Confirmed case of encephalitis caused by Murray Valley encephalitis virus infection in a horse

A 5-year-old Australian stock horse in Monto, Queensland, Australia, developed neurological signs and was euthanized after a 6-day course of illness. Histological examination of the brain and spinal cord revealed moderate to severe subacute, nonsuppurative encephalomyelitis. Sections of spinal cord stained positively in immunohistochemistry with a flavivirus-specific monoclonal antibody. Reverse transcription polymerase chain reaction assay targeting the envelope gene of flavivirus yielded positive results from brain, spinal cord, cerebrospinal fluid, and facial nerve. A flavivirus was isolated from the cerebrum and spinal cord. Nucleotide sequences obtained from amplicons from both tissues and virus isolated in cell culture were compared with those in GenBank and had 96-98% identity with Murray Valley encephalitis virus. The partial envelope gene sequence of the viral isolate clustered into genotype 1 and was most closely related to a previous Queensland isolate.

Murray Valley Encephalitis

Outbreaks of an acute, severe, encephalitic illness, clinically similar to Japanese and St. Louis encephalitis, occurred in rural areas of southeastern Australia in 1917, 1918, 1922, 1925, 1951, and 1974[1,9,14-16] and in north and northwestern Australia in 1981, 1993, and 2000.[8,12,41] Approximately 420 cases were reported in these nine outbreaks.[41] They are thought to represent a single entity for which various names (Australian X disease, Murray Valley encephalitis, Australian encephalitis) have been used. Twenty-two cases were diagnosed in the 5 years between 2007 and 2011; three were fatal, and one of the fatalities occurred in a Canadian tourist on return from a holiday in northern Australia. Case-fatality rates, as high as 70 percent in the early years,[9,11] declined to 20 percent in the 1974 outbreak and have remained at about this level since then.[5,10,12] However, significant residual neurologic disability occurs in as many as 50 percent of survivors.[10,12] The presence of this disease in Papua New Guinea was confirmed in 1956.[20] The causative virus was transmitted to experimental animals as early as 1918,[6,11] although those strains could not be maintained. The definitive isolation and characterization of Murray Valley encephalitis virus in 1951[19] led to epidemiologic studies that suggested its survival in bird-mosquito cycles in northern Australia but not in the area of epidemic occurrence in southern Australia.[1] Murray Valley encephalitis is caused by Murray Valley encephalitis virus. In an effort to dissociate a disease from a specific locality, the term Australian encephalitis was proposed by residents of Murray Valley for the disease caused by Murray Valley encephalitis virus. Some researchers subsequently have attempted to expand the term Australian encephalitis to include encephalitis caused by any Australian arbovirus. Because the term Australian encephalitis has no scientific validity and is ambiguous, it should not be used.

Biological, antigenic and phylogenetic characterization of the flavivirus Alfuy

Alfuy virus (ALFV) is classified as a subtype of the flavivirus Murray Valley encephalitis virus (MVEV); however, despite preliminary reports of antigenic and ecological similarities with MVEV, ALFV has not been associated with human disease. Here, it was shown that ALFV is at least 10(4)-fold less neuroinvasive than MVEV after peripheral inoculation of 3-week-old Swiss outbred mice, but ALFV demonstrates similar neurovirulence. In addition, it was shown that ALFV is partially attenuated in mice that are deficient in alpha/beta interferon responses, in contrast to MVEV which is uniformly lethal in these mice. To assess the antigenic relationship between these viruses, a panel of monoclonal antibodies was tested for the ability to bind to ALFV and MVEV in ELISA. Although the majority of monoclonal antibodies recognized both viruses, confirming their antigenic similarity, several discriminating antibodies were identified. Finally, the entire genome of the prototype strain of ALFV (MRM3929) was sequenced and phylogenetically analysed. Nucleotide (73%) and amino acid sequence (83 %) identity between ALFV and IMVEV confirmed previous reports of their close relationship. Several nucleotide and amino acid deletions and/or substitutions with putative functional significance were identified in ALFV, including the abolition of a conserved glycosylation site in the envelope protein and the deletion of the terminal dinucleotide 5'-CUOH-3' found in all other members of the genus. These findings confirm previous reports that ALFV is closely related to IMVEV, but also highlights significant antigenic, genetic and phenotypic divergence from MVEV. Accordingly, the data suggest that ALFV is a distinct species within the serogroup Japanese encephalitis virus.

Experimental Infection of Culex Annulirostris, Culex Gelidus, and Aedes Vigilax With a Yellow Fever/Japanese Encephalitis Virus Vaccine Chimera (Chimerivax TM-JE)

Australian mosquitoes from which Japanese encephalitis virus (JEV) has been recovered (Culex annulirostris, Culex gelidus, and Aedes vigilax) were assessed for their ability to be infected with the ChimeriVax-JE vaccine, with yellow fever vaccine virus 17D (YF 17D) from which the backbone of ChimeriVax-JE vaccine is derived and with JEV-Nakayama. None of the mosquitoes became infected after being fed orally with 6.1 log(10) plaque-forming units (PFU)/mL of ChimeriVax-JE vaccine, which is greater than the peak viremia in vaccinees (mean peak viremia = 4.8 PFU/mL, range = 0-30 PFU/mL of 0.9 days mean duration, range = 0-11 days). Some members of all three species of mosquito became infected when fed on JEV-Nakayama, but only Ae. vigilax was infected when fed on YF 17D. The results suggest that none of these three species of mosquito are likely to set up secondary cycles of transmission of ChimeriVax-JE in Australia after feeding on a viremic vaccinee.

Antibody reactivity to the transmembrane protein of the caprine arthritis encephalitis virus correlates with severity of arthritis: No evidence for the involvement of epitope mimicry

Serum and synovial antibody reactivities of caprine arthritis encephalitis virus (CAEV) infected goats were assessed by Western blotting against purified CAEV antigen and the greatest intensity of reactivity in the serum of arthritic goats was to the gp45 transmembrane protein (TM). The extracytoplasmic domain of the TM gene was cloned into a pGEX vector and expressed in Escherichia coil as a glutathione S transferase fusion protein (GST-TM). This clone was found to be 90.5 and 89.2% homologous to published sequences of CAEV TM gene. Serum of 16 goats naturally infected with CAEV were examined by Western blotting for reactivity to the fusion protein. Antibody reactivity to the GST-TM correlated with clinically detectable arthritis (R = 0.642, P ≤ 0.007). The hypothesis that the immune response to the envelope proteins of the CAEV contributes to the severity of arthritis in goats naturally infected with CAEV via epitope mimicry was tested. Antibodies from 5 CAEV infected goats were affinity purified against the GST-TM fusion protein and tested for cross-reactivity with a series of goat synovial extracts and proteogylcans. No serum antibody response or cross-reactivity of affinity purified antibodies could be detected. Peptides of the CAEV SU that were predicted to be linear epitopes and a similar heat shock protein 83 (HSP) peptide identified by database searching, were synthesized and tested for reactivity in CAEV goats using ELISA, in vitro lymphocyte proliferation and delayed type hypersensitivity (DTH) assays. Peripheral blood lymphocytes from 10 of 17 goats with long term natural CAEV infections proliferated in vitro in response to CAEV and in vivo 3 of 7 CAEV infected goats had a DTH reaction to CAEV antigen. However, none of the peptides elicited significant cell mediated immune responses from CAEV infected goats. No antibody reactivity to the SU peptides or HSP peptide was found. We observed that the antibody reactivity to the CAEV TM protein associated with severity of arthritis however epitope mimicry by the envelope proteins of CAEV is unlikely to be involved.

Heat shock protein 70 on Neuro2a cells is a putative receptor for Japanese encephalitis virus

Japanese encephalitis virus (JEV) envelope (E) protein has been shown to play a critical role in attachment to cells. However, the receptor interacting with envelope protein has not been conclusively identified. Using mouse neuroblastoma (Neuro2a) cells and purified JEV-E protein in `Virus Overlay Protein Binding Assay' followed by MALDI-TOF analysis, we identified `heat shock protein 70' (Hsp70) as a possible receptor for JEV. Indirect immunofluorescence and flow-cytometry analysis demonstrated localization of Hsp70 on Neuro2a cell surface. Co-immunoprecipitation followed by Western blot analysis reconfirmed the interaction between Hsp70 and JEV-E protein. Further, anti-Hsp70 polyclonal-antibodies were able to block JEV entry into Neuro2a cells. Additionally, using the bioinformatic tool - FTDOCK, clocking between the proteins was performed. Amongst six interacting structural poses studied one pose involving RGD motif on JEV-E and leucine(539) on Hsp70 displayed stable interaction. These observations indicate that Hsp70 serves as putative receptor for JEV in Neuro2A cells.

Transmission of Japanese Encephalitis Virus from the Black Flying Fox, Pteropus alecto, to Culex annulirostris Mosquitoes, Despite the Absence of Detectable Viremia.

To determine the potential role of flying foxes in transmission cycles of Japanese encephalitis virus (JEV) in Australia, we exposed Pteropus alecto (Megachiroptera: Pteropididae) to JEV via infected Culex annulirostris mosquitoes or inoculation. No flying foxes developed symptoms consistent with JEV infection. Anti-JEV IgG antibodies developed in 6/10 flying foxes exposed to infected Cx. annulirostris and in 5/5 inoculated flying foxes. Low-level viremia was detected by real-time reverse transcriptase polymerase chain reaction in 1/5 inoculated flying foxes and this animal was able to infect recipient mosquitoes. Although viremia was not detected in any of the 10 flying foxes that were exposed to JEV by mosquito bite, two animals infected recipient mosquitoes. Likewise, an inoculated flying fox without detectable viremia infected recipient mosquitoes. Although infection rates in recipient mosquitoes were low, the high population densities in roosting camps, coupled with migratory behavior indicate that flying foxes could play a role in the dispersal of JEV.

Baker's yeast expressing the Japanese encephalitis virus envelope protein on its cell surface: induction of an antigen-specific but non-neutralizing antibody response

Live recombinant Saccharomyces cerevisiae yeast expressing the envelope antigen of Japanese encephalitis virus (JEV) on the outer mannoprotein layer of the cell wall were examined for their ability to induce antigen-specific antibody responses in mice. When used as a modelantigen, parenteral immunization of mice with surface-expressing GFP yeast induced a strong anti-GFP antibody response in the absence of adjuvants. This antigen delivery approach was then used for a more stringent system, such as the envelope protein of JEV, which is a neurotropic virus requiring neutralizing antibodies for protection.Although 70% of cells were detected to express the total envelope protein on the surface by antibodies raised to the bacterially expressed protein, polyclonal anti-JEV antibodies failed to react with them. In marked contrast, yeast expressing the envelope fragments 238-398, 373-399 and 373-500 in front of a Gly-Ser linker were detected by anti-JEV antibodies as well as a monoclonal antibody but not by antibodies raised to the bacterially expressed protein. Immunization of mice with these surface-expressing recombinants resulted in a strong antibody response. However, the antibodies failed to neutralize the virus, although the fragments were selected based on neutralizing determinants.

Recognition of nonstructural protein-peptides by cytotoxic t-lymphocytes raised against japanese encephalitis-virus

We have previously reported that Lyt(2+) cytotoxic T lymphocytes (CTL) can be raised against Japanese encephalitis virus (JEV) in BALB/c mice. In order to confirm the presence of H-2K(d)-restricted CTL and to examine their cross-recognition of West Wile virus (WNV), we tested the capacity of anti-JEV CTL to lyse uninfected syngeneic target cells that were pulsed with synthetic peptides. The sequence of the synthetic peptides was predicted based upon the H-2K(d) binding consensus motif. We show here that preincubation of uninfected syngeneic targets (P388D1) with JEV NS1- and NS3-derived peptides [NS1 (891-899) and NS3 (1804-1812)], but not with JEV NS5-derived peptide [NS5 (3370-3378)], partially sensitized them for lysis by polyclonal anti-JEV CTL. These results indicate the CTL recognition of NS1- and NS3-derived peptides of JEV.

Virus-Specific Cytolytic Antibodies to Nonstructural Protein 1 of Japanese Encephalitis Virus Effect Reduction of Virus Output from Infected Cells

We demonstrate the presence of nonstructural protein 1 (NS1)-specific antibodies in a significant proportion of convalescent-phase human serum samples obtained from a cohort in an area where Japanese encephalitis virus (JEV) is endemic. Sera containing antibodies to NS1 but not those with antibodies to other JEV proteins, such as envelope, brought about complement-mediated lysis of JEV-infected BHK-21 cells. Target cells infected with a recombinant poxvirus expressing JEV NS1 on the cell surface confirmed the NS1 specificity of cytolytic antibodies. Mouse anti-NS1 cytolytic sera caused a complement-dependent reduction in virus output from infected human cells, demonstrating their important role in viral control. Antibodies elicited by JEV NS1 did not cross lyse West Nile virus- or dengue virus-infected cells despite immunoprecipitating the NS1 proteins of these related flaviviruses. Additionally, JEV NS1 failed to bind complement factor H, in contrast to NS1 of West Nile virus, suggesting that the NS1 proteins of different flaviviruses have distinctly different mechanisms for interacting with the host. Our results also point to an important role for JEV NS1-specific human immune responses in protection against JE and provide a strong case for inclusion of the NS1 protein in next generation of JEV vaccines.

Replication of Japanese encephalitis virus in mouse brain induces alterations in lymphocyte response

The experimental model using intracerebral (i.c.) challenge was employed in many studies evaluating the protection against disease induced by Japanese encephalitis virus (JEV). We investigated alterations in peripheral lymphocyte response caused by i.c. infection of mice with JEV. Splenocytes from the i.c.-infected mice showed suppressed proliferative response to concanavalin A (con A) and anti-CD3 antibody stimulation. At the same time, the expression of CD25 (IL-2R) and production of IL-2 was inhibited. Addition of anti-CD28 antibody restored the decreased anti-CD3 antibody-mediated proliferation in the splenocytes. Moreover, the number of con A-stimulated cells secreting IL-4 was significantly reduced in splenocytes from i.c.-infected mice. These studies suggested that the i.c. infection with JEV might involve additional immune modulation effects due to massive virus replication in the brain.

Japanese Encephalitis Virus Utilizes the Canonical Pathway To Activate NF-kappa B but It Utilizes the Type I Interferon Pathway To Induce Major Histocompatibility Complex Class I Expression in Mouse Embryonic Fibroblasts

Flaviviruses have been shown to induce cell surface expression of major histocompatibility complex class I (MHC-I) through the activation of NF-kappa B. Using IKK1(-/-), IKK2(-/-), NEMO-/-, and IKK1-/- IKK2-/- double mutant as well as p50(-/-) RelA(-/-) cRel(-/-) triple mutant mouse embryonic fibroblasts infected with Japanese encephalitis virus (JEV), we show that this flavivirus utilizes the canonical pathway to activate NF-kappa B in an IKK2- and NEMO-, but not IKK1-, dependent manner. NF-kappa B DNA binding activity induced upon virus infection was shown to be composed of RelA: p50 dimers in these fibroblasts. Type I interferon (IFN) production was significantly decreased but not completely abolished upon virus infection in cells defective in NF-kappa B activation. In contrast, induction of classical MHC-I (class 1a) genes and their cell surface expression remained unaffected in these NF-kappa B-defective cells. However, MHC-I induction was impaired in IFNAR(-/-) cells that lack the alpha/beta IFN receptor, indicating a dominant role of type I IFNs but not NF-kappa B for the induction of MHC-I molecules by Japanese encephalitis virus. Our further analysis revealed that the residual type I IFN signaling in NF-kappa B-deficient cells is sufficient to drive MHC-I gene expression upon virus infection in mouse embryonic fibroblasts. However, NF-kappa B could indirectly regulate MHC-I expression, since JEV-induced type I IFN expression was found to be critically dependent on it.

Detection and molecular epidemiology of tick-borne encephalitis virus infections

Pdf-file, link above

Cytotoxic T lymphocytes raised against Japanese encephalitis virus: effector cell phenotype, target specificity and in vitro virus clearance.

Several H-2 defined cell lines were examined for their ability to support infection and replication of Japanese encephalitis virus (JEV) before their use in in vitro and in vivo stimulation protocols for generating cytotoxic T lymphocytes (CTLs) against JEV. Among II different cell lines tested, two H-2(d) macrophage tumour lines (P388D1, RAW 264.7), an H-2(d) hybridoma (Sp2/0), an H-2K(k)D(d) neuroblastoma (Neuro 2a), and H-2(k) fibroblast cell line (L929) were found to support JEV infection and replication. These cell lines were used to generate anti-JEV CTLs by using in vivo immunization followed by in vitro stimulation of BALB/c mice. We observed that not only syngeneic and allogeneic infected cells but also JEV-infected xenogeneic cells could prime BALB/c mice for the generation of JEV-specific CTLs upon subsequent in vitro stimulation of splenocytes with JEV-infected syngeneic cells. Although infected xenogeneic cells were used for immunization, the anti-JEV effecters that were generated lysed infected syngeneic targets but not JEV-infected xenogeneic or allogeneic target cells in a 5h Cr-51 release assay. These anti-JEV effecters recognized syngeneic target cells infected with West Nile virus to a lesser extent and were shown to be Lyt-2.2(+) T cells. The results of unlabelled cold target competition studies suggested alterations in the cell surface expression of viral antigenic determinants recognized by these CTLs. We further demonstrate that the JEV-specific CTLs generated could virtually block the release of infectious virus particles from infected P388D1 and Neuro 2a cells in vitro.