Development of specific nucleic acid probes for the differentiation of porcine rotavirus serotypes

Abstract

A dot blot hybridization assay is described for the detection and differentiation of porcine rotavirus serotypes. Recombinant complementary DNA (cDNA) representing gene 9 (the gene encoding the neutralization antigens in VP7 glycoprotein) from OSU (porcine rotavirus serotype 1) and Gottfried (porcine rotavirus serotype 2) strains were used to determine the optimal hybridization conditions which allow specific detection of group A porcine rotaviruses. Probes were prepared by excision of the inserts from the recombinant plasmids and radiolabeling of cDNA with 32P by the random primer extension method. Probes were hybridized at various stringencies with viral RNA from different rotavirus serotypes bound to nylon membranes. Hybridization at low stringency (26% base pair mismatch for stable hybrid formation) had high sensitivity but low specificity. Hybridization at high stringency (16% base pair mismatch for stable hybrid formation) produced high specificity but decreased the sensitivity observed at low stringency. Probes were specific for rotavirus at both stringencies and did not hybridize with nucleic acids from other porcine viruses. Subgenomic gene 9 fragments were then tested to provide more specific probes. A 322 bp fragment from OSU gene 9 between nucleotides 382 and 704 and a 266 bp fragment from Gottfried gene 9 between nucleotides 230 and 496 were found to be specific as hybridization probes. These studies demonstrated the feasibility of the dot blot hybridization assay using subgenomic fragments of gene 9 to detect and differentiate serotypes of porcine rotavirus. Additional studies are warranted to further evaluate the sensitivity and the capability of these probes to detect porcine field isolates of the same serotype.

Applications of nucleic acid probes in veterinary infectious diseases

“…Nucleic acid probes have been used to detect viruses, bacteria, and parasites in clinical samples. Among viruses of veterinary importance detected by nucleic acid probes are adenovirus (Jouvenne et al, 1987 ), African swine fever virus (Tabares, 1987 ), blue tongue virus (Dangler et al, 1988 ), bovine viral diarrhea virus (Brock and Potgieter, 1990), enterovirus (Bruce et al, 1989), infectious bursal disease virus (Jackwood, 1990), foot-and-mouth disease virus (Rossi et al, 1988), porcine parvovirus (Ridpath et al, 1987;Harding and Molitor, 1988;Oraveerakul et al, 1989), pseudorabies virus (Gutekunst, 1979;Maes et al, 1990), rhinovirus (Gama et al, 1988), rotavirus (Dimitroy et al, 1985;Johnson et al, 1990;Rosen et al, 1990), and transmissible gastroenteritis virus (Shockley et al, 1987). Bacteria for which probes have been developed include Campylobacter spp.…”

“…(Zuerner and Bolin, 1990), Mycobacterium spp. (McFadden et al, 1988), and rotavirus groups, subgroups and serotypes (Dimitrov et al, 1985;Johnson et al, 1990;Rosen et al, 1990).…”

Genetic analysis of NSP1 genes of human rotaviruses isolated from neonates with asymptomatic infection

“…These conditions allowed hybridization with up to 17% mismatch (Bodkin & Knudson, 1985;Johnson et al, 1990). Probe DNA was prepared from cloned M37 gene 5 labelled with digoxigenin (DIG)-11-dUTP (Boehringer Mannheim) by random priming.…”

Expression and characterization of bovine rotavirus proteins in the baculovirus system

“…Rotavirus types based on VP4 have been designated as P types, whereas types based on VP7 have been referred to as G types. Monoclonal antibodies and nucleic acid probes have been successfully used to differentiate rotavirus G and P types (Johnson et al, 1990;Zaberezhny et al, 1991). A summary of rotaviruses with the major G and P types detected in animals is presented in Table 3.…”