A feline rotavirus G3P[9] carries traces of multiple reassortment events and resembles rare human G3P[9] rotaviruses.

The full-length genome sequence of a feline G3P[9] rotavirus (RV) strain, BA222, identified from the intestinal content of an adult cat, was determined. Strain BA222 possessed a G3-P[9]-I2-R2-C2-M2-A3-N1-T3-E2-H3 genomic constellation, differing substantially from other feline RVs. Phylogenetic analyses of each genome segment revealed common origins with selected animal and zoonotic human RVs, notably with rare multi-reassortant human G3P[9] RVs (Ita/PAI58/96 and Ita/PAH136/96). Altogether, the findings suggest that feline RVs are genetically diverse and that human RVs may occasionally originate either directly or indirectly (via reassortment) from feline RVs.

Interactions between rotavirus and gastrointestinal cells.

Rotaviruses are the leading cause of life-threatening diarrheal disease in infants and in young animals worldwide. The outcome of rotavirus infection of intestinal epithelial cells is more complex and involves induction of more diverse cellular responses than initially appreciated. Similar to bacteria, the pathogenesis of rotavirus-induced disease involves an enterotoxin, activation of the enteric nervous system and malabsorption, suggesting that common mechanisms of pathogenesis may exist between viral and bacterial pathogens.

Lapine rotaviruses of the genotype P[22] are widespread in Italian rabbitries.

An epidemiological survey was carried out to investigate the distribution of the VP7 and VP4 specificities of lapine rotaviruses (LRVs) in rabbitries from different geographical regions of Italy. Almost all the strains were characterized as P[22],G3, confirming the presence of the newly-recognized rotavirus P[22] VP4 allele in Italian rabbits. Only one P[14],G3 LRV strain was identified and two samples contained a mixed (P[14] + [22],G3) rotavirus infection. All the LRV strains analyzed exhibited a genogroup I VP6 specificity and a long dsRNA electropherotype. However, one of the P[14],G3 strains possessed a super-short pattern. Altogether, these data highlight the epidemiological relevance of the P[22] LRVs in Italian rabbitries.

Pathogenesis of rotavirus gastroenteritis.

The outcome of intestinal infection with rotaviruses is more complex than initially appreciated, and it is affected by a complex interplay of host and viral factors. Rotaviruses infect intestinal enterocytes, and the early events in infection are mediated by virus-epithelial cell interactions. Diarrhoea may be caused by several mechanisms including (i) malabsorption that occurs secondary to the destruction of enterocytes, (ii) villus ischaemia and activation of the enteric nervous system that may be evoked by release of a vasoactive agent from infected epithelial cells in the absence of significant pathologic lesions or enterocyte damage, and (iii) intestinal secretion stimulated by the intracellular or extracellular action of the rotavirus non-structural protein, NSP4, a novel enterotoxin and secretory agonist with pleiotropic properties. New studies of rotavirus infection of polarized intestinal epithelial cells show that rotaviruses infect cells differently depending on whether or not they require sialic acid for initial binding, and infection alters epithelial cell functions. NSP4 also affects epithelial cell function and interactions. NSP4 (i) induces an age- and dose-dependent diarrhoeal response in young rodents that is similar to virus-induced disease, (ii) stimulates a Ca(2+)-dependent cell permeability where the secretory response is age-dependent, and (iii) alters epithelial cell integrity. Antibody to NSP4 protects mouse pups from diarrhoea induced by homotypic and heterotypic viruses. These data support a new mechanism of rotavirus-induced diarrhoea whereby a viral enterotoxin triggers a signal transduction pathway that alters epithelial cell permeability and chloride secretion. This new information about how a gastrointestinal virus causes disease demonstrates common pathogenic mechanisms for viral and bacterial pathogens not previously appreciated. These results also suggest new approaches to prevent or treat rotavirus-induced diarrhoea.

Evaluation of rotavirus vaccines in small animal models.

The high morbidity and mortality of rotavirus (RV) infections has spurred the development of RV vaccines (1-13). Although children naturally infected with RV commonly undergo multiple infections, primary infections in children generally induce disease, and children are normally protected against severe disease during subsequent infections (1,2,6-8,14). For RV, the immunologic mechanisms responsible for protection are poorly understood, but antibody (Ab) in the intestine appears to be the primary mechanism of protection (2,15,16). Because RV is a localized enteric infection, and induction of intestinal mucosal immune responses was expected to be required for protection, live orally administered vaccines were pursued first. Vaccine development of the live attenuated vaccines proceeded to clinical trials in humans without prior animal testing. In August 1998, Rotashield™, a three dose, live attenuated tetravalent (TV), rhesus rotavirus (RRV) vaccine produced by Wyeth Lederle Vaccines and Pediatrics (West Henrietta, NY), was licensed. This vaccine shows promise, eliciting ∼80% protection against severe disease (6,7,12,17-19). The recent detection or emergence of new RV serotypes in humans suggests that incorporation of additional P-and G-serotypes into this vaccine may be necessary in the future (20-22). Additional concerns with the use of live attenuated vaccines include interference of vaccine replication by other enteric pathogens (common in children from the underdeveloped world); neutralization by maternal Ab; limited replication competence of animal strains, because of the host range restriction observed with RVs; and safety, because of the possibility of producing new virulent virus, emerging by reassortment of circulating wild-type (WT) virus with the vaccine virus.Development and testing of nonreplicating RV vaccines have also been pursued, and will be the focus of this chapter. The use of nonreplicating immunogens presents additional challenges beyond that of developing RV vaccines effective in young children against potential infection by multiple serotypes of RV. The nonreplicating immunogen must be able to induce protective immune responses against the target virus. Traditionally, nonreplicating vaccines have been thought to be poor inducers of mucosal immune responses and protection of the mucosa. Without amplification of the vaccine virus by replication, a high dose of nonreplicating immunogen may be required. To enhance the immune response to nonreplicating immunogens, development and testing of new adjuvants and/or delivery systems, and alternative routes of immunization to boost immunogenicity and protective efficacy, are needed. If administered orally, the nonreplicating immunogens must be stable in the digestive environments of the stomach and intestine.

Identification of group A porcine rotavirus strains bearing a novel VP4 (P) Genotype in Italian swine herds.

The VP4 gene of a G5 Italian porcine rotavirus strain, 344/04-1, was nontypeable by PCR genotyping. The amino acid sequence of the full-length VP4 protein had low identity (

Identification of a novel VP4 genotype carried by a serotype G5 porcine rotavirus strain.

Rotavirus genome segment 4, encoding the spike outer capsid VP4 protein, of a porcine rotavirus (PoRV) strain, 134/04-15, identified in Italy was sequenced, and the predicted amino acid (aa) sequence was compared to those of all known VP4 (P) genotypes. The aa sequence of the full-length VP4 protein of the PoRV strain 134/04-15 showed aa identity values ranging from 59.7% (bovine strain KK3, P8[11]) to 86.09% (porcine strain A46, P[13]) with those of the remaining 25 P genotypes. Moreover, aa sequence analysis of the corresponding VP8* trypsin cleavage fragment revealed that the PoRV strain 134/04-15 shared low identity, ranging from 37.52% (bovine strain 993/83, P[17]) to 73.6% (porcine strain MDR-13, P[13]), with those of the remaining 25 P genotypes. Phylogenetic relationships showed that the VP4 of the PoRV strain 134/04-15 shares a common evolutionary origin with porcine P[13] and lapine P[22] rotavirus strains. Additional sequence analyses of the VP7, VP6, and NSP4 genes of the PoRV strain 134/04-15 revealed the highest VP7 aa identity (95.9%) to G5 porcine strains, a porcine-like VP6 within VP6 genogroup I, and a Wa-like (genotype B) NSP4, respectively. Altogether, these results indicate that the PoRV strain 134/04-15 should be considered as prototype of a new VP4 genotype, P[26], and provide further evidence for the vast genetic and antigenic diversity of group A rotaviruses.

Sequence analysis of the VP7 and VP4 genes identifies a novel VP7 gene allele of porcine rotaviruses, sharing a common evolutionary origin with human G2 rotaviruses.

During an epidemiological survey encompassing several porcine herds in Saragoza, Spain, the VP7 and VP4 of a rotavirus-positive sample, 34461-4, could not be predicted by using multiple sets of G- and P-type-specific primers. Sequence analysis of the VP7 gene revealed a low amino acid (aa) identity with those of well-established G serotypes, ranging between 58.33% and 88.88%, with the highest identity being to human G2 rotaviruses. Analysis of the VP4 gene revealed a P[23] VP4 specificity, as its VP8* aa sequence was 95.9% identical to that of the P14[23],G5 porcine strain A34, while analysis of the VP6 indicated a genogroup I, that is predictive of subgroup I specificity. Analysis of the 10th and 11th RNA segments revealed close identity to strains of porcine and human origin, respectively. The relatively low overall aa sequence conservation (<89% aa) to G2 human rotaviruses, the lack of N-glycosylation sites that are usually highly conserved in G2 rotaviruses, and the presence of several amino acid substitutions in the major antigenic hypervariable regions hampered an unambiguous classification of the porcine strain 34461-4 as G2 serotype on the basis of sequence analysis alone. The identification of a borderline, G2-like, VP7 gene allele in pigs, while reinforcing the hypotheses of a tight relationship in the evolution of human and animal rotaviruses, provides additional evidence for the wide genetic/antigenic diversity of group A rotaviruses.

Serological and genomic characterization of two porcine rotaviruses with serotype G1 specificity.

Two porcine rotavirus strains, C60 and C95, which had been previously shown to be reactive in an enzyme-linked immunosorbent assay with serotype G1-specific monoclonal antibodies, were classified as G1 by cross-neutralization tests and on the basis of the homology of the sequenced VP7 gene. This report confirms that porcine rotavirus strains with a G1 serotype occur in nature.

Human and most animal rotavirus strains do not require the presence of sialic acid on the cell surface for efficient infectivity.

The outer capsid spike protein VP4 is the main rotavirus cell attachment protein, but the cellular receptor used by rotavirus to establish a productive infection remains unknown. Sialic acid (SA) residues on the cell surface have been shown to be required for efficient binding and infectivity of animal rotaviruses (ARVs), but not of human rotaviruses (HRVs). Since the SA dependence of only a limited number of strains has been tested to date, in this study a larger number of strains were tested to further investigate the involvement of SA in rotavirus infectivity. Following treatment of African green monkey kidney cell (MA104) monolayers with neuraminidase, productive infection of rotavirus was measured by immunofluorescence. The infectivity of all 14 HRVs tested was SA-independent. Ten of 15 ARVs tested were SA-independent, while only five were SA-dependent. These results indicate that most ARVs, like HRVs, infect permissive cells in an SA-independent manner, probably by a common cellular receptor.

Rotavirus and calicivirus infections of the gastrointestinal tract.

Virus infections of the gastrointestinal tract, leading to gastroenteritis, are a common problem in both developed and developing countries. Rotavirus and Norwalk-like viruses are the most common agents responsible for clinically severe disease in humans, and this paper focuses on new information about the mechanisms of pathogenesis and epidemiology of these two pathogens. Rotavirus-induced disease involves a viral enterotoxin and activation of the enteric nervous system, as well as malabsorption, suggesting that common mechanisms of pathogenesis may exist between viral and bacterial pathogens. Each gastrointestinal virus possesses unique molecular properties that can be exploited to discover new information about responses of cells of the gastrointestinal tract. Work continues toward making vaccines for rotavirus and Norwalk-like viruses.

Single point mutations may affect the serotype reactivity of serotype G11 porcine rotavirus strains: a widening spectrum?

A panel of single and double neutralization-resistant escape mutants of serotype G11 porcine rotavirus strains A253 and YM, selected with G11 monotype- and serotype-specific neutralizing monoclonal antibodies (MAbs) to VP7, was tested in neutralization assays with hyperimmune sera raised against rotavirus strains of different serotypes. Escape mutants with an amino acid substitution in antigenic region A (amino acids [aa] 87 to 101) resulting in a residue identical or chemically similar to those present at the same positions in serotype G3 strains, at positions 87 for strain A253 and 96 for strain YM, were significantly more sensitive than the parental strains to neutralization with sera against some serotype G3 strains. Also, one YM antigenic variant (YM-5E6.1) acquired reactivity by enzyme-linked immunosorbent assay with MAbs 159, 57/8, and YO-1E2, which react with G3 strains, but not with the serotype G11 parental strain YM. Cross-adsorption studies suggested that the observed cross-neutralization by the G3-specific sera was due to the sera containing antibodies reactive with the parental strain plus antibodies reactive with the epitope(s) on the antigenic variant that mimick the serotype G3 specific one(s). Moreover, antibodies reactive with antigenic region F (aa 235 to 242) of VP7 might also be involved since cross-reactivity to serotype G3 was decreased in double mutants carrying an additional mutation, which creates a potential glycosylation site at position 238. Thus, single point mutations can affect the serotype reactivity of G11 porcine rotavirus strains with both monoclonal and polyclonal antibodies and may explain the origin of rotavirus strains with dual serotype specificity based on sequence divergence of VP7.

Cross-reactive, serotype- and monotype-specific neutralization epitopes on VP7 of serotype G3 and G5 porcine rotavirus strains.

VP7 specific monoclonal antibodies raised against serotype G5 porcine rotavirus strains isolated in Venezuela showed either a serotype G5- or monotype-specific pattern of reactivity by neutralization against a panel of 53 group A rotavirus isolates representative of all established G serotypes. Monoclonal antibodies raised against two G3 porcine strains were either specific for a subset of porcine G3 strains or reactive with another subset of porcine G3 strains and with most G5 strains. Neither were reactive with G3 strains from other species. Analysis of neutralization resistant mutants selected with these monoclonal antibodies indicated that epitopes defined by cross-reactive, serotype- and monotype-specific monoclonal antibodies overlap functionally and that binding and neutralization by these antibodies depended on specific amino acid residues in the region A or C of VP7. Results indicate that a high degree of monotypic variation occurs among G5 and G3 porcine rotavirus strains and the existence of at least one common epitope shared by G5 and G3 porcine strains, in the major neutralization domain of these VP7s.

Simian rhesus rotavirus is a unique heterologous (non-lapine) rotavirus strain capable of productive replication and horizontal transmission in rabbits.

Simian rhesus rotavirus (RRV) is the only identified heterologous (non-lapine) rotavirus strain capable of productive replication at a high inoculum dose of virus (>10(8) p.f.u.) in rabbits. To evaluate whether lower doses of RRV would productively infect rabbits and to obtain an estimate of the 50% infectious dose, rotavirus antibody-free rabbits were inoculated orally with RRV at inoculum doses of 10(3), 10(5) or 10(7) p.f.u. Based on faecal virus antigen or infectious virus shedding, RRV replication was observed with inoculum doses of 10(7) and 10(5) p.f.u., but not 10(3) p.f.u. Horizontal transmission of RRV to one of three mock-inoculated rabbits occurred 4-5 days after onset of virus antigen shedding in RRV-infected rabbits. Rabbits infected at 10(7) and 10(5), but not 10(3), p.f.u. of RRV developed rotavirus-specific immune responses and were completely (100%) protected from lapine ALA rotavirus challenge. These data confirm that RRV can replicate productively and spread horizontally in rabbits. In attempts to elucidate the genetic basis of the unusual replication efficacy of RRV in rabbits, the sequence of the gene encoding the lapine non-structural protein NSP1 was determined. Sequence analysis of the NSP1 of three lapine rotaviruses revealed a high degree of amino acid identity (85-88%) with RRV. Since RRV and lapine strains also share similar VP7s (96-97%) and VP4s (69-70%), RRV might replicate efficiently in rabbits because of the high relatedness of these three gene products, each implicated in host range restriction.

Equine rotaviruses with G14 serotype specificity circulate among venezuelan horses.

Two group A rotavirus strains isolated from diarrheic foals in Venezuela were classified as belonging to G14 serotype by cross-neutralization tests and on the basis of the homology of the sequenced VP7 gene. This report confirms that rotavirus strains of G14 serotype specificity circulate among equine populations.

Comparative amino acid sequence analysis of the outer capsid protein VP4 from four lapine rotavirus strains reveals identity with genotype P[14] human rotaviruses.

The genes encoding the outer capsid VP4 proteins of four lapine rotavirus strains, three isolated in the US (ALA, C-11 and BAP-2) and one isolated in Japan (R-2) were sequenced, and the predicted amino acid (aa) sequence was compared to all known rotavirus genotypes. A high degree of aa identity (96.8-98.9%) was found among the American lapine strains, while the Japanese rotavirus strain R-2 shared less aa identity (89.5-90.0%) with the American strains. The four lapine rotaviruses shared the closest aa identity (90.6-94.9%) with the P[14] genotype, consisting of viruses isolated from humans in Italy, Finland and Thailand. These results indicate that the VP4 protein of the four lapine strains are genotype P14, and that among lapine strains there are possibly two subtypes, one represented by the American lapine strains and the other by the Japanese R-2 strain.

Characterization of neutralization epitopes on the VP7 surface protein of serotype G11 porcine rotaviruses.

Rotavirus strain A253, isolated from the faeces of a diarrhoeic piglet in Venezuela, was classified as serotype G11 by cross-neutralization studies and by comparison of the deduced amino acid sequence of the VP7 surface protein. The epitopes involved in neutralization of the two G11 porcine rotavirus strains A253 and YM were analysed using neutralization-resistant mutants selected with seven neutralizing monoclonal antibodies (MAbs), monotype-specific (M-) MAbs and serotype-specific (S-) MAbs, produced against VP7 of strain A253. Cross-neutralization tests and sequence analysis of the escape mutants selected from strains A253 and YM indicated the presence of two antigenic sites, one common to both M-MAbs and S-MAbs in region A (positions 87, 91 and 96) and the other defined by one S-MAb in region C (position 223). All A253 variants selected with M-MAbs and two S-MAbs, although having different amino acid substitutions, had a change at amino acid position 87, whereas YM variants involved residues 91 and 96, part of the same antigenic site. Compared to strain A253, the YM stain presents an amino acid substitution at position 87 and was not recognized by M-MAbs. These results suggest that in the VP7 of G11 serotype specificity, the amino acid at position 87 is an important component of a neutralization site associated with region A and the intraserotypic variation between strains A253 and YM may account for the selection of mutations at different positions by a single MAb.

Comparative amino acid sequence analysis of the major outer capsid protein (VP7) of porcine rotaviruses with G3 and G5 serotype specificities isolated in Venezuela and Argentina.

Seven porcine group A rotavirus strains isolated in Venezuela were shown to be antigenically related to serotype G3 (five strains) or to serotype G5 (two strains), whereas two strains isolated in Argentina were classified as serotype G5. The serological classification of eight of these strains was confirmed by sequence analysis of the gene encoding the VP7 glycoprotein. A high degree of homology was observed among strains belonging to the same G serotype, although some variations in the serotype-specific regions were detected among different strains. Comparison with the published VP7 amino acid sequences of serotype G3 indicated that most porcine rotavirus strains are more closely related to each other and to human rotavirus strains than to rotavirus strains isolated from other species. Amino acid sequence comparison among serotype G5 porcine strains revealed that Venezuelan porcine isolates were more closely related to the American strain OSU, while the Argentinian strains had a higher similarity to the Australian strain TRF-41. This report confirms the worldwide distribution of these G serotypes among the porcine population.

Identification of bovine rotaviruses in Venezuela: antigenic and molecular characterization of a bovine rotavirus strain.

Two serotypes of bovine group A rotaviruses were demonstrated by enzyme-linked immunoassay (ELISA) and polyacrylamide gel electrophoresis (PAGE) in 20 of 171 faecal samples collected from diarrhoeic calves in two dairy farms in Venezuela. By serotyping ELISA using G and P serotype-specific monoclonal antibodies, bovine rotaviruses (BRV) circulating on one farm were identified as serotype G6, while BRVs circulating on the other farm were identified as serotype G10. Only one BRV (033) could be successfully isolated in MA104 cells, and the nucleotide sequences of the VP7 and the VP8* trypsin-cleavage product of the VP4 were determined. Cross-neutralization tests and comparative sequence analysis showed that BRV 033 belonged to serotype G6 and genotype P1. This is the first report of BRVs identified in Venezuela.

Differential infection of polarized epithelial cell lines by sialic acid-dependent and sialic acid-independent rotavirus strains.

Infection of epithelial cells by some animal rotaviruses, but not human or most animal rotaviruses, requires the presence of N-acetylneuraminic (sialic) acid (SA) on the cell surface for efficient infectivity. To further understand how rotaviruses enter susceptible cells, six different polarized epithelial cell lines, grown on permeable filter membrane supports containing 0.4-microm pores, were infected apically or basolaterally with SA-independent or SA-dependent rotaviruses. SA-independent rotaviruses applied apically or basolaterally were capable of efficiently infecting both sides of the epithelium of all six polarized cell lines tested, while SA-dependent rotaviruses only infected efficiently through the apical surface of five of the polarized cell lines tested. Regardless of the route of virus entry, SA-dependent and SA-independent rotaviruses were released almost exclusively from the apical domain of the plasma membrane of polarized cells before monolayer disruption or cell lysis. The transepithelial electrical resistance (TER) of cells decreased at the same time, irrespective of whether infection with SA-independent rotaviruses occurred apically or basolaterally. The TER of cells infected apically with SA-dependent rotaviruses decreased earlier than that of cells infected basolaterally. Rotavirus infection decreased TER before the appearance of cytopathic effect and cell death and resulted in an increase in the paracellular permeability to [(3)H]inulin as a function of loss of TER. The presence of SA residues on either the apical or basolateral side was determined using a Texas Red-conjugated lectin, wheat germ agglutinin (WGA), which binds SA residues. WGA bound exclusively to SA residues on the apical surface of the cells, confirming the requirement for SA residues on the apical cell membrane for efficient infectivity of SA-dependent rotaviruses. These results indicate that the rotavirus SA-independent cellular receptor is present on both sides of the epithelium, but SA-dependent and SA-independent rotavirus strains infect polarized epithelial cells by different mechanisms, which may be relevant for pathogenesis and selection of vaccine strains. Finally, rotavirus-induced alterations of the epithelial barrier and paracellular permeability suggest that common mechanisms of pathogenesis may exist between viral and bacterial pathogens of the intestinal tract.