Peripheral Nerve Injury Induced by Japanese Encephalitis Virus in C57BL/6 Mouse.

Japanese encephalitis virus (JEV), with neurotoxic and neuroinvasive properties, is the major cause of human viral encephalitis in Asia. Although Guillain-Barré syndrome caused by JEV infections is not frequent, a few cases have been reported in recent years. To date, no existing animal model for JEV-induced peripheral nerve injury (PNI) has been established, and thus the pathogenic mechanism is not clarified. Therefore, an animal model is urgently required to clarify the correlation between JEV infection and PNI. In the present study, we used JEV GIb strain of NX1889 to establish a mouse model of JEV infection. The general neurological signs emerged on day 3 of modeling. The motor function continued to deteriorate, reaching a maximum at 8 to 13 days postinfection (dpi) and gradually recovered after 16 dpi. The injuries of 105 PFU and 106 PFU groups were the most severe. Transmission electron microscopy and immunofluorescence staining showed varying degrees of demyelination and axonal degeneration in the sciatic nerves. The electrophysiological recordings demonstrated the presence of demyelinating peripheral neuropathy with reduced nerve conduction velocity. The decreased amplitudes and the prolonged end latency revealed axonal-type motor neuropathy. Demyelination is predominant in the early stage, followed by axonal injury. The expression level of JEV-E protein and viral RNA was elevated in the injured sciatic nerves, suggesting that it may cause PNI at the early stage. Inflammatory cell infiltration and increased inflammatory cytokines indicated that neuroinflammation is involved in JEV-induced PNI. IMPORTANCE JEV is a neurotropic flavivirus belonging to the Flaviviridae family and causes high mortality and disability rates. It invades the central nervous system and induces acute inflammatory injury and neuronal death. Thus, JEV infection is a major global public health concern. Previously, motor dysfunction was mainly attributed to central nervous system damage. Our knowledge regarding JEV-induced PNI is vague and neglected. Therefore, a laboratory animal model is essential. Herein, we showed that C57BL/6 mice can be used to study JEV-induced PNI through multiple approaches. We also demonstrated that viral loads might be positively correlated with lesion severity. Therefore, inflammation and direct virus infection may be the putative mechanisms underlying JEV-induced PNI. The results of this study laid the foundation for further elucidation of the pathogenesis mechanisms of PNI caused by JEV.

Identification of a novel orthonairovirus from ticks and serological survey in animals near China-North korea border.

Emerging pathogenic tick-borne viruses (TBVs) have attracted a great deal of attention due to their significant impact on human and animal health. A novel orthonairovirus named Dadong virus (DDV) was isolated from Haemaphysalis concinna ticks in the Changbai Mountain region on the China-North Korea border. DDV can induce cytopathic effects in mammalian and human cell lines. Phylogenetic analysis showed that it belongs to the genus Orthonairovirus, family Nairoviridae, exhibiting 72.4%-81.3% nucleic acid identity to Tofla orthonairovirus, known to cause lethal infection in IFNAR KO mice. The first serological evidence of DDV circulating in cattle and mice was also obtained, with 4.0% (1/25) of cattle and 2.27% (1/44) of mice seropositive for DDV. Further investigations, including serological surveys using human samples, are required to assess the public health risk posed by DDV.

Novel Orthonairovirus Isolated from Ticks near China-North Korea Border.

We isolated a new orthonairovirus from Dermacentor silvarum ticks near the China-North Korea border. Phylogenetic analysis showed 71.9%-73.0% nucleic acid identity to the recently discovered Songling orthonairovirus, which causes febrile illness in humans. We recommend enhanced surveillance for infection by this new virus among humans and livestock.

Virome of Bat-Infesting Arthropods: Highly Divergent Viruses in Different Vectors.

Bats are reservoirs of important zoonotic viruses like Nipah and SARS viruses. However, whether the blood-sucking arthropods on the body surface of bats also carry these viruses and the relationship between viruses carried by the blood-sucking arthropods and viruses carried by bats have not been reported. This study collected 686 blood-sucking arthropods on the body surface of bats from Yunnan Province, China, between 2012 and 2015, and they included wingless bat flies, bat flies, ticks, mites, and fleas. The viruses carried by these arthropods were analyzed using a meta-transcriptomic approach, and 144 highly diverse positive-sense single-stranded RNA, negative-sense single-stranded RNA, and double-stranded RNA viruses were found, of which 138 were potentially new viruses. These viruses were classified into 14 different virus families or orders, including Bunyavirales, Mononegavirales, Reoviridae, and Picornavirales. Further analyses found that Bunyavirales were the most abundant virus group (84% of total virus RNA) in ticks, whereas narnaviruses were the most abundant (52 to 92%) in the bat flies and wingless bat flies libraries, followed by solemoviruses (1 to 29%) and reoviruses (0 to 43%). These viruses were highly structured based on the arthropod types. It is worth noting that no bat-borne zoonotic viruses were found in the virome of bat-infesting arthropod, seemingly not supporting that bat surface arthropods are vectors of zoonotic viruses carried by bats. IMPORTANCE Bats are reservoirs of many important viral pathogens. To evaluate whether bat-parasitic blood-sucking arthropods participate in the circulation of these important viruses, it is necessary to conduct unbiased virome studies on these arthropods. We evaluated five types of blood-sucking parasitic arthropods on the surface of bats in Yunnan, China, and identified a variety of viruses, some of which had high prevalence and abundance levels, although there is limited overlap in virome between distant arthropods. While most of the virome discovered here is potentially arthropod-specific viruses, we identified three possible arboviruses, including one orthobunyavirus and two vesiculoviruses (family Rhabdoviridae), suggesting bat-parasitic arthropods carry viruses with risk of spillage, which warrants further study.

Environmental factors and spatiotemporal distribution of Japanese encephalitis after vaccination campaign in Guizhou Province, China (2004-2016).

BACKGROUND: Although a vaccination campaign has been conducted since 2004, Japanese encephalitis (JE) is still a public health problem in Guizhou, one of the provinces with the highest incidence of JE in China. The aim of this study was to understand the spatiotemporal distribution of JE and its relationship with environmental factors in Guizhou Province in the post-vaccination era, 2004-2016. METHODS: We collected data on human JE cases in Guizhou Province from 2004 to 2016 from the national infectious disease reporting system. A Poisson regression model was used to analyze the relationship between JE occurrence and environmental factors amongst counties. RESULTS: Our results showed that the incidence and mortality of JE decreased after the initiation of vaccination. JE cases were mainly concentrated in preschool and school-age children and the number of cases in children over age 15 years was significantly decreased compared with the previous 10 years; the seasonality of JE before and after the use of vaccines was unchanged. JE incidence was positively associated with cultivated land and negatively associated with gross domestic product (GDP) per capita, vegetation coverage, and developed land. In areas with cultivated land coverage < 25%, vegetation coverage > 55%, and urban area coverage > 25%, the JE risk was lower. The highest JE incidence was among mid-level GDP areas and in moderately urbanized areas. CONCLUSIONS: This study assessed the relationship between incidence of JE and environmental factors in Guizhou Province. Our results highlight that the highest risk of JE transmission in the post-vaccination era is in mid-level developed areas.

Emerging Sand Fly-Borne Phlebovirus in China.

We isolated 17 viral strains capable of causing cytopathic effects in mammalian cells and death in neonatal mice from sand flies in China. Phylogenetic analysis showed that these strains belonged to the genus Phlebovirus. These findings highlight the need to control this potentially emerging virus to help safeguard public health.

Identification and genetic analysis of a totivirus isolated from the Culex tritaeniorhynchus in northern China.

Totiviridae, a viral family of double-stranded RNA (dsRNA) viruses, contain a single dsRNA genome 4.6-7.0 kb in length. Totiviridae were initially only known to infect fungi and other eukaryotes as well as plants, but an increase in totiviruses has been detected in insects, mosquitoes, and bats. Here, we describe the isolation and characterization of a strain belonging to the family Totiviridae isolated from Culex tritaeniorhynchus in Kenli, China, in 2016. We isolated a totivirus from field-collected mosquitoes in China by cell culture in Aedes albopictus C6/36 cells, identified the virus by morphological observation and complete genome sequencing, and characterized it by phylogenetic analysis. Transmission electron microscopy identified icosahedral, non-enveloped virus particles with a mean diameter of 35-40 nm. The genome was 7612 bp in length, including two open reading frames (ORFs). ORF1 (5058 nt) encodes the capsid protein, while ORF2 (2216 nt) encodes the viral RNA-dependent RNA polymerase (RdRp). Nucleotide and amino acid homology analysis of isolate showed higher levels of sequence identity with isolate CTV_NJ2 (China, 2010) with 94.87% nucleic acid identity and 97.32% amino acid identity. The isolate was designated C. tritaeniorhynchus totivirus KL (CTV-KL). This is the first identification of a totivirus in a C. tritaeniorhynchus in northern China. Analysis of the virus's morphology, characteristic and genome organization will further enrich our understanding of the molecular and biological characteristics of dsRNA Totiviridae viruses.

Genetic diversity of the Yokose virus, XYBX1332, isolated from bats (Myotis daubentonii) in China.

BACKGROUND: Yokose virus was first isolated from bats (Miniopterus fuliginosus) collected in Yokosuka, Japan, in 1971, and is a new member of the family Flaviviridae, genus Flavivirus. In this study, we isolated a Yokose virus from a serum sample of Myotis daubentonii (order Chiroptera, family Vespertilionidae) collected in Yunnan province, China in 2013. METHODS: The serum specimens of bat were used to inoculate in BHK-21 and Vero E6 cells for virus isolation. Then the viral complete genome sequence was obtained and was used for phylogenetic analysis performed by BEAST software package. RESULTS: The virus was shown to have cytopathic effects in mammalian cells (BHK-21 and Vero E6). Genome sequencing indicated that it has a single open reading frame (ORF), with a genome of 10,785 nucleotides in total. Phylogenetic analysis of the viral genome suggests that XYBX1332 is a Yokose virus (YOKV) of the genus Flavivirus. Nucleotide and amino acid homology levels of the ORF of XYBX1332 and Oita-36, the original strain of YOKV, were 72 and 82%, respectively. The ORFs of XYBX1332 and Oita-36 encode 3422 and 3425 amino acids, respectively. In addition, the non-coding regions (5'- and 3'-untranslated regions [UTRs]) of these two strains differ in length and the homology of the 5'- and 3'-UTRs was 81.5 and 78.3%, respectively. CONCLUSION: The isolation of YOKV (XYBX1332) from inland China thousands of kilometers from Yokosuka, Japan, suggests that the geographical distribution of YOKV is not limited to the islands of Japan and that it can also exist in the inland areas of Asia. However, there are large differences between the Chinese and Japanese YOKV strains in viral genome.

Arboviruses and their related infections in China: A comprehensive field and laboratory investigation over the last 3 decades.

Since the 1980s, a comprehensive field and laboratory investigation has been conducted throughout China, and a total of 29 virus species belonging to 7 families and 13 genera were identified through virological, morphological, and immunological methods, as well as whole-genome sequencing and molecular genetic analyses. Most of the virus isolates belong to 9 genera in the families Flaviviridae, Bunyaviridae, Togaviridae, and Reoviridae. Among them, 4 genera (Orthobunyavirus, Bunyavirus, Phlebovirus, and Nairovirus) belong to the family Bunyaviridae and 3 genera (Seadonavirus, Orbivirus, and Cypovirus) belong to the family Reoviridae. Analyses of the relationships between viruses and human/animal diseases indicated that Japanese encephalitis virus, dengue virus, severe fever with thrombocytopenia syndrome virus, tick-borne encephalitis virus, Crimean-Congo hemorrhagic fever virus, West Nile virus, and Tahyna virus can cause human and animal infections and disease epidemics in China. This review systematically introduces the current status of the diversity and geographical distribution of arboviruses and vectors in China. In addition, our results provide strong technical support for the prevention and control of arboviral diseases, the treatment of epidemics, and the early warning and prediction of diseases, and so they are significant for the control and prevention of arboviral diseases in Asia and around the world.

A Systems Vaccinology Approach Reveals Temporal Transcriptomic Changes of Immune Responses to the Yellow Fever 17D Vaccine.

In this study, we used a systems vaccinology approach to identify temporal changes in immune response signatures to the yellow fever (YF)-17D vaccine, with the aim of comprehensively characterizing immune responses associated with protective immunity. We conducted a cohort study in which 21 healthy subjects in China were administered one dose of the YF-17D vaccine; PBMCs were collected at 0 h and then at 4 h and days 1, 2, 3, 5, 7, 14, 28, 84, and 168 postvaccination, and analyzed by transcriptional profiling and immunological assays. At 4 h postvaccination, genes associated with innate cell differentiation and cytokine pathways were dramatically downregulated, whereas receptor genes were upregulated, compared with their baseline levels at 0 h. Immune response pathways were primarily upregulated on days 5 and 7, accompanied by the upregulation of the transcriptional factors JUP, STAT1, and EIF2AK2. We also observed robust activation of innate immunity within 2 d postvaccination and a durable adaptive response, as assessed by transcriptional profiling. Coexpression network analysis indicated that lysosome activity and lymphocyte proliferation were associated with dendritic cell (DC) and CD4+ T cell responses; FGL2, NFAM1, CCR1, and TNFSF13B were involved in these associations. Moreover, individuals who were baseline-seropositive for Abs against another flavivirus exhibited significantly impaired DC, NK cell, and T cell function in response to YF-17D vaccination. Overall, our findings indicate that YF-17D vaccination induces a prompt innate immune response and DC activation, a robust Ag-specific T cell response, and a persistent B cell/memory B cell response.

Identification and genetic analysis of Kadipiro virus isolated in Shandong province, China.

BACKGROUND: Kadipiro virus (KDV) belongs to the Reoviridae family, which consists of segmented, non-enveloped, double-stranded RNA viruses. It has previously been isolated from Culex, Anopheles, Armigeres and Aedes mosquitoes in Indonesia and China. Here, we describe the isolation and characterization of SDKL1625 from Anopheles sinensis mosquitoes in Shandong province, China. METHODS: In this study, we isolated Kadipiro virus in Aedes albopictus C6/36 cell culture and the complete genome sequencing was made by next generation sequencing. RESULTS: We isolated and characterized a Kadipiro virus from Anopheles sinensis mosquitoes in 2016 in Shandong province, China. Nucleotide and amino acid homology analysis of SDKL1625 showed higher levels of sequence identity with QTM27331 (Odonata, China, 2016) than with JKT-7075 (Culex fuscocephalus, Indonesia, 1981). The SDKL1625 has 86-97% amino acid identity with the JKT-7075, 88-99% amino acid identity with the QTM27331. Among the 12 fragments, VP1, VP2, VP4, VP6, VP7, VP9 and VP12 showed high amino acid identity (> 90%) and VP5 showed the lowest identity (86% and 88%). CONCLUSIONS: This is the first identification of KDV from mosquito in China. Virus morphology and genome organization were also determined, which will further enrich our understanding of the molecular biological characteristics of KDV and seadornaviruses.

Insights into the evolutionary history of Japanese encephalitis virus (JEV) based on whole-genome sequences comprising the five genotypes.

BACKGROUND: Japanese encephalitis virus (JEV) is the etiological agent of Japanese encephalitis (JE), one of the most serious viral encephalitis worldwide. Five genotypes have been classified based on phylogenetic analysis of the viral envelope gene or the complete genome. Previous studies based on four genotypes have reported that in evolutionary terms, genotype 1 JEV is the most recent lineage. However, until now, no systematic phylogenetic analysis was reported based on whole genomic sequence of all five JEV genotypes. FINDINGS: In this study, phylogenetic analysis using Bayesian Markov chain Monte Carlo simulations was conducted on the whole genomic sequences of all five genotypes of JEV. The results showed that the most recent common ancestor (TMRCA) for JEV is estimated to have occurred 3255 years ago (95% highest posterior density [HPD], -978 to-6125 years). Chronologically, this ancestral lineage diverged to produce five recognized virus genotypes in the sequence 5, 4, 3, 2 and 1. Population dynamics analysis indicated that the genetic diversity of the virus peaked during the following two periods: 1930-1960 and 1980-1990, and the population diversity of JEV remained relatively high after 2000. CONCLUSIONS: Genotype 5 is the earliest recognized JEV lineage, and the genetic diversity of JEV has remained high since 2000.

Distribution and phylogenetic analysis of Culex flavivirus in mosquitoes in China.

Culex flavivirus (CxFV) is an insect-specific virus of the genus Flavivirus. CxFV strains have been isolated from Cx. pipiens, Cx. quinquefasciatus, and other Cx. species in Asia, Africa, North America, Central America and South America. CxFV was isolated for the first time in China in 2006. As this is a novel flavivirus, we explored the distribution and genetic characteristics of Culex flavivirus in China. A total of 46,649 mosquitoes were collected in seven provinces between 2004 and 2012 and were analysed in 871 pools. 29 CxFV RNAs from Cx. pipiens, Cx. tritaeniorhynchus, Anopheles Sinensis, and Culex spp. tested positive for CxFV in real-time RT-PCR. 6 CxFV strains were isolated from Cx. species collected in Shandong, Henan, and Shaanxi provinces, while no virus or viral RNA was detected in samples from Sichuan, Chongqing, Hubei, and Fujian. Phylogenetic analysis of the envelope gene indicated that Chinese strains formed a robust subgroup of genotype 1, together with viruses from the United States and Japan. This study demonstrates that the geographic distribution of CxFV in China is widespread, but geographical boundaries to spread are apparent. Our findings suggest that CxFV can infect various mosquito species in nature.

Genome and evolution of Tibet orbivirus, TIBOV (genus Orbivirus, family Reoviridae).

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.

Incongruence between confirmed and suspected clinical cases of Japanese encephalitis virus infection.

Background: Japanese encephalitis (JE) is a notifiable infectious disease in China. Information on every case of JE is reported to the superior health administration department. However, reported cases include both laboratory-confirmed and clinically diagnosed cases. This study aimed to differentiate between clinical and laboratory-confirmed cases of Japanese encephalitis virus (JEV) infection, and improve the accuracy of reported JE cases by analyzing the acute-phase serum and cerebrospinal fluid of all reported JE cases in the Sichuan province from 2012 to 2022. Methods: All acute-phase serum and/or cerebrospinal fluid samples of the reported JE cases were screened for IgM(ImmunoglobulinM)to JEV using the enzyme-linked immunosorbent assay (ELISA), and the detection of the viral genes of JEV and 9 other pathogens including enterovirus (EV), using reverse transcription PCR was attempted. Epidemiological analyses of JE and non-JE cases based on sex, age, onset time, and geographical distribution were also performed. Results: From 2012 to 2022, 1558 JE cases were reported in the Sichuan province. The results of serological (JEV-specific IgM) and genetic testing for JEV showed that 81% (1262/1558) of the reported cases were confirmed as JEV infection cases (laboratory-confirmed cases). Among the 296 cases of non-JEV infection, 6 viruses were detected in the cerebrospinal fluid in 62 cases, including EV and the Epstein-Barr virus (EBV), constituting 21% (62/296) of all non-JE cases. Among the 62 non-JEV infection cases with confirmed pathogens, infections with EV and EBV included 17 cases each, herpes simplex virus (HSV-1/2) included 14 cases, varicella- zoster virus included 6 cases, mumps virus included 2 cases, and human herpes viruses-6 included 1 case. Additionally, there were five cases involving mixed infections (two cases of EV/EBV, one case of HSV-1/HSV-2, one case of EBV/HSV-1, and one case of EV/herpes viruses-6). The remaining 234 cases were classified as unknown viral encephalitis cases. Our analysis indicated that those aged 0-15 y were the majority of the patients among the 1558 reported JE cases. However, the incidence of laboratory-confirmed JE cases in the >40 y age group has increased in recent years. The temporal distribution of laboratory-confirmed cases of JE revealed that the majority of cases occurred from May to September each year, with the highest incidence in August. Conclusion: The results of this study indicate that there is a certain discrepancy between clinically diagnosed and laboratory-confirmed cases of JE. Each reported case should be based on laboratory detection results, which is of great importance in improving the accuracy of case diagnosis and reducing misreporting. Our results are not only important for addressing JE endemic to the Sichuan province, but also provide a valuable reference for the laboratory detection of various notifiable infectious diseases in China and other regions outside China.

Genetic Characteristics of Wuxiang Virus in Shanxi Province, China.

Wuxiang virus (WUXV) is the first sandfly-borne Phlebovirus isolated from Phlebotomus chinensis collected in China and has been established as a consistent viral presence in the local sandfly populations of both Wuxiang County and Yangquan City. However, its distribution in the Shanxi Province remains unclear. In this study, three novel WUXV strains were isolated from sandflies collected from Jiexiu City, Shanxi Province, China, in 2022. Subsequently, whole-genome sequences of these novel strains were generated using next-generation sequencing. The open reading frame (ORF) sequences of the WUXV strains from the three locations were subjected to gene analysis. Phylogenetic analysis revealed that WUXV belongs to two distinct clades with geographical differences. Strains from Wuxiang County and Yangquan City belonged to clade 1, whereas strains from Jiexiu City belonged to clade 2. Reassortment and recombination analyses indicated no gene reassortment or recombination between the two clades. However, four reassortments or recombination events could be detected in clade 1 strains. By aligning the amino acid sequences, eighty-seven mutation sites were identified between the two clades, with seventeen, sixty, nine, and one site(s) in the proteins RdRp, M, NSs, and N, respectively. Additionally, selection pressure analysis identified 17 positively selected sites across the entire genome of WUXV, with two, thirteen, one, and one site(s) in the proteins RdRp, M, NSs, and N, respectively. Notably, sites M-312 and M-340 in the M segment not only represented mutation sites but also showed positive selective pressure effects. These findings highlight the need for continuous nationwide surveillance of WUXV.

Peripheral nerve injury associated with JEV infection in high endemic regions, 2016-2020: a multicenter retrospective study in China.

Previously, we reported a cohort of Japanese encephalitis (JE) patients with Guillain-Barré syndrome. However, the evidence linking Japanese encephalitis virus (JEV) infection and peripheral nerve injury (PNI) remains limited, especially the epidemiology, clinical presentation, diagnosis, treatment, and outcome significantly differ from traditional JE. We performed a retrospective and multicenter study of 1626 patients with JE recorded in the surveillance system of the Chinese Center for Disease Control and Prevention, spanning the years 2016-2020. Cases were classified into type 1 and type 2 JE based on whether the JE was combined with PNI or not. A comparative analysis was conducted on demographic characteristics, clinical manifestations, imaging findings, electromyography data, laboratory results, and treatment outcomes. Among 1626 laboratory confirmed JE patients, 230 (14%) were type 2 mainly located along the Yellow River in northwest China. In addition to fever, headache, and disturbance of consciousness, type 2 patients experienced acute flaccid paralysis of the limbs, as well as severe respiratory muscle paralysis. These patients presented a greater mean length of stay in hospital (children, 22 years [range, 1-34]; adults, 25 years [range, 0-183]) and intensive care unit (children, 16 years [range, 1-30]; adults, 17 years [range, 0-102]). The mortality rate was higher in type 2 patients (36/230 [16%]) compared to type 1 (67/1396 [5%]). The clinical classification of the diagnosis of JE may play a crucial role in developing a rational treatment strategy, thereby mitigating the severity of the disease and potentially reducing disability and mortality rates among patients.

Phenotypic and molecular characteristics of plaque-purified MX10 virus, an Oriental-Australian genotype of Sindbis virus from Yunnan, China.

A previous investigation showed that MX10 virus, recently isolated in China, belongs to the Oriental-Australian (O/A) genotype of Sindbis virus (SINV) (Wang Jinglin, 2011, ATMH). Similar to the MRE16 isolate, the prototype O/A genotype of SINV, two derivate viruses with obviously different plaque morphologies were derived from MX10 virus, which were accordingly denoted as MX10-LP and MX10-SP. MX10-LP virus exhibited higher neurovirulence in neonatal mice than MX10-SP virus. Analysis of the complete genome revealed seven nucleotide differences between MX10-LP and MX10-SP. Compared with MRE16 virus, MRE16SP virus has a deletion of 30 aa in the E2 gene (200-229), which has been shown to be the molecular basis for the different plaque morphology. However, the MX10-SP virus did not have the 30-amino-acid deletion in the E2 gene. These results demonstrate that the molecular basis for the different plaque morphology of MX10 virus, the first strain of the O/A genotype of SINV isolated from China, is different from that of the prototype MRE16 virus.

Persistence of Tembusu Virus in Culex tritaeniorhynchus in Yunnan Province, China.

The Tembusu virus (TMUV), a member of the Flaviviridae family, can be transmitted via mosquitoes and cause poultry disease. In 2020, a strain of TMUV (YN2020-20) was isolated from mosquito samples collected in Yunnan province, China. In vitro experiments showed that TMUV-YN2020-20 produced a significant cytopathic effect (CPE) in BHK, DF-1, and VERO cells, while the CPE in C6/36 cells was not significant. Phylogenetic analysis revealed that the strain belonged to Cluster 3.2 and was closely related to the Yunnan mosquito-derived isolates obtained in 2012 and the Shandong avian-derived isolate obtained in 2014. Notably, TMUV-YN2020-20 developed five novel mutations (E-V358I, NS1-Y/F/I113L, NS4A-T/A89V, NS4B-D/E/N/C22S, and NS5-E638G) at loci that were relatively conserved previously. The results of this study demonstrate the continuous circulation and unique evolution of TMUV in mosquitoes in Yunnan province and suggest that appropriate surveillance should be taken.

Tracing the spatiotemporal phylodynamics of Japanese encephalitis virus genotype I throughout Asia and the western Pacific.

BACKGROUND: Japanese encephalitis virus (JEV; Flaviridae: Flavivirus) causes Japanese encephalitis (JE), which is the most important arboviral disease in Asia and the western Pacific. Among the five JEV genotypes (GI-V), GI has dominated traditional epidemic regions in the past 20 years. We investigated the transmission dynamics of JEV GI through genetic analyses. METHODS: We generated 18 JEV GI near full length sequences by using multiple sequencing approaches from mosquitoes collected in natural settings or from viral isolates obtained through cell culture. We performed phylogenetic and molecular clock analyses to reconstruct the evolutionary history by integrating our data with 113 publicly available JEV GI sequences. RESULTS: We identified two subtypes of JEV GI (GIa and GIb), with a rate of 5.94 × 10-4 substitutions per site per year (s/s/y). At present, GIa still circulates within a limited region, exhibited no significant growth, the newest strain was discovered in China (Yunnan) in 2017, whereas most JEV strains circulating belong to the GIb clade. During the past 30 years, two large GIb clades have triggered epidemics in eastern Asia: one epidemic occurred in 1992 [95% highest posterior density (HPD) = 1989-1995] and the causative strain circulates mainly in southern China (Yunnan, Shanghai, Guangdong, and Taiwan) (Clade 1); the other epidemic occurred in 1997 (95% HPD = 1994-1999) and the causative strain has increased in circulation in northern and southern China during the past 5 years (Clade 2). An emerging variant of Clade 2 contains two new amino acid markers (NS2a-151V, NS4b-20K) that emerged around 2005; this variant has demonstrated exponential growth in northern China. CONCLUSION: JEV GI stain circulating in Asia have shifted during the past 30 years, spatiotemporal differences were observed among JEV GI subclade. GIa is still circulating within a limited range, exhibite no significant growth. Two large GIb clades have triggered epidemics in eastern Asia, all JEV sequences identified in northern China during the past 5 years were of the new emerging variant of G1b-clade 2.