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PubMed1991

Homotypic and heterotypic serum and milk antibody to rotavirus in normal, infected and vaccinated horses.

The homotypic and heterotypic antibody response to rotavirus was determined in three pony mares and their foals. The normal concentrations of anti-rotavirus antibodies in mares' milk and mares' and foals' serum over the first 10 weeks post-partum were measured using IgA, IgG and rotavirus serotype-specific enzyme linked immunosorbent assays. Experimental infection of the foals with serotype 3 equine rotavirus produced a rapid, serotype-specific response which peaked 10 days after infection and a slower heterotypic response which peaked 32 days later. In contrast, vaccination of the mares with an inactivated, adjuvanted serotype 6 bovine rotavirus produced a heterotypic response similar to that of the homotypic response in both serum and milk, although the predominant response in serum was IgG, while in milk it was IgA. These results suggest that non serotype-restricted passive protection of foals against rotavirus may be achieved by parenteral vaccination of mares.

Animals↗
PubMedDate not supplied

Evaluation of assay systems for the detection of rotavirus in stool specimens.

We tested 41 rotavirus positive and 42 negative specimens as determined by electron microscopy. The assays systems used were an indirect NIH-ELISA, Meritec-Rotavirus, Virogen Rotatest, and Rotazyme II. Meritec and Virogen (latex agglutination assays) were the most sensitive tests, detecting 95% of the positive specimens. The NIH-ELISA detected 81% and Rotazyme detected 63%. Rotazyme was the most specific (100%), followed by the NIH-ELISA (95%) and the two latex agglutination systems (91%). To determine the level of rotavirus detection, we tested three systems against serial two-fold dilutions of ten positive stools. The NIH-ELISA detected rotavirus in an average dilution of 1:723. Rotazyme detected rotavirus in an average stool dilution of 1:366, and Meritec showed an average of 1:164. Rotavirus strains representing serotypes 1-4 were also tested. Meritec was able to detect all four serotypes. Virogen did not react with serotype 2 strains. The NIH-ELISA and rotazyme were unable to detect serotype 3. These data suggest that some latex agglutination assays may be a useful alternative to ELISAs in the clinical laboratory.

Child, Preschool↗
PubMed1988

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.

Age Factors↗
PubMed1991

Experience with serotyping rotavirus strains by reverse transcription and two-step polymerase chain reaction with generic and type-specific primers.

Six VP7 serotypes or G types (G1-G4, G8 and G9) occur in group A human rotaviruses. Gouvea et al. recently reported a novel G-typing method based on reverse transcription (RT)-polymerase chain reaction (PCR) amplification of the VP7 gene with type-specific primers [Gouvea, V. et al. (1990). Journal of Clinical Microbiology 28, 276-82]. When we followed their protocol, 40 (89%) of 45 faecal rotavirus specimens were typed into G1-G4 and G9. The five specimens that were untypeable by the RT-PCR method contained three G2 and two G4 rotavirus specimens which were identified by an ELISA using G type-specific monoclonal antibodies. On the other hand, the RT-PCR typing assay was able to determine the G type of the seven isolates that were untypeable by the ELISA. Of 33 faecal rotaviruses that were typed by both assays, 100% agreement of the result was observed. In addition, when applied to some animal rotaviruses, the RT-PCR method identified G3 feline and canine rotavirus strains. We conclude that further refinement of the RT-PCR assay is desirable in order to more readily type G2 and G4 strains, although this assay displayed an applicability to epidemiologic studies comparable with ELISAs.

DNA, Viral↗
PubMed2005

Phylogenetic analyses of human rotavirus in central Taiwan in 1996, 2001 and 2002.

BACKGROUND: Rotavirus epidemiology information is required for gastroenteritis disease control and prevention. Information gathered about the serotype distribution of rotaviruses isolated in Taiwan is of crucial significance, before a licensed rotavirus vaccine is introduced. OBJECTIVES: The purpose of the present study is to investigate the epidemiological diversity of rotaviruses in Taiwan. STUDY DESIGN: A total of 51 stool samples taken from cases of acute gastroenteritis were collected from three teaching hospitals in central Taiwan in 1996, 2001 and 2002. The samples were subjected to RT-PCR tests of VP7 gene of the human rotavirus group A, B, C. RESULTS: A total of 16 stool samples were detected positive by RT-PCR and 10 were sequence analyzed and classified into G1, G3, and G9 types. Compared with other HRV strains: the sequences of CS96-40 of G1 are similar to MVD9816 (identity rate 97.15% and 96.09%, respectively, from Uruguay); the sequences of CS02-01 of G3 are similar to 98-B31 (identity rate 98.93% and 98.72%, respectively, from Japan); the sequences of CS01-05, CS01-06, CS01-07, CS01-09, CS01-13, CS02-02, CS02-03, CS02-04 are very similar to other established G9 rotaviruses sequences (identity rate 96.85-99.88%), especially between CS02-04 and SP2737 (from Japan) with an identity rate of 99.88% and 100% nucleotide and amino acid, respectively. Except for CS01-06 strain, it is VR3, but not VR5, VR7 or VR8, that found to be the most frequent mutated amino acid regions of VP7 in these strains. CONCLUSIONS: Our findings are the first to document the high prevalence of G9 HRV strains in Taiwan, and suggest the re-emergence of G3 strains in central Taiwan since 1991. Epidemiological surveys carried out in this study suggest genotype shifts from type G1 before 1996, to G9 in 2001 and 2002 and the re-emergence of G3 type in 2002.

Antigens, Viral↗
PubMed2006

Genetic characterization of group A rotavirus strains circulating among children with acute gastroenteritis in Japan in 2004-2005.

A total of 752 fecal specimens collected from July 2004 to June 2005 from children with acute gastroenteritis in four localities in Japan (Maizuru, Tokyo, Sapporo, and Osaka) were screened for group A rotavirus by RT-PCR. It was found that 82 (10.9%) specimens were positive for group A rotavirus. The G-(VP7 genotypes) and P-(VP4 genotypes) types were further investigated. The P-types of 18 rotavirus strains, which could not be typed by RT-PCR, were determined by sequencing analysis. Of these, 94% (17/18) were P[8] with multiple point mutations at the VP4 primer-binding site. Another sample turned out to be a rare genotype P[9], which was closely related to feline rotavirus. The predominant genotype was G1P[8] (46.4%), followed by G3P[8] (32.9%) and G2P[4] (12.2%). A number of unusual combinations including, G1P[4] (1.2%), G2P[8] (1.2%), G3P[9] (1.2%), G1G3P[8] (1.2%), and G2G3P[8] (3.7%), were also detected. A new nomenclature of P[8] was proposed, in which worldwide rotavirus P[8] strains were classified into four sub-lineages, namely IA, IB, IIA, and IIB. A wide range of amino acid substitutions (up to 22) specific for P[8] lineages and sub-lineages were also identified. Interestingly, only short amino acid motifs located at positions 32-35, 121-135, and 195-236 of VP4 correctly defined the phylogenetic P[8] lineages and sub-lineages. Of note, at least two distinct clusters of rotavirus P[8] were co-circulating in the Japanese pediatric population studied.

Acute Disease↗
PubMed2004

Intranasal administration of an Escherichia coli-expressed codon-optimized rotavirus VP6 protein induces protection in mice.

We are developing rotavirus vaccines based on the VP6 protein of the human G1P[8] [corrected] [J. Virol. 73 (1999) 7574] CJN strain of rotavirus. One prototype candidate consisting of MBP::VP6::His6, a chimeric protein of maltose-binding protein, VP6 and hexahistidine, was expressed mainly as truncated polypeptides in Escherichia coli BL21(DE3) cells. A possible reason for this extensive truncation is the high frequencies of rare bacterial codons within the rotavirus VP6 gene. Expression of truncated recombinant VP6 was found to be reduced, and expression of complete VP6 protein was simultaneously increased, when the protein was expressed in Rosetta(DE3)pLacI E. coli cells that contain increased amounts of transfer RNAs for a selection of rare codons. The same observation was made when a synthetic codon-optimized CJN-VP6 gene was expressed in E. coli BL21 or Rosetta cells. To increase protein recovery, recombinant E. coli cells were treated with 8M urea. Denatured, full-length MBP::VP6::His6 protein was then purified and used for intranasal vaccination of BALB/c mice (2 doses administered with E. coli heat-labile toxin LT(R192G) as adjuvant). Following oral challenge with the G3P[16] [corrected] [J. Virol. 76 (2002) 560] EDIM strain of murine rotavirus, protection levels against fecal rotavirus shedding were comparable (P>0.05) between groups of mice immunized with denatured codon-optimized or native (not codon-optimized) immunogen with values ranging from 87 to 99%. These protection levels were also comparable to those found after immunization with non-denatured CJN VP6. Thus, expression of complete rotavirus VP6 protein was greatly enhanced by codon optimization, and the protection elicited was not affected by denaturation of recombinant VP6.

Administration, Intranasal↗
PubMed2005

Cloning, expression, and purification of recombinant bovine rotavirus hemagglutinin, VP8*, in Escherichia coli.

Rotavirus VP8* subunit is the minor trypsin cleavage product of the spike protein VP4, which is the major determinant of the viral infectivity and neutralization. To study the structure-function relationship of this fragment and to obtain type-specific reagents, substantial amounts of this protein are needed. Thus, full-length VP8* cDNA, including the entire trypsin cleavage-encoding region in gene 4, was synthesized and amplified by RT-PCR from total RNA purified from bovine rotavirus strain C486 propagated in MA104 cell culture. The extended VP8* cDNA (VP8ext) was cloned into the pGEM-T Easy plasmid and subcloned into the Escherichia coli expression plasmid pET28a(+). The correspondent 30 kDa protein was overexpressed in E. coli BL21(DE3)pLysS cells under the control of the T7 promoter. The identity and the antigenicity of VP8ext were confirmed on Western blots using anti-His and anti-rotavirus antibodies. Immobilized Ni-ion affinity chromatography was used to purify the expressed protein resulting in a yield of 4 mg of VP8ext per liter of induced E. coli culture. Our results indicate that VP8ext maintained its native antigenicity and specificity, providing a good source of antigen for the production of P type-specific immune reagents. Detailed structural analysis of pure recombinant VP8 subunit should allow a better understanding of its role in cell attachment and rotavirus tropism. Application of similar procedure to distinct rotavirus P serotypes should provide valuable P serotype-specific immune reagents for rotavirus diagnostics and epidemiologic surveys.

Animals↗
PubMed2004

Nasal immunization of mice with a rotavirus DNA vaccine that induces protective intestinal IgA antibodies.

DNA vaccination using a plasmid encoding the rotavirus inner capsid VP6 has been explored in the mouse model of rotavirus infection. BALB/c mice were immunized with a VP6 DNA vaccine by the intramuscular, nasal and oral routes. VP6 DNA vaccination by the nasal and oral routes induced the production of anti-VP6 IgA antibodies by intestinal lymphoid cells. Intramuscular DNA injection stimulated the production of serum anti-VP6 IgG but not serum anti-VP6 IgA antibodies. Protection against shedding of rotaviruses in stools after oral challenge with the murine EDIM rotavirus strain was investigated in the immunized mice. A significant reduction in the level of rotavirus antigen shedding was demonstrated in those mice immunized at mucosal surfaces, both orally and nasally, with the VP6 DNA vaccine. Intramuscular DNA immunization, which elicited serum anti-VP6 IgG responses but not virus-specific intestinal IgA antibodies, did not provide significant protection against rotavirus challenge.

Administration, Intranasal↗
PubMed2006

Immunity and correlates of protection for rotavirus vaccines.

Rotaviruses are the most common cause of severe, dehydrating diarrhea in children worldwide. The tremendous global incidence of rotavirus gastroenteritis, especially in developing countries, emphasizes the need for vaccines to prevent associated morbidity and mortality. However, immunity to rotavirus is not completely understood. At this time, total serum RV IgA, measured shortly after infection, appears to be the best marker of protection against rotavirus. This review describes the current understanding of rotavirus immunity, including mechanisms of protection against rotavirus from selected animal models, and correlates of protection associated with natural infection or vaccination from humans.

Animals↗
PubMed2005

Distribution of G (VP7) and P (VP4) genotypes in buffalo group A rotaviruses isolated in Southern Italy.

Group A rotaviruses are established agents of disease in buffalo calves. Early epidemiological studies in Italian buffalo herds revealed the predominance of strains with G8 specificity and detected strains with the rare, RRV-like, VP4 P[3] genotype. To acquire additional information on the VP4 and VP7 specificities of buffalo rotaviruses, a total of 125 fecal samples were collected from buffalo calves affected with diarrhoea, in seven dairy farms in Southern Italy. Rotaviruses were detected in 21 samples (16.8%) by an immunochromatographic assay and by reverse transcription-PCR (RT-PCR). Analysis of the VP7 gene revealed that 57% (12 of 21) of the isolates were G6, 23.8% were G8 (5 of 21) and 19% (4 of 21) were G10. Analysis of the VP4 revealed that 71.4% (15 of 21) of the isolates were P[5] and that 28.6% (6 of 21) were P[1]. The most common combination of G and P types was P[5],G6 (57%), followed by P[1],G10 (19%), P[5],G8 (14%) and P[1],G8 (9.5%). While P[5],G6 rotaviruses are very common in Italian bovine herds, the antigenic combination P[1],G10 is unusual and presumably derives from reassortment between P[1] and G10 strains, that appear to be more frequent in buffaloes and bovines, respectively. The presence of bovine-like G and P serotypes suggests that in Italy the epidemiology of buffalo rotaviruses overlaps the epidemiology of bovine rotaviruses, presumably because of the strict species affinity and/or of the intermingled distribution over the same geographical areas of the buffalo and bovine herds.

Animals↗
PubMed2004

Human infection with a P[14], G3 lapine rotavirus.

Group A rotaviruses are the main cause of severe diarrhoea in humans and animals throughout the world. We report the first description of a clinically apparent infection with a P[14], G3 rotavirus (strain B4106) in a hospitalized 6-year-old child. The VP7 gene of the B4106 strain had the closest sequence similarity (94% and 97% on the nucleotide and amino acid level, respectively) with strain 30/96 (P[14], G3), a lapine rotavirus isolated in an Italian rabbit in 1996 while the VP4 gene had the closest similarity with strain 30/96 on the nucleotide level (96%), and with lapine strains C-11 (P[14], G3) and Alabama (P[14], G3), isolated in the United States in the 1980s on the amino acid level (99%). The host restriction determinant gene NSP4 of B4106 was also most similar to lapine strain Alabama (95% nt identity and 97% aa identity). Phylogenetic analysis showed that the VP4, VP7, and NSP4 genes of the B4106 strain share a common evolutionary lineage with those of lapine rotavirus strains. We therefore hypothesize that a lapine rotavirus was able to cross the host species barrier and caused disease in a new host. The increasing detection of strains in humans that were previously believed to be restricted to animals raises questions whether interspecies transmission of rotaviruses is a common event in nature.

Antigens, Viral↗
PubMed2005

Evaluation of rotavirus dsRNA load in specimens and body fluids from experimentally infected juvenile macaques by real-time PCR.

We recently established a non-human primate model of rotavirus infection that is characterized by consistent and high levels of virus antigen shedding in stools. Here, we report that starting from post challenge day (PCD) 2, 6 x 10(3) to 1.5 x 10(6) copies of rotavirus double-stranded RNA per nanogram of total RNA were detected by real-time PCR in MA104 cells that were 48 h pre-incubated with filtered stool suspensions of three experimentally infected juvenile macaques. The peak of virus load was detected at PCD 4-5, followed by decreased load at PCD 6-11, and very low levels at PCD 12. Such a pattern corresponded to virus shedding in stools as reported recently based on enzyme-linked immunosorbent assay (ELISA) results. In addition, plasma and cerebrospinal fluids (CSF) from six infected animals were tested for the presence of rotavirus. Rotavirus extraintestinal escape was revealed in three out of six animals by a combination of real-time and nested PCR. However, very low quantities of detected viral RNA (approximately 20 copies/ng of total RNA) were not suggestive of viremia. Thus, the rhesus model of rotavirus infection can be exploited further in studies with vaccine candidates designed to prevent or abrogate rotavirus infection.

Animals↗
PubMed2004

Cloning and sequence analysis of dsRNA segments 5, 6 and 7 of a novel non-group A, B, C adult rotavirus that caused an outbreak of gastroenteritis in China.

A diarrhoeal outbreak among adults in China was caused by a new rotavirus, termed ADRV-N, that does not react with antisera directed against group A, B or C rotaviruses [Zhonghua Liu Xing Bing Xue Za Zhi (Chin. Epidemiol.) 19 (1998) 336]. ADRV-N can be propagated in cell cultures [Zhonghua Yi Xue Za Zhi (Natl. Med. J. China) 82 (2002) 14]. We present the complete sequences for ADRV-N genome segments 5 and 6, and a full ORF sequence of genome segment 7. The deduced amino acid sequences suggest that these segments encode NSP1, VP6 and NSP3, respectively. These three ADRV-N genome segments have a unique -ACCCC-3' terminal sequence. The 5'-GG- terminus of segments 5 and 6 is the same as that of other rotaviruses. The amino acid similarity between VP6 and NSP3 of ADRV-N and the cognate sequences of their closest counterpart, group B IDIR, was 37 and 35%, respectively. The ADRV-N NSP1 has a double-stranded RNA binding motif (DSRM) and a putative autoproteolytic cleavage motif upstream from the DSRM. The putative ADRV-N NSP3 has a truncated C-terminus compared to the cognate protein of group B rotaviruses. All the available data demonstrate that ADRV-N differs significantly from the known rotaviruses and strongly suggest that ADRV-N is the first recognized member of a new group of rotaviruses infecting humans.

Adult↗
PubMed2006

Heat shock enhances the susceptibility of BHK cells to rotavirus infection through the facilitation of entry and post-entry virus replication steps.

Rotavirus infection is known to induce several cellular stress proteins, although their possible involvement in the replication cycle of the virus has not been studied. In addition, the heat shock cognate protein hsc70 has been shown to function as a post-attachment receptor during virus entry. In this work we have studied the effect of heat shock on the susceptibility of cells to rotavirus infection. BHK cells, which are largely refractory to the virus, became about 100-fold more susceptible when heat-treated, while the rotavirus highly susceptible MA104 cells did not significantly modified their susceptibility upon heat stress, suggesting that heat shock induces factors that are rate-limiting the replication of rotaviruses in BHK but not in MA104 cells. The heat treatment was shown to facilitate the rotavirus infection of BHK cells at the penetration and post-penetration levels, and each of these stages seems to contribute comparably to the overall observed 100-fold increase in infectivity. Since the binding of the virus to the cell surface was not affected, the caloric stress probably facilitates the penetration and/or uncoating of the virus. The pathway of virus entry into heat-shocked BHK cells seems to be similar to that used in MA104 cells, since treatments that affect MA104 cell infection also affected rotavirus infectivity in heat-treated BHK cells.

Animals↗
PubMed2003

Oral immunization with rotavirus VP7 expressed in transgenic potatoes induced high titers of mucosal neutralizing IgA.

Rotaviruses (RV) are a common cause of severe diarrhea in young children, resulting in nearly one million deaths worldwide annually. Rotavirus VP7 was the rotavirus neutralizing protein. Previous study reported that VP7 DNA vaccine can induce high levels of IgG in mice but cannot protect mice against challenge (Choi, A.H., Basu, M., Rae, M.N., McNeal, M.M., Ward, R.L., 1998. Virology 250, 230-240). We found that rotavirus VP7 could maintain its neutralizing immunity when it was transformed into the potato genome. Mice immunized with the transformed tubers successfully elicited serum IgG and mucosal IgA specific for VP7. The mucosal IgA titer was as high as 1000, while serum IgG titer was only 600. Neutralizing assays indicated that IgA could neutralize rotavirus. These results indicate the potential usefulness of plants for production and delivery of edible rotavirus vaccines.

Administration, Oral↗
PubMed2003

Rotavirus infectious particles use lipid rafts during replication for transport to the cell surface in vitro and in vivo.

The pathway by which rotavirus is released from the cell is poorly understood but recent work has shown that, prior to cell lysis, rotavirus is released almost exclusively from the apical surface of the infected cell. By virtue of their unique biochemical and physical properties, viruses have exploited lipid rafts for host cell entry and/or assembly. Here we characterized the association of rhesus rotavirus (RRV) with lipid rafts during the rotavirus replication cycle. We found that newly synthesized infectious virus associates with rafts in vitro and in vivo. RRV proteins cosegregated with rafts on density gradients. Viral infectivity and genomic dsRNA also cosegregated with the raft fractions. Confocal microscopic analysis of raft and RRV virion proteins demonstrated colocalization within the cell. In addition, cholesterol depletion interfered with the association of RRV particles with rafts and reduced the release of infectious particles from the cell. Furthermore, murine rotavirus associates with lipid rafts in intestinal epithelial cells during a natural infection in vivo. Our results confirm the association of rotavirus infectious particles with rafts during replication in vitro and in vivo and strongly support the conclusion that this virus uses these microdomains for transport to the cell surface during replication.

Animals↗
PubMed1989

Passive immunisation of children with bovine colostrum containing antibodies to human rotavirus.

The efficacy of a 10-day course of bovine colostrum with high antibody titre against the four known human rotavirus serotypes in protecting children against rotavirus infection was examined in patients admitted to hospital. Children aged 3 to 15 months were blocked in pairs according to ward accommodation (ie, isolation or open area). Each block contained 1 treated and 1 control child. The allocation to treatment or control (an artificial infant formula) was randomised. 9 of 65 control children but none of 55 treated children acquired rotavirus infection during the treatment period (p less than 0.001). The importance of protecting against rotavirus infection was highlighted by the fact that parents of symptomatic rotavirus-positive children sought medical attention seven times more often than did parents of symptomatic rotavirus-negative children (p less than 0.05).

Administration, Oral↗