Hong Liu

Next-generation sequencing (NGS) has dramatically accelerated the identification of viral genomic sequences from arthropod vectors, uncovering an unprecedented diversity of potential arboviruses. Nevertheless, the majority of these novel viral genomic sequences (NVGS) remain uncharacterised genomic data without corresponding viral isolates, highlighting the discrepancy between sequence detection and biological validation. This gap impedes our understanding of the biological properties, host range, and pathogenic potential of these newly identified viruses. To bridge this gap, the review advocates for integrated strategies, including expanded cell culture screening, novel cell line development, and reverse genetics approaches. These are complemented by artificial intelligence (AI) for predictive viral characterization. In parallel, NGS can be combined with other sequencing strategies to obtain more complete and accurate viral whole-genome sequences. Validating NGS discoveries is essential for effective risk assessment and outbreak preparedness against emerging arboviral threats, with important implications for public health surveillance.

Metagenomic sequencing has become a cornerstone of pathogen discovery and emerging infectious disease surveillance, enabling unbiased detection of known and unknown viruses directly from clinical, animal, and environmental samples (1–5). However, the same sensitivity that underpins these approaches also increases susceptibility to laboratory-derived nucleic acid contaminants, which may be misinterpreted as evidence of novel viral circulation or cross-species transmission (6, 7). Such artifacts pose a particular challenge for early-warning systems, where spurious signals can complicate biological interpretation and downstream risk assessment.

Numerous studies have demonstrated that the Japanese encephalitis virus (JEV) is classified into five genotypes. Historically, JEV GIII and GI were the dominant strains before and after the 1990s, respectively. Recently, the non-dominant genotypes have been implicated in numerous JE outbreaks, posing significant public health challenges. This study conducted a comprehensive phylogenetic analysis of 126 JEVs covering five genotypes from 1938 to 2025 globally. Notably, it is the first study to conduct a time to most recent common ancestor (tMRCA) analysis of JEV GII. The findings indicate the common ancestor of JEV emerged approximately 1081 years ago, followed by the sequential evolution of GV, GIII, GI, GII, and GIV, which are estimated to have originated approximately 237, 184, 138, 130, and 113 years ago, respectively. The overall evolutionary rate of JEV was 3.3 × 10⁻⁴ substitutions/site/year, with genotype specific rates as follows: GI at 7.3 × 10⁻⁴, GII at 8.2 × 10⁻⁴, GIII at 4.4 × 10⁻⁵, GIV at 1.7 × 10⁻³, and GV at 2.2 × 10⁻³. Significant differences were found between the E protein of recent human-derived JEV strains across five genotypes and the vaccine strain. This aligns with evidence of the current JE vaccine’s reduced efficacy against new strains, underscoring the urgent need for broadly protective JE vaccines.

Arboviruses, transmitted by blood-sucking arthropods, are responsible for significant human and animal diseases, including fever, hemorrhagic fever, and encephalitis, posing a serious threat to global public health. Nevertheless, research on the mechanisms of arbovirus infection and the development of therapeutic interventions has been impeded. This delay is primarily due to the limitations inherent in current in vitro research models, including cell cultures and animal models. The simplicity of cell types and interspecies differences present significant obstacles to advancing our understanding of arbovirus infection mechanisms and the development of effective drugs. Human brain organoids, derived from human pluripotent stem cells or human embryonic stem cells and cultured in three-dimensional systems, more accurately replicate the extensive neuronal cellular diversity and key characteristics of human neurodevelopment. These organoids serve as an ideal model for investigating the intricate interactions between viruses and human hosts, and providing a novel platform for the development of antiviral drugs. In this review, we summarize how brain organoid models complement classical approaches to accelerate research into the infection mechanisms of arboviruses, with a particular focus on the types of neural cells, key factors, and cellular signaling pathways involved in the arbovirus infection of brain organoids that have been reported. Furthermore, we examine the development of brain organoids, address their current limitations, and propose future directions to enhance the application of brain organoids in the study of arboviral infectious diseases.

Tibet orbivirus (TIBOV) was first isolated from Anopheles maculatus mosquitoes in Xizang, China, in 2009. In recent years, more TIBOV strains have been isolated in several provinces across China, Japan, East Asia, and Nepal, South Asia. Furthermore, TIBOVs have also been isolated from Culex mosquitoes, and several midge species. Additionally, TIBOV neutralizing antibodies have been detected in serum specimens from several mammals, including cattle, sheep, and pigs. All of the evidence suggests that the geographical distribution of TIBOVs has significantly expanded in recent years, with an increased number of vector species involved in its transmission. Moreover, the virus demonstrated infectivity towards a variety of animals. Although TIBOV is considered an emerging orbivirus, detailed reports on its genome and molecular evolution are currently lacking. Thus, this study performed the whole-genome nucleotide sequencing of three TIBOV isolates from mosquitoes and midges collected in China in 2009, 2011, and 2019. Furthermore, the genome and molecular genetic evolution of TIBOVs isolated from different countries, periods, and hosts (mosquitoes, midges, and cattle) was systematically analyzed. The results revealed no molecular specificity among TIBOVs isolated from different countries, periods, and vectors. Meanwhile, the time-scaled phylogenetic analysis demonstrated that the most recent common ancestor (TMRCA) of TIBOV appeared approximately 797 years ago (95% HPD: 16-2347) and subsequently differentiated at least three times, resulting in three distinct genotypes. The evolutionary rate of TIBOVs was about 2.12 × 10^-3 nucleotide substitutions per site per year (s/s/y) (95% HPD: 3.07 × 10^-5, 9.63 × 10^-3), which is similar to that of the bluetongue virus (BTV), also in the Orbivirus genus. Structural analyses of the viral proteins revealed that the three-dimensional structures of the outer capsid proteins of TIBOV and BTV were similar. These results suggest that TIBOV is a newly discovered and rapidly evolving virus transmitted by various blood-sucking insects. Given the potential public health burden of this virus and its high infectious rate in a wide range of animals, it is significant to strengthen research on the genetic variation of TIBOVs in blood-feeding insects and mammals in the natural environment and the infection status in animals.

An outbreak of viral encephalitis caused by a Japanese encephalitis virus (JEV) genotype IV infection occurred in Australia between 2021 and 2022. A total of 47 cases and seven deaths were reported as of November 2022. This is the first outbreak of human viral encephalitis caused by JEV GIV since it was first isolated in Indonesia in the late 1970s. Here, a comprehensive phylogenetic analysis based on the whole genome sequences of JEVs revealed it emerged 1037 years ago (95% HPD: 463 to 2100 years). The evolutionary order of JEV genotypes is as follows: GV, GIII, GII, GI, and GIV. The JEV GIV emerged 122 years ago (95% HPD: 57–233) and is the youngest viral lineage. The mean substitution rate of the JEV GIV lineage was 1.145 × 10⁻³ (95% HPD values, 9.55 × 10⁻⁴, 1.35 × 10⁻³), belonging to rapidly evolving viruses. A series of amino acid mutations with the changes of physico-chemical properties located in the functional important domains within the core and E proteins distinguished emerging GIV isolates from old ones. These results demonstrate the JEV GIV is the youngest JEV genotype at a rapid evolution stage and has good host/vector adaptability for introduction to non-endemic areas. Thus, surveillance of JEVs is highly recommended.

The untranslated region (UTRs) of viral genome are important for viral replication and immune modulation. Japanese encephalitis virus (JEV) is the most significant cause of epidemic encephalitis worldwide. However, little is known regarding the characterization of the JEV UTRs. Here, systematic analyses of the UTRs of JEVs isolated from a variety of hosts worldwide spanning about 80 years were made. All the important cis-acting elements and structures were compared with other mosquito-borne Flaviviruses [West Nile virus (WNV), Yellow fever virus (YFV), Zika virus (ZIKV), Dengue virus (DENV)] and annotated in detail in the UTRs of different JEV genotypes. Our findings identified the JEV-specific structure and the sequence motif with unique JEV feature. (i) The 3’ dbsHP was identified as a small hairpin located in the DB region in the 3′ UTR of JEV, with the structure highly conserved among the JEV genotypes. (ii) The spacer sequence UARs of JEV consist of four discrete spacer sequences, whereas the UARs of other mosquito-borne Flaviviruses are continuous sequences. In addition, repetitive elements have been discovered in the UTRs of mosquito-borne Flaviviruses. The lengths, locations, and numbers of the repetitive elements of JEV also differed from other Flaviviruses (WNV, YFV, ZIKV, DENV). A 300 nt-length region located at the beginning of the 3′ UTR exhibited significant genotypic specificity. This study lays the basis for future research on the relationships between the JEV specific structures and elements in the UTRs, and their important biological function.

Introduction The best approach to preventing the importation of coronavirus disease 2019 (COVID-19) is enhancing the detection capacity at customs. The rapid detection is of utmost importance and therefore highly demanded. Methods We conducted a field validation study of a duplex real-time reverse transcription recombinase-aided amplification (RT-RAA) assay in Zhoushan and Hangzhou customs, in Zhejiang Province, China. The reverse transcriptase polymerase chain reaction (RT-PCR) assay kit routinely used at customs was used in parallel, and the duration the two methods took to complete a specific number of samples was compared. Results Among 506 samples collected, RT-RAA results were consistent with the RT-PCR results. The sensitivity and specificity were 100%, the total coincidence rate was 100%, and the Kappa value was 1 (P<0.05) for both methods. The RT-RAA kit took a significantly shorter time in testing the 20–200 samples than the RT-PCR kit. Discussion The RT-RAA detection method is more efficient and suitable for use at customs than RT-PCR assay to realize rapid customs clearance of 200 or fewer samples.