Molecular Characterization of HN1304M, a Cat Que Virus Isolated from Midges in China

The Cat Que orthobunyavirus has been found in mosquitoes, birds, pigs, and humans, suggesting its wide range of hosts and potential public health implications. During arbovirus surveillance in 2013, the HN1304M virus was isolated from naturally occurring Culicoides biting midges in Hunan Province, southern China. The virus was cytopathic to BHK-21 cells and showed stable passage, but was not cytopathic to C6/36 cells. Determination and analysis of the viral genome sequence revealed that HN1304M is an RNA virus with three gene segments, namely, L, M, and S. The nucleotide and amino acid sequence homologies of HN1304M to Cat Que viruses in the Manzanilla species complex were 90.3–99.4%, and 95–100%, respectively, while the homologies to other viruses in this species complex were 74–86.6% and 78.1–96.1%, respectively. A phylogenetic analysis of the viral genes revealed that HN1304M formed an evolutionary branch with other Cat Que viruses isolated from mosquitoes, pigs, birds, and humans, which was completely independent of the other viruses in this complex. The fact that the Cat Que virus was isolated from Culicoides suggests that biting midges may participate in the natural circulation of Cat Que viruses.


Introduction
According to the report of the International Committee on Taxonomy of Viruses (ICTV) published in 2021, the Bunyavirales order comprises a group of single-stranded The Manzanilla species complex includes Manzanilla, Ingwavuma, Mermet, and Cat Que viruses [8], as well as their hosts, including birds, pigs, monkeys, and humans [9][10][11]. Viruses of the Manzanilla species complex have also been isolated from mosquitoes in Vietnam and China [12][13][14]. As viruses of the Manzanilla species complex have been isolated from mosquitoes, birds, and various mammals, it is believed that these viruses follow a virus-mosquito-bird/mammal cycle in nature [15]. However, there are no reports of other blood-sucking arthropods that can serve as vectors for the circulation of viruses of the Manzanilla species complex in nature.
This study reports a virus strain, HN1304M, isolated from midge specimens collected in Hunan Province in southern China, during a study on blood-sucking arthropods, and the virus was found to be cytopathic to mammalian BHK-21cells. Determination and analysis of the whole-genome sequence of HN1304M revealed it to be a Cat Que virus of the Manzanilla species complex. This study is the first to report the isolation of a Cat Que virus from naturally occurring Culicoides biting midges. The study also suggested that midges may serve as a transmission vector of Cat Que viruses.
The midge specimens were divided into 20 batches, while the mosquito specimens were divided into 89 batches based on the time of collection, location, and type of arthropod and ground in the laboratory. The ground supernatant of the blood-sucking insects was parallelly inoculated into BHK-21 and C6/36 cell lines, and cytopathic changes were observed on a daily basis. One virus isolate (named HN1304M) was obtained from the Culicoides biting midge samples. Following continuous culture and observation, cytopathic changes appeared on the third day of inoculation of the BHK-21 cells (first generation) with the ground supernatant of the midge specimen containing the HN1304M virus, and the lesions reached +++ (75% of the cells exhibited marked cytopathic effect) on the fifth day of inoculation. The supernatant of the diseased cells was continuously passaged, and obvious cytopathic changes were observed in the cells during the second and third passages on the second day of inoculation. The cytopathic changes manifested as cell shrinkage and shedding and reached +++ on the third day of inoculation (Figure 1). It was observed that the HN1304M virus isolate can be stably passaged in BHK-21 cells; however, parallel inoculation of C6/36 cells with the ground supernatant of midges containing the HN1304M virus revealed that the HN1304M virus was not cytopathic to C6/36 cells. The suspension from the fourth passage of the BHK-21 was used as a viral stock and stored at −80 • C. The BHK-21 and C6/36 cells were also inoculated with the ground supernatants of other batches of mosquitoes and midges, and no cytopathic changes were observed.

Preliminary Identification of HN1304M
In order to identify the HN1304M virus isolate, arbovirus genera-specific primers (Flavivirus, Alphavirus, and Bunyavirus) and virus species-specific primers (Japanese encephalitis virus, Banna virus, Tibet orbivirus, Densovirus, Totivirus, and Oya virus) were used for polymerase chain reaction (PCR)-based detection of the cDNA prepared from the supernatant of the HN1304M virus isolate. The primers used are listed in Supplementary  Table S1. The PCR test for the Oya virus was positive, while all of the other PCR tests were negative. Sequencing of the PCR product revealed that the nucleotide sequence of the S gene segment of HN1304M was the most similar (95.0-99.1%) to that of the Cat Que viruses in the Simbu serogroup.

Nucleotide Sequence of the Viral Genome
Based on the genome sequences of the Cat Que viral strains in GenBank, we designed and synthesized amplification primers that cover the full-length gene sequence of the HN1304M virus (Supplementary Table S2). The cDNA prepared from the supernatant from the fourth passage of the BHK-21 cells infected by HN1304M was used as the template, and gene amplification primers were used for amplifying the viral gene sequence and determining its nucleotide sequence. The sequences of the coding regions of the HN1304M viral gene segments were finally obtained. The viral gene sequence information was registered in GenBank (registration numbers: OP129710, OP129711, and OP129712). The genome of the HN1304M virus contains three gene segments, including the L, M, and S segments. The coding region of the L segment has a nucleotide sequence length of 6786 nt and encodes an RNA-dependent RNA polymerase (RdRp) of 2261 aa, while the coding region of the M gene segment has a nucleotide length of 4302 nt and encodes a polyprotein of 1433 aa. The S segment contains two open reading frames (ORFs), and the sequence lengths of the coding regions in this gene segment are 702 nt and 291 nt, which encode a nucleoprotein (NP) of 233 aa and a nonstructural (NS) protein of 96 aa, respectively.

Nucleotide and Amino Acid Sequence Homology of HN1304M Viral Genome
In order to elucidate the molecular characteristics and differences between the HN1304M virus and other related viruses, the viral sequences of representative strains of the Manzanilla species complex, including the Mermet, Ingwavuma, Manzanilla, and Cat Que orthobunyaviruses, and sequences of all viruses related to Cat Que viruses were retrieved from GenBank (Supplementary Table S3). The nucleotide and amino acid

Nucleotide Sequence of the Viral Genome
Based on the genome sequences of the Cat Que viral strains in GenBank, we designed and synthesized amplification primers that cover the full-length gene sequence of the HN1304M virus (Supplementary Table S2). The cDNA prepared from the supernatant from the fourth passage of the BHK-21 cells infected by HN1304M was used as the template, and gene amplification primers were used for amplifying the viral gene sequence and determining its nucleotide sequence. The sequences of the coding regions of the HN1304M viral gene segments were finally obtained. The viral gene sequence information was registered in GenBank (registration numbers: OP129710, OP129711, and OP129712). The genome of the HN1304M virus contains three gene segments, including the L, M, and S segments. The coding region of the L segment has a nucleotide sequence length of 6786 nt and encodes an RNA-dependent RNA polymerase (RdRp) of 2261 aa, while the coding region of the M gene segment has a nucleotide length of 4302 nt and encodes a polyprotein of 1433 aa. The S segment contains two open reading frames (ORFs), and the sequence lengths of the coding regions in this gene segment are 702 nt and 291 nt, which encode a nucleoprotein (NP) of 233 aa and a nonstructural (NS) protein of 96 aa, respectively.

Nucleotide and Amino Acid Sequence Homology of HN1304M Viral Genome
In order to elucidate the molecular characteristics and differences between the HN1304M virus and other related viruses, the viral sequences of representative strains of the Manzanilla species complex, including the Mermet, Ingwavuma, Manzanilla, and Cat Que orthobunyaviruses, and sequences of all viruses related to Cat Que viruses were retrieved from GenBank (Supplementary Table S3). The nucleotide and amino acid sequence homology of HN1304M with the aforementioned viruses was determined using the online BLASTn and BLASTp alignment tools, respectively (https://blast.ncbi.nlm.nih.gov/Blast.cgi) (accessed on 1 August 2022). The results demonstrated that the nucleotide and amino acid sequences of the three gene segments of the HN1304M virus were similar to those of six strains of Cat Que viruses, including the original VN04-2108, GD18234, SC0806, JM1, NIV86209, and DHL10M107 isolates. The nucleotide and amino acid similarities between HN1304M and these six viral isolates were 90.3-99.4% and 95.0-100%, respectively (

Phylogenetic Analysis of the HN1304M Virus
In order to elucidate the molecular genetic evolutionary characteristics of the HN1304M virus isolated from the midge specimens, this study established a nucleotide sequence dataset of genes from representative strains of Clade A and Clade B viruses of the Simbu serogroup (Supplementary Table S3 (Figure 2A-C).
including the Manzanilla, Buttonwillow, Facey's Paddock, Utinga, and Orpouche lineages. The Manzanilla lineage further contained four viruses, namely, the original VN04-2108 isolate of the Cat Que virus, the Manzanilla orthobunyavirus virus (TRVL3586), the Ingwavuma orthobunyavirus virus (SA An 4165), and Mermet orthobunyavirus (AV 782). Seven viral isolates, including the HN1304M virus, comprised an evolutionary branch together with the original VN04-2108 isolate of the Cat Que virus, suggesting that these viruses belong to the Cat Que virus in the Manzanilla lineage (Figure 2A-C). During the Nipah virus outbreak in Malaysia in 1999, an orthobunyavirus (Oya virus) was isolated from ailing pigs [10]. Only a 365 nt partial sequence of the S gene segment of the virus is available in GenBank (accession number: JX983192). The 365 nt sequence of JX983192 was aligned with the sequences of the S genes of all corresponding viruses and phylogenetically analyzed in this study. The results demonstrated that the Oya virus that was isolated from ailing pigs in Malaysia in 1999, belonged to the branch of Cat Que viruses of the Manzanilla lineage and was closely related to the HN1304M virus isolated from the Culicoides biting midges in this study ( Figure 2D). This suggests a  Table S3). Phylogenetic analyses demonstrated that the Clade A viruses in the Simbu serogroup can be divided into five evolutionary lineages, including the Manzanilla, Buttonwillow, Facey's Paddock, Utinga, and Orpouche lineages. The Manzanilla lineage further contained four viruses, namely, the original VN04-2108 isolate of the Cat Que virus, the Manzanilla orthobunyavirus virus (TRVL3586), the Ingwavuma orthobunyavirus virus (SA An 4165), and Mermet orthobunyavirus (AV 782). Seven viral isolates, including the HN1304M virus, comprised an evolutionary branch together with the original VN04-2108 isolate of the Cat Que virus, suggesting that these viruses belong to the Cat Que virus in the Manzanilla lineage (Figure 2A-C). During the Nipah virus outbreak in Malaysia in 1999, an orthobunyavirus (Oya virus) was isolated from ailing pigs [10]. Only a 365 nt partial sequence of the S gene segment of the virus is available in GenBank (accession number: JX983192). The 365 nt sequence of JX983192 was aligned with the sequences of the S genes of all corresponding viruses and phylogenetically analyzed in this study. The results demonstrated that the Oya virus that was isolated from ailing pigs in Malaysia in 1999, belonged to the branch of Cat Que viruses of the Manzanilla lineage and was closely related to the HN1304M virus isolated from the Culicoides biting midges in this study ( Figure 2D). This suggests a represents mosquitoes; the Simbu serogroup (Supplementary Table S3). Phylogenetic analyses demonstrated that the Clade A viruses in the Simbu serogroup can be divided into five evolutionary lineages, including the Manzanilla, Buttonwillow, Facey's Paddock, Utinga, and Orpouche lineages. The Manzanilla lineage further contained four viruses, namely, the original VN04-2108 isolate of the Cat Que virus, the Manzanilla orthobunyavirus virus (TRVL3586), the Ingwavuma orthobunyavirus virus (SA An 4165), and Mermet orthobunyavirus (AV 782). Seven viral isolates, including the HN1304M virus, comprised an evolutionary branch together with the original VN04-2108 isolate of the Cat Que virus, suggesting that these viruses belong to the Cat Que virus in the Manzanilla lineage (Figure 2A-C). During the Nipah virus outbreak in Malaysia in 1999, an orthobunyavirus (Oya virus) was isolated from ailing pigs [10]. Only a 365 nt partial sequence of the S gene segment of the virus is available in GenBank (accession number: JX983192). The 365 nt sequence of JX983192 was aligned with the sequences of the S genes of all corresponding viruses and phylogenetically analyzed in this study. The results demonstrated that the Oya virus that was isolated from ailing pigs in Malaysia in 1999, belonged to the branch of Cat Que viruses of the Manzanilla lineage and was closely related to the HN1304M virus isolated from the Culicoides biting midges in this study ( Figure 2D). This suggests a represents midges; the Clade A including th ages. The M 2108 isolate Ingwavuma Seven viral i gether with ruses belong  Table S3). Phylogenetic analyses demonstrated that the Clade A viruses in the Simbu serogroup can be divided into five evolutionary lineages, including the Manzanilla, Buttonwillow, Facey's Paddock, Utinga, and Orpouche lineages. The Manzanilla lineage further contained four viruses, namely, the original VN04-2108 isolate of the Cat Que virus, the Manzanilla orthobunyavirus virus (TRVL3586), the Ingwavuma orthobunyavirus virus (SA An 4165), and Mermet orthobunyavirus (AV 782). Seven viral isolates, including the HN1304M virus, comprised an evolutionary branch together with the original VN04-2108 isolate of the Cat Que virus, suggesting that these viruses belong to the Cat Que virus in the Manzanilla lineage (Figure 2A-C). During the Nipah virus outbreak in Malaysia in 1999, an orthobunyavirus (Oya virus) was isolated from ailing pigs [10]. Only a 365 nt partial sequence of the S gene segment of the virus is available in GenBank (accession number: JX983192). The 365 nt sequence of JX983192 was aligned with the sequences of the S genes of all corresponding viruses and phylogenetically analyzed in this study. The results demonstrated that the Oya virus that was isolated from ailing pigs in Malaysia in 1999, belonged to the branch of Cat Que viruses of the Manzanilla lineage and was closely related to the HN1304M virus isolated from the Culicoides biting midges in this study ( Figure 2D). This suggests a represents birds; and the Simbu serogroup (Supplementary Table S3). Phylogenetic analyses demonstrated that the Clade A viruses in the Simbu serogroup can be divided into five evolutionary lineages, including the Manzanilla, Buttonwillow, Facey's Paddock, Utinga, and Orpouche lineages. The Manzanilla lineage further contained four viruses, namely, the original VN04-2108 isolate of the Cat Que virus, the Manzanilla orthobunyavirus virus (TRVL3586), the Ingwavuma orthobunyavirus virus (SA An 4165), and Mermet orthobunyavirus (AV 782). Seven viral isolates, including the HN1304M virus, comprised an evolutionary branch together with the original VN04-2108 isolate of the Cat Que virus, suggesting that these viruses belong to the Cat Que virus in the Manzanilla lineage (Figure 2A-C). During the Nipah virus outbreak in Malaysia in 1999, an orthobunyavirus (Oya virus) was isolated from ailing pigs [10]. Only a 365 nt partial sequence of the S gene segment of the virus is available in GenBank (accession number: JX983192). The 365 nt sequence of JX983192 was aligned with the sequences of the S genes of all corresponding viruses and phylogenetically analyzed in this study. The results demonstrated that the Oya virus that was isolated from ailing pigs in Malaysia in 1999, belonged to the branch of Cat Que viruses of the Manzanilla lineage and was closely related to the HN1304M virus isolated from the Culicoides biting midges in this study ( Figure 2D). This suggests a represents humans.
During the Nipah virus outbreak in Malaysia in 1999, an orthobunyavirus (Oya virus) was isolated from ailing pigs [10]. Only a 365 nt partial sequence of the S gene segment of the virus is available in GenBank (accession number: JX983192). The 365 nt sequence of JX983192 was aligned with the sequences of the S genes of all corresponding viruses and phylogenetically analyzed in this study. The results demonstrated that the Oya virus that was isolated from ailing pigs in Malaysia in 1999, belonged to the branch of Cat Que viruses of the Manzanilla lineage and was closely related to the HN1304M virus isolated from the Culicoides biting midges in this study ( Figure 2D). This suggests a potential relationship between the HN1304M virus isolated from midges and the Oya virus (JX983192) isolated from pigs during the Nipah virus outbreak in 1999.

Discussion
In this study, the HN1304M virus, which can cause mammalian cytopathies, was isolated from naturally occurring Culicoides biting midges collected from Hunan Province in southern China. The results of whole-genome molecular biology and phylogenetic analyses revealed that the HN1304M virus isolated from midges was a member of the Cat Que viruses in the Manzanilla species complex of the Simbu serogroup. Cat Que viruses have been previously isolated from mosquitoes [12][13][14], birds, humans [9,15], and pigs [10]; however, this study is the first to report the isolation of a Cat Que virus from Culicoides biting midges.
As aforementioned, Cat Que viruses have been isolated from mosquito samples in Vietnam [12], mosquito isolates in the Yunnan Province in China [13], mosquito isolates in Sichuan Province in China [14], birds (myna) and human patient specimens in India [9,15], midges (the HN1304M virus isolated in this study), and ailing pigs during the Nipah virus outbreak in Malaysia in 1999 [10]. These findings demonstrate that Cat Que viruses can be transmitted by mosquitoes and midges, and can infect pigs, poultry (birds), and humans ( Figure 2). Seroepidemiological analyses of Cat Que viruses have revealed positive IgM and IgG antibodies in serum samples collected from pigs in the Sichuan area of China, where the Cat Que virus (SC0806) was isolated. The positive rates of IgM and IgG antibodies were 21.98% (20/91) and 60.43% (55/91), respectively, while the positive rate of both antibodies was 7.53% (7/91). The findings demonstrated that infection with Cat Que viruses is common in locally raised pigs. Further analysis revealed that the positive rate of viral IgM antibodies was highest in locally raised piglets younger than 4 months of age, and the positive rate gradually decreased with increasing age, while the positive rate of viral IgG antibodies gradually increased with age, reaching 100% at 8 months of age [14]. The isolation of Cat Que viruses from ailing pigs, mosquitoes, and midges [10,[12][13][14], and the high positive rate of Cat Que viral IgM and IgG antibodies detected in the serum of domestic pigs [14] indicate that pigs are an important mammalian host for Cat Que viruses, while mosquitoes and midges serve as arthropod vectors of the virus. Therefore, the Cat Que virus, or even the Manzanilla species complex, could be transmitted following a virus-mosquito/midges-mammal (bird/pig) cycle in nature.

Specimen Collection
Specimens of mosquitoes were collected between 7 and 8 August 2013, from three sampling sites in Yizhang County of Chenzhou City in Hunan Province. The mosquitoes and midges were collected at night using mosquito lure lamps (Kung Fu Xiaoshuai, 12 V, 300 mA; Wuhan Lucky Star Environmental Protection Technology, Hubei, China). The specimens were collected from 16:30 h to 6:30 h the following day. Following a morphologybased classification, the specimens of mosquitos and midges were preserved in separate cryopreservation tubes based on the collection point and morphological classification, stored in liquid nitrogen, and transported to the laboratory on dry ice [14].

Virus Isolation
The samples were washed twice with grinding fluid (MEM supplemented with 5% penicillin-streptomycin (100 U/mL), 1% glutamine (30 g/L), and 1% sodium bicarbonate), following which 1.5 mL of grinding liquid was added, and the samples were ground for 3 min with a tissue grinder (Retsch Tissue Lyser, QIAGEN, Hilden, Germany) at a frequency of 25 times/s. After grinding, the sample was centrifuged at 12,000× g for 30 min at 4 • C. The C6/36 and BHK-21 cells were then separately inoculated with 100 µL of the supernatant of the grinding fluid used for culture, and cytopathy was observed on a daily basis [17].

Identification of Virus
Viral RNA was extracted from 140 µL of the cell culture supernatant of BHK-21 cells using an QIAamp Viral RNA Mini Kit (Qiagen), and the cDNA library was prepared using a Ready-To-Go kit (GE Healthcare, Little Chalfont, Buckinghamshire, UK).  Table S1) were used for PCR [18]. The viral