Recombinant VP7-based enzyme-linked immunosorbent assay for detection of immunoglobulin G antibodies to Colorado tick fever virus.
VP6, VP7, VP9, VP10, VP11, and VP12 of Colorado tick fever virus (CTF virus), a virus member of the genus Coltivirus, family Reoviridae, were expressed in bacteria with the pGEX-4T-2 vector. A partial sequence of VP7 (designated pVP7) was chosen to elaborate an enzyme-linked immunosorbent assay (ELISA) for detecting anti-CTF virus immunoglobulin G (IgG) antibodies in humans. This was based on two observations: (i) among all expressed proteins, pVP7 showed the highest immunoreactivity to an anti-CTF virus hyperimmune ascitic fluid; (ii) to provide the highest selectivity of antibody detection, the expressed sequence was chosen within a region which is highly divergent (49% amino acid identity) from the homologous sequence of another coltivirus, the Eyach virus. The pVP7 ELISA was evaluated with 368 serum samples from French blood donors and found to provide 98.1% specificity. Assays with the Calisher set of human serum samples, positive for anti-CTF virus antibodies (C. H. Calisher, J. D. Poland, S. B. Calisher, and L. A Warmoth, J. Clin. Microbiol. 22:84-88, 1985), showed that the pVP7 ELISA provided 100% sensitivity for the tested population. After elaboration of recombinant-protein-based ELISAs for diagnosis of infections with members of the viral genera Orbivirus, Orthoreovirus, and Rotavirus, it was shown that a recombinant protein could be used to detect antibodies to the human pathogen Colorado tick fever virus. (+info)
Termination and read-through proteins encoded by genome segment 9 of Colorado tick fever virus.
Genome segment 9 (Seg-9) of Colorado tick fever virus (CTFV) is 1884 bp long and contains a large open reading frame (ORF; 1845 nt in length overall), although a single in-frame stop codon (at nt 1052-1054) reduces the ORF coding capacity by approximately 40 %. However, analyses of highly conserved RNA sequences in the vicinity of the stop codon indicate that it belongs to a class of 'leaky terminators'. The third nucleotide positions in codons situated both before and after the stop codon, shows the highest variability, suggesting that both regions are translated during virus replication. This also suggests that the stop signal is functionally leaky, allowing read-through translation to occur. Indeed, both the truncated 'termination' protein and the full-length 'read-through' protein (VP9 and VP9', respectively) were detected in CTFV-infected cells, in cells transfected with a plasmid expressing only Seg-9 protein products, and in the in vitro translation products from undenatured Seg-9 ssRNA. The ratios of full-length and truncated proteins generated suggest that read-through may be down-regulated by other viral proteins. Western blot analysis of infected cells and purified CTFV showed that VP9 is a structural component of the virion, while VP9' is a non-structural protein. (+info)
Characterization of the stop codon readthrough signal of Colorado tick fever virus segment 9 RNA.
Diagnosis of Colorado tick fever virus infection by enzyme immunoassays for immunoglobulin M and G antibodies.
An immunoglobulin M (IgM) capture enzyme immunoassay technique was adapted for the detection of antibody to Colorado tick fever virus in sera from 84 individuals for whom diagnosis had been confirmed by virus isolation or neutralization test. Titers were compared with those for IgG and neutralizing antibodies in these Colorado tick fever cases. IgM antibody titers were higher than neutralizing antibody titers, but neither appeared until 1 to 2 weeks after the onset of illness. Neutralizing antibodies were detected earlier than IgM antibodies, and both were detected with greater frequency than IgG antibodies. Late-convalescent-phase sera contained both neutralizing and IgG antibodies, but IgM was all but undetectable by 2 months after onset. Although the neutralization test may remain the serological test of choice, the enzyme immunoassay for IgM antibody offers a simple and more rapid method of serodiagnosis; the enzyme immunoassay is, however, less sensitive than the neutralization test. Furthermore, because there was a sharp decline in IgM antibody after 45 days, the presence of IgM antibody in a single serum sample provides a basis for the presumptive serodiagnosis of recent Colorado tick fever virus infection. (+info)
Genetic relatedness of Colorado tick fever virus isolates by RNA-RNA blot hybridization.
The sequence relatedness of ten isolates of the Colorado tick fever (CTF) serogroup of orbiviruses was examined by RNA-RNA blot hybridization. The 12 dsRNA genome segments of each of the isolates were electrophoresed in a 10% polyacrylamide gel, the segments were transferred electrophoretically to membranes and hybridized to radiolabelled genomic RNA from CTF Florio mouse-adapted strain (CTF FMA) or CTF SS-18. All genome segments of the ten CTF viruses exhibited cross-hybridization signals with either CTF FMA or CTF SS-18, under conditions in which greater than or equal to 74% sequence homology was required to form stable hybrids. Although the dsRNA polyacrylamide gel profiles were unique for each isolate examined, the CTF genes did not exhibit sequence divergence as has been seen among other Orbivirus serogroups. These hybridization analyses suggest that the CTF gene pool is relatively homogeneous which may be a reflection of the lack of multiple serotypes of CTF strains in neutralization tests. Nevertheless, the hybridization signals of segments 4 and 6 were lighter than those of the other genes, indicating that these two genes exhibited the highest degree of sequence variability among these isolates. These data are compared with hybridization data on other Orbivirus serogroups. (+info)
Antigenic variants of Colorado tick fever virus.
Twenty strains of Colorado tick fever (CTF) virus, isolated from ticks, mammals and humans, and two antigenic relatives of CTF virus were compared in cross-neutralization tests. Viruses were tested using single-inoculation sera prepared in hamsters. Antigenic variation, as measured by differences detected in the neutralization test, was noted among the virus isolates identified as strains of CTF virus. The virus strains isolated from humans appeared to vary the most in serological reactions. The two antigenic relatives of CTF virus are clearly distinct from strains of CTF and are different from each other. Antigenic relationships between these two viruses were established using two sets of single-inoculation antisera and both complement fixation and neutralization tests. Six distinct antigenic variants of CTF virus isolated from humans and the virus strain from ticks (75V1906) that showed the least antigenic variation, were tested against 49 coded serum pairs from clinically diagnosed cases of CTF. Significant differences were noted in the number of convalescent-phase sera that reacted with each virus strain and in the number of seroconversions observed with each test virus strain. Convalescent phase sera that reacted with multiple virus strains often varied significantly in antibody titre from one virus strain to another. This indicates that, in some instances, antibody was probably produced in response to infection by different antigenic variants of CTF virus. (+info)
Inhibition of bluetongue and Colorado tick fever orbiviruses by selected antiviral substances.
The effects of four ribonucleic acid virus inhibitors were evaluated in cell cultures and in mice to determine inhibitory effects against bluetongue virus and Colorado tick fever virus (CTFV). Test compounds included 1-beta-D-ribofuranosyl-1,2,4-triazole-3-carboxamide (ribavirin), 3-deazaguanine, 3-deazauridine, and 9-(S)-(2,3-dihydroxypropyl)adenine. Ribavirin-2',3',5'-triacetate (ribavirin triacetate) was evaluated in vivo against CTFV. Inhibition of cytopathic effect and plaque reduction were used to evaluate antiviral activity. In cytopathic effect inhibition studies, bluetongue virus was markedly inhibited by 3-deazaguanine and 3-deazauridine in Vero cells with moderate inhibition by the other agents. Ribavirin and 3-deazaguanine markedly inhibited CTFV in MA-104 cells, 3-deazauridine was slightly less active, and 9-(S)-(2,3-dihydroxypropyl)adenine was negative. Ribavirin was less effective in Vero cells against CTFV. When mice were inoculated intracerebrally with CTFV and treated by a single intracerebral injection with drug, ribavirin triacetate increased the number of survivors, 3-deazaguanine increased mean survival time, and ribavirin was negative. Intraperitoneal treatment of infected mice with ribavirin triacetate for 1 week significantly increased the number of survivors and mean survival time, providing strong evidence that the agent is active across the blood-brain barrier. (+info)
The development of Colorado tick fever virus within cells of the haemopoietic system.
Electron microscopic examination of haemopoietic liver tissue from mice infected in utero or when newborn showed inclusions of Colorado tick fever virus within erythroblasts, reticulocytes and erythrocytes. Inclusions were also seen within erythroblastoid cells undergoing mitosis. Other evidence of virus replication within erythropoietic cells was the presence of intracytoplasmic and intranuclear fibres, which have been shown to be associated with Colorado tick fever virus replication. The findings reported here support the hypothesis that virus replication within infected erythropoietic cells occurs concurrently with differentiation of the infected cell, resulting in the presence of virions within erythrocytes. (+info)