Encephalitis Virus Strains Research Articles

Infection of mouse bone marrow-derived dendritic cells by live attenuated Japanese encephalitis virus induces cells maturation and triggers T cells activation

An attenuated Japanese encephalitis virus (JEV) strain SA14-14-2, generated from the wild strain SA14, is an effective live vaccine against JEV infection. It has led to a significant decrease in JEV infection around the world. Although it is highly effective, the mechanism for its robust immunity was not well investigated. In this study, the interaction of SA14-14-2 with bone marrow-derived dendritic cells (bmDCs) was investigated. Our results showed that the infection of bmDCs with SA14-14-2 resulted in viral replication and upregulation of bmDC maturation marker molecules (CD40, CD80, CD83 and MHC I). SA14-14-2 infection also stimulated the production of interferon-α (IFN-α), monocyte chemoattractant protein-1 (MCP-1/CCL2), tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) of bmDC. Both MLR and ELISPOT assay showed an enhanced allostimulatory capacity of SA14-14-2-infected bmDCs. Furthermore, the SA14-14-2-infected bmDCs impaired the expansion of Foxp3+ regulatory T (Treg) cells with immunosuppressive potential, suggesting that SA14-14-2 infection induced antiviral immunity rather than immunosuppression. Taken together, our results indicated that SA14-14-2 infection caused bmDC maturation, changed the expression profiles of several cytokines, and triggered T cell activation. This offered an insight in the immunologic mechanisms associated with the high efficiency of the SA14-14-2 vaccine.

Distribution of Far-Eastern tick-borne encephalitis virus subtype strains in the former Soviet Union

European and Asian viruses within the tick-borne encephalitis flavivirus complex are known to show temporal, spatial and phylogenetic relationships that imply a clinal pattern of evolution. However, the isolation of recognized Far-Eastern tick-borne encephalitis virus (TBEV) strains in the European region of the former Soviet Union (SU), i.e. thousands of kilometres west of the region in which they are considered endemic, appears to contradict this concept. Here, we present a parsimonious explanation for this apparent anomaly based on analysis of the dates and regions in which these non-endemic strains were isolated, together with their phylogenetic relationships and the records of redistribution of animals under the All-Union programme for acclimatization of game animals within the former SU. Our evidence supports the concept that the anomalous distribution of Far-Eastern TBEV strains in Europe and Siberia arose primarily as the result of the large-scale westward redistribution of game animals for economic purposes.

Molecular epidemiology of Japanese encephalitis virus circulating in South Korea, 1983-2005

We sequenced the envelope (E) gene of 17 strains of the Japanese encephalitis virus (JEV) isolated in South Korea in 1983-2005 and compared the sequences with those from previously reported strains. Our results show the remarkable genetic stability of the E gene sequence in Korean JEV strains. Five pairs of E gene sequences from 10 Korean strains were identical, despite geographical differences and a maximum five-year time span. Sequence comparisons with other Asian strains revealed that the Korean strains are closely related to those from China, Japan, and Vietnam. Genotype 3 strains were predominant in Korea before 1993, when genotype 1 strain K93A07 was first isolated. The two genotypes were detected simultaneously in 1994 but since then, only genotype 1 has been isolated in South Korea. Thus, the genotype change occurred according to the year of isolation rather than the geographical origin.

Molecular characteristics of the full-length genome of two new Japanese encephalitis virus isolated from mosquitoes in Hubei province

To sequence and analyze the complete genome of two new Japanese encephalitis virus (JEV) strains isolated from mosquitoes collected in Hubei province in 2008, and to understand the molecular biological characteristics of JEV in this area. RT-PCR was used to amplify the fragments of HBZG08-09 strain and HBZG08-55 strain with 16 pairs overlapping primers after they had been recovered and identified, then the full-length genome was obtained by sequencing and splicing. Biological sequence alignment, nucleotide and amino acid sequence analysis, phylogenetic analysis and analysis of amino acid differences were performed by the software of Clustal X (1.83), MegAlign, Mega (4.0) and Genedoc (3.2). The genome of two new strains were both 10 965 nucleotides in length with a single open reading frame from 96 to 10 392 coding for a 3432 amino acid poly-protein, the homology of nucleotide and amino acid sequence between two isolates were 98.2% and 99.7% respectively. Further study showed that the new strains were both belonging to genotype I. Two new strains were most closely related to isolates obtained from Henan and Zhejiang province in recent years. Compared with the live attenuated vaccine strain SA-14-14-2 in China, HBZG08-09 strain had 82 amino acid divergence; HBZG08-55 had 84 amino acid divergences. But the amino acid difference occurred in sites were not the key ones affecting the toxicity or antigenic of JEV. Two new JEV isolates were both belonging to genotype I, and the key sites of amino acid were not changed.

Genotyping and characterization of the geographical distribution of tick‐borne encephalitis virus variants with a set of molecular probes

A panel of deoxyoligonucleotide probes for studying the genetic variability and genotyping of Tick-borne encephalitis virus (TBEV) strains by molecular hybridization of nucleic acids (MHNA) was created. This panel allows to estimate the genetic structure of individual TBEV strains, as the targets for probes are both variable and genotype (subtype)-specific sequences of all TBEV genes. With the help of this panel using the method of molecular hybridization of nucleic acids 268 archived TBEV isolates were investigated and the distribution of its genotypes and subgenotypes of genotype 3 was made more precise in the territory of Eurasia. The conclusion made earlier has confirmed that five genotypes of TBEV co-circulate in Eastern Siberia. It is generally recognized that the Far Eastern (TBE-FE), European (TBE-Eu), and Siberian (TBE-Sib) genotypes are widespread and epidemiologically important. The fourth genotype is presented by only one isolate, TBE178-79, originated from Irkutsk region, Russia. The fifth genotype includes 10 isolates, 1 of them, TBE886-84, was found earlier and recognized as unique [Zlobin et al. (2001b): Vopr Virusol 1:12-16 (Russian)].

Ixodes ricinus tick saliva modulates tick-borne encephalitis virus infection of dendritic cells

Tick-borne encephalitis virus is an important human pathogen, naturally delivered into host skin via a tick bite. To examine the effects of the virus on dendritic cell biology, we cultured dendritic cells with two tick-borne encephalitis virus strains of different virulence in the presence of Ixodes ricinus tick saliva. Tick saliva treatment increased proportion of virus-infected cells, led to a decrease in virus-induced TNF-α and IL-6 production and to reduced virus-induced apoptosis. Our data indicate that tick saliva modulate virus-mediated alterations in dendritic cells, thus probably being involved in the early infection process in the host.

Phylogenetic analysis of poly and non structural protein in Japanese Encephalitis virus with other related viral families.

Japanese encephalitis (JE) causes inflammation of brain. The mortality rate due to JE is 30% while 10 -15 % of patients make full recovery. The disease spreads through infected mosquito bites breeding in rice fields and feeds on pigs, birds, and ducks. As proteins show important structure to function relationship the study was designed to carry out the identification of poly and non-structural proteins in the infective virus group using different strains of Japanese encephalitis virus i.e. JAOARS982, Nakayama Strain SA (V), Strain SA-14. With reference to non structural proteins we obtained protein sequences of the following Japanese encephalitis virus groups: Japanese encephalitis virus, Weatnile virus, Kunjin virus. Further comparative and phylogenetic analysis was performed to explore evolutionary relationship among these groups. Results of phylogeny of alignment score was found to be 375184 using multiple alignment, Jal view, ClustalW (1.83) and ClustalW2. However, the analysis among the non-structural proteins of Japanese Encephalitis Virus, Westnile Virus, and Kunjin Virus revealed the phylogeny alignment score to be 875 through multiple sequence alignment and Tree view respectively. Phylogenetic analysis revealed that these four strains are interrelated as well as showing high similarity with the other viruses of this group due to conserved regions among their sequences.

Genetic analysis of strains of Japanese Encephalitis Virus isolated from swine in central China

In this study, four strains of Japanese Encephalitis Virus (JEV) were isolated from the cerebrospinal fluid of aborted fetuses or stillborn piglets collected randomly from a number of piggeries in central China. The E genes were cloned by RT-PCR and sequenced. Phylogenetic analysis was performed with 48 JEV isolates previously reported in China and other countries, and showed that all four isolates can be classified into the subcluster of genotype III. The results strongly suggest that the genotype III of JEV is the major variant currently circulating in the swinery of central China.

Coding nucleotide sequences of tick-borne encephalitis virus strains isolated from human blood without clinical symptoms of infection

Genomes of four tick-borne encephalitis virus strains, isolated from the blood of the individuals after tick bites and causing no clinical symptoms of infection, were characterized. Analysis of translated polypeptides revealed 21 amino acid positions typical of this group of strains and distinguishing them from the other tickborne encephalitis virus strains of Far Eastern subtype examined earlier. Only three mutations led to substantial amino acid changes, which probably could affect the infection process severity. It is suggested that two associated mutations, deletion of amino acid 111 in the capsid protein C and substitution (Ser1534 --> Phe) in the NS3 protein influence strictly coordinated polyprotein processing, disturbing correct arrangement of viral particles. This process can result in the development of defect viral particles, containing no RNA. Mutation (Ser917 --> Gly) in nonstructural protein NS1 results in the substitution of hydrophilic amino acid, specific to highly virulent strains, by the hydrophobic one. This could influence the effectiveness of viral replication complex, thereby affecting the infectivity of tick-borne encephalitis virus strains.

Isolation and molecular characterization of a tick-borne encephalitis virus strain from a new tick-borne encephalitis focus with severe cases in Bavaria, Germany

Tick-borne encephalitis (TBE) is the most important viral infection transmitted by ticks in Central Europe. In Germany, where TBE was classified as a notifiable disease in 2001, a highly variable number of clinically apparent human cases was reported in the last few years, ranging from the lowest number of 238 in 2007 to a maximum of 546 in 2006. The dynamics of the virus and its vector tick remain poorly understood. We investigated a highly active TBE focus in south-eastern Germany where from 2003 to 2008 a total of 9 clinical human cases was diagnosed. Three out of these 9 cases were fatal indicating an unusually high mortality rate possibly due to a highly virulent TBEV strain. From 2005 till 2008, 2150 Ixodes ricinus ticks were collected and tested for the presence of TBE virus. Five TBEV-positive ticks were detected by real-time RT-PCR. A viable virus strain was isolated from one of the positive ticks sampled in 2005. This is the first TBE virus isolate from a tick in Germany for 30 years. Sequencing of the full-length genome of this virus strain (AS33) revealed 2 unique amino acid substitutions in the envelope protein known to play a role in the pathogenicity of TBE virus. Amplification of the envelope gene using 2 TBEV-PCR-positive ticks from 2006 also showed these particular mutations indicating that this TBE virus strain was present in at least 2 consecutive years. The entire sampling area was divided into smaller sectors for the exact location of TBEV-positive ticks. Virus-positive ticks were found to be randomly distributed throughout the investigated focus, which is used as recreational area by the local people.

Transmission Potential of Two Chimeric Western Equine Encephalitis Vaccine Candidates in Culex tarsalis

Western equine encephalitis virus (WEEV) is a zoonotic alphavirus that circulates in western North America between passerine birds and mosquitoes, primarily Culex tarsalis. Since it was isolated in 1930, WEEV has caused tens of thousands of equine deaths in addition to thousands of human cases. In addition because WEEV is a virus of agricultural importance in addition to a public health threat, we developed two live-attenuated chimeric vaccine candidates that have been shown to be immunogenic and efficacious in mouse models. Vaccine candidate strains were developed by inserting the structural protein genes of WEEV strain McMillan (McM) or CO92-1356 into a Sindbis virus (SINV) strain AR339 backbone. The SIN/McM chimera also derived the N-terminal half of its capsid gene from a North American eastern equine encephalitis virus (EEEV) strain FL39-939 (henceforth referred to as SIN/EEE/McM). Although these vaccines do not generate viremia in mice, we further assessed their safety by exposing Cx. tarsalis to artificial blood meals containing high viral titers of each vaccine candidate. Both viruses exhibited a decreased rate of infection, dissemination, and transmission potential compared with the parental alphaviruses. Specifically, SIN/CO92 infected 37% of mosquitoes and disseminated in 8%, but failed to reach the saliva of the mosquitoes. In contrast, the SIN/EEE/McM virus was unable to infect, disseminate, or be transmitted in the saliva of any mosquitoes. These findings suggest that both vaccine candidates are less competent than the parental strains to be transmitted by the primary mosquito vector, Cx. tarsalis, and are unlikely to be reintroduced into a natural WEEV transmission cycle.

Survival of Viral Biowarfare Agents in Disinfected Waters

Protecting civilian and military water supplies has received more attention since the United States began its war on terror in 2001. Both chlorine and bromine are used by branches of the U.S. military for disinfecting water supplies; however, limited data exists as to the effectiveness of these additives when used against viral biowarfare agents. The present study sought to evaluate the survival of selected viral biothreat agents in disinfected water. Disinfected water samples were spiked with vaccinia virus strain WR and Venezuelan equine encephalitis (VEE) virus strain TC-83 each separately to a final concentration of approximately 1 × 106 PFU/mL, and survival was assessed by plaque assay. Both viruses were inactivated by 1 mg/L free available chlorine (FAC) and 2mg/L total bromine within one hour. In conclusion, these results demonstrate that both chlorine and bromine are effective disinfectants against vaccinia virus and VEE strain TC-83 at the concentrations tested.

Molecular evolution of Japanese encephalitis virus isolates from swine in Oita, Japan during 1980–2009

In order to identify the patterns of genetic change of Japanese encephalitis virus (JEV) strains circulating in Oita, the complete envelope (E) gene has been sequenced for 35 isolates from swine in a 30-year span. Based on nucleotide and deduced amino acid sequences, the genetic variation was examined, phylogeny was estimated and selection pressures were also analyzed. This study demonstrated that the major genotype (G) of JEV isolates had shifted from GIII to GI in the mid-1990s in Oita. The intensities of selection acting on the Oita GIII and GI strains were found to be almost same. It suggests that the intensity of selection might not be the reason for such a genotype shift observed in Oita. Pairwise comparisons revealed the high conservation of the E gene at the protein level. Compared with the Oita GIII strains, all the Oita GI strains shared four amino acid changes at E129 (T–M), E222 (A–S), E327 (S–T) and E366 (A–S). Among all 70 JEV isolates involved in this paper, the GI strains shared only one amino acid change at E222 (A–S) in comparison with the GIII strains. No strong evidence for positive selection was found, the JEV evolution has generally been subject to strong purifying selection, but one ongoing evolutionary pathway was found to be under relaxed purifying selection in Oita. This study is a localized example of JEV molecular evolution in nature.

Origin and distribution of tick-borne encephalitis virus strains of the Siberian subtype in the Middle Urals, the north-west of Russia and the Baltic countries

Tick-borne encephalitis virus (TBEV) plays an important role in infectious human morbidity, particularly in Russia and the Middle Urals. The Siberian subtype of TBEV (S-TBEV) is dominant in the Middle Urals. Determining the origin of S-TBEV strains in this territory and also in the European part of Russia and the Baltic countries is very important for understanding the cause of its distribution. The surface glycoprotein E gene was partially sequenced in 165 S-TBEV isolates collected in the Middle Urals between 1966 and 2008. Nucleotide and amino acid sequence identity of the studied isolates is 94 and 97.4 %, respectively. Eighty per cent of them are represented by six clusters with identical amino acid sequences in the glycoprotein E fragment analysed. We have determined four types of isolate distribution in the explored territory: local, split, corridor and diffuse. The average rate of nucleotide substitutions per site year(-1) is estimated to be 1.56 x 10(-4). The age of the S-TBEV population was evaluated to be slightly less than 400 years. Phylogenetic analysis of the data and comparison with historical events indicate that the distribution of S-TBEV strains in the Middle Urals and the European part of Russia originated twice from different foci in western Siberia. This is related to the first land road into Siberia and the Trans-Siberian Way, which functioned at different times. The main reason for such rapid distribution of S-TBEV strains is the anthropogenic factor, i.e. human economic activity during the colonization of new territories in Siberia in the recent past.

Development of a novel monoclonal antibody with reactivity to a wide range of Venezuelan equine encephalitis virus strains

BackgroundThere is currently a requirement for antiviral therapies capable of protecting against infection with Venezuelan equine encephalitis virus (VEEV), as a licensed vaccine is not available for general human use. Monoclonal antibodies are increasingly being developed as therapeutics and are potential treatments for VEEV as they have been shown to be protective in the mouse model of disease. However, to be truly effective, the antibody should recognise multiple strains of VEEV and broadly reactive monoclonal antibodies are rarely and only coincidentally isolated using classical hybridoma technology.ResultsIn this work, methods were developed to reliably derive broadly reactive murine antibodies. A phage library was created that expressed single chain variable fragments (scFv) isolated from mice immunised with multiple strains of VEEV. A broadly reactive scFv was identified and incorporated into a murine IgG2a framework. This novel antibody retained the broad reactivity exhibited by the scFv but did not possess virus neutralising activity. However, the antibody was still able to protect mice against VEEV disease induced by strain TrD when administered 24 h prior to challenge.ConclusionA monoclonal antibody possessing reactivity to a wide range of VEEV strains may be of benefit as a generic antiviral therapy. However, humanisation of the murine antibody will be required before it can be tested in humans.Crown Copyright © 2009

A single mutation in the Japanese encephalitis virus E protein (S123R) increases its growth rate in mouse neuroblastoma cells and its pathogenicity in mice

We previously reported that the Japanese encephalitis virus (JEV) strain Mie/41/2002 has weak pathogenicity compared with the laboratory strain Beijing-1. To identify the determinants of its growth nature and pathogenicity, we produced intertypic viruses, rJEV(EB1-M41), rJEV(nEB1-M41) and rJEV(cEB1-M41), which contained the entire, the N-terminal, and the C-terminal half, respectively, of the Beijing-1 E region in the Mie/41/2002 background. The growth of rJEV(EB1-M41) in mouse neuroblastoma N18 cells and virulence in mice were similar to those of Beijing-1. rJEV(nEB1-M41) propagated in N18 cells to the same extent as did Beijing-1. Furthermore, we produced mutant viruses with single amino acid substitutions in the N-terminal half of the Mie/41/2002 E region. A Ser-123-Arg mutation in the Mie/41/2002 E protein exhibited significantly increased growth rate in N18 cells and virulence in mice. These results indicate that the position 123 in the E protein is responsible for determining the growth properties and pathogenicity of JEV.

Cloning and Sequence Analysis of the Full-Length Genome of Japanese encephalitis virus Strain SXBJ07 Isolated from Swine

A virus strain, showing cytopathic effect in BHK-21 cell, was isolated from swine brains in Shaanxi Province, China, in 2007. The isolate was confirmed as Japanese encephalitis virus (JEV) by immunofluorescence assay (IFA) and reverse transcription polymerase chain reaction (RT-PCR), and named SXBJ07. The complete nucleotide and deduced amino acid sequences of the JEV strain SXBJ07 were determined. Its single open reading frame has a total of 3 432 amino acid residues. An extensive E gene based phylogenetic analysis was performed, the result showed that SXBJ07 strain belongs to genotype I. Comparison of the SXBJ07 genomic sequence with those of the 24 fully sequenced JEV strains in published databases showed nucleotide homology ranging from 99.0 to 83.7%; amino acid homology ranged from 99.8 to 94.8%. Compared SXBJ07 with SA14-14-2 strain, the current live vaccine strain in China, the homology of amino acid in envelope gene was 97.0%; and there were amino acid substitutions in 13 sites of the active domains of E protein (E1-E411).

Pathogenesis of aerosolized Eastern Equine Encephalitis virus infection in guinea pigs

Mice and guinea pigs were experimentally exposed to aerosols containing regionally-distinct strains (NJ1959 or ArgM) of eastern equine encephalitis virus (EEEV) at two exclusive particle size distributions. Mice were more susceptible to either strain of aerosolized EEEV than were guinea pigs; however, clinical signs indicating encephalitis were more readily observed in the guinea pigs. Lower lethality was observed in both species when EEEV was presented at the larger aerosol distribution (> 6 μm), although the differences in the median lethal dose (LD50) were not significant. Virus isolation and immunohistochemistry indicated that virus invaded the brains of guinea pigs within one day postexposure, regardless of viral strain or particle size distribution. Immunohistochemistry further demonstrated that neuroinvasion occurred through the olfactory system, followed by transneuronal spread to all regions of the brain. Olfactory bipolar neurons and neurons throughout the brain were the key viral targets. The main microscopic lesions in infected guinea pigs were neuronal necrosis, inflammation of the meninges and neuropil of the brain, and vasculitis in the brain. These results indicate that guinea pigs experimentally infected by aerosolized EEEV recapitulate several key features of fatal human infection and thus should serve as a suitable animal model for aerosol exposure to EEEV.

Alphavirus replicon particle vaccines developed for use in humans induce high levels of antibodies to influenza virus hemagglutinin in swine: Proof of concept

A propagation-defective, single-cycle, alphavirus replicon particle (RP) system was used to produce two vaccines against human influenza virus A/Wyoming/03/2003 (H3N2). One vaccine was prepared from Venezeulan equine encephalitis virus (VEEV) strain 3014 and the other from VEEV strain TC-83. Both vaccines induced high antibody titers to the influenza hemagglutinin (HA) protein and illustrated the potential of using alphavirus RP influenza vaccines in swine.

Molecular smallpox vaccine delivered by alphavirus replicons elicits protective immunity in mice and non-human primates

Naturally occurring smallpox was eradicated as a result of successful vaccination campaigns during the 1960s and 1970s. Because of its highly contagious nature and high mortality rate, smallpox has significant potential as a biological weapon. Unfortunately, the current vaccine for orthopoxviruses is contraindicated for large portions of the population. Thus, there is a need for new, safe, and effective orthopoxvirus vaccines. Alphavirus replicon vectors, derived from strains of Venezuelan equine encephalitis virus, are being used to develop alternatives to the current smallpox vaccine. Here, we demonstrated that virus-like replicon particles (VRPs) expressing the vaccinia virus A33R, B5R, A27L, and L1R genes elicited protective immunity in mice comparable to vaccination with live-vaccinia virus. Furthermore, cynomolgus macaques vaccinated with a combination of the four poxvirus VRPs (4pox-VRP) developed antibody responses to each antigen. These antibody responses were able to neutralize and inhibit the spread of both vaccinia virus and monkeypox virus. Macaques vaccinated with 4pox-VRP, flu HA VRP (negative control), or live-vaccinia virus (positive control) were challenged intravenously with 5 × 10 6 pfu of monkeypox virus 1 month after the second VRP vaccination. Four of the six negative control animals succumbed to monkeypox and the remaining two animals demonstrated either severe or grave disease. Importantly, all 10 macaques vaccinated with the 4pox-VRP vaccine survived without developing severe disease. These findings revealed that a single-boost VRP smallpox vaccine shows promise as a safe alternative to the currently licensed live-vaccinia virus smallpox vaccine.