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.

Introduction Chikungunya virus (CHIKV) is an emerging and re-emerging infection that has occurred in many countries over several continents, including the Americas, eastern Africa, the eastern Indian Ocean islands, the western Indian Ocean islands, France, Italy, India, Singapore, Malaysia, Indonesia, and Thailand [1,2]. In Thailand, the reported of CHIKV outbreaks in the southern part of the country from the Bureau of Epidemiology, Department of Disease Control, Ministry of Public Health in 2008-2009 were affected more than 54,000 cases and the genetic studies found a characteristic alanine-to-valine mutation at amino acid residue 226 (A226V) of the E1 envelope glycoprotein of an East/Central/South/Africa (ECSA) genotype [3]. In 2013, from 109 blood samples of suspected patients obtained from Bueng Kan province, northeastern region of Thailand, 51 samples had evidence of CHIKV infection. Genetic characterization with 4 samples of this outbreak showed that CHIKV was belonged to the Indian Ocean Lineage (IOL) with A226V of an ECSA genotype [4]. Recently, outbreaks of CHIKV in 2020 have spread rapidly in Thailand, especially in metropolitan Bangkok, the capital of the country. The Bureau of Epidemiology, Ministry of Public Health, Thailand, described that a total of 10,905 cases of CHIKV infection were reported from 72 out of 77 provinces in 2020 [5]. Interestingly, 1,061 cases of CHIKV infection were investigated from Bangkok during this outbreak because only a few cases of CHIKV infection have been reported in Bangkok over the past decade. Chansaenroj et al. (2020) demonstrated that the complete genome sequences of CHIKV isolated from patients and an Aedes aegypti mosquito from this outbreak in southwest Bangkok belonged to an East/Central/South African (ECSA) lineage, with a mutation at A226V. However, the outbreak of CHIKV in 2019 was grouped into the ECSA genotypes but showed differences strains from previous outbreaks in Thailand in 2008-2009 and 2013 [6]. The Ae. aegypti mosquito is considered the main vector of CHIKV in this outbreak [7]. In addition, CHIKV RNA has been detected in both sexes of the vector mosquitoes, Ae. aegypti (L.) and Ae. albopictus Skuse [8]. Chompoosri et al. (2016) proved that IOL CHIKV could be transmitted vertically in both Ae. aegypti and Ae. albopictus under laboratory conditions up to F5 and F6 generations, respectively by using one-step qRT-PCR and CHIKV isolation in LLC-MK2 cell line [9]. However, several reports showed that CHIKV can be detected in Culex spp. mosquitoes. For example, glycerinated Culex mosquitoes in Newala district of Tanganyika [10] and Cx. quinquefasciatus in Reunion Island [11] have been found infected with CHIKV by virus isolation in C6/36 cells, RT-PCR, and sequencing. CHIKV has been isolated from female ornithophilic Cx. ethiopicus Edwards collected from an African field study [12,13]. In contrast, Cx. p. quinquefasciatus are not able to transmit the virus experimentally, with no infection detected in 19 mosquitoes by feeding on viremic vervet monkeys [14]. However, no studies have investigated the transmission capacity of Thai Cx. quinquefasciatus for CHIKV. Therefore, we studied Cx. quinquefasciatus from outbreak in Bangkok 2019 in the same area of CHIKV-positive male and female Ae. aegypti. The Cx. quinquefasciatus mosquito coexists with Ae. aegypti in urban and rural areas and may contribute to CHIKV transmission. In addition, wild-caught Cx. quinquefasciatus were evaluated for CHIKV using a nested-RT-PCR assay targeting the conserved E1 envelope protein genes, and blood-fed mosquitoes were evaluated by PCR. Finally, the replication of CHIKV was studied in laboratory strains of Cx. quinquefasciatus by using nested-RT-PCR, virus isolation in C6/36 cells, and immunofluorescence assay.

Ethics statement
The study was approved by the animal research ethics committee of Chulalongkorn University and adhered to the Animal Care and Use Protocol (CU-ACUP

CHIKV detection in field-caught Cx. quinquefasciatus by nested-RT-PCR
Individual whole mosquitoes were distinguished based on species and sex and were placed in 1.5 ml tubes with 1X phosphate-buffered saline (PBS). Each mosquito sample was ground in 300 μl of 1XPBS and the samples were centrifuged at 11000 g for 5 min. Then, 200 μl of the supernatant was mixed with 200 μl of 2X Minimum Essential Medium Eagle (MEM) medium (HyClone, USA) containing 2% heat-inactivated FBS (Gibco, USA), 2% penicillin (100 U/ml) and streptomycin (100 μg/ml) (Sigma-Aldrich, USA) for virus isolation, and the pellet was processed for viral RNA extraction using the Invisorb Spin Virus RNA Mini viral RNA extraction kit (STRATEC Molecular GmbH, Germany) according to the manufacturer's protocol." The extracted RNA samples were used immediately for CHIKV detection at the E1 region of the envelope protein genes, and the samples were maintained for long-term storage at -80˚C. Virus detection was performed by nested-RT-PCR as described by Intayot et al. (2019) [7]. In brief, the first amplification was performed using the Superscript III one-step RT-PCR kit (Invitrogen, USA) in a final volume of 25 μl of reaction mixture, containing 12.5 μl of 2X reaction mix, 0.7 μl of 10 μM each primer, 1 μl of SuperScript III RT/Platinum Taq Mix, 4.1 μl of sterile nuclease-free water, and 6.0 μl of RNA template. The reaction began with initial denaturation at 50˚C for 30 min, denaturation at 95˚C for 15 min, and followed by 40 cycles of 95˚C for 1 min, 64˚C for 1 min, 72˚C for 1 min, and the final extension at 72˚C for 10 min. The second round amplification, 2 μl of the first amplification step were amplified with the different set of primers in a final volume of 25 μl, consisted of 2.5 μl of 10X buffer, 2.5 μl of 25 mM MgCl 2 , 2.5 μl of 2 mM dNTP, 0.4 μl of 10 μM each primer, 0.2 μl of 1 U Taq polymerase (Thermo Fisher Scientific, USA), and 14.5 μl of sterile nuclease-free water. The reaction mixture was incubated at 95˚C for 3 min, followed by 40 cycles of 95˚C for 30 sec, 62˚C for 30 sec, 72˚C for 1 min, and the final step at 72˚C for 7 min. The amplified PCR products were electrophoresed through a 1.5% agarose gel, stained with ethidium bromide, and then visualized with Quantity one Quantification Analysis Software Version 4.5.2, Gel Doc EQ System (Bio-Rad, USA).

DNA cloning and sequencing
The PCR amplicons of CHIKV were directly ligated to pGEM-T Easy Vector (Promega, USA) using T4 DNA ligase, following the manufacturer's protocols. The recombinant DNA were transformed into competent of Escherichia coli DH5α cell and then screened the bacterial transformants using the blue-white colony system. The colonies suspected to carry the DNA fragment were cultured, and the plasmid DNA was isolated using the Invisorb Spin Plasmid Mini Kit (STRATEC molecular GmbH, Germany) according to the manufacturer's instructions. The purified plasmids were sent to a commercial company (Macrogen, South Korea) for direct DNA sequencing. Nucleotide sequences were analyzed by comparison with the Gen-Bank database using a BLAST program.

Phylogenetic analysis
The nucleotide sequences were aligned using BioEdit Sequence Alignment Editor Version 7.2.5 [15]. Phylogenetic trees were generated using the maximum-likelihood method with IQ-TREE on the IQ-TREE web server (http://iqtree.cibiv.univie.ac.at/) with 1000 ultrafast bootstrap replicates. The best fit model of substitution was identified using the auto function on the IQ-TREE web server [16].

CHIKV infection of mosquitoes and virus detection in mosquito tissues
Indian Ocean Lineage (IOL) CHIKV isolated from a Thai patient during a recent outbreak in central (PCHIK2019) Thailand was used in this study. The procedure for virus propagation and virus titration in the C6/36 cell line followed the methods described by Potiwat et al. (2011) [17]. Laboratory colonies of Ae. aegypti (generation > F 250 ) and Cx. quinquefasciatus (generation > F 300 ) mosquitoes were maintained under standard conditions as follows: 28 ± 2˚C, 65-85% relative humidity, and a 12/12-hour light/dark cycle. For CHIKV infection, 9.2×10 6 PFU/ml of PCHIK2019 from expired human blood from deidentified donors [18], which was obtained from the National Blood Center, Thai Red Cross Society, Bangkok, Thailand, was used. Five-day-old starved females Cx. quinquefasciatus and Ae. aegypti were allowed to feed on the blood containing CHIKV through an artificial apparatus for 60 min. Engorged females were maintained in a cage provided with a 5% sucrose and 5% vitamin B complex (w/v). Two days after the infectious blood meal, a black plastic bowl containing water was placed into the mosquito cage for 3 days to allow for oviposition. Fourteen mosquitoes were dissected to collect the midguts, ovaries, and salivary glands at 7, 10, 14, and 21 days post infection (dpi) for evaluation using immunofluorescence assay (IFA). After dissection, other tissues were individually transferred to a 1.5 ml microcentrifuge tube with 300 μl of 1XPBS following the above protocol for RNA extraction and virus isolation.

Evaluation of CHIKV RNA detection in blood-fed laboratory Cx. quinquefasciatus strain
Female Cx. quinquefasciatus or Ae. aegypti were allowed to feed on blood containing CHIKV (PCHIK2019) at 9.2×10 6 PFU/ml through an artificial feeding apparatus. Fully fed females were moved to a new cage and placed in an environmentally controlled room. Ten engorged mosquitoes were collected per day for virus detection. Individual mosquitoes were ground in 300 μl of 1XPBS, and the pellet was processed for viral RNA extraction. Two hundred microliters of the supernatant were mixed with 200 μl of 2X MEM medium and used for virus isolation. Mosquitoes were evaluated for CHIKV using nested-RT-PCR every day until the CHIKV RNA could not be detected in Cx. quinquefasciatus.

CHIKV detection and isolation in field-caught Cx. quinquefasciatus
We conducted CHIKV surveillance in mosquitoes collected in and around houses of confirmed CHIKV-infected patients at Chom Thong District, Bangkok, Thailand. A total of 43 Cx. quinquefasciatus adults were collected (16 males and 27 females) and Ae. aegypti were reported by Intayot et al. (2019) [7]. Eight females were positive for CHIKV RNA using nested-RT-PCR. Interestingly, all 8 positive samples showed undigested blood in the abdomen. A total of 8 positive samples were performed the sequences. All sequences consisted of 539 nucleotides in length. The BLAST result showed that the sequence also similar to CHIKV isolated from female Ae. aegypti in Bangkok, Thailand with 99-100% identity and full 100% coverage of the nucleotide sequences (Accession no. MN114297). All sequences of CHIKV in Cx. quinquefasciatus were submitted to the GenBank database under accession numbers MW271620-MW271627. The result of phylogenetic tree revealed that these CHIKV sequences were classed into the Indian Ocean clade, which belonged to the ECSA genotype ( Fig 1A); moreover, no presence of the E1: A226V in Indian Ocean clade of CHIKV showed in present study (Fig 1B). However, CHIKV isolation from the field-caught Cx. quinquefasciatus indicated no CPE at days 7, 10, and 14 in all positive samples. These findings suggested that there

PLOS ONE
No evidence of CHIKV replication in Cx. quinquefasciatus was insufficient evidence to conclude that there was no replication of CHIKV in field-caught Cx. quinquefasciatus. Therefore, we studied the evidence of IOL CHIKV infection in Cx. quinquefasciatus in laboratory conditions compared to Ae. aegypti.

Cx. quinquefasciatus compared to Ae. aegypti
We investigated the replication and distribution of IOL CHIKV in midguts, ovaries, salivary glands, and other tissues of Cx. quinquefasciatus and Ae. aegypti on various days post infection (7, 10, 14, 21 dpi). Replication and distribution of CHIKV were not observed in the midguts (Fig 2A), salivary glands (Fig 2B), ovaries (Fig 2C), and other tissues of Cx. quinquefasciatus by nested-RT-PCR, virus isolation, and IFA. In contrast, CHIKV infection was detected in the midguts, ovaries, salivary glands, and other tissues of Ae. aegypti as early as 7 dpi by nested-RT-PCR, virus isolation, and IFA. The maximum infection rate (midgut) and dissemination rate (other tissues) were found at 14 dpi and 10 dpi, respectively. For IFA assay, we examined the presence of CHIKV antigen in the cytoplasm of midgut cells at the posterior part as well as in gut-associated tracheoles at 7-21 dpi (Fig 2D). For transmission, CHIKV was first clearly detected in lateral lobes and the median lobe of the salivary glands at 7-21 dpi by IFA. All distal lobes of salivary glands showed the presence of some disrupted CHIKV and showed CHIKV antigens in cells of the salivary duct (Fig 2E), whereas we could not detect morphological damage in the proximal part of the lateral lobes. While vertical transmission was not found in Cx. quinquefasciatus, CHIKV antigen was visible in cells of CHIKV-infected ovaries and oviducts

PLOS ONE
No evidence of CHIKV replication in Cx. quinquefasciatus of Ae. aegypti, and CHIKV infection was demonstrated in a small number of oocytes in ovaries at 10 dpi (Fig 2F). From the other tissues, we separated the supernatant with MEM medium for virus culture in C6/36 cells and examined them by IFA. The results revealed that no evidence of CHIKV replication or CPE in cells treated with supernatant from Cx. quinquefasciatus (Fig 3A), whereas cells treated with supernatant from Ae. aegypti showed CPE at 7dpi ( Fig  3B).

Evaluation of CHIKV detected in the laboratory Cx. quinquefasciatus strain after CHIKV infected blood feeding
At days 1-3 after infection, the undigested blood in the abdomen of each individual Cx. quinquefasciatus was observed. Interestingly, by nested-RT-PCR, CHIKV RNA in Cx. quinquefasciatus was detected until 4 dpi, although undigested blood was not observed in all samples. CPE appeared in C6/36 cells at day 1 after infection from 1/10 (10%) Cx. quinquefasciatus samples, but infections from Ae. aegypti samples showed CPE since day 1 after CHIKV infection in all samples until up to 21 days (Table 1). This study concludes that CHIKV particles are therefore digested by the Cx. quinquefasciatus alongside the actual blood meal and are not replicated in C6/36 cells. However, we found clear evidence of replication of CHIKV in Ae. aegypti.

PLOS ONE
No evidence of CHIKV replication in Cx. quinquefasciatus quinquefasciatus are potential vectors for several diseases, such as Japanese encephalitis, chikungunya, Zika fever, West Nile fever, yellow fever, and filariasis [19,20]. Cx. quinquefasciatus is widely distributed in urban and rural areas of Thailand and shares similar patterns of habitats with Ae. aegypti during their life cycle because they have aquatic and terrestrial stages [21]. In this study, we collected Cx. quinquefasciatus from the same outbreak area in Bangkok, 2019, where CHIKV was detected in male and female Ae. aegypti [6,7]. Among 43 samples of Cx. quinquefasciatus, CHIKV RNA was detected in 8 female samples. We tried to demonstrate the viability of the virus in all nested-RT-PCR-positive samples through infection in C6/36 cells, but CPE was not observed. The supernatant collected from infected cells was also negative by nested-RT-PCR at different time-points. Several reports have suggested that Culex species clearly demonstrate the potential to be a vector for CHIKV, such as glycerinated Culex mosquitoes in Newala district of Tanganyika [10], Cx. quinquefasciatus in Reunion Island [11], and Cx. ethiopicus Edwards in an African field study [12,13]. Recently, Ribeiro Cruz et al.
(2020) revealed that CHIKV was isolated in pools of Ae. aegypti females and male and in Cx. quinquefasciatus females (n = 2); however, this report cannot speculate and assume that Cx. quinquefasciatus is participating in the CHIKV transmission. Moreover, the limitation of the study was not performed of salivary gland analysis, which could determine of vector competence [22]. In contrast, reports have shown evidence of a lack of competence of Cx. p. quinquefasciatus for CHIKV [14]. Laboratory studies were conducted with Cx. quinquefasciatus infected with IOL CHIKV isolated from a patient. The CHIKV isolate was not able to replicate in Cx. quinquefasciatus by virus isolation and IFA, and viral RNA was not detected by nested-PCR, in contrast to Ae. aegypti mosquitoes, which were used as a positive control. Moreover, we evaluated CHIKV in blood feed of female Cx. quinquefasciatus compared with Ae. aegypti. Undigested blood could be observed until 3 dpi in the abdomens of the mosquitoes, while CHIKV RNA in Cx. quinquefasciatus was detected until 4 dpi using nested-RT-PCR. In addition, CPE at day 4 was shown in 1 sample at day 1 after infection. There have been reports on the duration of blood nucleic acid detection in mosquitoes, such as 7 days post feeding in Cx. tarsalis Coquillett [23], up to 3 days after feeding in Cx. p. pipiens L. [24], only up to 2 days after feeding in Anopheles stephensi mosquito [25], and 3 days post feeding in Ae. aegypti [26]. However, the differences in the duration of detection of blood nucleic acid in mosquitoes depend on different digestive processes and mosquito species. We hypothesized that the positive results of CHIKV detection and CPE in Cx. quinquefasciatus during 1-4 dpi were due to the live virus remaining in the undigested infectious blood meal rather than CHIKV replication in Cx. quinquefasciatus.
Current virus detection techniques such as RT-PCR for the detection of viral genome, virus isolation or cell culture for infectious viral particle titration, and IFA for detection of viral antigen and for visualization and localization of target virus have been used for CHIKV detection in human samples [27]. In this study, we used E1 nested-RT-PCR, virus isolation, and IFA techniques to demonstrate CHIKV in a mosquito cell line and in the mosquitoes. This modified IFA protocol is simple, rapid and versatile for demonstration of the virus antigen and viral growth in infected cell and mosquitoes. This technique can also confirm true CHIKV infection and replication in mosquitoes. The IFA can also be used to detect CHIKV shortly after the initiation of infection in the mosquito midgut and demonstrate the virus dissemination in the mosquitoes. Therefore, IFA can be used as a tool for investigating CHIKV transmission in mosquitoes.
Our studies provided information to better understand the vector competence for current circulating CHIKV. Our findings indicated that Cx. quinquefasciatus is not a competent vector of CHIKV in Thailand. We assumed that CHIKV-infected female Cx. quinquefasciatus in the field might be maintaining the virus in patient blood because mosquitoes were collected from active CHIKV infected patients' homes. A report from Florida demonstrated CHIKV infection in Cx. p. quinquefasciatus, but it showed poor vector competence under laboratory conditions, with no transmission and dissemination of CHIKV in the mosquito [28]. This finding has important implications, as CHIKV-infected mosquitoes may not be able to transmit the virus. McIntosh et al. (1963) suggested that CHIKV infection and transmission did not occur in Cx. quinquefasciatus [29]. However, several factors could have affected the competency of the mosquitoes for virus transmission, such as the genetic variations in vectors between mosquito species, virus strains, doses of the virus for infection, and mosquito immunity and defense mechanisms [30][31][32]. The current study suggests that we can detect CHIKV RNA from fieldcollected Cx. quinquefasciatus, but transmission of virus does not occur in Cx. quinquefasciatus strains from Thailand. This Cx. quinquefasciatus species does not have competency to transmit CHIKV in either field or laboratory conditions. The real mechanism for why CHIKV is not able to replicate in Cx. quinquefasciatus should be investigated in the future for an improved understanding of virus-vector interaction.