Rotavirus genome segment 7 (NSP3) is a determinant of extraintestinal spread in the neonatal mouse

Abstract

We used the neonatal mouse model of rotavirus infection to study extraintestinal spread following oral inoculation. Five-day-old pups were inoculated with either SA11-Cl3, SA11-Cl4, SA11-4F, RRV, or B223. By using virus detection in the liver as a proxy determination for extraintestinal spread, rotavirus strains capable of extraintestinal spread at high frequency (rhesus rotavirus [RRV]) and very low frequency (SA11-Cl4) were identified. Both strains productively infected the gastrointestinal tract. Oral inoculation of mice with RRV/ SA11-Cl4 reassortants and determination of virus titers in the gut and liver revealed that the extraintestinal spread phenotype segregated with RRV genome segment 7 to a high level of significance (P = 10(-3)). RRV segment 7 also segregated with the growth of virus in the gut (P = 10(-5)). Although infection of the gut was clearly required for tropism to the liver, there was no correlation between virus titers in the gut and detection of virus in the liver. Five days after intraperitoneal administration to bypass the gut barrier to virus spread, RRV and SA11-Cl4 both were recovered in the liver. However, only RRV was found in the liver following subcutaneous inoculation, suggesting that this peripheral site presented a similar barrier to virus spread as the gut. Sequence analysis of segment 7 from parental RRV and SA11-Cl4 and selected reassortants showed that (i) amino acid differences were distributed throughout the coding sequences and not concentrated in any particular functional motif and (ii) parental sequence was preserved in reassortants. These data support the hypothesis that NSP3, coded for by genome segment 7, plays a significant role in viral growth in the gut and spread to peripheral sites. The mechanism of NSP3-mediated tropism is under investigation.

FIG. 1.

Alignment of the predicted amino acid sequences for RRV and SA11-Cl4 NSP3 (GenBank accession no. AY065842 and AY065843, respectively). Amino acid identity is noted by vertical lines, and similarity is noted by dots. Dashes indicate gaps in the sequence. Lines beneath the alignment denote NSP3 functional and structural domains: solid line (amino acids [aa] 4 to 149), RNA binding; dotted line (aa 150 to 206), dimerization; dashed line (aa 206 to 315), eIF4G1 binding; and double line (aa 160 to 250), coiled-coil domain. Previously published NSP3 sequences are shown for comparison (GenBank accession numbers: RRV, X81426 [33]; SA11-4F, M87502 [21]; SA11, X00355 [2]). Residues in lowercase differ from those in the sequences observed in this study. NSP3 sequences determined in this study are indicated by asterisks.