Protective immunity to Japanese encephalitis virus associated with anti-NS1 antibodies in a mouse model.

BACKGROUND: Japanese encephalitis virus (JEV) is a major mosquito-borne pathogen that causes viral encephalitis throughout Asia. Vaccination with an inactive JEV particle or attenuated virus is an efficient preventative measure for controlling infection. Flavivirus NS1 protein is a glycoprotein secreted during viral replication that plays multiple roles in the viral life cycle and pathogenesis. Utilizing JEV NS1 as an antigen in viral vectors induces a limited protective immune response against infection. Previous studies using E. coli-expressed JEV NS1 to immunize mice induced protection against lethal challenge; however, the protection mechanism through cellular and humoral immune responses was not described. RESULTS: JEV NS1 was expressed in and purified from Drosophila S2 cells in a native glycosylated multimeric form, which induced T-cell and antibody responses in immunized C3H/HeN mice. Mice vaccinated with 1 µg NS1 with or without water-in-oil adjuvant were partially protected against viral challenge and higher protection was observed in mice with higher antibody titers. IgG1 was preferentially elicited by an adjuvanted NS1 protein, whereas a larger load of IFN-γ was produced in splenocytes from mice immunized with aqueous NS1. Mice that passively received anti-NS1 mouse polyclonal immune sera were protected, and this phenomenon was dose-dependent, whereas protection was low or delayed after the passive transfer of anti-NS1 MAbs. CONCLUSION: The purified NS1 subunit induced protective immunity in relation with anti-NS1 IgG1 antibodies. NS1 protein efficiently stimulated Th1-cell proliferation and IFN-γ production. Protection against lethal challenge was elicited by passive transfer of anti-NS1 antisera, suggesting that anti-NS1 antibodies play a substantial role in anti-viral immunity.

Effect of the methyltransferase domain of Japanese encephalitis virus NS5 on the polymerase activity.

Japanese encephalitis virus (JEV) NS5 consists of an N-terminal guanylyltransferase/methyltransferase (MTase) domain and a C-terminal RNA-dependent RNA polymerase (RdRp) domain. We purified JEV NS5 from bacteria and examined its RdRp activity in vitro. It showed exclusive specificity for Mn(2+) and alkaline conditions (pH 8-10) for RdRp activity. It showed strong RdRp activity with dinucleotide primers, and the order of template strength was poly(U)>(I)>(A)>(C). It showed weak transcription activity without primers, but could not transcribe poly(I) without primers. It bound homopolymeric RNA templates, but weakly bound poly(C). The Km (µM) values were 22.13±1.11 (ATP), 21.94±3.88 (CTP), 21.27±1.23 (GTP), and 9.91±0.30 (UTP), indicating low substrate affinity. Vmax (/min) values were 0.216±0.017 (ATP), 0.781±0.020 (CTP), 0.597±0.049 (GTP), and 0.347±0.022 (UTP), indicating high polymerization activity. The RdRp domain alone did not show RdRp activity; a structural and functional interaction between the MTase and RdRp domains via 299-EHPYRTWTYH-308 (MTase domain) and 739-LIGRARISPG-748 (RdRp domain) was predicted, because mutations in the MTase domain affected RdRp activity.

Partial molecular characterization of alphaherpesviruses isolated from tropical bats.

Herpesviruses have previously been isolated from African and South-American bats. Recently, herpesviruses detected from European insectivorous bats (family Vespertilionidae) were classified molecularly as betaherpesviruses and gammaherpesviruses. In the current study, we performed PCR analyses targeting the UL30 catalytic subunit region of the DNA polymerase gene of the African and South American herpesviruses and new Malagasy and Cambodian herpesviruses isolated from bats, especially frugivorous bats from the families Pteropodidae and Phyllostomidae. The sequences obtained from the amplified products indicated that these isolates belonged to the genus Simplexvirus of the subfamily Alphaherpesvirinae. These results extend the taxonomic range of bat herpesviruses with the description of four members in the subfamily Alphaherpesvirinae. Furthermore, these data confirm and extend the geographical distribution of herpesvirus in bats to three more continents (Africa, South America and Asia) and indicate the presence of these viruses in frugivorous bats of the families Pteropodidae and Phyllostomidae.

Detection of Japanese encephalitis virus antibodies in bats in Southern China.

In this study, 336 bats of eight species were collected during 2004-2007 in six provinces of Southern China. Antibodies against Japanese encephalitis virus (JEV) were detected by ELISA in 43 of 336 (12.8%) serum samples. Among those ELISA-positive samples, 11 serum samples had neutralizing antibodies against JEV. No JEV was detected in brain and liver samples using specific real-time reverse transcriptase-polymerase chain reaction. This is the first report of JEV neutralizing antibodies in bats in China, which reinforces that bats can be involved in the life cycle of this virus.

Nipah virus in Lyle's flying foxes, Cambodia.

We conducted a survey in Cambodia in 2000 on henipavirus infection among several bat species, including flying foxes, and persons exposed to these animals. Among 1,072 bat serum samples tested by enzyme-linked immunosorbent assay, antibodies reactive to Nipah virus (NiV) antigen were detected only in Pteropus lylei species; Cynopterus sphinx, Hipposideros larvatus, Scotophilus kuhlii, Chaerephon plicata, Taphozous melanopogon, and T. theobaldi species were negative. Seroneutralization applied on a subset of 156 serum samples confirmed these results. None of the 8 human serum samples was NiV seropositive with the seroneutralization test. One virus isolate exhibiting cytopathic effect with syncytia was obtained from 769 urine samples collected at roosts of P. lylei specimens. Partial molecular characterization of this isolate demonstrated that it was closely related to NiV. These results strengthen the hypothesis that flying foxes could be the natural host of NiV. Surveillance of human cases should be implemented.

A virulence factor of myxoma virus colocalizes with NF-kappaB in the nucleus and interferes with inflammation.

NF-kappaB is one of the most important elements that coordinate stress-induced, immune, and inflammatory responses. Myxoma virus, a member of the Poxviridae family responsible for rabbit myxomatosis, codes for several factors that help its survival in the host. In this study, we focused on the product of the M150R gene. We show that the protein has nine ankyrin repeats (ANKs), with the eighth having a close similarity with the nuclear localization signal-containing ANK of I-kappaBalpha, which regulates NF-kappaB activity by sequestering it in the cytosol. Because the viral protein is targeted to the nucleus, it was named MNF, for myxoma nuclear factor. This localization was lost when the eighth ANK was removed. In tumor necrosis factor alpha-treated cells, MNF and NF-kappaB colocalized as dotted spots in the nucleus. In vivo experiments with a knockout virus showed that MNF is a critical virulence factor, with its deletion generating an almost apathogenic virus. Detailed histological examinations revealed an increase in the inflammatory process in the absence of MNF, consistent with the interference of MNF with the NF-kappaB-induced proinflammatory pathway. Because MNF has homologs in other poxviruses, such as vaccinia, cowpox, and variola viruses, this protein is probably part of a key mechanism that contributes to the immunogenic and pathogenic properties of these viruses.