Xiaoyan Gao

Emergence of Genotype I of Japanese Encephalitis Virus as the Dominant Genotype in Asia

ABSTRACT Japanese encephalitis virus (JEV), a mosquito-borne zoonotic pathogen, is one of the major causes of viral encephalitis worldwide. Previous phylogenetic studies based on the envelope protein indicated that there are four genotypes, and surveillance data suggest that genotype I is gradually replacing genotype III as the dominant strain. Here we report an evolutionary analysis based on 98 full-length genome sequences of JEV, including 67 new samples isolated from humans, pigs, mosquitoes, midges. and bats in affected areas. To investigate the relationships between the genotypes and the significance of genotype I in recent epidemics, we estimated evolutionary rates, ages of common ancestors, and population demographics. Our results indicate that the genotypes diverged in the order IV, III, II, and I and that the genetic diversity of genotype III has decreased rapidly while that of genotype I has increased gradually, consistent with its emergence as the dominant genotype.

Factors responsible for the emergence of arboviruses; strategies, challenges and limitations for their control

Slave trading of Africans to the Americas, during the 16th to the 19th century was responsible for the first recorded emergence in the New World of two arthropod-borne viruses (arboviruses), yellow fever virus and dengue virus. Many other arboviruses have since emerged from their sylvatic reservoirs and dispersed globally due to evolving factors that include anthropological behaviour, commercial transportation and land-remediation. Here, we outline some characteristics of these highly divergent arboviruses, including the variety of life cycles they have developed and the mechanisms by which they have adapted to evolving changes in habitat and host availability. We cite recent examples of virus emergence that exemplify how arboviruses have exploited the consequences of the modern human lifestyle. Using our current understanding of these viruses, we also attempt to demonstrate some of the limitations encountered in developing control strategies to reduce the impact of future emerging arbovirus diseases. Finally, we present recommendations for development by an international panel of experts reporting directly to World Health Organization, with the intention of providing internationally acceptable guidelines for improving emerging arbovirus disease control strategies. Success in these aims should alleviate the suffering and costs encountered during recent decades when arboviruses have emerged from their sylvatic environment.

Southernmost Asia Is the Source of Japanese Encephalitis Virus (Genotype 1) Diversity from which the Viruses Disperse and Evolve throughout Asia

Background Although a previous study predicted that Japanese encephalitis virus (JEV) originated in the Malaysia/Indonesia region, the virus is known to circulate mainly on the Asian continent. However, there are no reported systematic studies that adequately define how JEV then dispersed throughout Asia. Methodology/Principal Findings In order to understand the mode of JEV dispersal throughout the entire Asian continent and the factors that determine the dispersal characteristics of JEV, a phylogenetic analysis using Bayesian Markov chain Monte Carlo simulations was conducted on all available JEV E gene sequences in GenBank, plus strains recently isolated in China. Here we demonstrate for the first time that JEV lineages can be divided into four endemic cycles, comprising southern Asia, eastern coastal Asia, western Asia, and central Asia. The isolation places of the viruses in each endemic cycle were geographically independent regardless of years, vectors, and hosts of isolation. Following further analysis, we propose that the southernmost region (Thailand, Vietnam, and Yunnan Province, China) was the source of JEV transmission to the Asian continent following its emergence. Three independent transmission routes from the south to north appear to define subsequent dispersal of JEV. Analysis of JEV population dynamics further supports these concepts. Conclusions/Significance These results and their interpretation provide new insights into our understanding of JEV evolution and dispersal and highlight its potential for introduction into non-endemic areas.

Identification and isolation of Genotype-I Japanese Encephalitis virus from encephalitis patients

Historically, Japanese Encephalitis virus (JEV) genotype III (GIII) has been responsible for human diseases. In recent years, JEV genotype I (GI) has been isolated from mosquitoes collected in numerous countries, but has not been isolated from patients with encephalitis. In this study, we report recovery of JEV GI live virus and identification of JEV GI RNA from cerebrospinal fluid (CSF) of encephalitis patients in JE endemic areas of China. Whole-genome sequencing and molecular phylogenetic analysis of the JEV isolate from the CSF samples was performed. The isolate in this study is highly similar to other JEV GI strains which isolated from mosquitoes at both the nucleotide and deduced amino acid levels. Phylogenetic analysis based on the genomic sequence showed that the isolate belongs to JEV GI, which is consistent with the phylogenetic analysis based on the pre-membrane (PrM) and Glycoprotein genes. As a conclusion, this is the first time to isolate JEV GI strain from CSF samples of encephalitis patients, so continuous survey and evaluate the infectivity and pathogenecity of JEV GI strains are necessary, especially for the JEV GI strains from encephalitis patients. With respect to the latter, because all current JEV vaccines (live and inactivated are derived from JEV GIII strains, future studies should be aimed at investigating and monitoring cross-protection of the human JEV GI isolates against widely used JEV vaccines.

Tahyna virus and human infection, China.

In 2006, Tahyna virus was isolated from Culex spp. mosquitoes collected in Xinjiang, People’s Republic of China. In 2007, to determine whether this virus was infecting humans, we tested serum from febrile patients. We found immunoglobulin (Ig) M and IgG against the virus, which suggests human infection in this region.

Newly recognized mosquito-associated viruses in mainland China, in the last two decades

There are four principal arboviruses in mainland China. Two kinds of them are mosquito-borne viruses, namely Japanese encephalitis virus and dengue virus, which lead to Japanese encephalitis, and dengue fever/dengue hemorrhagic fever respectively; the other two are tick-borne viruses, namely tick-borne encephalitis virus and Crimean-Congo hemorrhagic fever virus (also known as Xinjiang hemorrhagic fever virus), which contribute to tick-borne encephalitis and Xinjiang hemorrhagic fever respectively. With exception of these four main arboviruses, many other mosquito-associated viruses have been isolated and identified in recent years. These newly isolated and identified mosquito-associated viruses are probably responsible for human and animal infections and diseases. The purpose of this review is to describe the newly isolated mosquito-associated viruses in mainland China which belong to five viral families, including their virological properties, phylogenetic relationships, serological evidence, as well as to appeal the public health concentration worldwide.

Vaccine Strategies for the Control and Prevention of Japanese Encephalitis in Mainland China, 1951–2011

Japanese encephalitis (JE) is arguably one of the most serious viral encephalitis diseases worldwide. China has a long history of high prevalence of Japanese encephalitis, with thousands of cases reported annually and incidence rates often exceeding 15/100,000. In global terms, the scale of outbreaks and high incidence of these pandemics has almost been unique, placing a heavy burden on the Chinese health authorities. However, the introduction of vaccines, developed in China, combined with an intensive vaccination program initiated during the 1970s, as well as other public health interventions, has dramatically decreased the incidence from 20.92/100,000 in 1971, to 0.12/100,000 in 2011. Moreover, in less readily accessible areas of China, changes to agricultural practices designed to reduce chances of mosquito bites as well as mosquito population densities have also been proven effective in reducing local JE incidence. This unprecedented public health achievement has saved many lives and provided valuable experience that could be directly applicable to the control of vector-borne diseases around the world. Here, we review and discuss strategies for promotion and expansion of vaccination programs to reduce the incidence of JE even further, for the benefit of health authorities throughout Asia and, potentially, worldwide.

Low Protective Efficacy of the Current Japanese Encephalitis Vaccine against the Emerging Genotype 5 Japanese Encephalitis Virus.

Background The current Japanese encephalitis (JE) vaccine derived from G3 JE virus (JEV) can induce protective immunity against G1–G4 JEV genotypes. However, protective efficacy against the emerging G5 genotype has not been reported. Methods/Principal Findings Using in vitro and in vivo tests, biological phenotype and cross-immunoreactions were compared between G3 JEV and G5 JEV (wild strains). The PRNT90 method was used to detect neutralizing antibodies against different genotypes of JEV in JE vaccine-immunized subjects and JE patients. In JE vaccine-immunized mice, the lethal challenge protection rates against G3 and G5 JEV wild strains were 100% and 50%, respectively. The seroconversion rates (SCRs) of virus antibodies against G3 and G5 JEV among vaccinated healthy subjects were 100% and 35%, respectively. All clinically identified JE patients showed high levels of G3 JEV neutralizing antibodies (≥1:10–1280) with positive serum geometric mean titers (GMTs) of 43.2, while for G5 JEV, neutralizing antibody conversion rates were only 64% with positive serum GMTs of 11.14. Moreover, the positive rate of JEV neutralizing antibodies against G5 JEV in pediatric patients was lower than in adults. Conclusions/Significance Low levels of neutralizing/protective antibodies induced by the current JE vaccine, based on the G3 genotype, were observed against the emerging G5 JEV genotype. Our results demonstrate the need for more detailed studies to reevaluate whether or not the apparent emergence of G5 JEV can be attributed to failure of the current vaccine to induce appropriate immune protectivity against this genotype of JEV.

High incidence of Japanese encephalitis, southern China.

To the Editor: Japanese encephalitis virus (JEV) remains a major source of illness and death in Asia (1). An estimated 67,900 cases occur each year in Asia; ≈33,900 cases— half the cases in the world—probably occur in the People’s Republic of China (2). However, because reporting is incomplete in most countries where JE incidence is high, these estimates are based on scarce data. In China, a study conducted during 2006–2007 in sentinel hospitals in 1 prefecture each in Shandong, Hubei, Guangxi, and Hebei Provinces (all in the eastern half of China) found that 9.2% of patients with acute meningitis and encephalitis had JEV; adjusted incidence for each prefecture was 0.08–1.58 cases per 100,000 population (3). Incidence in these 4 prefectures is lower than that among children <14 years of age in JE-endemic countries, where estimated incidence is 5.4 cases per 100,000 population (2). To assess the need for strengthening existing JE surveillance and vaccination programs, we conducted a populationbased study of JE incidence in 1 area of southern China. Dehong is a prefecture in western Yunnan Province, which borders Myanmar. The JE-susceptible population of Dehong Prefecture (residents <15 years of age) is 211,337. The 2 principal cities of Dehong Prefecture—Mangshi and Ruili—are busy commercial centers surrounded by areas of extensive rice cultivation. The mosquito vector of JEV, Culex tritaeniorhynchus, is predominant during summer (4). During 1988– 2007, JEV vaccination was available only at certain clinics and only for a fee; however, since 2008, vaccination with the live, attenuated SA 14–14–2 JEV vaccine (Chengdu Institute of Biologic Products, Chengdu, China) has been included in the national Expanded Program on Immunization at no charge. The recommendation for children is vaccination at 8 months and 2 years of age (5,6). To estimate JE incidence in Dehong Prefecture during January 1–December 31, 2010, we conducted an anonymous, unlinked study of all cases of encephalitis at the only 2 major children’s hospitals in the region, Dehong Prefecture Hospital in Mangshi and Ruili City Hospital in Ruili. All eligible patients admitted to these hospitals were included in the study. Inclusion criteria were age <15 years, residency in Dehong Prefecture, clinical diagnosis of encephalitis, lumbar puncture performed (routine for encephalitis patients at these 2 hospitals), and cerebrospinal fluid (CSF) pleocytosis. After routine testing was completed, leftover CSF and serum samples were stored at -70°C until further testing, which was all conducted at the Chinese Center for Disease Control and Prevention in Beijing. All CSF specimens were tested by viral culture in C6/36 and BHK21 cells (7) and tested for antibodies against JEV (3,4). Serum samples were tested for antibodies against JE virus, mumps virus, echoviruses, and coxsackieviruses (3,4,7). A case of JE was defined as illness in a person with IgM against JEV in CSF or serum. Clinical information was collected by using a standardized chart abstraction form. Linkages to personal identifiers were destroyed. A total of 189 eligible patients were enrolled, 150 from Mangshi and 39 from Ruili. Of these, 110 (58%) were male and 78 (41%) were <4 years of age. Enrollment peaked during summer (Figure). All patients were hospitalized within 6 days after symptom onset. A total of 22 (12%) patients were classified as having JE on the basis of IgM, in CSF for 21 and in serum for 1. Illness onset occurred during May–November (Figure); overall incidence was 10.4 cases per 100,000 children <15 years of age. Among these 22 children with JE, 11 were male; 20 were from rural areas; 14 were from Mangshi and 8 were from Ruili; and 5 were 0–1 years, 6 were 2–4 years, and 11 were 5–13 years of age. JEV vaccination history was infrequently recorded in the medical charts; however, JEV was more likely to be the cause of encephalitis among

Mosquitoes of Western Yunnan Province, China: seasonal abundance, diversity, and arbovirus associations.

Objective The western borderland between Yunnan Province, China, and Myanmar is characterized by a climate that facilitates year-round production of mosquitoes. Numerous mosquito-transmitted viruses, including Japanese encephalitis virus circulate in this area. This project was to describe seasonal patterns in mosquito species abundance and arbovirus activity in the mosquito populations. Methods Mosquitoes were collected in Mangshi and Ruili cities of Dehong Prefecture near the border of China and Burma in Yunnan Province, the Peoples Republic of China in 2010. We monitored mosquito species abundance for a 12-month period using ultraviolet light, carbon dioxide baited CDC light and gravid traps; and tested the captured mosquitoes for the presence of virus to evaluate mosquito-virus associations in rural/agricultural settings in the area. Results A total of 43 species of mosquitoes from seven genera were collected, including 15 Culex species, 15 Anopheles spp., four Aedes spp., three Armigeres spp., one Mimomyia spp., two Uranotaenia spp. and three Mansonia spp.. Species richness and diversity varied between Mangshi and Ruili. Culex tritaeniorhynchus, Culex quinquefasciatus, Anopheles sinensis and Anopheles peditaeniatus were the most abundant species in both sampling sites. Ultraviolet light traps collected more specimens than CDC light traps baited with dry ice, though both collected the same variety of mosquito species. The CDC gravid trap was the most effective trap for capture of Culex quinquefasciatus, a species underrepresented in light trap collections. A total of 26 virus strains were isolated, which included 13 strains of Japanese encephalitis virus, four strains of Getah virus, one strain of Oya virus, one strain from the orbivirus genus, and seven strains of Culex pipien pallens densovirus. Conclusions The present study illustrates the value of monitoring mosquito populations and mosquito-transmitted viruses year-round in areas where the climate supports year-round adult mosquito activity.