Deciphering the Virome of Culex vishnui Subgroup Mosquitoes, the Major Vectors of Japanese Encephalitis, in Japan

Abstract

Japanese encephalitis (JE) remains a public health concern in several countries, and the Culex mosquito plays a central role in its transmission cycle. Culex mosquitoes harbor a wide range of viruses, including insect-specific viruses (ISVs), and can transmit a variety of arthropod-borne viruses (arboviruses) that cause human and animal diseases. The current trend of studies displays enhanced efforts to characterize the mosquito virome through bulk RNA sequencing due to possible arbovirus–ISV interactions; however, the extent of viral diversity in the mosquito taxon is still poorly understood, particularly in some disease vectors. In this study, arboviral screening and RNA virome analysis of Culex tritaeniorhynchus and C. pseudovishnui, which are part of the Culex vishnui subgroup mosquitoes, were performed. Results from these two mosquito species, known as the major vectors of JE virus (JEV) in Asia, collected in three prefectures in Japan were also compared with the sympatric species C. inatomii. A total of 27 viruses, including JEV, were detected from these Culex mosquitoes. Molecular and phylogenetic analyses of the detected viruses classified 15 of the 27 viruses as novel species, notably belonging to the Flaviviridae, Rhabdoviridae, Totiviridae, and Iflaviridae families. The successful isolation of JEV genotype I confirmed its continuous presence in Japan, suggesting the need for periodic surveillance. Aside from JEV, this study has also reported the diversity of the RNA virome of disease vectors and broadened the knowledge on mosquito virome profiles containing both arbovirus and ISV. Mosquito taxon seemed to contribute largely to the virome structure (e.g., virome composition, diversity, and abundance) as opposed to the geographical location of the mosquito species. This study therefore offers notable insights into the ecology and evolution of each identified virus and viral family. To the authors’ knowledge, this is the first study to characterize the viromes of the major JE vectors in Japan.

Evaluating the competence of the primary vector, Culex tritaeniorhynchus, and the invasive mosquito species, Aedes japonicus japonicus, in transmitting three Japanese encephalitis virus genotypes

“…To address any effect from insect-specific viruses (ISV), 25 female mosquitoes from each colony were subjected to viral metagenomic screening and analysis by next-generation sequencing (NGS) as previously described by Faizah et al [ 34 ].…”

“…Homogenate was filtered, treated with DNase, and RNA eluted with 20 μl of RNase-free dH 2 O. Samples were subsequently subjected to RT-PCR by PrimeScript One-Step RT-PCR kit version 2 (Takara Bio) according to previously described RT-PCR protocol [ 34 ]. Primers used were gene specific primer sets provided in S1 Table [ 34 , 35 , 36 ] and electrophoresis was employed for the visualization.…”

“…Details of the three different JEV genotypes (GI, GIII, and GV) used in this study are provided in S2 Table [ 34 , 37 , 38 ]. GIII and GV were gifts from Dr. Shigeru Tajima, Department of Virology, National Institute of Infectious Diseases, Japan.…”

Highly Sensitive Virome Characterization of Aedes aegypti and Culex pipiens Complex from Central Europe and the Caribbean Reveals Potential for Interspecies Viral Transmission

“…Most RNA ISVs are classified in the families of Flaviviridae , Togaviridae , the order of Bunyavirales , and, recently, Mesoniviridae [ 6 ]. Despite being confined to the arthropod host, ISVs are medically relevant as they may alter arbovirus replication and transmission [ 6 , 9 , 10 , 11 , 12 ]. For example, coinfection with ISVs has been shown to attenuate arbovirus replication in Aedes cell lines in vitro, including West Nile virus, Japanese encephalitis virus, and Zika virus [ 13 , 14 ].…”

Ever-increasing viral diversity associated with the red imported fire ant Solenopsis invicta (Formicidae: Hymenoptera)

“…Nonetheless, like insect diversity, the diversity of viruses associated with insects is far from clear (1,3). While many studies have focused on viromes of arbovirus-transmitting insects, especially those involved in transmission of medically important viruses, such as mosquitoes (5)(6)(7)(8), other groups that are important pests either impacting agricultural or natural ecosystems, such as invasive ants, have been barely studied (9)(10)(11). In addition to contributing to better understanding of basic aspects of virus ecology and evolution, these studies may contribute to new opportunities to use viruses as tools to develop more sustainable insect control methods (12)(13)(14).…”