Young S. Lyoo

Immunogens of rotaviruses

Rotaviruses cause gastroenteritis in neonates of many animal species including cattle, swine, horses, dogs, cats, chickens and turkeys. Rotavirions are nonenveloped, are about 75 nm in diameter, have a double capsid, and contain 11 double-stranded RNA segments as their genome. Several antigenically distinct groups of rotaviruses have been identified and have been alphabetically designated as A through G. Group A rotaviruses were the first group of rotaviruses isolated and are the most commonly detected rotaviruses in diarrheic animals. Group A rotaviruses have two surface proteins, VP4 and VP7, both of which are important in serotype determination and in inducing neutralizing antibodies and protective immunity. Multiple serotypes of group A rotavirus based on glycoprotein VP7 (designated as G types) and based on VP4 (P types) have been identified. The immune response to rotaviruses is essentially serotype specific, however, cross-reactive or heterotypic epitopes have also been identified. Currently acceptable methods for immunogen quantitation include the induction of neutralizing antibody in host or laboratory animals. The in vivo efficacy of vaccines against rotavirus-associated gastroenteritis remains the standard method against which in vitro methods must be compared. Several animal models have been developed which could potentially be used in evaluating the efficacy of candidate vaccines. Monoclonal antibodies to rotavirus immunogens are also currently available and serve as valuable reagents for in vitro quantitation of rotaviral immunogens.

High-resolution chromatography of nucleic acids on the gen-pak fax column

High-performance liquid chromatography (HPLC) on a Gen-Pak FAX column has been used to separate and purify microgram amounts of single- and double-stranded DNA and RNA molecules. HPLC of mixtures of DNA restriction fragments showed that fragments within the size range 0.125-23.1 kilobase were easily resolved. Supercoiled (form I) plasmid DNA molecules were readily separated from single-stranded circular DNA of the same length and from various DNA conformational isomers including nicked (form II) and linear (form III) species. Topological isomers generated from supercoiled plasmid DNA molecules by DNA topoisomerase I exhibited different retention times than supercoiled molecules. Supercoiled (form I) DNA molecules were resolved from fully relaxed (form IV) molecules. Synthetic oligonucleotides of 74 and 128 nucleotides in length were separated from failure sequences, as well as from other contaminating synthesis products. Single-stranded circular M13mp18 DNA molecules sufficiently pure for use in automated DNA sequencing systems were prepared by HPLC on a Gen-Pak FAX column. HPLC was also used to fractionate linear double-stranded porcine rotavirus genomic RNA fragments into size classes between 0.3 and 3 kilobase. Finally, HPLC of unfractionated Escherichia coli tRNA molecules resolved multiple species. In all cases, HPLC on Gen-Pak FAX was carried out in phosphate or Tris buffers at neutral pH in the presence of sodium chloride. Columns were not damaged by repeated exposure to impure samples, provided they were cleaned frequently with sodium hydroxide and acetic acid. Although procedures for resolution of the various size ranges for each class of DNA and RNA molecules require further optimization, our preliminary data on the separations obtained, the moderate salt concentrations employed, and the durability of the matrix suggest that this column merits further study.

Prevalence of P types among porcine rotaviruses using subgenomic VP4 gene probes

Nucleic acid probes were developed to differentiate VP4 (P) types among porcine rotaviruses. These probes were then used to determine the relative prevalence of P types 6 (Gottfried-like) and 7 (OSU-like) in cultivated rotaviruses and field specimens. The variable regions between bases 205-551 of the VP4 gene of rotavirus strains OSU and Gottfried were amplified by the polymerase chain reaction and radiolabeled with 32P by random primer extension. Radiolabeled probes were tested in a dot blot hybridization assay. The subgenomic probes prepared from VP4 gene detected as little as 5 ng oF rotavirus RNA and were specific for differentiating the two porcine rotavirus P types. The probes were used to determine the P type of several reference rotavirus strains and recently cultivated porcine rotavirus strains. Eight of the 10 cultivated, previously untyped, rotaviruses isolates tested were of P type 7 (OSU-like). Two rotavirus strains neither reacted with OSU nor with the Gottfried probe, therefore, their P type could not be determined. Seventeen out of the 26 rotavirus (65.4%) field samples tested had a P type 6 (Gottfried-like) whereas 5 out of 26 (19.2%) had a P type 7 (OSU-like). Four of the 26 samples (15.4%) reacted neither with the OSU nor with the Gottfried probe and possibly represent previously unrecognized P types in swine. Data in this study suggests that (1) rotaviruses with P type 7 are most common among the cultivated rotaviruses, (2) rotaviruses with P type 6 are the most abundant type of rotavirus in natural infections and (3) rotaviruses with additional P types are also associated with diarrheic swine.