Combination of RT-PCR and proteomics for the identification of Crimean-Congo hemorrhagic fever virus in ticks

Abstract

Crimean-Congo hemorrhagic fever (CCHF) is an emerging tick-borne zoonotic disease caused by the CCHF virus (CCHFV). In this study, an experimental approach combining RT-PCR and proteomics was used for the identification and characterization of CCHFV in 106 ticks from 7 species that were collected from small ruminants in Greece. The methodological approach included an initial screening for CCHFV by RT-PCR followed by proteomics analysis of positive and control negative tick samples. This novel approach allowed the identification of CCHFV-positive ticks and provided additional information to corroborate the RT-PCR findings using a different approach. Two ticks, Dermacentor marginatus and Haemaphysalis parva collected from a goat and a sheep, respectively were positive for CCHFV. The sequences for CCHFV RNA segments S and L were characterized by RT-PCR and proteomics analysis of tick samples, respectively. These results showed the possibility of combining analyses at the RNA and protein levels using RT-PCR and proteomics for the characterization of CCHFV in ticks. The results supported that the CCHFV identified in ticks are genetic variants of the AP92 strain. Although the AP92-like strains probably do not represent a high risk of CCHF to the population, the circulation of genetically diverse CCHFV strains could potentially result in the appearance of novel viral genotypes with increased pathogenicity and fitness.

Detection of new Crimean–Congo haemorrhagic fever virus genotypes in ticks feeding on deer and wild boar, Spain

“…Tick RNA was analysed by a nested RT‐PCR targeting the CCHFV S segment as previously described (Fernández de Mera et al., 2017; Midilli et al., 2009) using the Access RT‐PCR System (Promega Corporation) and primers Eecf F1/Eecf R1 and Eecf F2/Eecf R2. The nested RT‐PCR conditions included a first step for retrotranscription of 45 min at 45°C.…”

Diagnostic tests for Crimean-Congo haemorrhagic fever: a widespread tickborne disease

“…There are many inhouse laboratory tests, some with primers designed for regional circulating strains (table 2). 53 59 85 86 Several commercial RT-PCR kits are available, typically with primers designed to target broad consensus sequences within the S segment, including several European Commission marked (CE) diagnostics and research use-only (RUO)-labelled products in stand-alone and multiplex test formats (online supplementary table S1).…”

“…Seroconversion in animals is a good indicator of CCHFV prevalence; when domestic animals in Turkey and Bulgaria were tested for CCHFV-specific IgG antibodies, the mean seroprevalence was 26% for Bulgaria and 57% for Turkey, with some provinces reporting seroprevalence of almost 90% 94. In both rural and urban settings, similar ‘random sampling’ surveillance programmes have been employed for ticks85 95–97 and other ruminants 98 99. However, routine reservoir/host monitoring is not broadly implemented, and surveillance is challenged by a lack of serodiagnostic tests suitable for large-scale animal testing,100 no clear guidance for standardised surveillance of CCHFV in the animal health sector, and the cost of routine implementation 6.…”

Proteomics analysis reveals heat shock proteins involved in caprine parainfluenza virus type 3 infection

“…This technique has been widely used to investigate proteome changes in cow, yak, buffalo, goat and camel milk [9], and peste des petits ruminants virus (PPRV)-infected Vero cells [10], based on the isobaric tags for relative and absolute quantification (iTRAQ) method. In addition, this technique has also been widely employed to examine the mechanisms of viral infection through comparative investigation of the proteome changes, for example, in the case of Crimean-Congo hemorrhagic fever virus (CCHFV) [11] and bovine respiratory syncytial viruses (BRSV) [12].…”