Robert F. Ramig

Pathogenesis of Intestinal and Systemic Rotavirus Infection

Rotaviruses are responsible for significant gastrointestinal disease, primarily in children <5 years of age and the young of other mammalian species. Each year rotaviruses cause approximately 111 million episodes of gastroenteritis in children, which result in 25 million visits to clinics, 2 million

Three-dimensional visualization of the rotavirus hemagglutinin structure

Abstract Three-dimensional structures of a native simian and reassortant rotavirus have been determined by electron cryomicroscopy and computer image processing. The structural features of the native virus confirm that the hemagglutinin spike is a dimer of VP4, substantiated by in vivo radiolabeling studies. Exchange of native VP4 with a bovine strain equivalent results in a poorly infectious reassortant. No VP4 spikes are detected in the three-dimensional reconstruction of the reassortant. The difference map between the two structures reveals a novel large globular domain of VP4 buried within the virion that interacts extensively with the intermediate shell protein, VP6. Our results suggest that assembly of VP4 precedes that of VP7, the major outer shell protein, and that VP4 may play an important role in the receptor recognition and budding process through the rough endoplasmic reticulum during virus maturation.

Identification of the gene coding for the hemagglutinin of reovirus

Abstract A genetic approach has been used to identify the gene which codes for the hemagglutinin of reovirus. Hemagglutination by reovirus serotypes is type specific; type 1 hemagglutinates human but not bovine erythrocytes and type 3 hemagglutinates bovine but not human erythrocytes. Using recombinants derived from mixed infections of reovirus types 1 and 3, we have determined that the hemagglutinating properties of reovirus are a function of the σ1 outer capsid polypeptide, the polypeptide encoded in the S1 dsRNA segment of the virus.

Trypsin Cleavage Stabilizes the Rotavirus VP4 Spike

ABSTRACT Trypsin enhances rotavirus infectivity by an unknown mechanism. To examine the structural basis of trypsin-enhanced infectivity in rotaviruses, SA11 4F triple-layered particles (TLPs) grown in the absence (nontrypsinized rotavirus [NTR]) or presence (trypsinized rotavirus [TR]) of trypsin were characterized to determine the structure, the protein composition, and the infectivity of the particles before and after trypsin treatment. As expected, VP4 was not cleaved in NTR particles and was cleaved into VP5∗ and VP8∗ in TR particles. However, surprisingly, while the VP4 spikes were clearly visible and well ordered in the electron cryomicroscopy reconstructions of TR TLPs, they were totally absent in the reconstructions of NTR TLPs. Biochemical analysis with radiolabeled particles indicated that the stoichiometry of the VP4 in NTR particles was the same as that in TR particles and that the VP8∗ portion of NTR, but not TR, particles is susceptible to further proteolysis by trypsin. Taken together, these structural and biochemical data show that the VP4 spikes in the NTR TLPs are icosahedrally disordered and that they are conformationally different. Structural studies on the NTR TLPs after trypsin treatment showed that spike structure could be partially recovered. Following additional trypsin treatment, infectivity was enhanced for both NTR and TR particles, but the infectivity of NTR remained 2 logs lower than that of TR particles. Increased infectivity in these particles corresponded to additional cleavages in VP5∗, at amino acids 259, 583, and putatively 467, which are conserved in all P serotypes of human and animal group A rotaviruses and also corresponded with a structural change in VP7. These biochemical and structural results show that trypsin cleavage imparts order to VP4 spikes on de novo synthesized virus particles, and these ordered spikes make virus entry into cells more efficient.

The effects of host age, virus dose, and virus strain on heterologous rotavirus infection of suckling mice

Seven-day-old suckling CD-1 mice, born to seronegative dams, were orally inoculated with a number of animal and human rotaviruses. Simian (SA11), rhesus (RRV), and bovine (B223) rotaviruses were found to replicate and cause severe disease. Canine (K9), bovine (B641), and human (Wa) rotaviruses either replicated minimally and caused minimal disease (K9, B641) or failed to replicate or cause disease (Wa). The features of SA11 infection of mice were examined in greater detail. Suckling mice were susceptible to infection and disease from 1 day of age to 13-15 days of age. Restriction of disease occurred at an earlier age (13 days) than restriction of replication (15 days). Dose-response studies in seven-day-old mice showed that virus replication and disease could be induced with doses as low as 1 x 10(2) pfu/mouse; however, both intestinal virus titers and severity of disease increased in parallel with virus dose. Intestinal virus replication appeared to be restricted in SA11 infections. Only at very low doses (1 x 10(2) pfu/mouse) did virus replication occur to levels above the inoculated dose. While light microscopic examination showed classical features of rotavirus infection in the ileum, electron microscopic examination revealed only the accumulation of large numbers of electron-lucent vacuoles in the ileal enterocytes of infected mice. Structures typical of rotavirus morphogenesis were not detected in enterocytes from mice infected with rotavirus SA11.

Genetics of reovirus: Identification of the ds RNA segments encoding the polypeptides of the μ and σ size classes

Abstract Recombinants derived from crosses between reovirus serotypes 1, 2, and 3 were utilized to identify the ds RNA segments encoding polypeptides of the μ and σ size classes. The results indicate the following assignments: M1 ds RNA encodes polypeptide μ2, M2 encodes μ1, M3 encodes μNS, S1 encodes σ1, S2 encodes σ2, S3 encodes σNS, and S4 encodes σ3. In addition polypeptide species μ2 has been conclusively identified as a structural component of the reovirus core. A new nomenclature for the viral polypeptides is discussed.

A genetic map of reovirus I. Correlation of genome RNAs between serotypes 1, 2, and 3

Abstract The double-stranded genome RNAs of recombinants between reovirus serotypes 1, 2, and 3 were examined by polyacrylamide-gel electrophoresis. Analysis of deletions and replacements in the recombinants allowed construction of a map of the serotypes correlating genome segments providing functions interchangeable between the serotypes. The relative migration rates of segments M1 and M2 of type 3 are reversed between the traditional Tris-acetate-buffered gel system and the Tris-glycine gel system used here. In the Tris-glycine system, the genome segments of serotype 1 correspond to type 3 in order of increasing electrophoretic mobility except for S3 and S4 which are reversed. In serotype 2 all segments except M1 and M2 and S3 and S4 correspond in order of increasing electrophoretic mobility. The migration of these two segment pairs is reversed in type 2 relative to type 3. A map is presented correlating the migration of genome segments of types 1, 2, and 3 in both the Tris-glycine- and the Tris-acetate-buffered systems. The nomenclature of the genome segments is standardized to that which appears in the literature. In addition, these data demonstrate that recombinants arise by physical reassortment of genome segments between parents.

A genetic map of reovirus. II. Assignment of the double-stranded RNA-negative mutant groups C, D, and E to genome segments.

Abstract The double-stranded RNA (dsRNA) of recombinants derived from crosses of the dsRNA-negative, temperature-sensitve (ts) mutants of reovirus type 3 and reovirus serotypes 1 or 2 were examined by polyacrylamide gel electrophoresis. Analysis of deletions and replacements in the recombinants allowed identification of genome segments containing the ts lesions. In this way the location of the mutation of the group C prototype mutant tsC(447) os genome segment S2, that of the group D prototype mutant tsD(357) is genome segment L1, and that of the group E prototype mutant tsE(320) is genome segment S3. In addition the location of the temperature-sensitive lesion of serotype 2 is genome segment S1.

A Lymphatic Mechanism of Rotavirus Extraintestinal Spread in the Neonatal Mouse

ABSTRACT We used the neonatal mouse model of rotavirus infection and virus strains SA11-clone 4 (SA11-Cl4) and Rhesus rotavirus (RRV) to examine the mechanism of the extraintestinal spread of viruses following oral inoculation. The spread-competent viruses, RRV and reassortant R7, demonstrated a temporal progression from the intestine, to the terminal ileum, to the mesenteric lymph nodes (MLN), and to the peripheral tissues. SA11-Cl4 was not found outside the intestine. Reassortant virus S7, which was unable to reach the liver in previous studies (E. C. Mossel and R. F. Ramig, J. Virol. 76:6502-6509, 2002), was recovered from 60% of the MLN, suggesting that there are multiple determinants for the spread of virus from the intestine to the MLN. Phenotypic segregation analysis identified RRV genome segment 6 (VP6) as a secondary determinant of the spread of virus to the MLN (P = 0.02) in reassortant viruses containing segment 7 from the spread-incompetent parent. These data suggest that in the orally infected neonatal mouse, the extraintestinal spread of rotavirus occurs via a lymphatic pathway, and the spread phenotype is primarily determined by NSP3 and can be modified by VP6.

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.