivirus (AeFV, 1), Chaoyang virus (CHAOV, 1), Culex flavivirus (CxFV, 17), Hanko virus (HANKV, 2), QuangBinh virus (QBV, 22), and Yunnan Culex flavivirus (YNCxFV, 2). Whole genomes of one strain each of GETV, CxFV, CHAOV, and AeFV were successfully amplified. Phylogenetic analysis revealed that the new JEV strains detected in the Shanghai and Hubei Provinces belong to the GI-b strain and are phylogenetically close to the NX1889 strain (MT134112) isolated from a patient during a JE outbreak in Ningxia in 2018. GETVs were found in Inner Mongolia, Hubei, and Hainan and belonged to Group III. They were closely related to strains isolated from swine. HANKV was recorded for the first time in China and other ISFVs were newly detected at several sentinel sites. The bias-corrected maximum likelihood estimation value for JEV in Jinshan, Shanghai was 4.52/1,000 (range 0.80–14.64). Hence, there is a potential risk of a JEV epidemic in that region.ConclusionGI-b is the dominant circulating JEV genotype in nature and poses a health risk to animals and humans. The potential threat of widespread GETV distribution as a zoonosis is gradually increasing. The present study also disclosed the dispersion and host range of ISFVs. These findings highlight the importance of tracing the movements of the vectors and hosts of mosquito-borne pathogens in order to prevent and control arbovirus outbreaks in China.