Identification of Phasi Charoen-Like Phasivirus in Field Collected Aedes aegypti from Karnataka State,India

Abstract

Background:

A wide range of insect-specific viruses (ISVs) have been reported worldwide. There are no studies from India that have reported ISVs. The current study describes the identification of Phasi Charoen-like virus (PCLV) from Aedes aegypti mosquito-pools from six districts of Karnataka state, India.

Materials and Methods:

During the Chikungunya virus (CHIKV) outbreak in the Bangalore Urban district in 2019, using conventional PCR, it was found that both human and mosquito samples were positive for CHIKV. For retrieve the complete genome sequence, mosquito samples were subjected to next generation sequencing (NGS) analysis and PCLV was also found. During 2019, as part of a vector-borne disease surveillance, we received 50 mosquito pool samples from 6 districts of the state, all of them were subjected to NGS to identify PCLV.

Results:

The A. aegypti mosquito-pools samples were subjected to the NGS platform that led to identification of an ISV, PCLV. PCLV was identified in 26 A. aegypti mosquito-pools collected from 6 districts. We also found mixed infection of PCLV with the Dengue virus (DENV; genotypes 1 and 3) and CHIKV from five pools. The nucleotide identity for the L gene of Indian PCLV sequences ranged between 97.1% and 98.3% in comparison with the Thailand sequences.

Conclusions:

To the best of our knowledge, this is the first report of PCLV dual infection with DENV and CHIKV in India. The present study confirms the presence of PCLV in A. aegypti mosquitoes from Karnataka state. The study adds India in the global geographical distribution of PCLV.

Introduction

Arthropod-borne viruses, mainly transmitted by mosquitoes, ticks, and midges, have become a significant public health concern as they cause significant mortality and morbidity among both humans and animals. Application of next-generation sequencing (NGS) technology and bioinformatic tools led to the finding that mosquitoes are naturally infected with a wide range of viruses (Iwashita et al. 2018). Over the past 15 years, many novel insect-specific viruses (ISVs) were discovered and reported from different parts of the world (Junglen et al. 2009, Quan et al. 2009, Yamao et al. 2009, Marklewitz et al. 2013, Vasilakis et al. 2013, Auguste et al. 2014, Chandler et al. 2014, Hobson-Peters et al. 2016). ISVs come under virus families traditionally associated with human arboviral pathogens, which include the Bunyavirales order and families such as Flaviviridae, Togaviridae, Reoviridae, Rhabdoviridae, and Phenuiviridae (Bolling et al. 2015, Nouri et al. 2018, Öhlund et al. 2019). India is one of the mega biodiversity countries in the world with a wide range of arthropod species that have evidenced multiple arthropod vector-borne diseases. To date, ISVs have not been identified or reported from any part of India. This study describes the accidental identification of an ISV in Aedes aegypti pools from six districts of Karnataka state, India.

During an outbreak in June 2019, in Mantapa village in Bangalore district, we found that both human and mosquito samples were positive for Chikungunya virus (CHIKV) by conventional PCR. We wanted to confirm the presence of CHIKV in both human and mosquito samples, so we subjected both samples to NGS analysis. During NGS, we accidentally found the Phasi Charoen-like virus (PCLV) in a mosquito sample. This had given us clue to screen for PCLV in all mosquito-pools samples collected during that period.

Materials and Methods

As a part of the vector-borne disease surveillance, mosquito samples from 50 pools were collected between June and August 2019 from Bangalore Urban, Bangalore Rural, Chikkaballapura, Kolar, Tumkur, and Ramanagara districts of Karnataka state. The state entomologist collected mosquito pool samples from suspect houses and the exterior of their house structure manually, using a mechanical aspirator. Inside the home, total catch was done using 2% pyrethrum extract spray. The state entomologist had identified and separated the different mosquito-pools species-wise and gender-wise. A. aegypti was identified using a hand lens or stereomicroscope based on its appearance, having a narrow and typically black body, unique patterns of light and dark scales on the abdomen and thorax, and alternating light and dark bands on the legs. A sample consisting of 10 to 20 mosquitoes was collected in a tube, where male and female mosquitoes were separated. Then, live mosquitoes were transported in test tubes plugged with cotton (soaked with glucose water) at ambient temperature within 2–4 h of collection.

Mosquitoes were frozen in liquid nitrogen and crushed with the help of a mortar and pestle. This was followed by homogenization in lysis buffer with proteinase K solution by pipetting. RNA extraction from mosquitoes was performed using the QIAamp^® Viral RNA Mini Kit (Qiagen, Hilden, Germany) with minor modifications (homogenized sample was passed twice in the extraction column). RNA was eluted in 50 μL of nuclease-free water. These were tested for Dengue virus (DENV) and CHIKV by RT-PCR. The actin-1 primer (Staley et al. 2010) was used as an internal control to test the quality of RNA from mosquito pool samples. The degenerate PCR primers (ChikF: forward primer 5′-CAACTTGCCCAGCTGATCTC-3′ and ChikR: reverse primer 5′-GGATGGCAAG ACTCCACTCT-3′) for CHIKV were designed based on the E gene segment from GenBank (accession no. AF369024) with support of an earlier report (Parida et al. 2007). RT-PCR for the primer pairs of DENV was carried out using standard primer pairs (Menting et al. 2011): DENVF: forward primer 5′-GGTTAGAGGAGACCCCTCCC-3′ and DENVR: reverse primer 5′-GAGACAGCAGGATCTCTGGTCT-3′. PCR conditions are maintained as per the following steps: a reverse transcription step at 50°C for 30 min, followed by 95°C for 15 min for predenaturation, and a 40-cycle repeat of denaturation at 95°C for 30 s, annealing at 55°C for 30 s, and an extension at 72°C for 30 s. The PCR fragment is separated with the agarose gel electrophoresis method and final stain visualized by the gel documentation system (Bio-Rad) to determine the band size.

To get the complete viral genome sequence from the mosquito samples, they were referred to the Indian Council of Medical Research-National Institute of Virology, Pune, for NGS analysis. Ribosomal depletion was carried out to remove host ribosomal RNA from the extracted RNA using the NEBNext^® rRNA Depletion Kit (New England Biolabs). RNA libraries were prepared from the samples received using the method described in earlier published literature (Yadav et al. 2019). These RNA libraries were quantified and loaded on the MiniSeq Illumina machine (Qiagen). The paired-end reads generated from the files were analyzed using the CLC Genomics Workbench, version 11.0. Reference-based mapping, as well as de novo assemblies, was performed to identify the agents present in the samples. A maximum likelihood tree was generated with a general time-reversible model and bootstrap replication of 1000 cycles using MEGA software, version 7.0 (Pennsylvania State University). Furthermore, percent nucleotide identity (PNI) was also retrieved using MEGA software.

Results

Of 50 A. aegypti mosquito pool samples subjected to the NGS platform, 26 revealed the presence of ISV. Analysis of the generated contigs led to identification of the Phasi Chareon-like virus (PCLV), an ISV in A. aegypti (Table 1).

Table 1.

Summary of the Chikungunya and Dengue Virus RT-PCR Tests from Mosquito Pools and Identified Virus using the Next-Generation Sequencing Analysis During Year 2019

Sr. no.	Mosquito ID	Mosquito species	No. of mosquitoes	Gender	Month of collection	District of collection	CHIKV RT-PCR results	DENV RT-PCR results	NGS analysis report
1	19-5119-1	Aedes aegypti	20:17	Female Male	June	Ramanagara	Neg	Neg	PCLV
2	19-5119-2	A. aegypti	11	Male	June	Ramanagara	Neg	Neg	PCLV
3	19-5177-1	A. aegypti	10	Male	June	Ramanagara	Neg	Neg	NA
4	19-5739-1	A. aegypti	12	Female	June	Bangalore Urban	Pos	Neg	NA
5	19-5739-2	A. aegypti	14	Female	June	Bangalore Urban	Pos	Neg	PCLV
6	19-5890-1	A. aegypti	10	Male	July	Kolar	Neg	Neg	NA
7	19-5897-1	A. aegypti	20:10	Female Male	July	Chikkaballapura	Neg	Pos	Dengue 1 (partial) and PCLV
8	19-5897-2	A. aegypti	11	Male	July	Chikkaballapura	Neg	pos	NA
9	19-6114-1	A. aegypti	20:20	Female Male	July	Chikkaballapura	Pos	Neg	Chikungunya and PCLV
10	19-6114-2	A. aegypti	10	Male	July	Chikkaballapura	Neg	Neg	PCLV
11	19-6330-1	A. aegypti	12	Female	July	Bangalore Urban	Neg	Neg	PCLV (partial)^a
12	19-6330-2	A. aegypti	10	Male	July	Bangalore Urban	Neg	Neg	NA
13	19-6330-3	A. aegypti	13	Female	July	Bangalore Urban	Neg	Neg	NA
14	19-6586-1	A. aegypti	20	Female	July	Bangalore Rural	Neg	Neg	NA
15	19-6586-2	A. aegypti	18	Male	July	Bangalore Rural	Neg	Neg	NA
16	19-6586-3	A. aegypti	20:11	Female Male	July	Tumkur	Neg	Neg	NA
17	19-6586-4	A. aegypti	16	Female	July	Chikkaballapura	Neg	Neg	NA
18	19-6586-5	A. aegypti	10	Male	July	Chikkaballapura	Pos	Neg	NA
19	19-6788-1	A. aegypti	11	Male	July	Bangalore Urban	Neg	Neg	PCLV
20	19-6788-2	A. aegypti	19	Female	July	Bangalore Urban	Neg	Neg	PCLV
21	19-6789-1	A. aegypti	10	Female	July	Bangalore Urban	Neg	Neg	NA
22	19-6904-1	A. aegypti	10	Male	July	Kolar	Pos	Neg	Chikungunya and PCLV
23	19-6904-2	A. aegypti	12	Female	July	Kolar	Neg	Neg	NA
24	19-6904-3	A. aegypti	16	Male	July	Kolar	Neg	Neg	NA
25	19-6945-1	A. aegypti	12	Female	July	Chikkaballapura	Neg	Neg	PCLV
26	19-6945-2	A. aegypti	20:11	Female Male	July	Bangalore Rural	Neg	Neg	NA
27	19-6945-3	A. aegypti	10	Male	July	Bangalore Rural	Neg	Neg	PCLV
28	19-7102-1	A. aegypti	12	Male	August	Bangalore Urban	Neg	Neg	NA
29	19-7168-1	A. aegypti	17	Female	August	Chikkaballapura	Neg	Neg	NA
30	19-7168-2	A. aegypti	14	Female	August	Chikkaballapura	Neg	Pos	Dengue 1 (partial) and PCLV
31	19-7168-3	A. aegypti	14	Male	August	Chikkaballapura	Neg	Neg	PCLV
32	19-7168-4	A. aegypti	19	Female	August	Chikkaballapura	Neg	Neg	PCLV
33	19-7168-5	A. aegypti	17	Female	August	Kolar	Neg	Neg	PCLV
34	19-7282-1	A. aegypti	10	Female	August	Tumkur	Neg	Neg	PCLV
35	19-7282-2	A. aegypti	13	Female	August	Tumkur	Neg	Pos	PCLV
36	19-7282-3	A. aegypti	17	Male	August	Tumkur	Neg	Neg	PCLV
37	19-7352-1	A. aegypti	13	Female	August	Chikkaballapura	Neg	Neg	NA
38	19-7352-2	A. aegypti	10	Male	August	Chikkaballapura	Neg	Neg	PCLV
39	19-7352-3	A. aegypti	20:13	Female Male	August	Chikkaballapura	Neg	Pos	Dengue 3 (partial) and PCLV
40	19-7352-4	A. aegypti	12	Male	August	Chikkaballapura	Neg	Neg	PCLV
41	19-7475-1	A. aegypti	20:18	Female Male	August	Kolar	Neg	Neg	PCLV
42	19-7475-2	A. aegypti	20:10	Female Male	August	Kolar	Neg	Neg	NA
43	19-7475-3	A. aegypti	15	Male	August	Kolar	Neg	Neg	NA
44	19-7475-4	A. aegypti	10	Female	August	Kolar	Neg	Neg	NA
45	19-7475-5	A. aegypti	12	Male	August	Kolar	Neg	Neg	PCLV
46	19-7475-6	A. aegypti	10	Female	August	Kolar	Neg	Pos	NA
47	19-7475-7	A. aegypti	14	Female	August	Kolar	Neg	Neg	NA
48	19-7475-8	A. aegypti	16	Female	August	Ramanagara	Neg	neg	NA
49	19-7475-9	A. aegypti	13	Male	August	Ramanagara	Neg	Neg	PCLV
50	19-7475-10	A. aegypti	11	Female	August	Ramanagara	Neg	Neg	PCLV

Partial sequence, hence not retrieved and submitted to GenBank.

NA, not available; CHIKV, Chikungunya virus; DENV, Dengue virus; NGS, next-generation sequencing; PCLV, Phasi Charoen-like virus.

NGS analysis of 50 mosquito-pools samples reports that CHIKV and PCLV were present together in 2 pools, Dengue serotype 1 and PCLV were present together in two pools, and Dengue serotype 3 and PCLV were present together in one pool. PCLV alone was identified in 21 pools. Twenty-four pools reported no viral RNA through NGS analysis. For details, refer Table 1.

Chikkaballapura districts reported 11 mosquito pools positive for PCLV, of them, 3 pools had Dengue and PCLV together and 1 pool had CHIKV and PCLV together. The Kolar district reported four mosquito pools positive for PCLV, of them, one pool had CHIKV and PCLV together. The Ramanagara district reported PCLV from four mosquito pools, Tumkur district reported three pools positive for PCLV, and Bangalore Urban and Rural districts reported PCLV from two mosquito pools each. Refer Table 1 and Fig. 1 for more information.

FIG. 1.

Karnataka state map showing mosquito pools positive for PCLV. The numbers in brackets indicate the number of mosquito pools found positive in each district. PCLV, Phasi Charoen-Like Virus.

Reference-based assembly was performed to retrieve the complete genome of the PCLV, DENV, and CHIKV. The percentages of relevant reads mapped and the genome retrieved are given in Table 2. Phylogenetic analysis was performed for Phasi Charoen-Like Virus (PCLV) (L, M, and S segments), DENV, and CHIKV using the reference sequences downloaded from GenBank. It was observed that the CHIKV retrieved from the current study clustered with the South/Eastern/Central African genotype of the CHIKV (Fig. 2B). It shared a PNI of 98.9% with the Indian CHIKV sequence (accession no. KX619422). The three DENV sequences clustered in two different groups, identifying two sequences to be of the DENV-1 genotype and one as DENV-3 (Fig. 2A). The DENV-1 sequences shared a PNI of ∼98.5% with the DENV-1 sequence of Singapore (accession no. MF033253). Figure 3A–C depicts the phylogenetic tree for the L, M, and S segments of the PCLV, respectively. It is observed that all these segments are clustered with the PCLV of the Thailand strain. The nucleotide identity for the PCLV L gene ranged between 97.1% and 98.3%, while for the M gene and S gene, it ranged between 96.4–97.8% and 95.3–97.3%, respectively. PNI values for the different segments of PCLVs retrieved during the study for the Thailand PCLV strain (accession no. KX619422) are given in Table 3.

FIG. 2.

Phylogenetic tree of DENV and CHIKV sequences retrieved from the study. The tree was built with the maximum likelihood method algorithm and sequences of our study, which was designated by the accession number (GenBank) reference sequences and our findings of sequences for (A) DENV and (B) CHIKV using the general time-reversible model. A bootstrap replication of 1000 cycles was performed to assess the statistical robustness. Indian sequences are marked in bold face. CHIKV, Chikungunya virus; DENV, Dengue virus.

FIG. 3.

Phylogenetic tree of PCLV segments retrieved from the study. The tree was built with the maximum likelihood method algorithm and sequences of our study, which was designated by the accession number (GenBank) reference sequences and our findings of sequences for (A) the PCLV L segment, (B) PCLV M segment, and (C) PCLV S segment using the general time-reversible model. A bootstrap replication of 1000 cycles was performed to assess the statistical robustness. Indian sequences are marked in bold face.

Table 2.

The Percentage of Relevant Reads and the Genome Retrieved for Different Segments of the Phasi Charoen-Like Virus with Respect to the Phasi Charoen-Like Virus from Thailand

Mosquito ID	Total reads	L gene			M gene			S gene
Mosquito ID	Total reads	Accession no.	Relevant reads	% Genome covered	Accession no.	Relevant reads	% Genome covered	Accession no.	Relevant reads	% Genome covered
19-5897-1	864612.0	MN866219	387	99.7	MN866244	415	98.7	MN866269	179	98.5
19-6114-1	756118.0	MN866220	1597	99.8	MN866245	1537	99.0	MN866270	1065	99.0
19-6904-1	785960.0	MN866221	2926	99.8	MN866246	6624	98.9	MN866271	3440	99.0
19-7168-2	839892.0	MN866222	3606	99.9	MN866247	5336	98.9	MN866272	2232	99.0
19-7282-2	843634.0	MN866223	12,618	99.8	MN866248	13,957	98.8	MN866273	6633	99.0
19-7352-3	945572.0	MN866224	4170	99.9	MN866249	6473	99.8	MN866274	4243	99.3
19-5119-1	1034250.0	MN866225	6617	100.0	MN866250	12,248	99.1	MN866275	2400	99.5
19-5119-2	886708.0	MN866226	5613	100.0	MN866251	9783	100.0	MN866276	2380	99.6
19-5890-1	938090.0	MN866243	4582	100.0	MN866252	6522	99.6	MN866277	2852	98.9
19-6114-2	832956.0	MN866227	714	99.8	MN866256	689	98.5	MN866278	371	98.5
19-6788-1	826010.0	MN866228	1831	99.4	MN866266	1201	98.8	MN866279	1102	98.6
19-6788-2	750042.0	MN866229	13,214	99.9	MN866253	9939	98.9	MN866280	3252	98.9
19-6945-1	681210.0	MN866230	3288	99.7	MN866263	5180	98.8	MN866281	2126	99.6
19-6945-3	656534.0	MN866231	5667	99.8	MN866260	7752	98.8	MN866282	2907	99.5
19-7282-3	682946.0	MN866232	12,130	99.9	MN866264	11,979	98.9	MN866283	11115	99.0
19-7352-2	675574.0	MN866242	5935	99.8	MN866259	7118	100.0	MN866284	2765	99.6
19-7352-4	755826.0	MN866233	4001	99.7	MN866258	4889	98.9	MN866285	3639	99.0
19-7475-1	951588.0	MN866234	14,618	99.9	MN866268	29,593	98.9	MN866286	24935	99.7
19-7475-5	912200.0	MN866235	3074	99.7	MN866255	4191	98.8	MN866287	2776	99.7
19-7475-9	786314.0	MN866236	6327	99.8	MN866265	12,649	98.9	MN866288	2648	98.7
19-7475-10	790222.0	MN866237	4667	99.9	MN866254	4478	98.8	MN866289	3412	99.0
19-7168-3	840468.0	MN866238	4892	99.9	MN866257	5780	99.1	MN866290	2085	99.9
19-7168-4	721300.0	MN866239	1487	99.7	MN866261	1811	98.8	MN866291	935	98.7
19-7168-5	789084.0	MN866240	2482	99.8	MN866267	3273	98.9	MN866292	3409	98.8
19-7282-1	3362808.0	MN866241	24,923	99.9	MN866262	13,861	98.9	MN866293	7164	99.6

Table 3.

Percent Nucleotide Identity of the Indian Phasi Charoen-Like Virus with Respect to the L, M, and S Segments of the Phasi Charoen-Like Virus from Thailand, 2008

Accession no. of the L gene	KM001085.1	Accession no. of the M gene	KM001086.1	Accession no. of the S gene	KM001087.1
MN866241	97.4	MN866262	97.8	MN866293	97.3
MN866240	97.1	MN866267	96.8	MN866292	96.4
MN866239	96.8	MN866261	97.0	MN866291	95.3
MN866238	97.0	MN866257	97.3	MN866290	97.1
MN866237	96.4	MN866254	96.6	MN866289	96.4
MN866236	96.9	MN866265	96.9	MN866288	95.6
MN866235	97.2	MN866255	96.9	MN866287	96.9
MN866234	97.1	MN866268	97.2	MN866286	96.5
MN866233	96.4	MN866258	96.7	MN866285	95.8
MN866242	96.2	MN866259	96.7	MN866284	96.5
MN866232	96.6	MN866264	96.9	MN866283	96.5
MN866231	96.9	MN866260	96.2	MN866282	95.6
MN866230	96.8	MN866263	96.9	MN866281	95.3
MN866229	96.5	MN866253	96.4	MN866280	96.5
MN866228	97.3	MN866266	96.8	MN866279	96.4
MN866227	95.7	MN866256	96.7	MN866278	96.6
MN866243	97.2	MN866252	96.8	MN866277	96.5
MN866226	96.8	MN866251	96.9	MN866276	95.6
MN866225	96.8	MN866250	96.9	MN866275	95.6
MN866224	96.6	MN866249	96.7	MN866274	96.1
MN866223	96.7	MN866248	95.0	MN866273	96.5
MN866222	96.8	MN866247	95.1	MN866272	96.0
MN866221	96.5	MN866246	96.0	MN866271	96.7
MN866220	96.3	MN866245	94.6	MN866270	96.4
MN866219	96.7	MN866244	95.2	MN866269	94.7
NC_040494.1_Kaisodi_virus_isolate_G14132	36.6	NC_040493.1_Kaisodi_virus_isolate_G14132	28.3	NC_040492.1_Kaisodi_virus_isolate_G14132	31.0
NC_038262.1_Phasi_Charoen-like_virus_isolate_Rio	96.2	NC_038261.1_Phasi_Charoen-like_virus_isolate_Rio	96.2	NC_038263.1_Phasi_Charoen-like_virus_isolate_Rio	42.9
NC_038257.1_Badu_virus_isolate_TS6347	58.5	NC_038258.1_Badu_virus_isolate_TS6347	50.0	NC_038259.1_Badu_virus_isolate_TS6347_segment_S_complete_sequence	49.7
NC_031317.1_Wutai_Mosquito_virus_strain_QN3-5	55.5	NC_031318.1_Wutai_Mosquito_virus_strain_QN3	51.0	NA	NA
NC_031138.1_Huangpi_Tick_virus_2_strain_H114-17	36.5	NC_031139.1_Huangpi_Tick_virus_2_strain_H114-17	28.2	NA	NA
NC_027140.1_Bhanja_virus_strain_ibAr2709	35.0	NC_027141.1_Bhanja_virus_strain_ibAr2709	28.9	NC_027142.1_Bhanja_virus_strain_ibAr2709	28.5
NC_005214.1_Uukuniemi_virus	35.9	NC_005220.1_Uukuniemi_virus	27.0	NC_005221.1_Uukuniemi_virus	36.0
MN053781.1_strain_Ab-AAM_Guadeloupe_2016	87.3	MN053782.1_strain_Ab-AAM_Guadeloupe_2016	96.0	MN053783.1_strain_Ab-AAM_Guadeloupe_2016	84.6
MN053778.1_strain_Ab-AAF_Guadeloupe_2016	87.3	MN053779.1_strain_Ab-AAF_Guadeloupe_2016	95.9	MN053780.1_strain_Ab-AAF_Guadeloupe_2016	93.4
MN053775.1_strain_PB-AAF-5_Guadeloupe_2017	86.8	MN053776.1_strain_PB-AAF-5_Guadeloupe_2017	95.5	MN053777.1_strain_PB-AAF-5_Guadeloupe_2017	59.7
MN053772.1_strain_PB-AAF-1-5_Guadeloupe_2017	86.8	MN053773.1_strain_PB-AAF-1-5_Guadeloupe_2017	95.0	MN053774.1_strain_PB-AAF-1-5_Guadeloupe_2017	94.8
MN053769.1_strain_PB-AAF-1-4_Guadeloupe_2017	96.1	MN053770.1_strain_PB-AAF-1-4_Guadeloupe_2017	94.9	MN053771.1_strain_PB-AAF-1-4_Guadeloupe_2017	60.5
MN053766.1_strain_PB-AAF-1-3_Guadeloupe_2017	86.6	MN053767.1_strain_PB-AAF-1-3_Guadeloupe_2017	95.7	MN053768.1_strain_PB-AAF-1-3_Guadeloupe_2017	87.9
MN053763.1_strain_Ab-AAM-1-5_Guadeloupe_2017	86.3	MN053764.1_strain_Ab-AAM-1-5_Guadeloupe_2017	85.9	MN053765.1_strain_Ab-AAM-1-5_Guadeloupe_2017	89.7
MN053760.1_strain_Ab-AAM-1-3_Guadeloupe_2017	87.3	MN053761.1_strain_Ab-AAM-1-3_Guadeloupe_2017	95.1	MN053762.1_strain_Ab-AAM-1-3_Guadeloupe_2017	90.9
MN053757.1_strain_Ab-AAF-1-5_Guadeloupe_2017	87.4	MN053758.1_strain_Ab-AAF-1-5_Guadeloupe_2017	95.5	MN053759.1_strain_Ab-AAF-1-5_Guadeloupe_2017	94.1
MN053754.1_strain_Ab-AAM-5_Guadeloupe_2017	87.2	MN053755.1_strain_Ab-AAM-5_Guadeloupe_2017	95.0	MN053756.1_strain_Ab-AAM-5_Guadeloupe_2017	92.3
MN053751.1_strain_PB-AAM-5_Guadeloupe_2017	96.2	MN053752.1_strain_PB-AAM-5_Guadeloupe_2017	95.3	MN053753.1_strain_PB-AAM-5_Guadeloupe_2017	93.6
MN053748.1_strain_Ab-AAM-1-4_Guadeloupe_2017	87.4	MN053749.1_strain_Ab-AAM-1-4_Guadeloupe_2017	95.5	MN053750.1_strain_Ab-AAM-1-4_Guadeloupe_2017	94.5
MN053745.1_strain_Ab-AAF-5_Guandeloupe_2017	96.1	MN053746.1_strain_Ab-AAF-5_Guadeloupe_2017	95.8	MN053747.1_Ab-AAF-5_Guadeloupe_2017	92.7
KR003786.1_Brazil_2012	96.2	KR003784.1_Brazil_2012	96.2	MH310081.1_isolate_CC_A_USA_2015	94.7

Discussion

Karnataka state is a biodiversity hotspot, with a high prevalence of various mosquito species. Vector-borne diseases such as Dengue and Chikungunya are reported every year in this state (Yergolkar et al. 2006, Lakshmi et al. 2008). During June to August 2019, mosquito pool samples were collected from six southern districts of Karnataka state as part of the surveillance. After conventional PCR detection of CHIKV and DENV in mosquito samples, we subjected the same samples to complete genome sequence analysis for identifying CHIKV and DENV; interestingly, we identified PCLV.

For decades, insect-specific bunyaviruses have been gaining attention in various parts of the globe with increased newer virus detection among mosquito species. Among Aedes species, new ISVs identified were the Cumuto and Wallerfield viruses from Trinidad; Dezidougou, Jonchet, Ferak, and Cavally viruses from Côte d'Ivoire; Badu virus from Australia; Guapiacu virus from Brazil; Phasi Charoen virus; and PCLV from Thailand (Junglen et al. 2009, Yamao et al. 2009, Vasilakis et al. 2013, Auguste et al. 2014, Chandler et al. 2014, Marklewitz et al. 2015, Hobson-Peters et al. 2016, de Oliveira Ribeiro et al. 2021).

Accidental identification of the PCLV suggests the potential of unidentified viruses in Karnataka state, India. Studies have reported high prevalence of ISVs in Aedes species, such as the novel flavivirus in the Caribbean and PCLV in Grenada, suggesting the presence of high diversity of unknown viruses in mosquito species (Jeffries et al. 2020, Ramos-Nino et al. 2020). Several studies have reported that insect-specific bunyaviruses such as PCLV have been identified in Aedes mosquito species (Schultz et al. 2018, Shi et al. 2019, Ramos-Nino et al. 2020). In vitro studies have shown PCLV growth in Aedes cell lines (Aa23 cells) (Schultz et al. 2018). Studies from mosquito surveillance and in vitro studies have reported that PCLV and arbovirus together can infect Aedes species (Schultz et al. 2018, Shi et al. 2019, Ramos-Nino et al. 2020). In the present study, all PCLVs were identified from Aedes mosquito pools.

Mosquito pools from Kolar and Chikkaballapura districts have reported both PCLV and CHIKV, and pools from the Chikkaballapura district also reported PCLV and Dengue together. A study on mosquito surveillance from Brazil reported that the Aedes mosquito pool was positive for PCLV and CHIKV (Cunha et al. 2020). In vitro studies with Aedes (Aa23) cell lines have reported dual infection of PCLV and DENV (Schultz et al. 2018). There are several laboratory studies related to newly identified ISV interaction with arboviruses in mosquitoes, trying to understand dual-host tropism (Kenney et al. 2014, Goenaga et al. 2015, Nasar et al. 2015, Jeffries et al. 2020). However, it is not understood whether the newly identified ISVs in mosquito species are hidden arboviruses that may gain potential or lose the ability to infect vertebrate hosts (Calisher and Higgs 2018, Jeffries et al. 2020). The evolutionary relationship between ISVs and arboviruses is not quite understood. The present study findings broaden our understanding of the diversity and geographical distribution of PCLVs and their associations with DENV and CHIKV in Aedes mosquito species in our country.

Conclusions

This study reports first-time identification of PCLV in India as well as PCLV cocirculation with DENV and CHIKV in Aedes mosquitoes. Detailed exploration of the presence of ISVs in other parts of the state and country is crucial to know the distribution patterns, differences in genetic sequences, and their ancestral origins. Further in vitro studies on wild Aedes mosquitoes are needed to assess the impact of PCLV on the mosquito life cycle and vector competency.

Footnotes

Acknowledgments

The authors gratefully acknowledge the epidemiologist and entomologist from the Directorate of Health and Family Welfare Services, Bangalore, Karnataka state for their support in the collection of the mosquito-pools. The authors would also like to acknowledge Ms. Rutuja Kharde for the technical help provided for sequence submission. Authors are thankful to Dr. Atanu Basu, Scientist ‘G’ and Head of the Department, Electron Micropsy, and Dr. Alagarasu, Scientist “D,” Dengue Department, ICMR-National Institute of Virology Pune for providing critical comments on the manuscript.

Authors' Contributions

A.M., P.D.Y., and D.P.S. conceived, planned, and designed the study; A.M., D.A.N., P.D.Y., M.R., S.P., T.M., S.M., M.M.J., and P.O.P. were involved in collecting information, summarizing data, and writing the draft; A.M., P.D.Y., S.M., M.M.J., and D.P.S. were involved in critical analysis and correction of the draft; and A.M., D.A.N., P.D.Y., M.R., S.P., T.M., S.M., D.P.S., M.M.J., and P.O.P prepared the final draft. All authors approved the final draft of the manuscript.

Author Disclosure Statement

No conflicting financial interests exist.

Funding Information

Financial support was provided by intramural funding from the Indian Council of Medical Research-National Institute of Virology, Pune, India. The idea and concept are the sole responsibility of the authors and do not represent the views of the funding agency.

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