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.

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.

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.

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.

Molecular analysis of the VP7, VP4, VP6, NSP4, and NSP5/6 genes of a buffalo rotavirus strain: identification of the rare P[3] rhesus rotavirus-like VP4 gene allele.

We report the detection and molecular characterization of a rotavirus strain, 10733, isolated from the feces of a buffalo calf affected with diarrhea in Italy. Strain 10733 was classified as a P[3] rotavirus, as the VP8* trypsin cleavage product of the VP4 protein revealed a high amino acid identity (96.2%) with that of rhesus rotavirus strain RRV (P5B[3]), used as the recipient virus in the human-simian reassortant vaccine. Analysis of the VP7 gene product revealed that strain 10733 possessed G6 serotype specificity, a type common in ruminants, with an amino acid identity to G6 rotavirus strains ranging from 88 to 98%, to Venezuelan bovine strain BRV033, and Hungarian human strain Hun4. Phylogenetic analysis based on the VP7 gene of G6 rotaviruses identified at least four lineages and an apparent linkage between each lineage and the VP4 specificity, suggesting the occurrence of repeated interspecies transmissions and genetic reassortment events between ruminant and human rotaviruses. Moreover, strain 10733 displayed a bovine-like NSP4 and NSP5/6 and a subgroup I VP6 specificity, as well as a long electropherotype pattern. The detection of the rare P[3] genotype in ruminants provides additional evidence for the wide genetic and antigenic diversity of group A rotaviruses.

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.

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.

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.

Antigenic and molecular analyses reveal that the equine rotavirus strain H-1 is closely related to porcine, but not equine, rotaviruses: interspecies transmission from pigs to horses?

We have sequenced the genes encoding the inner capsid protein VP6 and the outer capsid glycoprotein VP7 of the subgroup (SG) I equine rotavirus strain H-1 (P9[7], G5). The VP6 and VP7 proteins of the equine rotavirus strain H-1 shared a high degree of sequence and deduced amino acid identity with SG I porcine strains and serotype G5 porcine strains, respectively. Previous sequence analyses of the genes encoding the outer capsid spike protein VP4 and the nonstructural proteins NSP1 and NSP4 of equine H-1 strain also revealed a high degree of sequence and deduced amino acid homology with the prototype porcine rotavirus strain OSU (P9[7], G5). We have also confirmed and extended the VP4 and VP7 antigenic relatedness of equine rotavirus strain H-1 to porcine strains of P9[7] and G5 serotype specificities isolated in the United States, Venezuela, Argentina, and Australia based on cross-neutralization studies. In addition, the pathogenicity of tissue culture-adapted equine H-1, H-2, FI-14, FI-23, and L338, and porcine OSU rotavirus strains was compared in the neonatal mouse model. The 50% diarrhea dose (DD50) of equine H-1 was similar to that of porcine OSU and equine H-2 and L338 strains, while the DD50 of equine H-2 was > or = 50 or 315-fold lower than those of equine FI-14 or FI-23, respectively. Our sequence comparison of NSP4 of the rotavirus strains tested potentially identified amino acid residue 136, within the variable region spanning amino acids 130 to 141, as playing a role in virulence. Taken together, there is strong support to suggest that the equine rotavirus strain H-1 may represent an example of interspecies transmission from pigs to horses.

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.

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.

Identification of an epitope common to genogroup 1 "norwalk-like viruses".

A panel of 10 monoclonal antibodies (MAbs) to recombinant Norwalk virus (NV) capsid protein were tested in competition enzyme-linked immunosorbent assays. Patterns of competition indicated that these MAbs recognize six to eight epitopes covering five nonoverlapping regions of the capsid protein. A single epitope, recognized by NV MAbs NV3901, NV3912, and NV2461 was found to occur in the majority of genogroup 1 (G1) but not genogroup 2 (G2) "Norwalk-like viruses" (NLVs). This observation supports the subdivision of human NLVs into two genogroups and provides an assay for the rapid identification of G1 NLVs in fecal specimens.

Species specificity and interspecies relatedness of NSP4 genetic groups by comparative NSP4 sequence analyses of animal rotaviruses.

Previous sequence analyses of the rotavirus nonstructural NSP4 from human and some animal rotavirus strains revealed the presence of three distinct NSP4 alleles or genetic groups. To examine the species of origin relatedness and diversity of NSP4, the nucleotide and deduced amino acid sequences of the gene encoding the NSP4 from 15 animal rotavirus strains of porcine, equine, bovine, lapine and canine origin were determined and compared to human and other animal strains sequenced previously. Lapine and equine strains were shown to belong to the NSP4 genotype A. Murine NSP4 sequences formed a previously unrecognized fourth distinct NSP4 genotype (genotype D) that was more divergent compared to NSP4 genotype A, B, and C than the latter three are among each other. Within NSP4 genotypes, strains isolated from rabbits, horses, cows (genotype A) and pigs (genotype B) clustered according to species of origin, suggesting a conserved pattern of evolution within species. NSP4 sequence comparison among one wildtype and two tissue culture-adapted lapine strains, known to cause disease in neonatal rabbits, failed to identify amino acid changes within the variable region spanning amino acids 130 to 141, suggesting that disease in rabbits is the result of the lapine virus infection and replication, including production of the NSP4 enterotoxin.

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.

Heterotypic protection and induction of a broad heterotypic neutralization response by rotavirus-like particles.

The recognition that rotaviruses are the major cause of life-threatening diarrheal disease and significant morbidity in young children has focused efforts on disease prevention and control of these viruses. Although the correlates of protection in children remain unclear, some studies indicate that serotype-specific antibody is important. Based on this premise, current live attenuated reassortant rotavirus vaccines include the four predominant serotypes of virus. We are evaluating subunit rotavirus vaccines, 2/6/7-VLPs and 2/4/6/7-VLPs, that contain only a single VP7 of serotype G1 or G3. In mice immunized parenterally twice, G3 virus-like particles (VLPs) induced a homotypic, whereas G1 VLPs induced a homotypic and heterotypic (G3) serum neutralizing immune response. Administration of three doses of G1 or G3 VLPs induced serum antibodies that neutralized five of seven different serotype test viruses. The inclusion of VP4 in the VLPs was not essential for the induction of heterotypic neutralizing antibody in mice. To confirm these results in another species, rabbits were immunized parenterally with two doses of 2/4/6/7-VLPs containing a G3 or G1 VP7, sequentially with G3 VLPs followed by G1 (G3/G1) VLPs, or with live or psoralen-inactivated SA11. High-titer homotypic serum neutralizing antibody was induced in all rabbits, and low-level heterotypic neutralizing antibody was induced in a subset of rabbits. The rabbits immunized with the G1 or G3/G1 VLPs in QS-21 were challenged orally with live G3 ALA rotavirus. Protection levels were similar in rabbits immunized with homotypic G3 2/4/6/7-VLPs, heterotypic G1 2/4/6/7-VLPs, or G3/G1 2/4/6/7-VLPs. Therefore, G1 2/4/6/7-VLPs can induce protective immunity against a live heterotypic rotavirus challenge in an adjuvant with potential use in humans. Following challenge, broad serum heterotypic neutralizing antibody responses were detected in rabbits parenterally immunized with G1, G3/G1, or G3 VLPs but not with SA11. Immunization with VLPs may provide sufficient priming of the immune system to induce protective anamnestic heterotypic neutralizing antibody responses upon subsequent rotavirus infection. Therefore, a limited number of serotypes of VLPs may be sufficient to provide a broadly protective subunit vaccine.

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.

Expression and self-assembly of Grimsby virus: antigenic distinction from Norwalk and Mexico viruses.

A cDNA obtained from Grimsby virus (GRV), a Norwalk-like virus, purified from a stool sample of a symptomatic adult associated with a gastroenteritis outbreak in the United Kingdom, was used to obtain the complete nucleotide sequence of the second open reading frame (ORF2). The ORF2 sequence of GRV predicts a capsid of 539 amino acids (aa) which exhibits aa identities of 96% to Lordsdale virus, 67% to Mexico virus (MXV), and 43% to Norwalk virus (NV). The GRV capsid protein was expressed in insects cells by using a recombinant baculovirus, and the resulting virus-like particles (VLPs) possessed a protein with an apparent molecular weight of 58,000. Hyperimmune antisera raised against purified GRV, MXV, and NV VLPs were tested in an indirect enzyme-linked immunosorbent assay (ELISA) against GRV, NV, and MXV VLPs, revealing that GRV is antigenically distinct from both NV and MXV. The antigenic specificity of the GRV-hyperimmune antiserum was confirmed in an antigen capture ELISA using GRV-, NV-, or MXV-containing fecal specimens. The expression of the GRV capsid protein has, for the first time, allowed the antigenic comparison of three distinct recombinant Norwalk-like viruses.

Rotavirus disease, but not infection and development of intestinal histopathological lesions, is age restricted in rabbits.

The rabbit model of rotavirus infection has proved to be useful for assessing active immunity and protection after infection or vaccination with virus or virus-like particles. One limitation of the rabbit model is that after experimental infection of rabbits, clinical diarrhea is not routinely induced. Lack of diarrhea in the rabbit model has been proposed to be due to the fluid absorptive capability of the cecum or attenuation of virus strains through tissue culture adaptation. To test whether a wild-type lapine rotavirus strain BAP (BAPwt) isolated from diarrheic rabbits would cause disease on passage in rabbits, 1-, 2-, 10-, and 16-week-old rabbits were orally inoculated with BAPwt, its tissue culture-adapted counterpart strain (BAP-2), tissue culture-adapted lapine strain ALA, or PBS. Lapine rotavirus infection in 1-week-old, but not >/=2-week-old, rabbits resulted in the development of disease characterized by soft, wet, yellow-to-brownish-green partially formed-to-liquid stools observed only at the time of virus antigen shedding. The level and duration of virus shedding after infection were prolonged in 1-week-old rabbits compared with rabbits >/=2 weeks of age. Although diarrhea was not observed beyond the first 2 weeks of life, histopathological changes, including villus shortening and fusion, increased vacuolation of epithelial cells, and mononuclear infiltration of the lamina propria, were observed throughout the small intestine between 12 and 120 h after ALA infection in 1-week-old, 1- to 2-month-old, and 11-month-old rabbits. In 11-month-old rabbits, onset of intestinal damage appeared to be slightly delayed, was less severe, and was not observed in the duodenum. There were no differences in the immune responses to rotavirus infection in rabbits of different age groups (1 week to 5 years of age). All lapine rotavirus-inoculated rabbits seroconverted and were protected from virus challenge at 28 days postinoculation. Like in mice, rotavirus disease is age restricted in rabbits.

Subunit rotavirus vaccine administered parenterally to rabbits induces active protective immunity.

Virus-like particles (VLPs) are being evaluated as a candidate rotavirus vaccine. The immunogenicity and protective efficacy of different formulations of VLPs administered parenterally to rabbits were tested. Two doses of VLPs (2/6-, G3 2/6/7-, or P[2], G3 2/4/6/7-VLPs) or SA11 simian rotavirus in Freund's adjuvants, QS-21 (saponin adjuvant), or aluminum phosphate (AlP) were administered. Serological and mucosal immune responses were evaluated in all vaccinated and control rabbits before and after oral challenge with 10(3) 50% infective doses of live P[14], G3 ALA lapine rotavirus. All VLP- and SA11-vaccinated rabbits developed high levels of rotavirus-specific serum and intestinal immunoglobulin G (IgG) antibodies but not intestinal IgA antibodies. SA11 and 2/4/6/7-VLPs afforded similar but much higher mean levels of protection than 2/6/7- or 2/6-VLPs in QS-21. The presence of neutralizing antibodies to VP4 correlated (P < 0.001, r = 0.55; Pearson's correlation coefficient) with enhanced protection rates, suggesting that these antibodies are important for protection. Although the inclusion of VP4 resulted in higher mean protection levels, high levels of protection (87 to 100%) from infection were observed in individual rabbits immunized with 2/6/7- or 2/6-VLPs in Freund's adjuvants. Therefore, neither VP7 nor VP4 was absolutely required to achieve protection from infection in the rabbit model when Freund's adjuvant was used. Our results show that VLPs are immunogenic when administered parenterally to rabbits and that Freund's adjuvant is a better adjuvant than QS-21. The use of the rabbit model may help further our understanding of the critical rotavirus proteins needed to induce active protection. VLPs are a promising candidate for a parenterally administered subunit rotavirus vaccine.