Serologic and molecular diagnosis of Colorado tick fever viral infections.

Abstract

Abstract. 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) virusinfected 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.Colorado tick fever (CTF) is a severe tick-borne disease that is usually self-limited. This disease occurs in 11 western states in the United States and has also been reported in Canada. 1 Yearly, 200-400 cases are reported throughout the endemic states in the United States. 1,2 The clinical manifestations appear following the bite of the infected adult Rocky Mountain wood tick Dermacentor andersoni 3 and were first reported in the Rocky Mountain region. 1 It has an abrupt onset with flu-like manifestations and some complications of the disease have been reported, particularly in children. [4][5][6][7] Since the first reports in 1875 to reports of Keiffer and Becker in 1906 and 1926, respectively, 8 on a tick bite-generated biphasic fever having clinical symptoms that does not conform to those of Rocky Mountain spotted fever (RMSF), CTF was recognized as being a separate clinical entity from RMSF. Furthermore, the isolation and characterization of the causative agent, CTF virus, by Florio and others 9 in the 1940s established the viral etiology of the disease. In 1990, the genus Coltivirus was created within the family Reoviridae, encompassing those viruses isolated from vertebrates and invertebrates exhibiting a 12-segment double-stranded (ds) RNA genome. At present, this genus includes the CTF virus, the Eyach virus isolated in Germany in 1976, 10 the two Eyach antigenic variants Ar/T577 and Ar/T578 that were isolated in France in 1981 11 , and other members isolated in Indonesia 12 and China. 13The Eyach virus has been suspected to be involved in some neurologic manifestations since antibodies to this virus were detected in sera of patients with polyradiculonevritis. 14 The diagnosis of CTF virus infection is made using various procedures. Isolation and characterization by animal inoculation and neutralization were reported to be the most reliable techniques. 8,15 However, serologic detection by ELI-SA has also been developed. 15 Moreover, many strains were reported 16, 17 that could be detected by RN...

Comparative sequence analysis of American, European and Asian isolates of viruses in the genus Coltivirus.

“…Primers designed from the sequences of segments 9-12 of the Florio strain (Attoui et al, 1997(Attoui et al, , 1998 of CTF virus permitted PCR amplification of these segments from the R-1575, 69V28 and S6-14-03 viruses. The amplified sequences had the same lengths as those of the Florio strain and exhibited high degrees of nucleic acid and deduced protein identities.…”

“…The extracted viral dsRNAs from CTF strains R-1575 and 69V28, Eyach, AR578 and S6-14-03 viruses were resuspended in diethylpyrocarbonate-treated water. Reverse transcription was carried out as previously described in the presence of random hexanucleotides and the MMuLV Superscript CEIC reverse transcriptase (Attoui et al, 1998). PCR primers were designed from the sequences genomic segments 9-12 of the Florio strain (N-7180) of CTF virus with the help of the Oligo software program (National Biosciences).…”

“…Primer sequences are shown in Table 1. The PCR protocol using segment-specific primers is described elsewhere (Attoui et al, 1998). Briefly, the reaction was carried out in a volume of 100 µl and the amplification mixture included : 10 mM Tris-HCl (pH 8n8), 50 mM KCl, 1n5 mM MgCl # , 0n1 % Triton X-100, 0n2 mM of each dNTP, 20 µl cDNA solution, 1 µM of each primer and 2n5 U Taq DNA polymerase.…”

The Biological Survey of Canada: A Personal History

“…Infection by this virus has been diagnosed by a variety of serological procedures including complement fixation, indirect immunofluoresence, IgM capture enzyme-linked immunosorbent assays (ELISAs), and plaque reduction neutralisation (Attoui et al 1998). Reverse transcription PCR has been used to detect viral RNA in acute samples of blood collected within five days of illness (Lambert et al 2007).…”

Recombinant VP6-based enzyme-linked immunosorbent assay for detection of immunoglobulin G antibodies to Eyach virus (genus Coltivirus)

“…This protein was highly reactive to human sera infected with CTFV and to a mouse hyper-immune ascitic fluid to CTFV. The ELISA test based on this protein (Mohd Jaafar et al, 2003) was validated using a panel of human sera, some of which were earlier found positive for CTFV antibodies by complement fixation, detection of IgG and IgM and virus neutralisation (Calisher et al, 1985;Attoui et al, 1998). Hence, it was logical to choose the homologous protein of EYAV (VP6) as a target for selectively detecting antibodies to EYAV.…”