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.

Serologic and molecular diagnosis of Colorado tick fever viral infections.

Molecular and serologic methods usable for the biological diagnosis of Coltivirus infection are reported. We designed a multiplex reverse transcription-polymerase chain reaction system that allowed the simultaneous and specific amplification of three genomic segments from as little as 0.01 plaque-forming units. Another system in the S2 viral segment permitted the differential diagnosis of American and European viral isolates. We also discuss some improvements of previous ELISAs, and the results obtained with paired sera from Colorado tick fever (CTF) virus-infected individuals. Western blot analysis was developed that allowed the detection of antibodies to a 38-kD viral protein in all tested sera. It also enabled the detection of anti-CTF virus antibodies in ELISA-negative sera. Specific IgM antibodies against a synthetic viral peptide could be detected in sera at the acute stage of the infection. Together, these results should permit the diagnosis of Coltivirus infection at any stage of the pathology.

Experimental Colorado tick fever virus infection in Colorado mammals.

One hundred and twenty-four small mammals of six species were inoculated with three strains of Colorado tick fever (CTF) virus to define viremia and neutralizing (N) antibody responses. Adult Eutamias minimus and Eutamias embrinus, and juvenile Peromyscus maniculatus and Spermophilus lateralis, were highly susceptible to development of viremic infection. Adult S. lateralis and P. maniculatus were moderately susceptible (greater than or equal to 50% viremic). Five Sylvilagus nuttalli did not become viremic following experimental inoculation. Spermophilus richardsoni was also relatively resistant (less than or equal to 50% viremic). The longest duration of viremia (mean 15.8 days) and highest peak viremia levels (mean peak titer 10(3.9 plaque-forming units per ml) occurred in E. minimus. Adult E. umbrinus, juvenile S. lateralis, and juvenile P. maniculatus had moderate viremias. Adult S. lateralis and S. richardsoni often had short viremias during which virus was only intermittently detectable. N antibody production was most rapid in E. minimus in comparison with other species. In addition, N antibody persisted for 1 year in this species. In other species, many animals lost detectable antibody 5-11 months after infection. No significant differences were found in patterns of infection between three CTF virus strains. We conclude that of the six species inoculated, E. minimus is the best experimental host for CTF virus.

Immunoperoxidase staining for detection of Colorado tick fever virus, and a study of congenital infection in the mouse.

Indirect immunoperoxidase (IP) staining was evaluated for sensitivity and specificity in detecting Colorado tick fever (CTF) virus antigen in infected cell cultures and infected mouse tissues, and then was applied to a study of congenital CTF infection in mice. The sensitivity of IP staining was comparable to that of immunofluorescence staining in detecting CTF antigen in infected cell cultures. Endogenous peroxidase activity of mouse tissues caused nonspecific reactivity in the IP system, but this could be abolished by treatment with sodium azide and hydrogen peroxide without destroying CTF antigen. Offspring of mice infected with CTF virus during the 2nd week of pregnancy showed a highly significant increase in the incidence of stillbirths and neonatal deaths as compared with offspring of uninfected controls. CTF antigen or virus was demonstrable in only a low proportion (7%) of embryos, ill newborns or stillborns examined, but a high proportion of mice examined at a time when maternal antibody would be lost (6 and 12 weeks) showed CTF antibody, indicating a higher incidence of infection. IP staining showed potential for use in studies of viral pathogenesis in the mouse model.

Tick-borne diseases in the United States: Rocky Mountain spotted fever and Colorado tick fever. A review.

The historical, clinical, ecological, and epidemiological features of Rocky Mountain spotted fever and Colorado tick fever, the two important tick-borne diseases in the United States, are reviewed. Rocky Mountain spotted fever, once considered a disease of the past, has again become a measurable public health problem. Its nationwide incidence has steadily increased since 1960 and has reached record proportions in 1976. The various factors responsible for this trend as well as for the mortality rates, which in spite of availability of effective antibiotics ranges from 5 to 10%, are discussed. Education of the public about ticks and their potential role as vectors of Rickettsia rickettsii and/or Colorado tick fever virus, and about the clinical manifestations of Rocky Mountain spotted fever, is considered the best means for preventing high incidence and mortality from these diseases.

Arbovirus infections in man in British Columbia.

During the summer of 1971, the first laboratory-proved cases of acute encephalitis in man due to any of the known arboviruses occurred in the south-central region of British Columbia. Five human cases of encephalitis with two deaths were diagnosed; three of these patients, including one of the fatalities, were proven in the laboratory to have contracted western equine encephalitis.During 1968 and 1969, a human serum survey undertaken in approximately 2000 life-long residents of the province discovered low levels of hemagglutinin-inhibiting and/or complement-fixing as well as neutralizing antibodies for western equine encephalitis, St. Louis encephalitis, Powassan encephalitis, California encephalitis and Colorado tick fever. Evidence of recent sub-clinical infection was detected in some cases.