Population genetic structure of Culex tritaeniorhynchus in different types of climatic zones in China.

BACKGROUND: Culex tritaeniorhynchus is widely distributed in China, from Hainan Island in the south to Heilongjiang in the north, covering tropical, subtropical, and temperate climate zones. Culex tritaeniorhynchus carries 19 types of arboviruses. It is the main vector of the Japanese encephalitis virus (JEV), posing a serious threat to human health. Understanding the effects of environmental factors on Culex tritaeniorhynchus can provide important insights into its population structure or isolation patterns, which is currently unclear. RESULTS: In total, 138 COI haplotypes were detected in the 552 amplified sequences, and the haplotype diversity (Hd) value increased from temperate (0.534) to tropical (0.979) regions. The haplotype phylogeny analysis revealed that the haplotypes were divided into two high-support evolutionary branches. Temperate populations were predominantly distributed in evolutionary branch II, showing some genetic isolation from tropical/subtropical populations and less gene flow between groups. The neutral test results of HNQH (Qionghai) and HNHK(Haikou) populations were negative (P < 0.05), indicating many low-frequency mutations in the populations and that the populations might be in the process of expansion. Moreover, Wolbachia infection was detected only in SDJN (Jining) (2.24%), and all Wolbachia genotypes belonged to supergroup B. To understand the influence of environmental factors on mosquito-borne viruses, we examined the prevalence of Culex tritaeniorhynchus infection in three ecological environments in Shandong Province. We discovered that the incidence of JEV infection was notably greater in Culex tritaeniorhynchus from lotus ponds compared to those from irrigation canal regions. In this study, the overall JEV infection rate was 15.27 per 1000, suggesting the current risk of Japanese encephalitis outbreaks in Shandong Province. CONCLUSIONS: Tropical and subtropical populations of Culex tritaeniorhynchus showed higher genetic diversity and those climatic conditions provide great advantages for the establishment and expansion of Culex tritaeniorhynchus. There are differences in JEV infection rates in wild populations of Culex tritaeniorhynchus under different ecological conditions. Our results suggest a complex interplay of genetic differentiation, population structure, and environmental factors in shaping the dynamics of Culex tritaeniorhynchus. The low prevalence of Wolbachia in wild populations may reflect the recent presence of Wolbachia invasion in Culex tritaeniorhynchus.

Population Genetics of Culex tritaeniorhynchus (Diptera: Culicidae) in Türkiye.

PURPOSE: Mosquitoes are important vectors of pathogens that can affect humans and animals. Culex tritaeniorhynchus is an important vector of arboviruses such as Japanese encephalitis virus, West Nile virus among various human and animal communities. These diseases are of major public health concern and can have huge economic and health burdens in prevalent countries. Although populations of this important mosquito species have been detected in the Mediterranean and Aegean regions of Türkiye; little is known about its population structure. Our study is to examine the population genetics and genetic composition of Cx. tritaeniorhynchus mosquitoes collected from several localities using cytochrome oxidase subunit I (COI) and the NADH dehydrogenase subunit 5 genes (ND5). This is the first extensive study of Cx. tritaeniorhynchus in the mainland Türkiye with sampling spanning many of provinces. METHODS: In this study, DNA extraction, amplification of mitochondrial COI and ND5 genes and population genetic analyses were performed on ten geographic populations of Culex tritaeniorhynchus in the Aegean and Mediterranean region of Türkiye. RESULTS: Between 2019 and 2020, 96 samples were collected from 10 geographic populations in the Aegean and Mediterranean regions; they were molecularly analyzed and 139 sequences (50 sequence for COI and 89 sequence for ND5) were used to determine the population structure and genetic diversity. For ND5 gene region, the samples produced 24 haplotypes derived from 15 variable sites and for COI gene region, 43 haplotypes were derived from 17 variable sites. The haplotype for both gene regions was higher than nucleotide diversity. Haplotype phylogeny revealed two groups present in all populations. AMOVA test results show that the geographical populations were the same for all gene regions. Results suggest that Cx. tritaeniorhynchus is a native population in Türkiye, the species is progressing towards speciation and there is no genetic differentiation between provinces and regions. CONCLUSION: This study provides useful information on the molecular identifcation and genetic diversity of Cx. tritaeniorhynchus; these results are important to improve mosquito control programs.

Infection, dissemination, and transmission of lumpy skin disease virus in Aedes aegypti (Linnaeus), Culex tritaeniorhynchus (Giles), and Culex quinquefasciatus (Say) mosquitoes.

Lumpy skin disease virus (LSDV) is a transboundary viral disease in cattle and water buffaloes. Although this Poxvirus is supposedly transmitted by mechanical vectors, only a few studies have investigated the role of local vectors in the transmission of LSDV. This study examined the infection, dissemination, and transmission rates of LSDV in Aedes aegypti, Culex tritaeniorhynchus, and Culex quinquefasciatus following artificial membrane feeding of 102.7, 103.7, 104.7 TCID50/mL LSDV in sheep blood. The results demonstrated that these mosquito species were susceptible to LSDV, with Cx tritaeniorhynchus exhibiting significantly different characteristics from Ae. aegypti and Cx. quinquefasciatus. These three mosquito species were susceptible to LSDV. Ae. aegypti showed it as early as 2 days post-infection (dpi), indicating swift dissemination in this particular species. The extrinsic incubation period (EIP) of LSDV in Cx. tritaeniorhynchus and Cx. quinquefasciatus was 8 and 14 dpi, respectively. Ingestion of different viral titers in blood did not affect the infection, dissemination, or transmission rates of Cx. tritaeniorhynchus and Cx. quinquefasciatus. All rates remained consistently high at 8-14 dpi for Cx. tritaeniorhynchus. In all three species, LSDV remained detectable until 14 dpi. The present findings indicate that, Ae. aegypti, Cx. tritaeniorhynchus, and Cx. quinquefasciatus may act as vectors during the LSDV outbreak; their involvement may extend beyond being solely mechanical vectors.

Geographical Characteristics of Culex tritaeniorhynchus and Culex orientalis Microbiomes in Korea.

Mosquitoes, the primary vectors of arboviruses, harbor a diverse microbiome that plays a crucial role in their development, immunity, and vector competence. The composition of the mosquito microbiome is heavily influenced by the environment and habitats. Therefore, identifying the relationship between the habitat and the mosquito's microbial community can improve the overall understanding of mosquito biology. However, The microbiome profiles of Culex tritaeniorhynchus and Culex orientalis, known as transmission vectors of the Japanese encephalitis virus, are poorly understood. Using 16S rRNA Illumina sequencing, we hereby investigated the microbial profiles in these two mosquito species collected in several areas in the Republic of Korea. Thirty-six prevalent bacterial families were identified from these mosquito species. The microbial composition variations were primarily influenced by the mosquito collecting sites. Moreover, species biomarkers were identified by utilizing the regional specificity of the mosquito microbiome. Based on the microbiome profiles representing high similarity, Culex orientalis may share an ecological niche with Culex tritaeniorhynchus.

The identification and genetic characteristics of Quang Binh virus from field-captured Culex tritaeniorhynchus (Diptera: Culicidae) from Guizhou Province, China.

BACKGROUND: Mosquitoes carry a variety of viruses that can cause disease in humans, animals and livestock. Surveys for viruses carried by wild mosquitoes can significantly contribute to surveillance efforts and early detection systems. In addition to mosquito-borne viruses, mosquitoes harbor many insect-specific viruses (ISVs). Quang Binh virus (QBV) is one such example, categorized as an ISV within the Flavivirus genus (family Flaviviridae). QBV has been specifically documented in Vietnam and China, with reports limited to several mosquito species. METHODS: The homogenate obtained from female mosquitoes was cultured on C6/36 (Aedes albopictus) and BHK-21 (baby hamster kidney) cell lines. Positive cultures were identified by reverse transcription-polymerase chain reaction (RT‒PCR) with taxon- or species-specific primers. Next-generation sequencing was employed to sequence the complete genomes of the identified positive samples. Subsequently, phylogenetic, gene homology, molecular evolutionary and genetic variation analyses were conducted. RESULT: In 2021, a total of 32,177 adult female mosquitoes were collected from 15 counties in Guizhou Province, China. The predominant mosquito species identified were Culex tritaeniorhynchus, Armigeres subalbatus and Anopheles sinensis. Among the collected mosquitoes, three positive cultures were obtained from Cx. tritaeniorhynchus pools, revealing the presence of Quang Binh virus (QBV) RNA sequences. Phylogenetic analysis indicated that the three Guizhou isolates, along with the prototype isolate from Vietnam, formed distinct branches. These branches were primarily closely related to other QBV isolates reported in China. Comparative analysis revealed a high degree of nucleotide and amino acid homology between the Guizhou isolates and both Vietnamese and other indigenous Chinese isolates. Additionally, nonsynonymous single-nucleotide variants (SNVs) were observed in these strains compared to the QBV prototype strain. CONCLUSION: This study represents the first report of QBV presences in Cx. tritaeniorhynchus mosquitoes in Guizhou Province, China. Phylogenetic tree analysis showed that the three Guizhou isolates were most closely related to the QBV genes found in China. In addition, the study of the genetic characteristics and variation of this virus provided a deeper understanding of QBV and enriched the baseline data of these insect-specific flaviviruses (ISFVs).

Identification and Molecular Characteristics of a Novel Single-Stranded RNA Virus Isolated from Culex tritaeniorhynchus in China.

Hubei mosquito virus 2 (HMV2) is a novel mosquito virus that was first identified in 2016 in Hubei Province, China. Until now, HMV2 has been shown to be endemic in some areas of China and Japan, but its biological characteristics, epidemiology, and pathogenicity are not yet known. This report describes the detection of HMV2 in mosquitoes that were collected in Shandong Province in 2019 and presents the first isolation and molecular characterization of the virus. In this study, a total of 2,813 mosquitoes were collected and then divided into 57 pools, according to location and species. qRT-PCR and nested PCR were performed to confirm the presence of HMV2, and its genomic features, phylogenetic relationships, growth characteristics, and potential pathogenicity were further analyzed. The results showed that HMV2 was detected in 28 of the 57 mosquito pools and that the minimum infection rate (MIR) for HMV2 was 1.00% (28/2,813). A HMV2 strain and 14 viral partial sequences were obtained from the HMV2-positive pools, including one complete genome sequence. A phylogenetic analysis revealed that HMV2 from Shandong Province shared over 90% identity with other reported isolates and was closely related to the Culex inatomii luteo-like virus. IMPORTANCE Our study provided important epidemiological evidence for the epidemic of HMV2 in Shandong Province. Here, we report the first isolation and molecular characteristics of this virus and enrich our knowledge of the distribution of HMV2 in China.

Detection of Culex tritaeniorhynchus Giles and Novel Recombinant Strain of Lumpy Skin Disease Virus Causes High Mortality in Yaks.

Lumpy skin disease virus (LSDV) is capable of causing transboundary diseases characterized by fever, nodules on the skin, mucous membranes, and inner organs. The disease may cause emaciation with the enlargement of lymph nodes and sometimes death. It has had endemic importance in various parts of Asia in recent years, causing substantial economic losses to the cattle industry. The current study reported a suspected LSDV infection (based on signs and symptoms) from a mixed farm of yak and cattle in Sichuan Province, China. The clinical samples were found positive for LSDV using qPCR and ELISA, while LSDV DNA was detected in Culex tritaeniorhynchus Giles. The complete genome sequence of China/LSDV/SiC/2021 was determined by Next-generation sequencing. It was found that China/LSDV/SiC/2021 is highly homologous to the novel vaccine-related recombinant LSDV currently emerging in China and countries surrounding China. Phylogenetic tree analysis revealed that the novel vaccine-associated recombinant LSDV formed a unique dendrograms topology between field and vaccine-associated strains. China/LSDV/SiC/2021 was found to be a novel recombinant strain, with at least 18 recombination events via field viruses identified in the genome sequence. These results suggest that recombinant LSDV can cause high mortality in yaks, and its transmission might be due to the Culex tritaeniorhynchus Giles, which acts as a mechanical vector.

Vector competence of three species of mosquitoes to Ingwavuma virus (Manzanilla orthobunyavirus), a new bunyavirus found circulating in India.

Ingwavuma virus (INGV), a mosquito-borne arbovirus reported from Africa and Southeast Asia has been found circulating in India as evidenced by virus isolation and antibody prevalence. INGV is now classified as Manzanilla orthobunyavirus belonging to family Peribunyaviridae. The virus is maintained in nature in a pig-mosquito-bird cycle. Human infection has been confirmed by virus isolation and detection of neutralizing antibodies. A study was initiated to determine the vector competence of Aedes aegypti, Culex quinquefasciatus, and Cx tritaeniorhynchus mosquitoes to INGV due to their high prevalence in India. Mosquitoes were oral fed on viraemic mice and INGV dissemination to legs, wings, salivary glands (saliva) was studied alongwith virus growth kinetics. The three mosquitoes replicated INGV with maximum titers of 3.7, 3.7 and 4.7log10TCID50/ml respectively and maintained the virus till 16th day post infection. However, vector competence and horizontal transmission to infant mouse was demonstrated only by Cx quinquefasciatus mosquitoes. Vertical or trans-ovarial transmission of INGV could not be demonstrated in the mosquito during the study. Though no major outbreak involving humans has been reported yet, the potential of the virus to replicate in different species of mosquitoes and vertebrates including humans pose a threat to public health should there be a change in its genome.

Blood meal source identification and RNA virome determination in Japanese encephalitis virus vectors collected in Ishikawa Prefecture, Japan, show distinct avian/mammalian host preference.

In Asia, Culex mosquitoes are of particular interest because of their role in maintaining endemic mosquito-borne viral diseases, including the Japanese encephalitis virus (JEV). Nonetheless, host-feeding preferences, along with naturally infecting RNA viruses in certain Culex species, remain understudied. In this study, selected blood-fed mosquitoes were processed for avian and mammalian blood meal source identification. Concurrently, cell culture propagation and high-throughput sequencing (HTS) approaches were used to determine the RNA virome of Culex mosquitoes collected in Ishikawa Prefecture, Japan. The identification of blood meal sources from wild-caught Culex spp. revealed that Culex (Culex) tritaeniorhynchus Giles, 1901, has a robust preference toward wild boar (62%, 26/42), followed by heron (21%, 9/42). The other two species, Culex (Oculeomyia) bitaeniorhynchus Giles, 1901, and Culex (Culex) orientalis Edwards, 1921, showed a distinct preference for avian species, including migratory birds. From the HTS results, 34 virus sequences were detected, four of which were newly identified virus sequences of unclassified Aspiviridae, Qinviridae, Iflaviridae, and Picornaviridae. The absence of observable cytopathic effects in mammalian cells and phylogenetic analysis suggested that all identified virus sequences were insect-specific. Further investigations involving other mosquito populations collected in different areas are warranted to explore previously unknown vertebrate hosts that may be linked to JEV dispersal in nature.

Evaluating the mosquito host range of Getah virus and the vector competence of selected medically important mosquitoes in Getah virus transmission.

BACKGROUND: The Getah virus (GETV) is a mosquito-borne Alphavirus (family Togaviridae) that is of significant importance in veterinary medicine. It has been associated with major polyarthritis outbreaks in animals, but there are insufficient data on its clinical symptoms in humans. Serological evidence of GETV exposure and the risk of zoonotic transmission makes GETV a potentially medically relevant arbovirus. However, minimal emphasis has been placed on investigating GETV vector transmission, which limits current knowledge of the factors facilitating the spread and outbreaks of GETV. METHODS: To examine the range of the mosquito hosts of GETV, we selected medically important mosquitoes, assessed them in vitro and in vivo and determined their relative competence in virus transmission. The susceptibility and growth kinetics of GETVs in various mosquito-derived cell lines were also determined and quantified using plaque assays. Vector competency assays were also conducted, and quantitative reverse transcription-PCR and plaque assays were used to determine the susceptibility and transmission capacity of each mosquito species evaluated in this study. RESULTS: GETV infection in all of the investigated mosquito cell lines resulted in detectable cytopathic effects. GETV reproduced the fastest in Culex tritaeniorhynchus- and Aedes albopictus-derived cell lines, as evidenced by the highest exponential titers we observed. Regarding viral RNA copy numbers, mosquito susceptibility to infection, spread, and transmission varied significantly between species. The highest vector competency indices for infection, dissemination and transmission were obtained for Cx. tritaeniorhynchus. This is the first study to investigate the ability of Ae. albopictus and Anopheles stephensi to transmit GETV, and the results emphasize the role and capacity of other mosquito species to transmit GETV upon exposure to GETV, in addition to the perceived vectors from which GETV has been isolated in nature. CONCLUSIONS: This study highlights the importance of GETV vector competency studies to determine all possible transmission vectors, especially in endemic regions.

Global Distribution of Culex tritaeniorhynchus and Impact Factors.

Culex tritaeniorhynchus is the primary vector of Japanese encephalitis (JE) and has a wide global distribution. However, the current and future geographic distribution maps of Cx. tritaeniorhynchus in global are still incomplete. Our study aims to predict the potential distribution of Cx. tritaeniorhynchus in current and future conditions to provide a guideline for the formation and implementation of vector control strategies all over the world. We collected and screened the information on the occurrence of Cx. tritaeniorhynchus by searching the literature and online databases and used ten algorithms to investigate its global distribution and impact factors. Cx. tritaeniorhynchus had been detected in 41 countries from 5 continents. The final ensemble model (TSS = 0.864 and AUC = 0.982) indicated that human footprint was the most important factor for the occurrence of Cx. tritaeniorhynchus. The tropics and subtropics, including southeastern Asia, Central Africa, southeastern North America and eastern South America, showed high habitat suitability for Cx. tritaeniorhynchus. Cx. tritaeniorhynchus is predicted to have a wider distribution in all the continents, especially in Western Europe and South America in the future under two extreme emission scenarios (SSP5-8.5 and SSP1-2.6). Targeted strategies for the control and prevention of Cx. tritaeniorhynchus should be further strengthened.

Japanese encephalitis virus genotype III from mosquitoes in Tarlac, Philippines.

Objectives: The aim of this study was to investigate the presence of Japanese encephalitis virus (JEV) in a rice-farming community in the Philippines and to determine its implications regarding the epidemiology of viral encephalitides in the Asia-Pacific Region. Methods: Mosquitoes were collected monthly from animal-baited traps close to flooded rice fields in two barangays (villages) in the Municipality of San Jose, Tarlac Province in Luzon, from May 2009 to July 2010. Virus was detected by nested reverse transcription PCR. Phylogenetic analysis of the amplified virus envelope gene was done using the maximum-likelihood method. Results: A total of 28 700 known vector mosquitoes were collected, namely Culex vishnui, Culex fuscocephala, Culex tritaeniorhynchus, and Culex gelidus. JEV genotype III was detected in C. tritaeniorhynchus, belonging to the same genotype but form a different clade from those reported in the 1980s and in 2020 in this country. Conclusions: Japanese encephalitis is associated with rice cultivation and the presence of infected mosquitoes in Tarlac, Philippines. It remains to be seen whether the observed genetic shift of genotype III to genotype I in Asia will in time have an impact on the epidemiology of Japanese encephalitis in the Philippines. For long-term disease control, regular surveillance and Japanese encephalitis immunization in children and travelers in high risk areas are recommended.

Metavirome Analysis of Culex tritaeniorhynchus Reveals Novel Japanese Encephalitis Virus and Chikungunya Virus.

To explore the Culex tritaeniorhynchuses-specific virome, 6400 C. tritaeniorhynchuses were collected in Honghe autonomous prefecture, China. Abundant virus sequences were obtained from 28 viral families using metavirome sequencing. Herein, several viruses in C. tritaeniorhynchuses virome were verified using the PCR technique, which covers Japanese encephalitis virus (JEV), Getah virus, and even Chikungunya virus (CHIKV). Seven JEV gene sequences were amplified successfully, of which JEV-China/CT2016E-1 shared the highest homology with the known JEV sequence isolated in Korea, 1946, with at least 96.1% nucleotide (nt) identity, which belonged to genotype III. Nine CHIKV gene sequences were amplified, which shared the highest with at least 93.0% nt identity with CHIKV from Thailand isolated in 2007, which was assigned to genotype Asian. Remarkably, CHIKV was isolated from C. tritaeniorhynchus in China for the first time. It was initially confirmed that the isolated virus CHIKV-China/CT2016-1 may increase infectivity after passaging in Vero cells from BHK-21 cells. Collectively, our study reveals the diversity, properties, and potential virus susceptibility dynamics of the C. tritaeniorhynchus virome and sheds new perspectives on the viral ecology in other important biological vectors.

Culex vishnui (Diptera: Culicidae): An Overlooked Vector of Arboviruses in South-East Asia.

Culex vishnui Theobald, 1901, a main vector of Japanese encephalitis virus (JEV), is widely distributed in the Oriental region where it often accounts for a great part of the culicid fauna. This species also has been found naturally infected with at least 13 other arboviruses of medical and veterinary importance. Females blood feed predominantly upon pigs and birds, but may readily bite cattle and humans. Because of its abundance, medical importance, and presence throughout ecological gradients among urban, peri-urban, and rural areas, Cx. vishnui potentially may serve as a bridge vector transmitting viruses from natural and wild hosts to humans. Being zoo- and anthropophagic, omnipresent in the Oriental region, and presenting strong resistance to many insecticide families, this overlooked mosquito species may pose a serious health risk in one of the most densely populated regions of the world.

Utilization of Pomelo (Citrus maxima) Peel Waste into Bioactive Essential Oils: Chemical Composition and Insecticidal Properties.

The wastes generated during the post-harvest handling of various agricultural commodities is rather under-utlilized. The peels of citrus fruits are often discarded as waste. Citrus peels are rich in essential oils and exhibit toxicity towards various insect species. The essential oils are also an eco-friendly option for insect pest management. The Citrus maxima peel essential oil (CMEO), a waste product, characterized it, and evaluated its potential for insect pest management. The major terpenoids present in CMEO are Limonene and α-Pinene. The CMEO displayed potentials in controlling the insect pests via contact and fumigant toxicity. Moreover, CMEO showed significant larvicidal activities against Culex tritaeniorhynchus and Aedes aegypti species of mosquitoes; however, Armigeres subalbatus was more resistant. The biological safety of the essential oil was also tested against the stored seeds, where no significant inhibition of seed germination was noticed compared to the control. Utilizing a waste product such as citrus peel for pest management can achieve the dual objective of waste utilization and eco-friendly pest management. Overall, the CMEO is therefore found to be a bioactive essential oil extracted from the wastes of pomelo (C. maxima).

Characterization and mosquitocidal potency of a Bacillus thuringiensis strain of rice field soil of Burdwan, West Bengal, India.

Bacillus thuringiensis is the most popular mosquitocidal bacteria, strains of which are effective against almost all mosquito larvae. It has host specificity and thus, has no adverse effect on non-target species of the ecosystem. Culex tritaeniorhynchus, a vector of Japanese encephalitis (JE), breeds in vast area of rice fields in Burdwan district of West Bengal, India, which has already confronted JE epidemic. Entomological investigation and ecological studies on this vector mosquito showed that JE epidemic may reoccur anytime in the area. A strain of Bt (BU55) was isolated from rice field soil, efficacy was tested against Cx. tritaeniorhynchus and mosquitocidal role was confirmed against Cx. quinquefascistus also. The LC50 of Bacillus thuringiensis BU55 against Cx. tritaeniorhynchus and Cx. quinquefascistus after 72 h was 8.59 ml (final dose 2.49 x107 CFU/ml) and 7.52 ml (final dose 2.20 x 107 CFU/ml), respectively. Insecticidal crystal protein profile of BU55 produced 136.89, 64.80, 43.45, 33.65 and 26.98 kDa bands. Among them 136.89, 64.29, 26.98 kDa proteins are comparable to actual toxins viz. Cry1Ac (138.3 kDa, Lepidoptera specific), Cry4D (68.0 kDa, Diptera specific) and Cyt (27.4 kDa, Diptera specific). The results clearly showed that the Bt strain is a potent dipteran larvicide and can be used against the JE vectors to control the disease.

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.

Shape relatedness between geographic populations of Culex tritaeniorhynchus, the primary vector of Japanese encephalitis virus: A landmark study.

BACKGROUND: Japanese encephalitis is a severe disease of acute encephalitis, with children and the elderly primarily affected, and with mortality rates reaching over 25%. The virus is transmitted mainly by species of the Culex (Culex) vishnui subgroup, primarily the widely spread Cx. tritaeniorhynchus Giles. The latter is known as a highly migratory mosquito which moves with airflow over large distances. We explored the geometric variation of the wing venation among distant areas of its geographic distribution. Our working hypothesis was that shape variation across geography could reveal known past and present migratory routes. MATERIALS METHODS: We compared the wing venation geometry of 236 female Culex tritaeniorhynchus from different locations in the Madagascan (La Reunion), Oriental (Thailand, Vietnam) and Paleartic (Japan) regions. To ascertain the taxonomic signal of the wing venation we also used two species as relative outgroups, Cx. whitmorei and Cx. brevipalpis. RESULTS: In spite of an increasing morphometric variation as expected with larger geographic dispersion, our Cx. tritaeniorhynchus samples were clustered as a single species when considered relative to other Culex species. The relationships between geographic sites of Cx. tritaeniorhynchus globally conformed with an isolation by distance model. The shape homogeneity of our Palearctic samples (Japan) contrasted with some heterogeneity observed in the Oriental region (Thailand, Vietnam), and could be related to the different regimes of wind trajectories in these regions. CONCLUSION: The average shape variation of Culex tritaeniorhynchus disclosed a separation between Madagascan, Oriental and Palearctic regions in accordance with geography. The wing venation not only could reflect geography, it also contained a clear taxonomic signal separating three Culex species. Within Cx. tritaeniorhynchus, a contrasting pattern of shape variation between the Palearctic and the Oriental regions is tentatively explained by the influence of wind trajectories.

Genetic structure of Culex tritaeniorhynchus (Diptera: Culicidae) based on COI DNA barcodes.

Culex tritaeniorhynchus Gile is a major vector of Japanese encephalitis in China. The population genetics study is crucial as it helps understanding the epidemiological aspects of mosquito-brone diseases and improving vector control measures. Here, the genetic population structure of C. tritaeniorhynchus in the mainland China were estimated using the cytochrome c oxidase subunit 1 (COI) DNA barcodes region. 485 individuals of C. tritaeniorhynchus were collected from 38 sampling sites in 21 geographic populations in the mainland China. In total, 485 sequences were used to explore the population structure and genetic diversity. The results showed that the populations of C. tritaeniorhynchus had high haplotype diversity (Hd = 0.98, with 303 haplotypes), low nucleotide diversity (p = 0.02245) and high gene flow (Nm = 47.11) with two maternal lineages and four groups. An AMOVA indicated that 98.8% of the total variation originated from variation within populations. In addition, the population genetic structure exhibited by C. tritaeniorhynchus filling the vacant of the genetic structure in the mainland China. Human activities may also assist mosquito movement and migration. Gene flow among the populations of C. tritaeniorhynchus can facilitate the spread of insecticide resistance genes over geographical areas, and it will be a challenging for controlling the populations.

Vector competence of Culex tritaeniorhynchus and Culex quinquefasciatus (Diptera: Culicidae) for duck Tembusu virus transmission.

Duck Tembusu virus (DTMUV), an emerging infectious disease in ducks, was detected in Culex (Cx.) tritaeniorhynchus mosquitoes collected from a duck farm; however, the exact role of mosquitoes in the ecology of DTMUV in Thailand remains unclear. Vector competence of Cx. tritaeniorhynchus and Cx. quinquefasciatus was examined for DTMUV. Cx. tritaeniorhynchus mosquitoes were allowed to feed on four levels (102, 103, 104, and 105 TCID50/mL) of DTMUV, while Cx. quinquefasciatus were allowed to feed on two levels (104 and 105 TCID50/mL) of DTMUV. Infection rates in Cx. tritaeniorhynchus were 1.6, 10.2, 35.8, and 59.3% after feeding on 102, 103, 104, and 105 TCID50/mL of DTMUV, respectively, while dissemination and transmission were 20.3 and 16.9% after feeding on 105 TCID50/mL of DTMUV. Infection rates in Cx. quinquefasciatus were 2.5 and 2.3% after feeding on 104 and 105 TCID50/mL of DTMUV, respectively, with no virus dissemination and transmission found in all tested mosquitoes. Another study was conducted to examine the transovarial transmission of DTMUV in Cx. tritaeniorhynchus. Mosquitoes were allowed to feed on blood meal infected with 105 TCID50/mL of DTMUV. Each blood-fed mosquito was isolated and allowed to lay eggs. After oviparity, the mosquitoes were tested for DTMUV infection; 43 DTMUV infected and 37 non-infected female mosquitoes with eggs were included. A total of 182 F1 progeny from DTMUV infected mosquitoes and 145 F1 progeny from non-infected mosquitoes were tested for DTMUV but all were negative. Findings indicated the potential role of Cx. tritaeniorhynchus in the DTMUV transmission cycle in duck farms in Thailand. No transovarial transmission of DTMUV was found in Cx. tritaeniorhynchus.