Mosquito-borne viruses are an important component of emerging infectious diseases and pose notably global public health risks. Increasing international exchange poses greater challenges for epidemic control in border areas. Yunnan Province in China has a subtropical monsoon climate and a complex and diverse environment rich in natural resources, which is suitable for many plants and animals, providing a habitat for mosquitoes to survive and breed. In addition, conditions are favorable for the spread of mosquito-borne viruses. Over the last half-century, mosquitoes belonging to more than 10 families have been identified in Yunnan Province. The abundance of species and the suitability of the environment for host viruses increase the risk of complex viruses circulating in this region. In addition, Yunnan Province has the highest abundance and most widely distributed density of mosquito-borne viruses in China compared to other provinces (Atoni et al., 2020). The southeastern part of Yunnan Province shares borders with several neighboring countries. Owing to the different living habits and medical conditions of people in neighboring countries, the prevalence of pathogens varies from country to country. Since mosquitoes can migrate long distances (Huestis et al., 2019), and due to the expanding distribution of mosquitoes affected by climate change (Watts et al., 2018), Yunnan is at high risk of mosquito-borne pathogen outbreaks imported from neighboring countries. For instance, in 2013, an unexpected large outbreak of dengue fever was reported in Xishuangbanna Dai Autonomous Prefecture in Yunnan Province, whose evolutionary analysis showed that this dengue virus may have originated from neighboring countries (Zhang et al., 2014). Therefore, the abundance, distribution, and pathogenicity of circulating mosquito-borne viruses in border areas deserve more in-depth attention in the context of the development and implementation of disease prevention and control strategies. However, previous studies on arboviruses in Yunnan have mainly focused on the Sino–Myanmar and Sino–Laos border areas (Wang et al., 2011; Feng et al., 2012; Zeng et al., 2018; Hameed et al., 2020; Fang et al., 2021), with fewer studies being conducted on the Sino–Vietnam border area (Zhou et al., 2009). The northern part of Vietnam, bordering Yunnan, is an endemic area for dengue fever and the Japanese encephalitis virus (JEV), and an outbreak of the Zika epidemic occurred there during 2016–2017 (Jakobsen et al., 2019; Nealon et al., 2019; Nguyen-Tien et al., 2019, 2021). People living in the areas around Sino–Vietnam border may be at an increased risk of cross-border infectious diseases. In this study, we conducted a survey of mosquito and arbovirus species in the residential areas of Malipo and Funing counties of Wenshan Prefecture, Sino–Vietnam border area in Yunnan from 2020 to 2021, to monitor and describe the distribution of vectors and arboviruses in this area.

In our study, 39 species of mosquitoes from eight genera were collected near residential areas along the Sino–Vietnam border, mainly from livestock pens and nearby standing water containers and wood/bamboo forests. A total of 10,800 mosquitoes from 29 species were used for virus isolation. Culex tritaeniorhynchus accounted for 69.62% (7,519/10,800), and Anopheles sinensis accounted for 17.9% (1,936/10,800), and these species were the two most abundant species in this study. A total of 537 mosquito pools were mixed, and 62 viral isolates with CPE in C6/36 cells were isolated. Viruses from the isolates were classified into 11 categories based on the virus-specific primers used for RT-PCR. Each of the 61 viral isolates was identified with one viral category, whereas one viral isolate was identified with the co-existence of BAV and NDiV. We revealed the abundance of mosquito species, and the diversity of the pathogens they carry, in the vicinity of inhabited areas along the Sino–Vietnamese border.

Of the several important viruses that may become vertebrate pathogens, both TMUV and GETV were found initially in Malaysia and have been recently found in many places in China (Hamel et al., 2021; Li et al., 2022). TMUV can cause encephalitis and neurological disorders in birds and induce severe egg drop syndrome in ducks (Yu et al., 2022). TMUV was reported to cause epidemics affecting ducks in China in 2010, leading to huge economic losses and attracting widespread attention (Cao et al., 2011). A wide range of avian species, including geese, chickens, house sparrows, and pigeons, was confirmed to be naturally infected by TMUV (Tang et al., 2013a, 2015; Chen et al., 2014; Yan et al., 2017, 2022; Zhu et al., 2022), and infections in human have been demonstrated by serological tests or the presence of RNA (Wolfe et al., 2001; Tang et al., 2013b). Due to the lack of widespread surveillance, the virus is mostly reported in Thailand, Malaysia, and China, with few reports of TMUV in Vietnam (Hamel et al., 2021). The sequence of TMUVs we found at the Sino–Vietnam border was highly similar to that of avian TMUVs, and a possibility of the potential for local avian outbreaks cannot be ruled out. GETV is a mosquito-borne arbovirus, and GETVs have been shown to cause fever, rash, edema of the hind legs, and swelling of the lymph nodes in horses; GETVs cause miscarriages and other symptoms in pigs (Li et al., 2022). GETVs are widely distributed in the Asian continent and have been found in various animal specimens. These viruses have been isolated in 13 countries and have gradually spread from the tropics to temperate and even cold climate zones (Li et al., 2022). All GETVs are divided into four groups (GI–GIV) based on phylogenetic analysis of the E2 gene sequence. The GIII group members have gradually become the most numerous isolates of GETV, and this group bears the most responsibility for causing animal diseases, with a widely distributed lineage and a trend of gradual expansion (Li et al., 2022). The GETV identified in this study was classified as GIII. TMUVs and GETV have both been isolated from mosquitoes in corrals. The presence of these viruses at the Sino–Vietnam border could pose a threat to the local poultry and pig farming industries.

BAV and YUOV were originally identified in the Yunnan region, and we confirmed that both viruses are still circulating in this region. BAV was first isolated from patients with encephalitis in Yunnan Province (Xu et al., 1990). and has been found in many locations in China, Vietnam (Nabeshima et al., 2008), Indonesia (Liu et al., 2016), and other areas. Compared with other known segmented double-stranded RNA viruses, BAV has a significantly higher nucleotide substitution rate, which gives BAV the capacity to adapt quickly to a new environment and host (Liu et al., 2016). Rearrangement events between different BAV genotypes have also been observed (Nabeshima et al., 2008). The BAVs we isolated were classified as subgroup A2, which comprises strains from South China and Vietnam (Xia et al., 2018). A previous study claimed that BAV strains from the same latitudes were almost 100% identical (Liu et al., 2016) yet we observed that two branches of BAVs, with approximately 8% nucleotide sequence divergence in the tested region, co-existed in both Malipo and Funing counties. Our findings extend the knowledge of the diversity of BAV distributions. YUOV was identified for the first time in the Yunnan province in Culex tritaeniorhynchus (Attoui et al., 2005), and YUOV has been isolated from vertebrates in Japan, Peru, and North America (Attoui et al., 2009; Murota et al., 2021; Viadanna et al., 2021). IgM and IgG against YUOV were detected in the serum of one patient at the China–Myanmar–Laos border in Yunnan Province, providing evidence of YUOV infection in the area (Wang et al., 2011). Our findings prove that YUOV is present in Wenshan Prefecture on the Sino–Vietnam border, a potential human pathogen that should be investigated in this area.

In addition, six MSVs and one Totivirus, which could produce CPEs in C6/36 cells, were identified. MSVs are commonly neglected as they cannot cause infections in humans and have almost no economic impact on the farming industry. The MSVs identified in this study accounted for approximately 60% (38/62) of the isolates. TANAV, NTANAV, MRV, Totivirus, and NDiV have been previously isolated in China and could be tested using primers designed to detect, which have no more than 5% sequence divergence to known viruses. The other two viruses, DKNV and SWKV, were identified for the first time in China using next-generation sequencing (NGS) and confirmed using RT-PCR. DKNV and SWKV were discovered in Vietnam and Malaysia, respectively (Kuwata et al., 2013; Sadeghi et al., 2017). Currently, DKNV and SWKV have only one sequence of genetic information available in GenBank. In addition to the location where they were originally found, we found these two viruses on the Sino–Vietnam border. This finding extends the current knowledge regarding the distribution of DKNV and SWKV. We speculate that these viruses may have a broader distribution in Asia.

We collected a large number of samples from multiple locations along the Sino–Vietnam border for virus isolation, followed by molecular identification of the isolates to determine the viral species, which is an accepted protocol for identifying the presence of viruses. However, the method still has limitations, as viruses that are less abundant or more difficult to isolate may be overlooked. For example, in another virus detection study on JEV, we amplified 1,500 bp of the E gene of JEV in raw samples rather than in culture supernatants. In addition, some isolable viruses may be overlooked due to experimental design and targeting issues. In this study, 32.26% (20/62) of isolates were not identified based on the primers we initially designed but were ultimately identified as DKNV or SWKV using RT-PCR with redesigned primers based on NGS data. Mosquito virome studies using large-scale metagenome sequencing have shown many advantages over the traditional virus identification methods (da Silva et al., 2021), providing an unbiased description of the viruses in the entire sample. However, this approach still has a notable limitation. Determining whether the viruses identified in NGS data can infect host cells or whether they are the main cause of any observed cellular CEP is difficult. We found the presence of Kaiowa and Guato viruses in our NGS data. However, we also identified them in the negative control cells using RT-PCR. Therefore, we defined them as viruses naturally carried by the host cells or those that did not cause cellular CEP, and they were not described in this study. We used NGS to identify viral isolates and reconfirmed the results using RT-PCR, which improved the accuracy of the assays to some extent. Viral surveillance and screening by multiple modalities (e.g., virus isolation, macro-genome sequencing, and antibody-level testing) are useful and essential for additional confirmation and early warning of emerging pathogens.

In summary, we revealed the abundance of mosquito species and the diversity of viruses they carry in the vicinity of inhabited areas along the Sino–Vietnamese border. Conducting a more extensive regional viral surveillance is necessary to reflect the changing epidemics of mosquito-borne viruses.

The data presented in the study are deposited in the GenBank repository, accession number OP675480-OP675514 and OP858811-OP858851.

YTo and XG conceived and designed this study. FT and JH performed the experiments, analyzed the data, and prepared the initial draft of the manuscript. SS, ZC, YTa, ML, and CH contributed to sampling. All authors contributed to the article and approved the submitted version.

This research was supported by the National Natural Science Foundation of China (grant no. 81560548), the National Key Research and Development Program of China (grant nos. 2018YFA0903000 and 2021YFC0863400), and the Key Project of Beijing University of Chemical Technology (no. XK1803-06).

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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