Flaviviruses are known to cause a variety of diseases in humans in different parts of the world. There are very limited numbers of antivirals to combat flavivirus infection, and therefore new drug targets must be explored. The flavivirus NS2B-NS3 proteases are responsible for the cleavage of the flavivirus polyprotein, which is necessary for productive viral infection and for causing clinical infections; therefore, they are a promising drug target for devising novel drugs against different flaviviruses. This review highlights the structural details of the NS2B-NS3 proteases of different flaviviruses, and also describes potential antiviral drugs that can interfere with the viral protease activity, as determined by various studies. Moreover, optimized in vitro reaction conditions for studying the NS2B-NS3 proteases of different flaviviruses may vary and have been incorporated in this review. The increasing availability of the in silico and crystallographic/structural details of flavivirus NS2B-NS3 proteases in free and drug-bound states can pave the path for the development of promising antiflavivirus drugs to be used in clinics. However, there is a paucity of information available on using animal cells and models for studying flavivirus NS2B-NS3 proteases, as well as on the testing of the antiviral drug efficacy against NS2B-NS3 proteases. Therefore, on the basis of recent studies, an effort has also been made to propose potential cellular and animal models for the study of flavivirus NS2B-NS3 proteases for the purposes of exploring flavivirus pathogenesis and for testing the efficacy of possible drugs targets, in vitro and in vivo.

Japanese encephalitis (JE) is a vaccine-preventable disease caused by the Japanese encephalitis virus (JEV), which is primarily prevalent in Asia. JEV is a Flavivirus, classified into a single serotype with five genetically distinct genotypes (I, II, III, IV, and V). JEV genotype III (GIII) had been the most dominant strain and caused numerous outbreaks in the JEV endemic countries until 1990. However, recent data shows the emergence of JEV genotype I (GI) as a dominant genotype and it is gradually displacing GIII. The exact mechanism of this genotype displacement is still unclear. The virus can replicate in mosquito vectors and vertebrate hosts to maintain its zoonotic life cycle; pigs and aquatic wading birds act as an amplifying/reservoir hosts, and the humans and equines are dead-end hosts. The important role of pigs as an amplifying host for the JEV is well known. However, the influence of other domestic animals, especially birds, that live in high abundance and close proximity to the human is not well studied. Here, we strive to briefly highlight the role of birds in the JEV zoonotic transmission, discovery of birds as a natural reservoirs and amplifying host for JEV, species of birds susceptible to the JEV infection, and the proposed effect of JEV on the poultry industry in the future, a perspective that has been neglected for a long time. We also discuss the recent in vitro and in vivo studies that show that the newly emerged GI viruses replicated more efficiently in bird-derived cells and ducklings/chicks than GIII, and an important role of birds in the JEV genotype shift from GIII to GI.

Background: Japanese encephalitis virus (JEV) has a significant impact on public health. An estimated three billion people in 'at-risk’ regions remain unvaccinated and the number of unvaccinated individuals in certain Asian countries is increasing. Consequently, there is an urgent need for the development of novel therapeutic agents against Japanese encephalitis. Nitazoxanide (NTZ) is a thiazolide anti-infective licensed for the treatment of parasitic gastroenteritis. Recently, NTZ has been demonstrated to have antiviral properties. In this study, the anti-JEV activity of NTZ was evaluated in cultured cells and in a mouse model. Methods: JEV-infected cells were treated with NTZ at different concentrations. The replication of JEV in the mock- and NTZ-treated cells was examined by virus titration. NTZ was administered at different time points of JEV infection to determine the stage at which NTZ affected JEV replication. Mice were infected with a lethal dose of JEV and intragastrically administered with NTZ from 1 day post-infection. The protective effect of NTZ on the JEV-infected mice was evaluated. Findings: NTZ significantly inhibited the replication of JEV in cultured cells in a dose dependent manner with 50% effective concentration value of 0.12 ± 0.04 μg/ml, a non-toxic concentration in cultured cells (50% cytotoxic concentration = 18.59 ± 0.31 μg/ml). The chemotherapeutic index calculated was 154.92. The viral yields of the NTZ-treated cells were significantly reduced at 12, 24, 36 and 48 h post-infection compared with the mock-treated cells. NTZ was found to exert its anti-JEV effect at the early-mid stage of viral infection. The anti-JEV effect of NTZ was also demonstrated in vivo, where 90% of mice that were treated by daily intragastric administration of 100 mg/kg/day of NTZ were protected from a lethal challenge dose of JEV. Conclusions: Both in vitro and in vivo data indicated that NTZ has anti-JEV activity, suggesting the potential application of NTZ in the treatment of Japanese encephalitis.

Metagenomic analysis of mosquito-borne and mosquito-specific viruses is useful to understand the viral diversity and for the surveillance of pathogens of medical and veterinary importance. Yunnan province is located at the southwest of China and has rich abundance of mosquitoes. Arbovirus surveillance is not conducted regularly in this province particularly at animal farms, which have public health as well as veterinary importance. Here, we have analyzed 10 pools of mosquitoes belonging toCulex tritaeniorhyncus,Aedes aegypti,Anopheles sinensis, andArmigeres subalbatusspecies, collected from different animal farms located at Yunnan province of China by using metagenomic next-generation sequencing technique. The generated viral metagenomic data reveal that the viral community matched by the reads was highly diverse and varied in abundance among animal farms, which contained more than 19 viral taxonomic families, specific to vertebrates, invertebrates, fungi, plants, protozoa, and bacteria. Additionally, a large number of viral reads were related to viruses that are non-classified. The viral reads related to animal viruses included parvoviruses, anelloviruses, circoviruses, flaviviruses, rhabdoviruses, and seadornaviruses, which might be taken by mosquitoes from viremic animal hosts during blood feeding. Notably, the presence of viral reads matched with Japanese encephalitis virus, Getah virus, and porcine parvoviruses in mosquitoes collected from different geographic sites suggested a potential circulation of these viruses in their vertebrate hosts. Overall, this study provides a comprehensive knowledge of diverse viral populations present at animal farms of Yunnan province of China, which might be a potential source of diseases for humans and domestic animals.

Japanese encephalitis virus (JEV) genotype dominance has shifted to genotype I (GI) from genotype III (GIII) in China as demonstrated by molecular epidemiological surveillance. In this study, we performed a serological survey in JEV-non-vaccinated pigs to confirm JEV genotype shift at the sero-epidemiological level. The average ratio of GI/GIII infection was 1.87, suggesting co-circulation of GI and GIII infections with GI infection being more prevalent in pigs in China. To gain an insight into the reasons for this JEV genotype shift, the replication kinetics of seven recently-isolated JEV isolates including three GI strains and four GIII strains were compared in mosquito C6/36 cells, chicken fibroblast cells (DF-1) and porcine iliac artery endothelial cells (PIEC). We observed that GI strains replicated more efficiently than GIII strains in DF-1 and PIEC cells, particularly in DF-1 cells with titers reaching 22.9–225.3 fold higher than GIII strains. This shows an enhanced replication efficiency of GI viruses in avian cells. To examine this enhanced replication efficiency in vivo, young domestic ducklings were used as the animal model and inoculated with GI and GIII strains at day 2 post-hatching. We observed that GI-inoculated ducklings developed higher viremia titers and displayed a comparatively longer viremic duration than GIII-inoculated ducklings. These results conform to the hypothesis of an enhanced replication efficiency for GI viruses in birds. There are 36 amino acid differences between GI and GIII viruses, some of which may be responsible for the enhanced replication efficiency of GI viruses in birds. Based on these findings, we speculated that the enhanced replication of GI viruses in birds would have resulted in higher exposure and therefore infection in mosquitoes, which could result in an increased transmission efficiency of GI viruses in the birds-mosquitoes-birds enzootic transmission cycle, thereby contributing to JEV genotype shift. Japanese encephalitis virus (JEV) causes encephalitis in humans and reproductive disorder in pigs. The enzootic transmission cycle of JEV is maintained in nature by several species of mosquitoes and vertebrates including birds and pigs. In recent years, JEV genotype I (GI) replaced genotype IIII (GIII) as the dominant genotype in Asian countries. Genotype shift has an impact on disease control, and understanding the reasons for this shift will offer valuable insight into avenues for future disease control. Therefore, we compared the replication efficiencies of GI and GIII viruses in vitro and in vivo. We observed that GI viruses show higher replication titers in avian cells and higher viremia levels in young domestic ducklings than GIII viruses, suggesting an enhanced replication efficiency of GI viruses in birds. Based on these findings, we speculated that the enhanced replication of GI viruses in birds could provide increased mosquito infection, leading to an increase in the birds-mosquitoes-birds transmission cycle, thereby contributing to JEV genotype shift.