Comparative analysis of midgut bacterial communities in Chikungunya virus-infected and non-infected Aedes aegypti Thai laboratory strain mosquitoes.

Chikungunya virus (CHIKV) poses a significant global health threat, re-emerging as a mosquito-transmitted pathogen that caused high fever, rash, and severe arthralgia. In Thailand, a notable CHIKV outbreak in 2019-2020 affected approximately 20,000 cases across 60 provinces, underscoring the need for effective mosquito control protocols. Previous studies have highlighted the role of midgut bacteria in the interaction between mosquito vectors and pathogen infections, demonstrating their ability to protect the insect from invading pathogens. However, research on the midgut bacteria of Aedes (Ae.) aegypti, the primary vector for CHIKV in Thailand remains limited. This study aims to characterize the bacterial communities in laboratory strains of Ae. aegypti, both infected and non-infected with CHIKV. Female mosquitoes from a laboratory strain of Ae. aegypti were exposed to a CHIKV-infected blood meal through membrane feeding, while the control group received a non-infected blood meal. At 7 days post-infection (dpi), mosquito midguts were dissected for 16S rRNA gene sequencing to identify midgut bacteria, and CHIKV presence was confirmed by E1-nested RT-PCR using mosquito carcasses. The study aimed to compare the bacterial communities between CHIKV-infected and non-infected groups. The analysis included 12 midgut bacterial samples, divided into three groups: CHIKV-infected (exposed and infected), non-infected (exposed but not infected), and non-exposed (negative control). Alpha diversity indices and Bray-Curtis dissimilarity matrix revealed significant differences in bacterial profiles among the three groups. The infected group exhibited an increased abundance of bacteria genus Gluconobacter, while Asaia was prevalent in both non-infected and negative control groups. Chryseobacterium was prominent in the negative control group. These findings highlight potential alterations in the distribution and abundance of gut microbiomes in response to CHIKV infection status. This study provides valuable insights into the dynamic relationship between midgut bacteria and CHIKV, underscoring the potential for alterations in bacterial composition depending on infection status. Understanding the relationships between mosquitoes and their microbiota holds promise for developing new methods and tools to enhance existing strategies for disease prevention and control. This research advances our understanding of the circulating bacterial composition, opening possibilities for new approaches in combating mosquito-borne diseases.

Genetic diversity and phylogenetic analyses of Asian lineage Zika virus whole genome sequences derived from Culex quinquefasciatus mosquitoes and urine of patients during the 2020 epidemic in Thailand.

Zika virus (ZIKV), a mosquito-borne flavivirus, has been continually emerging and re-emerging since 2010, with sporadic cases reported annually in Thailand, peaking at over 1000 confirmed positive cases in 2016. Leveraging high-throughput sequencing technologies, specifically whole genome sequencing (WGS), has facilitated rapid pathogen genome sequencing. In this study, we used multiplex amplicon sequencing on the Illumina Miseq instrument to describe ZIKV WGS. Six ZIKV WGS were derived from three samples of field-caught Culex quinquefasciatus mosquitoes (two males and one female) and three urine samples collected from patients in three different provinces of Thailand. Additionally, successful isolation of a ZIKV isolate occurred from a female Cx. quinquefasciatus. The WGS analysis revealed a correlation between the 2020 outbreak and the acquisition of five amino acid changes in the Asian lineage ZIKV strains from Thailand (2006), Cambodia (2010 and 2019), and the Philippines (2012). These changes, including C-T106A, prM-V1A, E-V473M, NS1-A188V, and NS5-M872V, were identified in all seven WGS, previously linked to significantly higher mortality rates. Furthermore, phylogenetic analysis indicated that the seven ZIKV sequences belonged to the Asian lineage. Notably, the genomic region of the E gene showed the highest nucleotide diversity (0.7-1.3%). This data holds significance in informing the development of molecular tools that enhance our understanding of virus patterns and evolution. Moreover, it may identify targets for improved methods to prevent and control future ZIKV outbreaks.

Description of potential vectors of zoonotic filarial nematodes, Brugia pahangi, Setaria digitata, and Setaria labiatopapillosa in Thai mosquitoes.

Filariasis is classified as a vector-borne zoonotic disease caused by several filarial nematodes. The disease is widely distributed in tropical and subtropical regions. Understanding the relationship between mosquito vectors, filarial parasites, and vertebrate hosts is therefore essential for determining the probability of disease transmission and, correspondingly, developing effective strategies for prevention and control of diseases. In this study, we aimed to investigate the infection of zoonotic filarial nematodes in field-caught mosquitoes, observe the potential vectors of filaria parasites in Thailand using a molecular-based survey, conduct a study of host-parasite relationship, and propose possible coevolution of the parasites and their hosts. Mosquitoes were collected around cattle farms in Bangkok, Nakhon Si Thammarat, Ratchaburi, and Lampang provinces from May to December 2021 using a CDC Backpack aspirator for 20-30 minutes in each area (intra-, peri-, and wild environment). All mosquitoes were identified and morphologically dissected to demonstrate the live larvae of the filarial nematode. Furthermore, all samples were tested for filarial infections using PCR and sequencing. A total of 1,273 adult female mosquitoes consisted of five species: 37.78% Culex quinquefasciatus, 22.47% Armigeres subalbatus, 4.71% Cx. tritaeniorhynchus, 19.72% Anopheles peditaeniatus, and 15.32% An. dirus. Larvae of Brugia pahangi and Setaria labiatopapillosa were found in Ar. subalbatus and An. dirus mosquitoes, respectively. All mosquito samples were processed by PCR of ITS1 and COXI genes for filaria nematode species identification. Both genes showed that B. pahangi was found in four mosquitoes of Ar. subalbatus from Nakhon Si Thammarat, S. digitata was detected in three samples of An. peditaeniatus from Lampang, and S. labiatopapillosa was detected in one of An. dirus from Ratchaburi. However, filarial nematodes were not found in all Culex species. This study infers that this is the first data regarding the circulation of Setaria parasites in Anopheles spp. from Thailand. The phylogenetic trees of the hosts and parasites are congruent. Moreover, the data could be used to develop more effective prevention and control strategies for zoonotic filarial nematodes before they spread in Thailand.

Genetic characterization of chikungunya virus isolates from Aedes aegypti mosquitoes collected during a recent outbreak in Bangkok, Thailand.

Chikungunya virus (CHIKV) is a mosquito-borne emerging pathogen that is transmitted to humans through the bite of female Aedes mosquitoes. CHIKV infection has become a major public health concern worldwide, as it has a significant impact on the healthcare system. Since 2004, the virus has emerged in Africa and subsequently spread to countries located near the Indian Ocean, including India, and to Europe, the Americas, and Asia. In Thailand, a large CHIKV outbreak occurred during 2008-2009 and was caused by a virus originating from the east/central/south African (ECSA) CHIKV genotype. Since then, the ECSA genotype of CHIKV has continued to circulate and has caused sporadic cases in different areas in Thailand. Approximately 20,000 reported cases have been confirmed by the Bureau of Epidemiology, Ministry of Public Health, Thailand, from January 1, 2018 to July 31, 2020. However, the causes of this CHIKV re-emergence remain unclear. To obtain a better understanding of CHIKV circulation during the recent outbreak in Bangkok, Thailand, complete genome analysis of CHIKV isolates from field-caught mosquitoes collected in outbreak areas was performed. A total of 28 Ae. aegypti samples (21 females and 7 males) were collected, and individual mosquitoes were used for CHIKV detection and isolation. Eleven of 28 (39.29%) female and three of 28 (10.71%) male mosquitoes were positive for CHIKV by E1 nested RT-PCR. Four CHIKV isolates were successfully isolated from four female Ae. aegypti mosquitoes. Based on complete genome analysis, several amino acid substitutions were identified in the protein coding region. The E1:K211E and E2:V264A mutations in the background of the E1:226A mutation were observed in all four CHIKV isolates. An important observation was the presence of one amino acid substitution, leading to an E1:K245R change. This mutation was found in all four CHIKV isolates from mosquitoes in this study and in Thai patients described previously. Additionally, phylogenetic analysis indicated that the four CHIKV isolates belonged to the Indian Ocean clade of the ECSA genotype. The results obtained in this study provide detailed information on the molecular characteristics and evolution of currently circulating CHIKV strains in Thailand, which are useful for developing prevention and control strategies.

Molecular detection of Indian Ocean Lineage Chikungunya virus RNA in field collected Culex quinquefasciatus Say from Bangkok, Thailand but no evidence of virus replication.

Following an outbreak of chikungunya virus (CHIKV) infections in Thailand in 2019, numerous cases of CHIKV infection have been diagnosed in Bangkok, the capital of the country. In our previous investigation of the vectors for disease transmission, we found natural infection of CHIKV in both male and female Aedes aegypti mosquitoes collected from the outbreak areas in Bangkok. Some reports mentioned the detection of CHIKV in Culex mosquitoes. In Thailand, the Culex quinquefasciatus Say mosquito is a common species found in urban and rural settings that coexists with Ae. aegypti. However, the role of Cx. quinquefasciatus mosquitoes in the spread of the Indian Ocean Lineage (IOL) of CHIKV in Thailand has never been investigated. In this study, Cx. quinquefasciatus were collected (16 males and 27 females) from an outbreak area in Bangkok. Eight of the 27 in field-caught female Cx. quinquefasciatus were positive for IOL CHIKV RNA, and 99-100% identity and full 100% coverage of sequences similar to CHIKV isolated from female Ae. aegypti in Bangkok, Thailand, whereas viral RNA was not detected in male samples using nested-RT-PCR. To determine whether CHIKV is able to replicate in Cx. quinquefasciatus, the laboratory strain of Cx. quinquefasciatus was allowed to feed on blood containing IOL CHIKV isolated from patient serum. The nested-RT-PCR, virus isolation, and immunofluorescence assay (IFA) were performed for CHIKV detection and replication. The results showed that CHIKV RNA was detected in Cx. quinquefasciatus until day 4 post infection. CHIKV did not produce any remarkable signs of infection, dissemination, or transmission in Cx. quinquefasciatus, and cytopathic effect (CPE) was not observed in C6/36 cells when infected with supernatant obtained from Cx. quinquefasciatus at days 7, 10, 14, and 21 post infection when compared to Ae. aegypti. The data from this study infer that CHIKV may be detected in Cx. quinquefasciatus but that the mosquito is not able to transmit CHIKV in Thailand.

Genetic Characterization of Chikungunya Virus in Field-Caught Aedes aegypti Mosquitoes Collected during the Recent Outbreaks in 2019, Thailand.

Chikungunya virus (CHIKV) is a mosquito-borne virus belonging to the genus Alphavirus. The virus is transmitted to humans by the bite of infected female Aedes mosquitoes, primarily Aedes aegypti. CHIKV infection is spreading worldwide, and it periodically sparks new outbreaks. There are no specific drugs or effective vaccines against CHIKV. The interruption of pathogen transmission by mosquito control provides the only effective approach to the control of CHIKV infection. Many studies have shown that CHIKV can be transmitted among the Ae. aegypti through vertical transmission. The previous chikungunya fever outbreaks in Thailand during 2008-2009 were caused by CHIKV, the East/Central/South African (ECSA) genotype. Recently, there have been 3794 chikungunya cases in 27 provinces reported by the Bureau of Epidemiology of Health Ministry, Thailand during 1 January-16 June 2019; however, the cause of the re-emergence of CHIKV outbreaks is uncertain. Therefore, the aims of this study were to detect and analyze the genetic diversity of CHIKV infection in field-caught mosquitoes. Both female and male Ae. aegypti were collected from endemic areas of Thailand, and CHIKV detection was done by using E1-nested RT-PCR and sequencing analysis. A total of 1646 Ae. aegypti samples (900 females and 746 males) were tested. CHIKV was detected in 54 (3.28%) and 14 samples (0.85%) in female and male mosquitoes, respectively. Seventeen samples of female Ae. aegypti collected from the Ubon Ratchathani, Chiang Rai, Chiang Mai, Nakhon Sawan, and Songkhla provinces found mutation at E1: A226V. Interestingly, E1: K211E mutation was observed in 50 samples collected from Nong Khai, Bangkok, Prachuap Khiri Khan, and Krabi. In addition, the phylogenetic tree indicated that CHIKV in Ae. aegypti samples were from the Indian Ocean Clade and East/South African Clade. Both clades belong to the ECSA genotype. The information obtained from this study could be used for prediction, epidemiological study, prevention, and effective vector control of CHIKV. For instance, a novel CHIKV strain found in new areas has the potential to lead to a new outbreak. Health authorities could plan and apply control strategies more effectively given the tools provided by this research.

Vertical transmission of Zika virus in Culex quinquefasciatus Say and Aedes aegypti (L.) mosquitoes.

Several mosquito species have been described as vectors for the Zika virus (ZIKV), such as those in the Aedes, Anopheles, Mansonia and Culex genera. Our previous survey studies were found the ZIKV RNA positive in both male, female and larvae of Culex quinquefasciatus Say and Aedes aegypti (L.) mosquitoes collected from active ZIKV infected patients' homes in Thailand. Therefore, the aims of this study were to investigate whether ZIKV could be vertically transmitted in Cx. quinquefasciatus, Ae. aegypti and Ae. albopictus. Laboratory and field colonies of these mosquito species were maintained and artificially fed with ZIKV in human blood. Fully engorged mosquitoes (F0) were selected and reared for the vertical transmission study. The subsequent mosquito generations were fed with human blood without the virus. ZIKV in the mosquitoes was detected by hemi-nested RT-PCR and sequencing. C6/36 cells were used to isolate ZIKV from samples that tested positive by hemi-nested RT-PCR. Moreover, ZIKV was identified by immunocytochemical staining 7 days after infection in several organs of infected F0 females, including the salivary glands, midguts, yoke granules and facet cells of the eye. The localization of the ZIKV antigen was identified by the presence of the specific antibody in the salivary glands, midguts, yoke granules and facet cells. ZIKV was detected in female and male Cx. quinquefasciatus until the F6 and F2 generations, respectively. The isolated virus showed cytopathic effects in C6/36 cells by 5 days postinfection. The results suggested that the vertical transmission of ZIKV occurs in Cx. quinquefasciatus in the laboratory. However, we were able to detect the presence of ZIKV in Ae. aegypti in only the F1 generation in both male and female mosquitoes, and Ae. albopictus mosquitoes were not able to vertically transmit the virus at all. Data obtained from this study could be valuable for developing a better understanding of the role of Cx. quinquefasciatus as a potential vector for ZIKV transmission in Thailand and may be useful in creating more effective mosquito vector control strategies in the future.

Molecular Epidemiology and Genetic Diversity of Zika Virus from Field-Caught Mosquitoes in Various Regions of Thailand.

Zika virus (ZIKV) infection is an emerging and re-emerging arbovirus disease that is transmitted to humans through the bite of infected mosquitoes. ZIKV infections were first described in Thailand in 1954 from the sera of indigenous residents and several travelers returning from Thailand in 2014. However, reported cases in Thailand have been increasing since 2015 and 2016, and epidemiological information about the vectors of ZIKV is unclear. We investigated the molecular epidemiology and genetic diversity of ZIKV from mosquitoes collected from different geographic regions experiencing ZIKV outbreaks in Thailand. Polymerase chain reaction was used to amplify the non-structural protein (NS5) gene of ZIKV, which was then sequenced. A total of 1026 mosquito samples (626 females, 367 males, and 33 larvae) were collected from active ZIKV patients' houses. ZIKV was detected in 79 samples (7.7%), including Aedes aegypti (2.24% female, 1.27% male, and 0.19% larvae), Culex quinquefasciatus (1.85% female, 1.66% male, and 0.29% larvae), and Armigeres subalbatus (0.1% female and 0.1% male), whereas no ZIKV was detected in Aedes albopictus. Phylogenetic analysis of the 79 positive samples were classified into two clades: Those closely related to a previous report in Thailand, and those related to ZIKV found in the Americas. This is the first report of the detection of ZIKV in Ae. aegypti, Cx. quinquefasciatus, and Ar. subalbatus mosquitoes, and genetic variations of ZIKV in the mosquitoes collected from several geographic regions of Thailand were examined. Detection of ZIKV in male and larval mosquitoes suggests that vertical transmission of ZIKV occurred in these mosquito species. This study provides a more in-depth understanding of the patterns and epidemiologic data of ZIKV in Thailand; the data could be used for future development of more effective prevention and control strategies of ZIKV in Thailand.