Development of a latex agglutination method for diagnosis of rotavirus infections

Rotavirus is a major cause of morbidity and mortality among children with gastroenteritis. Since the discovery of rotaviruses, several techniques have been used for their laboratory diagnosis; those included Electron Microscopy [EM] and enzyme immunoassay. These methods, however, are expensive and not readily available everywhere. We have developed a technique which can be used for routine diagnosis of rotavirus gastroenteritis. Purified simian rotavirus, SA1 1, was injected into rabbits and the y-globulin fraction of antisera was purified and used for coating of latex beads. The prepared sensitizied latex was then used for agglutination test on fecal samples. 94 stool samples from infants with acute gastroenteritis were tested by [EM], enzyme immunoassay and Latex Agglutination [LA] method. The sensitivity of enzyme immune assay and [LA] were 92.5% and 90%, respectively; the specificity of both tests was 98.1% as compared with [EM]. Latex Agglutination Test [LAT] is a simple and relatively inexpensive test which can be used for diagnosis of rotavirus gastroenteritis in diagnostic laboratories and health centers

Expression of nonstructural glycoprotein NSP4 of SA11 Simian rotavirus in escherichia coli and production of antibody aginst it

Rotavirus non structural glycoprotein NSP4 can induce diarrhea in newborn mice. It has been suggested that NSP4 may be a key determinant for rotavirus pathogenesis and a target for vaccine development. In order to study the biological and morphological role of NSP4 a large amount of the purified protein and antibody against it are required. Simian rotavirus SA11 was propagated in BSCI cell, purified on cesium chloride gradients, and its genomic RNA was extracted. A cDNA from RNA segment 10 was synthesized and amplified by RT-PCR. cDNA fragment was cloned into plasmid vectors. The recombinant plasmid was characterized by restriction enzyme and sequencing. Construction of expression plasmid containing nsp4 gene was performed and expression of NSP4 was demonstrated by SDS-PAGE, Western blot, ELISA and IF using polyclonal antibody against NSP4 from SA11 infected BSC1 cells. A polyclonal antiserum against purified recombinant NSP4 was raised in rabbit; which was reacted with NSP4 in BSCI cells infected with SA11 rotavirus. These results indicated successful expression of the full-length NSP4 in E.coli to produce antibody against it and to study its biological activities

Sodium valproate-induced potentiation of antiherpetic effect of acyclovir

Sodium valproate [VPA], an anticonvalsant drug, has been reported to stimulate viral replication. A combination therapy with VPA and acyclovir [ACV] is used for the treatment of herpesvirus encephalitis, the commonest sporadic encephalitis of viral origin. To determine a possible interaction between VPA and ACV leading to a modification of antiviral activity of ACV. Cultured Hela cells were treated with 5 micro M of ACV and various concentrations of VPA followed by infection with herpes simplex virus type 1 [HSV-1]. Virus replication was monitored by quantal assay. Further investigations comprised electron microscopy, immunoperoxidase and immunoblot procedures. Possible chemical interaction between VPA and ACV was studied by nuclear magnetic resonance [NMR] spectrometer. Combined treatment of infected cells with ACV and VPA revealed 50- to 250-fold potentiation of antiviral activity of ACV by increasing VPA concentrations. Examination by NMR spectrometer showed a strong chemical interaction between amino groups of ACV and carboxyl part of VPA. The present in vitro studies should be paralleled by appropriate in vivo investigations, and if substantiated, a combination therapy with ACV and VPA may supersede single ACV therapy for herpesvirus encephalitis. Further studies are thus needed to establish which of VPA metabolites or newly-formed compounds is accountable for augmentation of antiviral effect of ACV