Computational Prediction of Usutu Virus E Protein B Cell and T Cell Epitopes for Potential Vaccine Development.

Usutu virus (family Flaviviridae), once confined to Africa, has emerged in Europe a decade ago. The virus has been spreading throughout Europe at a greater pace mostly affecting avian species. While most bird species remain asymptomatic carriers of this virus, few bird species are highly susceptible. Lately, Usutu virus (USUV) infections in humans were reported sporadically with severe neuroinvasive symptoms like meningoencephalitis. As so much is unknown about this virus, which potentially may cause severe diseases in humans, there is a need for more studies of this virus. In this study, we have used computational tools to predict potential B cell and T cell epitopes of USUV envelope (E) protein. We found that amino acids between positions 68 and 84 could be a potential B cell epitope, while amino acids between positions 53 and 69 could be a potential major histocompatibility complex (MHC) class I- and class II-restricted T cell epitope. By homology 3D modeling of USUV E protein, we found that the predicted B cell epitope was predominantly located in the coil region, while T cell epitope was located in the beta-strand region of the E protein. Additionally, the potential MHC class I T cell epitope (LAEVRSYCYL) was predicted to bind to nearly 24 human leucocyte antigens (HLAs) (IC50 ≤5000 nm) covering nearly 86.44% of the Black population and 96.90% of the Caucasoid population. Further in vivo studies are needed to validate the predicted epitopes.

Seroprevalence of West Nile and Usutu viruses in military working horses and dogs, Morocco, 2012: dog as an alternative WNV sentinel species?

A serosurvey of 349 military working horses and 231 military working dogs was conducted in ten sites in Morocco in 2012. This survey revealed a high level of exposure of these animals to flaviviruses: seroprevalence rates of 60% in horses and of 62% in dogs were observed using a competitive West Nile virus (WNV) enzyme-linked immunosorbent assay (cELISA). Seroneutralization test results showed that the majority of cELISA-positive results were due to exposure to WNV. Further assays conducted in vaccinated horses with a DIVA (Differentiating Infected from Vaccinated Animals) test indicated that anti-WNV antibodies had been stimulated through WNV natural infection. Moreover, in both species, seroneutralization tests suggested an exposure to Usutu virus (USUV). Data analysis did not show any significant difference of cELISA seropositivity risk between horses and dogs. Dogs may thus represent an interesting alternative to equines for the serological surveillance of WNV or USUV circulation, especially in areas where equine vaccination precludes passive surveillance (based on the detection of West Nile fever cases) in horses.

The West Nile virus-like flavivirus Koutango is highly virulent in mice due to delayed viral clearance and the induction of a poor neutralizing antibody response.

UNLABELLED: The mosquito-borne West Nile virus (WNV) is responsible for outbreaks of viral encephalitis in humans, horses, and birds, with particularly virulent strains causing recent outbreaks of disease in eastern Europe, the Middle East, North America, and Australia. Previous studies have phylogenetically separated WNV strains into two main genetic lineages (I and II) containing virulent strains associated with neurological disease. Several WNV-like strains clustering outside these lineages have been identified and form an additional five proposed lineages. However, little is known about whether these strains have the potential to induce disease. In a comparative analysis with the highly virulent lineage I American strain (WNVNY99), the low-pathogenicity lineage II strain (B956), a benign Australian strain, Kunjin (WNVKUN), the African WNV-like Koutango virus (WNVKOU), and a WNV-like isolate from Sarawak, Malaysia (WNVSarawak), were assessed for neuroinvasive properties in a murine model and for their replication kinetics in vitro. While WNVNY99 replicated to the highest levels in vitro, in vivo mouse challenge revealed that WNVKOU was more virulent, with a shorter time to onset of neurological disease and higher morbidity. Histological analysis of WNVKOU- and WNVNY99-infected brain and spinal cords demonstrated more prominent meningoencephalitis and the presence of viral antigen in WNVKOU-infected mice. Enhanced virulence of WNVKOU also was associated with poor viral clearance in the periphery (sera and spleen), a skewed innate immune response, and poor neutralizing antibody development. These data demonstrate, for the first time, potent neuroinvasive and neurovirulent properties of a WNV-like virus outside lineages I and II. IMPORTANCE: In this study, we characterized the in vitro and in vivo properties of previously uncharacterized West Nile virus strains and West Nile-like viruses. We identified a West Nile-like virus, Koutango virus (WNVKOU), that was more virulent than a known virulent lineage I virus, WNVNY99. The enhanced virulence of WNVKOU was associated with poor viral clearance and the induction of a poor neutralizing antibody response. These findings provide new insights into the pathogenesis of West Nile virus.

Evaluation of chimeric DNA vaccines consisting of premembrane and envelope genes of Japanese encephalitis and dengue viruses as a strategy for reducing induction of dengue virus infection-enhancing antibody response.

Neutralizing antibodies induced by dengue virus (DENV) infection show viral infection-enhancing activities at sub-neutralizing doses. On the other hand, preimmunity against Japanese encephalitis virus (JEV), a congener of DENV, does not increase the severity of DENV infection. Several studies have demonstrated that neutralizing epitopes in the genus Flavivirus are mainly located in domain III (DIII) of the envelope (E) protein. In this study, chimeric premembrane and envelope (prM-E) gene-based expression plasmids of JEV and DENV1 with DIII substitution of each virus were constructed for use as DNA vaccines and their immunogenicity evaluated. Sera from C3H/He and ICR mice immunized with a chimeric gene containing DENV1 DIII on a JEV prM-E gene backbone showed high neutralizing antibody titers with less DENV infection-enhancing activity. Our results confirm the applicability of this approach as a new dengue vaccine development strategy.

Prophylactic Administration of Gut Microbiome Metabolites Abrogated Microglial Activation and Subsequent Neuroinflammation in an Experimental Model of Japanese Encephalitis.

Short-chain fatty acids (SCFAs) are gut microbial metabolic derivatives produced during the fermentation of ingested complex carbohydrates. SCFAs have been widely regarded to have a potent anti-inflammatory and neuro-protective role and have implications in several disease conditions, such as, inflammatory bowel disease, type-2 diabetes, and neurodegenerative disorders. Japanese encephalitis virus (JEV), a neurotropic flavivirus, is associated with life threatening neuro-inflammation and neurological sequelae in infected hosts. In this study, we hypothesize that SCFAs have potential in mitigating JEV pathogenesis. Postnatal day 10 BALB/c mice were intraperitoneally injected with either a SCFA mixture (acetate, propionate, and butyrate) or PBS for a period of 7 days, followed by JEV infection. All mice were observed for onset and progression of symptoms. The brain tissue was collected upon reaching terminal illness for further analysis. SCFA-supplemented JEV-infected mice (SCFA + JEV) showed a delayed onset of symptoms, lower hindlimb clasping score, and decreased weight loss and increased survival by 3 days (p < 0.0001) upon infection as opposed to the PBS-treated JEV-infected animals (JEV). Significant downregulation of inflammatory cytokines TNF-α, MCP-1, IL-6, and IFN-Υ in the SCFA + JEV group relative to the JEV-infected control group was observed. Inflammatory mediators, phospho-NF-kB (P-NF-kB) and iba1, showed 2.08 ± 0.1 and 3.132 ± 0.43-fold upregulation in JEV versus 1.19 ± 0.11 and 1.31 ± 0.11-fold in the SCFA + JEV group, respectively. Tissue section analysis exhibited reduced glial activation (JEV group─42 ± 2.15 microglia/ROI; SCFA + JEV group─27.07 ± 1.8 microglia/ROI) in animals that received SCFA supplementation prior to infection as seen from the astrocytic and microglial morphometric analysis. Caspase-3 immunoblotting showed 4.08 ± 1.3-fold upregulation in JEV as compared to 1.03 ± 0.14-fold in the SCFA + JEV group and TUNEL assay showed a reduced cellular death post-JEV infection (JEV-6.4 ± 1.5 cells/ROI and SCFA + JEV-3.7 ± 0.73 cells/ROI). Our study critically contributes to the increasing evidence in support of SCFAs as an anti-inflammatory and neuro-protective agent, we further expand its scope as a potential supplementary intervention in JEV-mediated neuroinflammation.

Usutu virus growth in human cell lines: induction of and sensitivity to type I and III interferons.

The mechanisms of Usutu virus (USUV) pathogenesis are largely unknown. The aim of this study was to evaluate the sensitivity of USUV to interferon (IFN) and the capacity of USUV to stimulate IFN production. Initial experiments were conducted to characterize the susceptibility of human cell lines to USUV infection and to evaluate the single-growth cycle replication curve of USUV. Results indicate that USUV is able to infect a variety of human cell lines, completing the replication cycle in Hep-2 and Vero cells within 48 h. Pre-treatment of cells with types I and III IFNs significantly inhibited the replication of USUV. However, the inhibitory effects of IFNs were considerably less if IFN was added after viral infection had been initiated. Also, USUV weakly induced types I and III IFNs.

Low Usutu virus seroprevalence in four zoological gardens in central Europe.

BACKGROUND: Usutu virus (USUV), a mosquito-borne flavivirus of the Japanese encephalitis virus antigenic group, caused bird die-offs in Austria, Hungary and Switzerland between 2001 and 2009. While the zoological gardens of Vienna and Zurich recorded USUV-associated mortality in different species of birds during this period, incidences in Budapest were limited to areas outside the zoo, and in the greater Basel area avian mortality due to USUV infection was not observed at all. The objectives of this investigation were to gain insight into USUV infection dynamics in captive birds in zoos with varying degrees of virus exposure and to study differences in susceptibility to USUV of different species of birds. RESULTS: 372 bird sera were collected between October 2006 and August 2007. The samples were tested in parallel by hemagglutination inhibition (HI) and 90% plaque reduction neutralization tests (PRNT-90). 8.75%, 5.3% and 6.59% of birds in the zoos of Vienna, Zurich and Basel, respectively, showed USUV-specific antibodies by PRNT-90. No antibodies to USUV were detected in birds of the Budapest zoo. The order Strigiformes (owls) exhibited the highest USUV-seroprevalence, compared to other orders of birds. CONCLUSIONS: USUV seems not to pose an imminent threat to zoo bird populations in central Europe at the moment. Depending on a variety of especially environmental factors, however, this may change at any time in the (near) future, as experienced with West Nile virus (WNV). It is therefore strongly suggested to continue with combined WNV and USUV surveillance activities in affected areas.

Retrospective identification of human cases of West Nile virus infection in Austria (2009 to 2010) by serological differentiation from Usutu and other flavivirus infections.

There is increasing evidence for the spread of West Nile virus (WNV) in southern, eastern and central Europe. In parallel, another flavivirus, the antigenically closely related Usutu virus, was introduced from Africa and first detected in Austria (2001), followed by Spain (2003), Hungary (2005), Italy (2006), Switzerland (2006) and Germany (2007). In Austria, human WNV infections have not previously been documented, although the virus was isolated from birds and detected in mosquitoes in 2008 and 2009. We therefore conducted a retrospective search for human cases of WNV infection using serum and cerebrospinal fluid samples collected from patients with central nervous system (CNS) disease in the summers of 2009, 2010 and 2011. Although all samples were negative for WNV by polymerase chain reaction, quantitative evaluation of standardised antibody assays with purified flavivirus antigens (including Usutu virus, which cross-reacts with WNV even in neutralisation assays) provided serological evidence for three autochthonous WNV infections in Austria: two in 2009 and one in 2010. Our data highlight the importance of raising awareness of WNV infections in Austria and neighbouring countries and suggest including testing for this infection in routine diagnostic practice of CNS diseases.

Detection of Usutu virus infection in a healthy blood donor from south-west Germany, 2012.

From September 2011 until November 2012, 31 serum samples from German patients with clinically suspected acute Usutu virus (USUV) infections were tested for USUV-specific antibodies. All samples tested negative. In addition, 4,200 serum samples from healthy blood donors from south-west Germany were collected in January 2012 and also analysed for the presence of specific antibodies. One sample tested positive for USUV-IgG and -IgM. Thus, the seroprevalence of USUV antibodies in healthy blood donors from south-west Germany was low in January 2012.

[Long-term seroprotection against Japanese encephalitis using an inactivated vaccine (Jevax)]. / Séroprotection à long terme avec le vaccin inactivé contre l'encéphalite japonaise (Jevax).

Japanese encephalitis vaccine (Jevax) is an inactivated vaccine using the Nakayama viral strain. Until 2007, Jevax was the only Japanese encephalitis vaccine available in France but the duration of seroprotection after vaccination and exact timing of booster injections was unclear for travelers from non-endemic areas. The purpose of this report is to describe the results of a retrospective study in which neutralizing antibody levels were measured in 71 subjects previously vaccinated with Jevax. All subjects underwent testing at the Pasteur Institute Medical Center as part of preparation for humanitarian missions to endemic Japanese encephalitis areas in 2005-2006. A neutralizing antibody level greater than or equal to 20 was considered as protective. Findings showed that 49 of the 71 subjects (69%) still had protective antibody levels at a median of 4 years after the last Jevax immunization. In multivariate analysis, the only factor correlated with long-term seroprotection was the total number of vaccinations received. Based on these findings, it was concluded that long-term seroprotection after Jevax vaccination requires repeated booster injections even in subjects frequently exposed to the virus. No correlation was found between seroprotection and the interval between the booster injections.

Neutralization Potency of Sera from Vietnamese Patients with Japanese Encephalitis (JE) against Genotypes I and V JE Viruses.

Japanese encephalitis virus (JEV) is classified into 5 genotypes (GI, GII, GIII, GIV, and GV), and the GI and GIII strains are the most widely distributed in JE endemic areas. In recent years, GV JEV has been detected in China and Korea, suggesting that GV JEV may invade other JE endemic areas, including Vietnam, and that more attention should be paid to the JEV strains circulating in these areas. In this study, we investigated the neutralization ability of the sera collected from 22 Vietnamese patients with JE who lived in northern Vietnam against the GI and GV JEV strains. In most cases, the ratios of the titer against GV to that against GI (GV:GI) were equal to or less than 1:4. However, the titer against GV JEV was equivalent (1:1) to that against GI JEV in only a few cases, and no serum had a ratio higher than 1:1. Thus, our results did not show convincing evidence that GV JEV was emerging in northern Vietnam in 2014.

Activation of type I interferon antiviral response in human neural stem cells.

BACKGROUND: Neural stem cells (NSCs) residing in the central nervous system play an important role in neurogenesis. Several viruses can infect these neural progenitors and cause severe neurological diseases. The innate immune responses against the neurotropic viruses in these tissue-specific stem cells remain unclear. METHODS: Human NSCs were transfected with viral RNA mimics or infected with neurotropic virus for detecting the expression of antiviral interferons (IFNs) and downstream IFN-stimulated antiviral genes. RESULTS: NSCs are able to produce interferon-ß (IFN-ß) (type I) and λ1 (type III) after transfection with poly(I:C) and that downstream IFN-stimulated antiviral genes, such as ISG56 and MxA, and the viral RNA sensors RIG-I, MDA5, and TLR3, can be expressed in NSCs under poly(I:C) or IFN-ß stimulation. In addition, our results show that the pattern recognition receptors RIG-I and MDA5, as well as the endosomal pathogen recognition receptor TLR3, but not TLR7 and TLR8, are involved in the activation of IFN-ß transcription in NSCs. Furthermore, NSCs infected with the neurotropic viruses, Zika and Japanese encephalitis viruses, are able to induce RIG-I-mediated IFN-ß expression. CONCLUSION: Human NSCs have the ability to activate IFN signals against neurotropic viral pathogens.

Serological evidence of continuing high Usutu virus (Flaviviridae) activity and establishment of herd immunity in wild birds in Austria.

Usutu virus (USUV), family Flaviviridae, has been responsible for avian mortality in Austria from 2001 to 2006. The proportion of USUV-positive individuals among the investigated dead birds decreased dramatically after 2004. To test the hypothesis that establishment of herd immunity might be responsible, serological examinations of susceptible wild birds were performed. Blood samples of 442 wild birds of 55 species were collected in 4 consecutive years (2003--2006). In addition, 86 individuals from a birds of prey rehabilitation centre were bled before, at the peak, and after the 2005 USUV transmission season in order to identify titre dynamics and seroconversions. The haemagglutination inhibition test was used for screening and the plaque reduction neutralization test for confirmation. While in the years 2003 and 2004 the proportion of seropositive wild birds was <10%, the percentage of seroreactors raised to >50% in 2005 and 2006. At the birds of prey centre, almost three quarters of the owls and raptors exhibited antibodies before the 2005 transmission season; this percentage dropped to less than half at the peak of USUV transmission and raised again to almost two thirds after the transmission season. These data show a from year to year continuously increasing proportion of seropositive wild birds. The owl and raptor data indicate significant viral exposure in the previous season(s), but also a number of new infections during the current season, despite the presence of antibodies in some of these birds. Herd immunity is a possible explanation for the significant decrease in USUV-associated bird mortalities in Austria during the recent years.

Explaining Usutu virus dynamics in Austria: model development and calibration.

Usutu virus (USUV), a flavivirus of the Japanese encephalitis virus complex, was for the first time detected outside Africa in the region around Vienna (Austria) in 2001 by Weissenböck et al. [Weissenböck, H., Kolodziejek, J., Url, A., Lussy, H., Rebel-Bauder, B., Nowotny, N., 2002. Emergence of Usutu virus, an African mosquito-borne flavivirus of the Japanese encephalitis virus group, central Europe. Emerg. Infect. Dis. 8, 652-656]. USUV is an arthropod-borne virus (arbovirus) circulating between arthropod vectors (mainly mosquitoes of the Culex pipiens complex) and avian amplification hosts. Infections of mammalian hosts or humans, as observed for the related West Nile virus (WNV), are rare. However, USUV infection leads to a high mortality in birds, especially blackbirds (Turdus merula), and has similar dynamics with the WNV in North America, which, amongst others, caused mortality in American robins (Turdus migratorius). We hypothesized that the transmission of USUV is determined by an interaction of developing proportion of the avian hosts immune and climatic factors affecting the mosquito population. This mechanism is implemented into the present model that simulates the seasonal cycles of mosquito and bird populations as well as USUV cross-infections. Observed monthly climate data are specified for the temperature-dependent development rates of the mosquitoes as well as the temperature-dependent extrinsic-incubation period. Our model reproduced the observed number of dead birds in Austria between 2001 and 2005, including the peaks in the relevant years. The high number of USUV cases in 2003 seems to be a response to the early beginning of the extraordinary hot summer in that year. The predictions indicate that >70% of the bird population acquired immunity, but also that the percentage would drop rapidly within only a couple of years. We estimated annually averaged basic reproduction numbers between R (0)=0.54 (2004) and 1.35 (2003). Finally, extrapolation from our model suggests that only 0.2% of the blackbirds killed by USUV were detected by the Austrian USUV monitoring program [Chvala, S., Bakonyi, T., Bukovsky, C., Meister, T., Brugger, K., Rubel, F., Nowotny, N., Weissenböck, H., 2007. Monitoring of Usutu virus activity and spread by using dead bird surveillance in Austria, 2003-2005. Vet. Microbiol. 122, 237-245]. These results suggest that the model presented is able to quantitatively describe the process of USUV dynamics.

Survival of mice immunized with monoclonal antibodies against glycoprotein E of Japanese encephalitis virus before or after infection with Japanese encephalitis, West Nile, and Dengue viruses.

In the present study, the effect of monoclonal antibodies (MAbs) against glycoprotein E (gE) of Japanese encephalitis virus (JEV) strain 733913 administered 1 day before or 2 days after intracerebral (i.c.) challenge with West Nile virus (WNV) strain 68856 or Dengue virus (DENV-2) strain P23085, was studied in mice. Furthermore, two JEV strains belonging to group II (strains 641686 and 691004) that have lost reactivity against virus-specific MAbs were also used in passive immunization experiments. MAbs as ascitic fluids were administered intraperitoneally (i.p.) in mice. Hemagglutination-inhibition- (HAI) positive JEV-specific (Hs-3) MAbs given 2 days after the virus infection showed reduced mortality along with increased survival of mice challenged with WNV or with DENV-2. Also the HAI-positive flavivirus cross-reactive (Hx) MAbs produced a marginal increase in the survival of mice challenged with both JEV strains 641686 and 691004 belonging to the group II. As the MAbs reacting with HAI-positive JEV-specific (Hs) and HAI-negative JEV-specific (NHs) epitopes were neutralizing and protective in mice against JEV strain 733913 challenge, the results indicated presence of the cross-protection phenomenon that might be occurring in some of the localities endemic for the three closely related flaviviruses.

Recent change of the antigenicity and genotype of Japanese encephalitis viruses distributed on Okinawa Island, Japan.

In this study, five isolates of Japanese encephalitis virus (JEV) were obtained from swine serum samples collected on Okinawa Island, Japan, between 2002 and 2003. All five JEV isolates belonged to genotype 1, and JEV isolates obtained from the island before 1992 were genotype 3. Genotype 1 was known to be distributed from northern Thailand to Cambodia and recently expanded to Australia, Vietnam, the Republic of Korea, and Japan. However, phylogenetic analysis showed that the source of the newly emerging genotype 1 in Asia is different from that in Australia. Sero-epidemiologic investigations showed that serum samples collected from 1985 to 1988 from JEV-immune swine neutralized both the Naha Meat 54 strain (1985 JEV Okinawan isolate from swine, genotype 3) and the Oki 431S strain (2002 JEV Okinawan isolate from swine, genotype 1), and many samples collected in 2002 neutralized the Oki 431S strain but not the Naha Meat 54 strain. These results strongly suggest that the genotype and antigenicity of JEV on Okinawa Island have changed significantly over the past decade.

Immunogenicity of a Live Attenuated Chimeric Japanese Encephalitis Vaccine as a Booster Dose After Primary Vaccination With Live Attenuated SA14-14-2 Vaccine: A Phase IV Study in Thai Children.

This single-group study investigated the immunogenicity and safety of a booster dose of the recently licensed live attenuated chimeric Japanese encephalitis vaccine in 50 healthy children (1-5 years old) who were primed with the live attenuated SA14-14-2 vaccine. A strong anamnestic response was induced 28 days postbooster: geometric mean titer, 9144 (95% confidence interval: 7365-11353); and seroprotection rate, 49 of 49 (100%) children.

Accuracy estimation of an indirect ELISA for the detection of West Nile Virus antibodies in wild birds using a latent class model.

West Nile virus (WNV) and Usutu virus (USUV), genus Flavivirus, are members of the Japanese encephalitis virus antigenic complex, and are maintained primarily in an enzootic cycle between mosquitoes and birds. WNV is zoonotic, and poses a threat to public health, especially in relation to blood transfusion. Serosurveillance of wild birds is suitable for early detection of WNV circulation, although concerns remain to be addressed as regards i) the type of test used, whether ELISA, virus neutralization test (VNT), plaque reduction neutralization test (PRNT), ii) the reagents (antigens, revealing antibodies), iii) the different bird species involved, and iv) potential cross-reactions with other Flaviviruses, such as USUV. The authors developed an indirect IgG ELISA with pan-avian specificity using EDIII protein as antigen and a monoclonal antibody (mAb 1A3) with broad reactivity for avian IgG. A total of 140 serum samples were collected from juvenile European magpies (Pica pica) in areas where both WNV and USUV were co-circulating. The samples were then tested using this in-house ELISA and VNT in parallel. Estimation of test accuracy was performed using different Bayesian two latent class models. At a cut-off set at an optical density percentage (OD%) of 15, the ELISA showed a posterior median of diagnostic sensitivity (DSe) of 88% (95%PCI: 73-99%) and a diagnostic specificity (DSp) of 86% (95%PCI: 68-99%). At this cut-off, ELISA and VNT (cut-off 1/10) performances were comparable: DSe=91% (95%PCI: 79-99%), and DSp=77% (95%PCI: 59-98%). With the cut-off increased to 30 OD%, the ELISA DSe dropped to 78% (95%PCI: 52-99%), and the DSp rose to 94% (95%PCI: 83-100%). In field conditions, the cut-off that yields the best accuracy for the ELISA appears to correspond to 15 OD%. In areas where other Flaviviruses are circulating, however, it might be appropriate to raise the cut-off to 30 OD% in order to achieve higher specificity and reduce the detection of seropositive birds infected by other Flaviviruses, such as USUV.

Detection of seroconversion to West Nile virus, Usutu virus and Sindbis virus in UK sentinel chickens.

We previously reported evidence of West Nile virus (WNV) circulation in UK birds, probably introduced by migratory birds from overseas. We now demonstrate WNV-specific seroconversion in sentinel chickens raised on an English farm. Maternal neutralizing antibodies to WNV in hatchlings declined within three weeks. During the following months, healthy chickens developed WNV neutralizing antibodies that were confirmed by immunoblotting and indirect immunofluorescence tests using WNV antigens. The proportion of seropositive chickens was higher for WNV than for Usutu virus or Sindbis virus. Attempts to isolate infectious virus or to detect viral RNA in the sera, failed.

[Influence of JEV E-HSP70 (Mycobacterium tuberculosis) fusion protein on immune response in BALB/c mice].

JEV infection can cause severe central nerve system disease which result in high mortality or developing permanent neurological sequelae in more than half of the survivors. The envelope (E) protein of JEV is the major antigen peptide fused to it. A recombinant hsp70 protein expression vector pPICZalpha-E-HSP70 in methylotrophic yeast Pichia pastoris was developed that permits major antigenic segment of JEV E protein fused to the amino terminus of M. tuberculosis hsp70. This core vector avoided inclusion bodies formed in Escherichia coli and complex purification. Moreover,it ruled out contamination of LPS. Two other vectors pPICZalpha-E and pPICZalpha-HSP70 were also constructed. The two vectors were constructed by routine molecular technique. All vectors were transformed into yeast X-33 by electroporation. Expression of the fusion protein in yeast was induced by the addition of methanol every 24 hours and analysed by SDS-PAGE and western blot. Major antigenic segment of E protein was produced at a yield of 290 mg per litter of culture, hsp70 protein at a yield of 178 mg per litter of culture and E-HSP70 fusion protein at a yield of 33 mg per litter of culture in methylotrophic yeast Pichia pastoris. To examine cell and body immune response after BALB/c mice were immunized with E-hsp70 fusion protein expressed in Pichia pastoris, there were three groups with ten mice in each group. 5.7 microg (50pmol) of E-hsp70 fusion protein, 2.2 microg (50pmol) major antigenic segment of E protein and a mixture of hsp70 and major antigenic segment of E protein (1:1) including 3.5 microg (50pmol) Hsp70 and 2.2 microg (50pmol) major antigenic segment of JEV E protein were used per mouse i.p. on day 0 and day 21. The production of mIL-2 was quantitated by semi-quantitative RT-PCR. Besides, proliferation of lymphocytes was measured by MTT and titers of antibody was determined by ELISA. These data show that the fusion protein is a more powerful antigen than major antigenic segment of JEV E protein. So it also illustrates the effectiveness of hsp70 in eliciting a humoral and cellular response to an attached molecule in the absence of adjuvant and affirms the potential utility of hsp70 in vaccine development.