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.

Quantitation of poliovirus antigens in inactivated viral vaccines by enzyme-linked immunosorbent assay using animal sera and monoclonal antibodies.

Recent advances in methods for the manufacture of inactivated poliovirus vaccines have resulted in increased vaccine immunogenicity. In conjunction with this capability it is important to have available highly sensitive and quantitative potency assays. The potential suitability of enzyme-linked immunoassay (ELISA) was evaluated using animal sera with neutralizing antibodies or neutralizing monoclonal antibodies for antigen detection in potency tests. The monoclonal antibodies developed, which bound D antigen but not C antigen, were neutralizing unless relatively weakly reactive. Those that bound C antigen only were non-neutralizing. Those that bound both C and D antigens were sometimes neutralizing. D-specific and D/C-specific neutralizing monoclonal antibodies against type-2 poliovirus protected mice on passive immunization against paralytic disease and death from the MEF strain virus. Potency measurements by ELISA using either D-specific neutralizing monoclonal antibodies or type-specific goat sera for antigen detection were sensitive and precise. Tests using C-specific monoclonal antibodies for antigen detection indicated that increased C antigen content may result in falsely elevated reactivities of animal sera with some vaccines. Monoclonal antibodies may be useful ELISA reagents for IPV potency testing.

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.

Rapid diagnosis of Rift Valley fever: a comparison of methods for the direct detection of viral antigen in human sera.

Human sera collected during the 1987 Rift Valley fever (RVF) epidemic in the Senegal River basin were analysed using three enzyme immunoassays to establish the best method for rapid diagnosis of RVF. A biotin-avidin-enhanced antigen detection method utilizing monoclonal antibodies proved most sensitive. Eighty-two viremic human sera were tested, and this assay detected antigen in 29.3% of the samples.

Capsid protein precursor is one of two initiated products of translation of poliovirus RNA in vitro.

Previous studies in our laboratory have demonstrated that cell-free systems translating the Mahoney strain of poliovirus type I RNA utilize two unique initiation sites. In this study, defective-interfering particles of poliovirus, which contain deletions in the region encoding the capsid proteins, are shown to initiate translation of proteins in vitro at these same two sites. Both the standard virus and the defective-interfering virus RNA direct the synthesis of two polypeptides labeled with n-formyl-methionine (fmet) at their amino termini. The size of the smaller fmet polypeptide synthesized in vitro by the defective virus appears identical in size to that of the standard virus. However, the larger-molecular-weight fmet polypeptide is reduced in size from 115,000 to 69,000 daltons. This correlates exactly with the reduced size of the precursor to the capsid proteins synthesized by the defective virus in vivo and with the size of the deletion in the defective virus RNA (1,200 bases). This provides genetic evidence that the 115,000-dalton fmet polypeptide synthesized into vitro by the standard virus is NCVP1a, the precursor to the coat proteins. Although the identity of the small (5,000 to 10,000 daltons) fmet polypeptide is not clear, several lines of evidence enable us to exclude the possibility that it is VP4, the smallest viral capsid protein.

Two initiation sites for translation of poliovirus RNA in vitro: comparison of LSc and Mahoney strains.

Previous studies in our laboratory provided evidence that the initiation of translation by the Mahoney strain of poliovirus type 1 RNA in vitro occurs at two unique sites. This study shows that the LSc strain of poliovirus type 1, a multistep, temperature-sensitive mutant of the Mahoney strain, also utilizes two sites for the initiation of translation in vitro. Incorporation of formyl-[35S]methionine into the amino terminus of newly synthesized polypeptides revealed the production of two labeled tryptic peptides which are identical in size and electrophoretic mobility with those produced by Mahoney virus. The polypeptides containing amino-terminal label showed similar patterns on sodium dodecyl sulfate-acrylamide gels, although one of the LSc polypeptides had a slightly faster mobility. The relative proportion of initiation at each site varied with the magnesium concentration for both viruses, but the LSc strain favored initiation at one site more so than did the Mahoney strain.

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.