Arboviral infections have natural epidemiological characteristics and are mostly zoonotic diseases, making it difficult to predict epidemiological trends and posing significant risks to public health and safety [21]. Climate change, urbanization, geographic expansion of vectors, and frequent cross-border exchanges of people and goods contribute to the rising incidence of emerging, re-emerging, and imported arboviruses, escalating the risk of arboviral epidemics [22–24]. Therefore, it is necessary to comprehensively enhance the ability to respond to the threats and challenges posed by arboviruses, particularly by bolstering active monitoring of arbovirus, in order to clarify the virus spectrum and distribution, high-risk populations and animals, cross species transmission, as well as the genome evolution and mutation. Recently, NGS has revolutionized viral metagenomics, significantly accelerating the discovery of arbovirus and providing a powerful tool for understanding unknown arbovirus [25]. In this research, we identified a previously unreported mosquito-borne virus, Huajieling virus, in Culex quinquefasciatus and Armigeres subalbatus from Shandong, China. After genome-wide analysis and homology analysis of the virus-encoded proteins, it was classified as a new member of the family Caliciviridae within the order Picornavirales.
By analyzing the positive rate of mosquitoes carrying viruses (MIR = 0.17%, 7/3960), we can infer that Huajieling virus is widespread among local mosquitoes in Yantai City. This is also consistent with our previous research results on Hubei mosquito virus 2 (HMV2) in the Shandong region[26]. This suggests that the virus has adapted to the local habitat and formed a continuous and stable viral life cycle. In addition, we found the presence of Huajieling virus in two different species of mosquitoes, that the virus can spread and be transmitted through a variety of mosquito species, indicating that the vector of this virus is not singular. The virus can spread and be transmitted through a variety of mosquito species, and we cannot rule out the possibility that it can be present in other mosquito species. This also implies that Huajieling virus may have a wider distribution range and stronger transmission ability. Meanwhile, the present study found that there was no statistically significant difference in the viral carriage rates of the two mosquitoes, suggesting that Huajieling virus can survive in both mosquitoes, and that the virus does not have any obvious preference or predilection for mosquito species.
The results of animal serum virus nucleic acid testing indicate that cattle can be infected by the Huajieling virus and may serve as a natural host of the virus, but its role in virus transmission and epidemic needs further investigation to confirm. In our opinion, the next step is to conduct a prospective cohort study of cattle in the region to observe the duration of viremia in cattle and the production of related antibodies, to determine whether cattle play the role of amplification hosts in the virus cycle. Furthermore, it is worth further investigation whether the Huajieling virus can infect other mammals, and even humans.
Through whole genome sequence analysis, we found that Huajie virus is a single-stranded sense RNA virus with a small genome and simple structure, containing only one ORF. It encodes a structural protein called Calicivirus coat protein and two nonstructural proteins, 2OG Fe (II) oxygenase superfamily and RDRP proteins. After comparative analysis, we found that the similarity match between these three proteins and the currently known viral proteins is not more than 43%, indicating that Huajieling virus is likely a novel mosquito-borne virus. Our findings add a new member to the family of mosquito-borne viruses, but unraveling the full extent of the life cycle characteristics of this virus requires further research.
In terms of viral taxonomy, we attempted to localise the virus by analyzing and comparing the homology of the three proteins encoded by the virus. It was found that the 2OG-Fe(II) oxygenase superfamily was poorly classified, even in many bacteria, where the protein had a high similarity, leading to an inability to further accurately classify and localize the protein. However, by comparing the Calicivirus coat protein and RDRP, we found that both of them have relatively conserved properties under the family Caliciviridae, order Picornavirales. Currently, there are 10 genera of viruses in the family Caliciviridae, including Bavovirus, Lagovirus, and so on, and the rest are unclassified viruses. We performed phylogenetic analyses of these two proteins with virus-related proteins of the above 10 genera, and found that Huajieling virus clustered with Wufeng shrew picorna-like virus 43, and both of them were most closely related to Stoat valovirus 1 of genus Valovirus and Fathead minnow calicivirus of genus Minovirus. This suggests that these viruses share a large evolutionary homology and may have originated from a common viral ancestor. However, based on the current results of protein evolution analysis, we are unable to classify Huajieling virus accurately to a specific genus, which may involve gene recombination or intermediate viruses between these two genera.
In addition, in this study, we amplified three partial nucleic acid sequences of Huajieling virus from mosquitoes at different sampling sites in this area, and their nucleotide homology exceeded 99%. This indicates that the virus strains prevalent in the local epidemic area are the same strain, and its genetic properties are relatively stable, with no significant nucleic acid sequence mutations.
Eventually, some limitations of our study cannot be ignored. First, the blood in the mosquito's body is not fully digested before tissue grinding. Therefore, the Huajieling virus we detected may have originated from the blood of animals or humans. Second, no live virus was successfully isolated. However, this result cannot be interpreted as evidence of an absence of virus, and may be due to extremely low copy numbers of live virus in the sample or inappropriate cell lines.