Identification of Group A Rotavirus Genes Associated with Virulence of a Porcine Rotavirus and Host Range Restriction of a Human Rotavirus in the Gnotobiotic Piglet Model

Abstract

Rotaviruses are the leading cause of severe diarrhea in infants and young children worldwide. Thus, the development of an effective rotavirus vaccine is a major public health goal. This study was performed to identify the gene or genes responsible for rotavirus virulence or host range restriction and attenuation in a natural host. Such knowledge could have an important bearing on the selection of candidate live vaccine strains. We addressed this issue by analyzing the response of gnotobiotic piglets to orally administered porcine x human rotavirus reassortants. It was possible to determine (i) which porcine rotavirus genes were required for the induction of diarrhea, and (ii) which human rotavirus genes are associated with the host range restriction because the parental porcine rotavirus (SB-1A strain) caused diarrhea in piglets, whereas the parental human rotavirus (DS-1 strain) was attenuated in piglets. Substitution of the 3rd (VP3) or 4th (VP4) or 9th (VP7) or 10th (NS28 (NSP4)) gene of the avirulent human strain for the corresponding gene of porcine rotavirus that was virulent for gnotobiotic piglets yielded viral reassortants that failed to induce diarrhea. Further analysis indicated that reassortants which possessed only one, two, or three of these porcine rotavirus genes on a background of human rotavirus genes also failed to induce diarrhea. However, diarrhea was induced when all four of these porcine rotavirus genes were included in a reassortant in which the remaining seven genes were derived from the human rotavirus. These observations suggest that it may be possible to attenuate wild-type human rotavirus strains that are virulent for humans by selective genetic reassortment with an animal rotavirus strain that is attenuated for humans.

Molecular characterization of human rotavirus vaccine strain CDC-9 during sequential passages in vero cells

“…Since CDC-9 demonstrated robust growth in Vero cells, as evidenced by high titer and efficient production of triple-layered particles, this strain is being evaluated as a candidate for an inactivated rotavirus vaccine or a live oral vaccine that could be developed by manufacturers in resource-rich or emerging developing countries. Despite VP3 and three other genes have been linked to the virulence and pathogenicity of rotavirus, 47 the molecular mechanisms for attenuation have not been clearly defined. Our results demonstrated 8 aa changes in several genes, including VP4, of CDC-9 during the passages in cells.…”

A Lack of Consistent Amino Acid Substitutions in NSP4 between Rotaviruses Derived from Diarrheal and Asymptomatically‐Infected Kittens

“…Thus, with regard to the amino acid identities, these NSP4s were closely related to each other and were not grouped according to the different clinical categories from which each strain originated. The feline rotavirus strains used in this study were also grouped into either G3P5 [3] strains or G3P3 [9] strains (Table 1). Irrespective of these serotypic groupings, however, the NSP4 sequences were homogeneous (Table 2).…”

“…Strains are grouped and boxed according to the symptom (diarrheal or asymptomatic) of the cat from which each strain was isolated. Sequence identities between every pair of G3P3 [9] strains are slightly shaded, whereas sequence identities between G3P5 [3] strains are more densely shaded.…”

“…By calculating a variability index at each amino acid position of the NSP4 protein, we have previously shown that sequence variation increases immediately downstream from the toxic peptide region and remains high toward the carboxy-terminus of the NSP4, with the greatest sequence variation occurring between residues 135 and 141 (7). In this regard, it deserves mention that all P3 [9] strains possess Ile-Lys-Pro at amino acid residues 136-168, whereas all P5 [3] strains possess Ala-Thr-Ser at these residues (Fig. 1).…”

“…Indeed, pathogenicity and virulence are complex characteristics (5), and evidence is available in the literature that several gene segments are involved in these characteristics. For example, in the gnotobiotic piglet model, VP3, VP4, VP7, and NSP4 were all shown to be associated with diarrhea induction by single-gene substitution reassortants between a virulent porcine rotavirus strain (SB-l A) and a human rotavirus strain (DS-l) that is not pathogenic for pigs (9). Thus, involvement of other genes may explain the outcome of infection in cats from which these 10 feline rotaviruses were isolated.…”