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Abstract
Objective: To analyze the molecular characteristics of the full-length genome of a newly isolated genotype V Japanese encephalitis virus (JEV) strain (XZ0934) in China and the first genotype V JEV strain (Muar) isolated in Malaya 60 years ago. Methods: Several softwares including ClustalX 2.0.9, DNAStar 7.1, Bioedit 7.2.5 and MEGA6.06 were used to conduct sequence alignments and phylo-genetic analysis. Results: The full-length genomes of XZ0934 strain (isolated in Tibet, China in 2009) and Muar strain (isolated in Malaya in 1952) were composed of 10 983 and 10 988 nucleotides, respectively. The XZ0934 strain was highly similar with the Muar strain showing the homology of 90.6% in nucleotide (nt) and 98.3% in amino acid (aa). The open reading frame (ORF) of the two genotype V JEV strains encoded 3433 aa while the ORF of other four genotypes (I-IV) (10 299 nt) encoded 3432 aa. Compared with JEV strains of other genotypes, a serine were inserted into the NS4A gene of JEV strains genotype V and 10-14 nucleotides were inserted into the downstream of the ORF stop codon in 3'-untranslated region. Phylogenetic analysis of E sequences of all JEV strains genotyped I-V revealed that in the cluster of genotype V, XZ0934 and 10-1827 (isolated from mosquitoes in South Korea, 2010) stains formed a branch and were divergent from that of Muar strain indicating that there were molecular genetic differences among genotype V JEV strains after a 60 years hiatus. Conclusion: The two genotype V JEV strains showed high levels of identity in nucleotide sequences and amino acid sequences with serine insertion in the NS4A gene. However, there were molecular genetic differences between genotype V JEV strains isolated after a 60 years hiatus.
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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.
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.
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.
Japanese encephalitis virus (JEV) genotype V reemerged in Asia (China) in 2009 after a 57-year hiatus from the continent, thereby emphasizing a need to increase regional surveillance efforts. Genotypic characterization was performed on 19 JEV-positive mosquito pools (18 pools of Culex tritaeniorhynchus and 1 pool of Cx. bitaeniorhynchus) from a total of 64 positive pools collected from geographically different locations throughout the Republic of Korea (ROK) during 2008 and 2010.
Two regions of the JEV genome were sequenced from 19 pools; the envelope gene and the nonstructural protein 5 (NS5)/3'-untranslated region (UTR). Eighteen pools of Culex tritaeniorhynchus and one pool of Cx. bitaeniorhynchus were positive for genotype I and genotype V, respectively. Sequence alignment of the complete E gene from Cx. bitaeniorhynchus showed high amino acid similarity (98.8%) to the Muar strain, characterized as the first report of genotype V, isolated from an encephalitis patient in Malaysia in 1952.
This study represents the first report of JEV genotype V in the ROK. The reemergence of genotype V in Asia (China and ROK) after more than a half-century and its discovery in Cx. bitaeniorhynchus, a mosquito species previously unknown to carry JEV in the ROK, emphasizes the need for enhanced JE surveillance to monitor the dynamics of JEV strains within the region. Future findings may have implications with regard to JEV vaccination/prevention strategies.
Japanese encephalitis (JE) is a global public health issue that has spread widely to more than 20 countries in Asia and has extended its geographic range to the south Pacific region including Australia. JE has become the most important cause of viral encephalitis in the world. Japanese encephalitis viruses (JEV) are divided into five genotypes, based on the nucleotide sequence of the envelope (E) gene. The Muar strain, isolated from patient in Malaya in 1952, is the sole example of genotype V JEV. Here, the XZ0934 strain of JEV was isolated from Culex tritaeniorhynchus, collected in China. The complete nucleotide and amino acid sequence of XZ0934 strain have been determined. The nucleotide divergence ranged from 20.3% to 21.4% and amino acid divergence ranged from 8.4% to 10.0% when compared with the 62 known JEV isolates that belong to genotype I-IV. It reveals low similarity between XZ0934 and genotype I-IV JEVs. Phylogenetic analysis using both complete genome and structural gene nucleotide sequences demonstrates that XZ0934 belongs to genotype V. This, in turn, suggests that genotype V JEV is emerging in JEV endemic areas. Thus, increased surveillance and diagnosis of viral encephalitis caused by genotype V JEV is an issue of great concern to nations in which JEV is endemic.
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.
Japanese encephalitis (JE), a vector-borne viral disease, is endemic to large parts of Asia and the Pacific. An estimated 3 billion people are at risk, and JE has recently spread to new territories. Vaccination programs, increased living standards, and mechanization of agriculture are key factors in the decline in the incidence of this disease in Japan and South Korea. However, transmission of JE is likely to increase in Bangladesh, Cambodia, Indonesia, Laos, Myanmar, North Korea, and Pakistan because of population growth, intensified rice farming, pig rearing, and the lack of vaccination programs and surveillance. On a global scale, however, the incidence of JE may decline as a result of large-scale vaccination programs implemented in China and India.
Since it emerged in Japan in the 1870s, Japanese encephalitis has spread across Asia and has become the most important cause of epidemic encephalitis worldwide. Four genotypes of Japanese encephalitis virus (JEV) are presently recognized (representatives of genotypes I to III have been fully sequenced), but its origin is not known. We have determined the complete nucleotide and amino acid sequence of a genotype IV Indonesian isolate (JKT6468) which represents the oldest lineage, compared it with other fully sequenced genomes, and examined the geographical distribution of all known isolates. JKT6468 was the least similar, with nucleotide divergence ranging from 17.4 to 19.6% and amino acid divergence ranging from 4.7 to 6.5%. It included an unusual series of amino acids at the carboxy terminus of the core protein unlike that seen in other JEV strains. Three signature amino acids in the envelope protein (including E327 Leu-->Thr/Ser on the exposed lateral surface of the putative receptor binding domain) distinguished genotype IV strains from more recent genotypes. Analysis of all 290 JEV isolates for which sequence data are available showed that the Indonesia-Malaysia region has all genotypes of JEV circulating, whereas only more recent genotypes circulate in other areas (P < 0.0001). These results suggest that JEV originated from its ancestral virus in the Indonesia-Malaysia region and evolved there into the different genotypes which then spread across Asia. Our data, together with recent evidence on the origins of other emerging viruses, including dengue virus and Nipah virus, imply that tropical southeast Asia may be an important zone for emerging pathogens.
Sixty-two new Japanese encephalitis virus (JEV) isolates were obtained from mosquitoes, biting midges, human cerebrospinal fluid and human blood samples in China during 2002-2005. The E and prM genes were sequenced and phylogenetic analyses were performed with 38 JEV other isolates from China and 36 JEV strains from other countries. Phylogenetic trees based on the E and prM gene sequences were similar. The results indicate that: (i) recent JEV isolates from China are divided into two genotypes, genotype 1 and genotype 3; (ii) recent JEV isolates from China are grouped into the same clusters within genotypes 1 and 3; and (iii) genotype 1 JEV strains have been isolated in China since 1979, whilst genotype 3 JEV strains were isolated before the 1970s. The results suggest that genotype 1 JEV was introduced to China around 1979 and that JEV strains belonging to genotypes 1 and 3 circulate in China.
To determine the molecular characterization of full-length genome of Japanese encephalitis virus (JEV) genotype V.
The full-length nucleotide sequences of JEV strains isolated from different locations and sources were used in sequence and phylogenetic analysis.
The full-length genome of genotypes V JEV, XZ0934, and Muar strain were composed of 10 983 and 10 988 nucleotides respectively and shared a lower level of identity with JEV genotypes I-IV, ranging from 78.4% (G I, KV1899) to 79.7% (G III, JaGAr01), for the nucleotide sequences, and from 90.0% (G I, KV1899) to 91.8% (G III, JaGAr01) for the amino acid sequences. The open reading frame (ORF) of JEV genotype V spanned nucleotides 96 to 10 397 and encoded 3 433 amino acids. Interestingly, a comparison with JEV genotype I-IV revealed that 3 nucleotides (encoded with a serine residue) were inserted in the NS4A gene of JEV genotype V, and the insertion of nucleotides was also found in downstream of the ORF stop codon in 3'-untranslated region. Moreover, numerous amino acid mutations were observed in 3 functional domains of the E gene of JEV genotype V.
The molecular characterization of JEV genotype V is significantly different from that of the known genotypes I-IV. The mutations located in the coding region and the non-coding region may be molecular markers of JEV genotype V and warrant further studies to determine their effects on biology and immunogenicity of genotype V strains.
Japanese encephalitis virus (JEV) is the most important cause of epidemic encephalitis worldwide but its origin is unknown. Epidemics of encephalitis suggestive of Japanese encephalitis (JE) were described in Japan from the 1870s onwards. Four genotypes of JEV have been characterised and representatives of each genotype have been fully sequenced. Based on limited information, a single isolate from Malaysia is thought to represent a putative fifth genotype. We have determined the complete nucleotide and amino acid sequence of Muar strain and compared it with other fully sequenced JEV genomes. Muar was the least similar, with nucleotide divergence ranging from 20.2 to 21.2% and amino acid divergence ranging from 8.5 to 9.9%. Phylogenetic analysis of Muar strain revealed that it does represent a distinct fifth genotype of JEV. We elucidated Muar signature amino acids in the envelope (E) protein, including E327 Glu on the exposed lateral surface of the putative receptor binding domain which distinguishes Muar strain from the other four genotypes. Evolutionary analysis of full-length JEV genomes revealed that the mean evolutionary rate is 4.35 × 10(-4) (3.4906 × 10(-4) to 5.303 × 10(-4)) nucleotides substitutions per site per year and suggests JEV originated from its ancestral virus in the mid 1500s in the Indonesia-Malaysia region and evolved there into different genotypes, which then spread across Asia. No strong evidence for positive selection was found between JEV strains of the five genotypes and the E gene has generally been subjected to strong purifying selection.
A novel Japanese encephalitis (JE) attenuated live vaccine virus SA14-14-2 was licensed for commercial application in China in 1989. Since then this vaccine has been widely used in China and other countries in Asia, and no vaccine associated encephalitis case was reported. The neurovirulence of the SA14-14-2 was tested in JE susceptible laboratory animals, such as mice, monkeys, hamsters and athymic nude mice. The results showed that the attenuated virus strain was avirulent to these animals by intracerebral inoculation (i.c.) or intraperitoneal inoculation (i.p.). Studies on the neuroattenuation stability revealed that no reversion after 17 times tissue culture passages or one i.c. sucking mice passage. Mosquito infection studies indicated that after one mosquito intrathoracical passage, the progeny viruses in the infected mosquitoes were unable to cause sucking mice or weanling mice disease. Molecules characteristics' studies of the SA14-14-2 virus strain showed that there are 57-61 nucleotide changes and 24-31 amino acid substitutions, eight substitutions in the E protein gene are the critical amino acid related to the virus attenuation. The E gene sequence studies have showed that the 8 critical amino acids were not changed after 22 passages in tissue cultures or one passage in mosquitoes. Comparison of the full-length sequence to the parental SA14 virus has revealed that after 22 passages in the tissue cultures, only 8 nucleotides changed leading to 4 amino acid substitutions. However they were not the reverse mutation and none of the 8 critical residues changed. The homology of the nucleotide and amino acid between the virus of passage 22 and the primary seed virus in Genbank was 99.93% and 99.88% respectively. The above results demonstrated that the SA14-14-2 virus is highly attenuated for the various JE susceptible animals. The attenuated phenotypes and the genetic characteristics of the SA14-14-2 strain are highly stable after multiple in vitro passages or mosquitoes infection. Therefore the safety of the live JE vaccine is due to a high degree of neuroattenuation and a number of stable phenotypically and genetically characteristics, suggesting that reversion to neurovirulence of the vaccine strain would be highly unlikely.
The immunological characteristics of 26 strains of Japanese encephalitis virus (JEV) isolated in Japan and Malaya between 1935 and 1966 have been investigated mainly by the antibody-absorption variant of the haemagglutination-inhibition test, and to a certain extent also by conventional haemagglutination-inhibition and complement-fixation tests. The antibody-absorption technique shows promise as a routine method for the immunotyping of JEV.At present, two immunotypes can be distinguished. One comprises 2 strains, Nakayama-NIH and I-58, and is designated as the I-58 immunotype. The other immunotype, JaGAr 01, comprises 17 strains which share the characteristics of the JaGAr 01 strain, including one subline of the Nakayama strain, Nakayama-Yakken. The Nakayama-RFVL strain was found to have the characteristics of both immunotypes. The I-58 immunotype differs more markedly from related arboviruses, such as the Murray Valley encephalitis virus and the West Nile Eg101 strain, than does the JaGAr 01 immunotype.Evidence is presented which suggests that a given JEV strain can change immunotype on repeated passage through mice.
Based on the reactivities of monoclonal antibodies, we have reported previously that 25 Japanese encephalitis virus (JEV) strains fell into five antigenic groups: Nakayama, Beijing-1, Kamiyama, 691004 and Muar. In the present study, we compared the nucleotide and deduced amino acid sequences of the structural proteins of these strains. Four strains, Nakayama-RFVL, Beijing-1, Kamiyama and 691004, shared > 96.4% identity in the nucleotide sequence and > 94.7% identity in the amino acid sequence. However, the Muar strain had only an 80.3-87.7% homology for nucleotides and an 85.7-93.5% homology for amino acids, compared with the other four strains. The following strain-specific amino acid differences were found in the envelope protein: two in Nakayama-RFVL; three in Beijing-1; one in Kamiyama; five in 691004; 42 in Muar. These findings showed that the Muar strain was the most structurally different from the other four strains, and that it would be possible to compare antigenic differences among the JEV strains by using monoclonal antibodies which showed different specificities.
Haemorrhagic disease, encephalitis, biphasic fever, flaccid paralysis, and jaundice are typical manifestations of diseases in human beings after infections by mosquito-borne or tick-borne flaviviruses such as yellow fever, dengue, West Nile, St Louis encephalitis, Japanese encephalitis, tick-borne encephalitis, Kyasanur Forest disease, and Omsk haemorrhagic fever. Although the characteristics of these viruses are well defined, they are still unpredictable with increases in disease severity, unusual clinical manifestations, unexpected methods of transmission, long-term persistence, and the discovery of new species. This Seminar will compare the epidemiological and clinical features of the medically important flaviviruses, consider the effect of human activity on their evolution and dispersal, and draw attention to new findings and some of the unanswered questions, unresolved issues, and controversies that remain.