Hailin Zhang

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.

Isolation of Kyasanur Forest Disease Virus from Febrile Patient, Yunnan, China

We recently determined that Nanjianyin virus, isolated from serum of a patient in Yunnan Province, China, in 1989, is a type of Kyasanur Forest disease virus. Results of a 1987–1990 seroepidemiologic investigation in Yunnan Province had shown that residents of the Hengduan Mountain region had been infected with Nanjianyin virus.

Identification of Diverse Alphacoronaviruses and Genomic Characterization of a Novel Severe Acute Respiratory Syndrome-Like Coronavirus from Bats in China

ABSTRACT Although many severe acute respiratory syndrome-like coronaviruses (SARS-like CoVs) have been identified in bats in China, Europe, and Africa, most have a genetic organization significantly distinct from human/civet SARS CoVs in the receptor-binding domain (RBD), which mediates receptor binding and determines the host spectrum, resulting in their failure to cause human infections and making them unlikely progenitors of human/civet SARS CoVs. Here, a viral metagenomic analysis of 268 bat rectal swabs collected from four counties in Yunnan Province has identified hundreds of sequences relating to alpha- and betacoronaviruses. Phylogenetic analysis based on a conserved region of the RNA-dependent RNA polymerase gene revealed that alphacoronaviruses had diversities with some obvious differences from those reported previously. Full genomic analysis of a new SARS-like CoV from Baoshan (LYRa11) showed that it was 29,805 nucleotides (nt) in length with 13 open reading frames (ORFs), sharing 91% nucleotide identity with human/civet SARS CoVs and the most recently reported SARS-like CoV Rs3367, while sharing 89% with other bat SARS-like CoVs. Notably, it showed the highest sequence identity with the S gene of SARS CoVs and Rs3367, especially in the RBD region. Antigenic analysis showed that the S1 domain of LYRa11 could be efficiently recognized by SARS-convalescent human serum, indicating that LYRa11 is a novel virus antigenically close to SARS CoV. Recombination analyses indicate that LYRa11 is likely a recombinant descended from parental lineages that had evolved into a number of bat SARS-like CoVs. IMPORTANCE Although many severe acute respiratory syndrome-like coronaviruses (SARS-like CoVs) have been discovered in bats worldwide, there are significant different genic structures, particularly in the S1 domain, which are responsible for host tropism determination, between bat SARS-like CoVs and human SARS CoVs, indicating that most reported bat SARS-like CoVs are not the progenitors of human SARS CoV. We have identified diverse alphacoronaviruses and a close relative (LYRa11) to SARS CoV in bats collected in Yunnan, China. Further analysis showed that alpha- and betacoronaviruses have different circulation and transmission dynamics in bat populations. Notably, full genomic sequencing and antigenic study demonstrated that LYRa11 is phylogenetically and antigenically closely related to SARS CoV. Recombination analyses indicate that LYRa11 is a recombinant from certain bat SARS-like CoVs circulating in Yunnan Province.

Japanese encephalitis viruses from bats in Yunnan, China.

Genome sequencing and virulence studies of 2 Japanese encephalitis viruses (JEVs) from bats in Yunnan, China, showed a close relationship with JEVs isolated from mosquitoes and humans in the same region over 2 decades. These results indicate that bats may play a role in human Japanese encephalitis outbreaks in this region.

Characterization of a novel G3P[3] rotavirus isolated from a lesser horseshoe bat: a distant relative of feline/canine rotaviruses

ABSTRACT Bats are considered important animal reservoirs for many viruses pathogenic to humans. An approach based on viral metagenomics was used to study gut specimens from 78 insectivorous bats in Yunnan Province, China. Seventy-four reads were found to be related to group A rotavirus (RVA). Further reverse transcription-PCR screening and viral isolation on cell cultures confirmed the presence of a novel RVA strain, named RVA/Bat-tc/MSLH14/2012/G3P[3], in 1 (6%) of 16 lesser horseshoe bats. Full genomic sequencing analyses showed that MSLH14 possessed the genotype constellation G3-P[3]-I8-R3-C3-M3-A9-N3-T3-E3-H6, which is akin to human and animal rotaviruses believed to be of feline/canine origin. Phylogenetic analysis indicated that VP7 was most closely related to bovine RVA strains from India, whereas VP4 was most closely related to an unusual human RVA strain, CMH222, with animal characteristics isolated in Thailand. The remaining gene segments were only distantly related to a range of animal RVA strains, most of which are believed to be related to feline/canine RVAs. Experimental infection showed that bat RVA strain MSLH14 was highly pathogenic to suckling mice, causing 100% mortality when they were inoculated orally with a titer as low as 5 × 102 50% tissue culture infective doses. As this virus is not closely related to any known RVA strain, it is tempting to speculate that it is a true bat RVA strain rather than a virus transmitted between species. However, further screening of bat populations, preferably juvenile animals, will be crucial in determining whether or not this virus is widely distributed in the bat population.

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.

Distribution of Mosquitoes and Mosquito-Borne Arboviruses in Yunnan Province near the China―Myanmar―Laos Border

Economic development and increased tourism in the southern region of Yunnan Province in China, adjacent to several countries in Southeast Asia, has increased the likelihood of import and export of vectors and vector-borne diseases. We report the results of surveillance of mosquitoes and mosquito-borne arboviruses along the border of China-Myanmar-Laos in 2005 and 2006, and information associating several arboviruses with infections and possibly disease in local human populations. Seventeen mosquito species representing four genera were obtained, and 14 strains of mosquito-borne viruses representing six viruses in five genera were isolated from Culex tritaeniorhynchus. In addition, IgM against Japanese encephalitis virus, Sindbis virus, Yunnan orbivirus and novel Banna virus was detected in acute-phase serum samples obtained from hospitalized patients with fever and encephalitis near the areas where the viruses were isolated. This investigation suggests that Japanese encephalitis virus, Sindbis virus, and lesser-known arboviruses circulate and may be infecting humans in the China-Myanmar-Laos border region.

Human Infection with West Nile Virus, Xinjiang, China, 2011

To the Editor: West Nile virus (WNV) is a mosquito-borne flavivirus in the Japanese encephalitis serocomplex of the family Flaviviridae (1). It has been reported in Africa, Asia, Europe, Australia, and North America, and is recognized as the most globally widespread mosquito-borne flavivirus (2). Isolation of WNV has previously been attempted in China, Japan and South Korea; however, no virus has been isolated (3–5). We report isolation of WNVs from mosquitoes in Xinjiang Uyghur Autonomous Region in western China. We also provide evidence of WNV human infections confirmed by IgM ELISA and seroconversion by 90% plaque reduction neutralization tests of paired serum samples obtained from persons with febrile illness and viral encephalitis in 2011.

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

Isolation and identification of a natural reassortant mammalian orthoreovirus from least horseshoe bat in China.

Background Mammalian orthoreoviruses (MRVs) have a wide geographic distribution and can infect virtually all mammals. Infections in humans may be either symptomatic or asymptomatic. This study describes the isolation and identification of a natural reassortant MRV from least horseshoe bats (Rhinolophus pusillu) in China, referred to as RpMRV-YN2012. Methods and Results The RpMRV-YN2012 was obtained from urine samples of Rhinolophus pusillus by cell culture. Negative-staining electron microscopy revealed that RpMRV-YN2012 was a non-enveloped icosahedral virus with ∼75 nm in diameter. Polyacrylamide gel electrophoresis (PAGE) migration patterns of the genome segments showed that RpMRV-YN2012 contained 10 segments in a 3:3:4 arrangement. The whole genome sequence of RpMRV2012 was determined. The consensus terminal sequences of all segments of 5’-GCUAh…yUCAUC-3’ (h = A, U or C; y = C or U) were similar to the MRV species within the genus Orthoreovirus. Its evolution and evidence of genetic reassortment were analyzed by sequence comparison and phylogenetic analysis. The results showed that RpMRV-YN2012 is a novel serotype 2 MRV that may have originated from reassortment among bat, human, and/or pig MRV strains which associated with diarrhea, acute gastroenteritis and necrotizing encephalopathy in animals and humans. Conclusions RpMRV-YN2012 is a novel bat reassortant MRV, which may have resulted from a reassortment involving MRVs known to infect humans and animals. It is necessary to identify whether RpMRV-YN2012 is associated with diarrhea, acute gastroenteritis and necrotizing encephalopathy in clinical patients. In addition, we should carefully monitor its evolution and virulence in real time.