Characterization of mouse neuronal Ca2+/calmodulin kinase II inhibitor alpha

We have overexpressed an 8.5-kDa mouse Ca2+/calmodulin kinase II inhibitor a protein (mCaMKIIN alpha) in Escherichia coli and demonstrate that the recombinant protein is a potent inhibitor of Ca2+/calmodulin kinase 11 (CaMKII) in vitro. However, antibodies raised against recombinant mCaMKIIN alpha. react with an similar to 37-kDa protein present in mouse brain. The pattern of expression of the similar to 37-kDa protein is similar to that of mCaMKIIN alpha mRNA as both are expressed in normal but not Japanese encephalitis virus (JEV)-infected mouse brain. Subcellular localization studies indicate that the similar to 37-kDa protein is present in the post-synaptic density (PSD) where mCaMKII alpha is known to perform key regulatory functions. We conclude that the similar to 37-kDa protein identified in this study is mCaMKIIN alpha. and its localization in the PSD indicates a novel role for this protein in the regulation of neuronal CaMKII alpha. (c) 2007 Elsevier B.V. All rights reserved.

Identification of protein interaction regions of VINC/NEAT1/Men epsilon RNA

The virus inducible non-coding RNA (VINC) was detected initially in the brain of mice infected with Japanese encephalitis virus (JEV) and rabies virus. VINC is also known as NEAT1 or Men epsilon RNA. It is localized in the nuclear paraspeckles of several murine as well as human cell lines and is essential for paraspeckle formation. We demonstrate that VINC interacts with the paraspeckle protein, P54nrb through three different protein interaction regions (PIRs) one of which (PIR-1) is localized near the 50 end while the other two (PIR-2, PIR-3) are localized near the 30 region of VINC. Our studies suggest that VINC may interact with P54nrb through a novel mechanism which is different from that reported for protein coding RNAs. (C) 2010 Federation of European Biochemical Societies. Published by Elsevier B. V. All rights reserved.

Detection and characterisation of circulating cysticercal antigens in human cerebrospinal fluid

With biotin labelled and unlabelled immunoglobulin fraction of anticysticercal antibodies raised in rabbits, tandem-enzyme linked immunosorbent assay (T-ELISA), capture-dot immunobinding assay (C-DIA) and reverse passive haemagglutination (RPHA) tests were developed for the detection of cysticercal antigens. The sensitivity levels were respectively, 9 ng ml−1, 2 ng ml−1 and 45 ng ml−1. All three methods were of equal specificity as none of the antigens of Mycobacterium tuberculosis, Japanese encephalitis virus and Echinococcus granulosus reacted with anticysticercal IgG. Cysticercal antigens were detected in the cerebrospinal fluid (CSF) of confirmed neurocysticercosis at sensitivity levels of 91·6% by T-ELISA, 83·33% by C-DIA and 75% by RPHA and specificity levels of >93%. Western analysis of these antigens in CSF showed mainly antigens of 64–68 kDa and 24–28 kDA. By crossed immunoelectrophoresis (CIE) with an intermediate gel technique, five circulating antigens were found to be released from scolex and fluid.

Mosquito larvae borne viruses from the central travancore region of kerala: isolation and characterization

The present study was initiated when several massive outbreaks of Chikungunya, Dengue and Japanese Encephalitis were frequently reported across the State of Kerala. Multiple symptoms persisted among the affected individuals and the public health officials were in search of aetiological agents responsible for the out breaks and, other than clinical samples no resources were available. In this context, a study was undertaken to focus on mosquito larvae to investigate the viruses borne by them which remain silently prevalent in the environment. The study was not a group specific investigation limited to either arbovirus or enterovirus, but had a broad spectrum approach. The study encompassed the viral pathogens that could be isolated, their impact when passaged through cell lines, growth kinetics, titer of the working stocks in specific cell line, the structure by means of transmission electron microscopy(TEM), the one step growth and molecular characterization using molecular tools.

Applications of pox virus vectors to vaccination: an update.

Recombinant pox viruses have been generated for vaccination against heterologous pathogens. Amongst these, the following are notable examples. (i) The engineering of the Copenhagen strain of vaccinia virus to express the rabies virus glycoprotein. When applied in baits, this recombinant has been shown to vaccinate the red fox in Europe and raccoons in the United States, stemming the spread of rabies virus infection in the wild. (ii) A fowlpox-based recombinant expressing the Newcastle disease virus fusion and hemagglutinin glycoproteins has been shown to protect commercial broiler chickens for their lifetime when the vaccine was administered at 1 day of age, even in the presence of maternal immunity against either the Newcastle disease virus or the pox vector. (iii) Recombinants of canarypox virus, which is restricted for replication to avian species, have provided protection against rabies virus challenge in cats and dogs, against canine distemper virus, feline leukemia virus, and equine influenza virus disease. In humans, canarypox virus-based recombinants expressing antigens from rabies virus, Japanese encephalitis virus, and HIV have been shown to be safe and immunogenic. (iv) A highly attenuated vaccinia derivative, NYVAC, has been engineered to express antigens from both animal and human pathogens. Safety and immunogenicity of NYVAC-based recombinants expressing the rabies virus glycoprotein, a polyprotein from Japanese encephalitis virus, and seven antigens from Plasmodium falciparum have been demonstrated to be safe and immunogenic in early human vaccine studies.

West Nile virus vaccines

West Nile virus (WNV) is a mosquito-borne flavivirus that is emerging as a global pathogen. In the last decade, virulent strains of the virus have been associated with significant outbreaks of human and animal disease in Europe, the Middle East and North America. Efforts to develop human and veterinary vaccines have taken both traditional and novel approaches. A formalin-inactivated whole virus vaccine has been approved for use in horses. DNA vaccines coding for the structural WNV proteins have also been assessed for veterinary use and have been found to be protective in mice, horses and birds. Live attenuated yellow fever WNV chimeric vaccines have also been successful in animals and are currently undergoing human trials. Additional studies have shown that immunisation with a relatively benign Australian variant of WNV, the Kunjin virus, also provides protective immunity against the virulent North American strain. Levels of efficacy and safety, as well as logistical, economic and environmental issues, must all be carefully considered before vaccine candidates are approved and selected for large-scale manufacture and distribution.

NS1 protein secretion during the acute phase of West Nile virus infection

The West Nile virus (WNV) nonstructural protein NS1 is a protein of unknown function that is found within, associated with, and secreted from infected cells. We systematically investigated the kinetics of NS1 secretion in vitro and in vivo to determine the potential use of this protein as a diagnostic marker and to analyze NS1 secretion in relation to the infection cycle. A sensitive antigen capture enzyme-linked immunosorbent assay (ELISA) for detection of WNW NS1 (polyclonal-ACE) was developed, as well as a capture ELISA for the specific detection of NS1 multimers (4G4-ACE). The 4G4-ACE detected native NS1 antigens at high sensitivity, whereas the polyclonal-ACE had a higher specificity for recombinant forms of the protein. Applying these assays we found that only a small fraction of intracellular NS1 is secreted and that secretion of NS1 in tissue culture is delayed compared to the release of virus particles. In experimentally infected hamsters, NS1 was detected in the serum between days 3 and 8 postinfection, peaking on day 5, the day prior to the onset of clinical disease; immunoglobulin M (IgM) antibodies were detected at low levels on day 5 postinfection. Although real-time PCR gave the earliest indication of infection (day 1), the diagnostic performance of the 4G4-ACE was comparable to that of real-time PCR during the time period when NS1 was secreted. Moreover, the 4G4-ACE was found to be superior in performance to both the IgM and plaque assays during this time period, suggesting that NS1 is a viable early diagnostic marker of WNV infection.

Identification of new flaviviruses in the Kokobera virus complex

Novel flavivirus isolates from mosquitoes collected in northern Australia were analysed by partial genomic sequencing, monoclonal antibody-binding assays and polyclonal cross-neutralization tests. Two isolates were found to be antigenically distinct from, but related to, viruses of the Kokobera virus complex, which currently contains Kokobera (KOKV) and Stratford (STRV) viruses. Nucleotide sequence comparison of two separate regions of the genome revealed that an isolate from Saibai Island in the Torres Strait in 2000 (TS5273) was related closely to KOKV and STRV, with 74-80 and 75-76% nucleotide similarity, respectively. An isolate from mainland Cape York in 1998 (CY1014) was found to be more divergent from KOKV and STRV, with