Detection of Rift Valley fever virus in mosquitoes by RT-PCR.

A reverse transcriptase-polymerase chain reaction (RT-PCR) assay to detect Rift Valley fever (RVF) virus RNA in experimentally infected mosquitoes was developed. The specificity of the assay was evaluated with three other phleboviruses; sandfly fever Sicilian (Sabin), sandfly fever Naples (Sabin) and Punta Toro (MSP 3) viruses. The relative sensitivity of the assay, determined by using RVF virus RNA extracted from serial dilutions of virus culture, was approximately 50 plaque forming units. This sensitivity level was 100-fold higher when a nested PCR procedure was used. When the RT-PCR assay was used with coded samples of intrathoracically-infected and uninfected mosquito, the assay detected the virus in all infected mosquitoes. With this assay, it was possible to detect RVF virus RNA in a single infected mosquito in the background of 10, 25 or 50 uninfected mosquitoes.

Comparison of hantavirus isolates using a genus-reactive primer pair polymerase chain reaction.

RNA of more than 40 hantavirus isolates, originating from rodents and humans of widely separated geographical areas, was copied to cDNA using reverse transcriptase and amplified by polymerase chain reaction (PCR). A genus-reactive oligonucleotide primer pair, flanking a 365 bp region of the G2 glycoprotein gene, was chosen for genus-reactive PCR. DNA products were digested with 20 restriction endonucleases and cleavage patterns were analysed. For strains of known sequence, the restriction patterns observed were consistent with those predicted from sequence data, demonstrating that the amplified products originated from target virus RNA. Further analyses suggested that all amplified viruses could be easily typed into one of five restriction patterns using only five enzymes. The categories identified by restriction analysis of PCR-amplified cDNA corresponded with serogroups established by plaque-reduction neutralization tests. This method may greatly simplify the identification of new hantavirus isolates.

Isolation of Rift Valley fever virus from human peripheral blood mononuclear cells: Mauritanian epidemic.

During the Mauritanian Rift Valley fever (RVF) epidemic of 1987, peripheral blood mononuclear cells (PBMC) were studied from 78 sick patients. RVF virus (RVFV) was isolated in 5 cases, on Aedes pseudoscutellaris AP61, from both PBMC and serum. Among the 78 cases studied, RVF was proven in 19 cases (24.3%) by specific IgM detection, and in 12 cases (15.3%) by virus isolation from serum, of which 3 also exhibited anti-RVF IgM. Among the 5 PBMC-positive RVFV cases, 2 strains were isolated in the presence of specific IgM from patients presenting with neurologic signs. These observations raised the question as to the role of specific IgM in cellular infection, and suggest that, in certain cases, mononuclear cells may promote RVFV dissemination into brain cells. Further investigations need to be undertaken to determine the RVFV receptor expressed on PBMC membranes.

Rift Valley fever infection of rhesus monkeys: implications for rapid diagnosis of human disease.

Rhesus monkeys inoculated with Rift Valley fever (RVF) virus provide a model in which serial observations of serum viral antigen and antibodies can be made. In 9 non-fatal and 3 fatal infections, either antigen or IgM enzyme-linked immunosorbent assay (ELISA) antibodies were detected in every serum sample during the acute phase. Furthermore, viral nucleic acid could be detected by filter hybridization in most samples taken on days 1 to 3. Circulation of significant quantities of viral RNA provides an additional approach to the diagnosis and study of RVF.

Assessment of an rDNA probe filter hybridization assay for the detection of Rift Valley fever virus RNA in human serum samples from the Mauritanian epidemic.

The Rift Valley fever virus (RVFV) epidemic that occurred in southern Mauritania during the 1987 rainy season provided a unique opportunity to test and evaluate a recently developed, M-segment-specific, nucleic acid filter hybridization assay on a large collection of infected human serum samples. It afforded the opportunity to compare the procedure with two other methods for detecting virus: virus isolation and antigen detection by ELISA. The filter hybridization procedure employed a polyethylene-glycol-precipitation and proteinase-K-digestion sample treatment step developed specifically for preparing serum samples for hybridization. The procedure was less sensitive for detecting RVFV in the Mauritanian human viremic samples than in sera from experimentally infected monkeys used to evaluate this procedure. It was also less sensitive than an antigen detection procedure used to test the Mauritanian samples. However, we were able to detect virus RNA in a significant proportion of the virus-isolation-positive samples. Advances in sample preparation, labelling and detection procedures, and hybridization methods will improve the sensitivity, precision and ease of use of this assay and increase its value as a diagnostic tool.

Complementation Analysis of Metabolite-Resistant Mutations with Forced Heterokaryons of NEUROSPORA CRASSA.

The double mutant strain pyr-3 arg-12(s) is a prototroph because a common precursor of arginine and pyrimidine is supplied by the arginine pathway. Growth of this strain is inhibited by exogenous citrulline or arginine. Citrulline-resistant mutants of this strain were selected, and they resulted from modifier mutations at other loci. Forced heterokaryons were used to study complementation among these modifiers. Since the complementation test requires the scoring of non-growth as the positive result, there was concern that variations in nuclear ratios could give erroneous results. This possibility does not seem significant, since groups of mutants established by complementation correspond with groups established by physiological, enzymatic, and recombinational measurements.-The technique has revealed that the most frequently mutated loci are arg-1 and what is probably un-3. Arg-1 mutations affect the conversion of citrulline to argininosuccinate, while un-3 mutations reduce the citrulline uptake rate. Since most of these mutations are of the intracistronic complementing type, a complementation map was constructed for most of the affected loci. The high proportion of complementors in each map can be explained by assuming that partially functioning gene products are more likely to complement with each other than are those which are nonfunctional.