Hong Liu ¹ Minghua Li ^2,3,4,5 Guodong Liang ^6*

• ¹ Shandong Provincial Research Center for Bioinformatic Engineering and Technique, School of Life Sciences and Medicine, Shandong University of Technology, Zibo, China
• ² Department of Pathology, University of Texas Medical Branch, Galveston, TX, United States
• ³ Center for Biodefense and Emerging Infectious Diseases, Department of Pathology, John Sealy School of Medicine, University of Texas Medical Branch at Galveston, Galveston, United States
• ⁴ Center for Tropical Diseases, University of Texas Medical Branch at Galveston, Galveston, United States
• ⁵ Institute for Human Infections and Immunity, University of Texas Medical Branch at Galveston, Galveston, United States
• ⁶ National Institute for Viral Disease Control and Prevention (China CDC), Beijing, China

agents of human and animal diseases (Karabatsos, 1985). Notably, all these 504 viruses were isolated using traditional cell culture techniques and they can replicate in various insect or mammalian cell lines (such as C6/36, BHK, and VERO) or animals (such as suckling mice and chicken embryos), leading to the cytopathic effects (CPE) and animal death. The biological characters of these virus isolates including viral titers, plaque characteristics, replication dynamics, and morphological features have been intensively investigated. Studies have also been conducted to evaluate the pathogenesis of each virus by using relevant animal models and measuring neutralizing antibodies against the virus in human serum. Further,these viruses are classified into different families, genera, species, and even strains or types based on the antigen-antibody reactions. Over the past two decades, emerging and re-emerging arboviruses such as Zika virus (ZIKV), Rift Valley fever virus (RVFV), Dengue virus (DENV), Chikungunya virus (CHIKV), Yellow fever virus (YFV), West Nile virus (WNV) and Japanese encephalitis virus (JEV) are distributed globally and cause severe disease in humans, posing a significant threat to public health. These viruses isolated from cell culture function as critical tools for the development of vaccines and other antiviral therapeutics against arboviral infections. A significant number of "Uncultured viruses" have been discovered through next- The rapid progress in next-generation sequencing (NGS) technology and bioinformatics approaches has facilitated the identification of a significant number of viral nucleotide sequences across diverse vertebrate and invertebrate samples. These sequences include known viral sequences from previously identified virus isolates (such as ZIKV, DENV) and a substantial amount of novel viral sequence data that has not been reported before.In this paper, the newly identified viral sequences obtained through next-generation sequencing, which do not have existing viral isolates as references, are termed "uncultured viruses". The discovery of "uncultured viruses" using next-generation sequencing does not focus solely on the presence or absence of CPE but rather on the identification of novel viral sequences. Advances in research methodologies such as Virome and Metatranscriptomics have led to discovering numerous novel viral sequences that were previously unknown. For example, a recent study revealed 1445 new RNA virus sequences from 220 animal specimens across 9 animal phyla (Shi et al., 2016), a milestone described as ' unveiling the mystery of the RNA virosphere.' (York, 2016).This special issue also features an article that discusses the results of next-generation sequencing on tick samples (Wang et al., 2023), significantly enhancing our understanding of viral communities in ticks. Researchers can explore the long-term interaction between RNA viruses and their vertebrate hosts by leveraging "Uncultured virus" sequence information. Moreover, this approach facilitates tracing the origins and evolution of vertebrate viruses (Shi et al., 2018). The discovery of numerous Most importantly, whether or not these "uncultured viruses" can cause diseases in humans and animals and their impact on public health remain to be elucidated. Strengthening the study on the biological significance of "uncultured viruses"Previous studies indicate that 40% of the "uncultured viruses" detected through next-generation sequencing originate from eukaryotes such as humans, arthropods, domestic animals, bats, and monkeys (Cobbin et al., 2021). Over the past 20 years, viruses such as ZIKV, DENV, RVFV,CHIKV and other arboviruses transmitted by Aedes aegypti have been circulating all over the world, causing significant disease burdens. Therefore, it is anticipated that the next epidemic may arise from a blood-sucking insect-transmitted virus, particularly an arbovirus transmitted by Aedes aegypti (Hotez and Murray, 2017). Consequently, the future research effort should focus on virus isolates obtained from various blood-sucking arthropods such as mosquitoes, midges, ticks, and sandflies. We previously inoculated C6/36 cells with the supernatants of homogenized 785 mosquito pools collected from northern China in 2019. Despite two rounds of viral isolation using C6/36 cells, no viral isolates were obtained (data not shown). We then performed next-generation sequencing on the pooled mosquito samples and identified 129 novel viruses from 32 families (He, et al,2021). The recently identified "uncultured viruses" by other research groups may also contain a number of novel arboviruses that are distinct from previously isolated viruses and can infect both humans and animals.Furthermore, it is possible that the viral pathogens responsible for next emerging disease "X diseases" may originate from these "uncultured viruses". Therefore, the study of "uncultured viruses" particularly uncultured arboviruses needs to be further strengthened in the field. Due to the absence of viral isolates as references for "uncultured viruses", it is currently challenging to determine their relationship with the prevalent arboviruses circulating in the natural environment.Furthermore, experimental verification of their potential infectivity in humans and animals remains elusive. Consequently, it is recommended to leverage artificial intelligence to analyze the extensive datasets of various "uncultured viruses" and their associations with diverse blood-sucking vectors and host animals within the established natural circulation cycles of arboviruses is recommended. This approach aims to identify the closest reservoirs and host animals, and their relationships, to predict viruses with potential public health implications based on the viral sequences for further investigation. Bunyaviridae, Sarviridae, Filoviridae, and the newly discovered Jingmen tick virus (JMTV), belonging to the Flaviviridae family. These viral families include numerous viruses that cause diseases in humans and animals, such as Congo-Crimean hemorrhagic fever virus (CCHFV), a member of the Bunyaviridae family. The potential threat to public health of the newly discovered "uncultured viruses" in these viral families remains a scientific concern for virologists and public health experts. Studies have demonstrated the detection of the complete genome sequence of a novel virus, South Bay virus, SBV, in ticks (Ixodes scapularis) collected from the northeastern region of the United States (Tokarz et al., 2014). SBV, classified within the genus Nairovirus of the Bunyaviridae family, shares significant genetic similarity with CCHFV. Viral sequence data obtained from ticks collected from Yunnan Province, southwestern China, showed an overlap of over 80% with known viral information. Notably, these specimens from Yunnan Province were simultaneously inoculated into two different cell lines (C6/36 and BHK), yet no viral strain was successfully isolated (Xu et al., 2022). Together, these data suggest ticks in both the United States and China, harbor a novel "uncultured virus" with significant public health implications. Whether or not SBV is transmissible to humans and cause human disease are unknown. The lack of live viral isolates impedes the further investigation of the relationship between these viruses and the associated human and animal diseases, as well as their public health implications. Therefore, molecular virology and molecular cloning techniques such as reverse genetics is useful to generate 'rescued viruses' for studying important "uncultured viruses." This approach can also be applied to evaluate the vector competence of the virus and blood-sucking insects, host susceptibility to the virus, and the viral pathogenesis in humans and animals. These strategies can provide insights into the public health implications of important "uncultured viruses". Sandfly specimens collected from central China were subjected to viral isolation using BHK and C6/36 cells and next-generation sequencing. While only the previously isolated sandfly viruses were obtained using cell culture, the parallel next-generation sequencing identified 78 RNA viruses across 15 families, in which 66 viruses are novel (Wang et al., 2022). These findings indicate that the current cell lines used for virus isolation are not sensitive enough for isolating and culturing "uncultured viruses". Although BHK, VERO and C6/36 cells are the most commonly used cell lines that are sensitive to various animal viruses and insect viruses, including arboviruses, making them effective for isolating a wide range of arboviruses (Karabatsos, 1985;Liang et al., 2018), these cell lines are ineffective in isolating "uncultured viruses" from mosquito and sandfly specimens. There is an urgent need to develop novel cell lines for isolating and culturing of "uncultured viruses".Developing such new culture system requires the collaboration between cell biologists, virologists, and other relevant experts. This editorial discusses the substantial amount of viral sequence data including arbovirus sequences generated in recent years, with the aim of enhancing comprehension of the biological features of these viral sequences.This study will provide valuable information and contribute to the prevention and control of arboviruses and their associated diseases. To achieve these goals, the collaborative research across multiple scientific disciplines, particularly between bioinformaticians and virologists is needed.Furthermore, multiple experimental approaches should be integrated and utilized, such as concurrently culturing the same batch of specimens in diverse cell lines for viral isolation and nextgeneration sequencing analyses. This approach can enhance the acquisition of virus isolates and viral sequence information from the same sample. Finally, the authors of this editorial would like to express their gratitude to all the authors and reviewers who contributed to this special issue, as well as to Frontiers in Cellular and Infection Microbiology for providing a platform for the special issue on arbovirus infection: pathogenesis, molecular genetics, evolution, and diagnosis.