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Background ROTAVAC® (nHRV), derived naturally from the human 116E rotavirus (RV) neonatal strain, was licensed in India in 2015 based on promising results of a phase 3, safety and efficacy vaccine trial. As a pre-requisite for WHO prequalification, we compared the immunogenicity and safety of ROTAVAC® to those of a WHO-prequalified, Rotarix®. Methods We conducted a multicentre, open-labeled, randomized phase 4 clinical trial where 464 infants, 6–8 weeks of age were equally randomized to receive as licensed, the complete regimen of ROTAVAC® (3 doses; Group I) or Rotarix® (2 doses; Group II). Antibody responses (serum anti-RV Immunoglobulin A [IgA]) were measured by enzyme-linked immunosorbent assay (ELISA). The primary analysis was an assessment of non-inferiority of ROTAVAC® to Rotarix® for geometric mean concentration (GMC) for infants who received the complete regimen of either vaccine. Results The GMC for Group I was 20.4 (95%CI: 17.6, 23.6) and that for Group II was 24.8 (95%CI: 20.3, 30.3), the GMC ratio was 0.82 (95% CI: 0.64, 1.05), thus meeting the non-inferiority criterion. Site-wise analysis of GMC titres revealed that one site had a peculiar pre-vaccination titre affecting only ROTAVAC® post-vaccination GMCs. Seroconversion rates were 35.3% (95%CI: 29.0, 41.9) and 31.0% (95%CI: 25.1, 37.4) for Groups I and Group II, respectively. There was no substantive difference in safety profiles between both vaccines. Conclusions The complete regimen of ROTAVAC® demonstrated immunological non-inferiority to the complete regimen of Rotarix® with a clinically acceptable safety profile. Because the demand for RV vaccines is increasing as more countries are expanding their immunization schedules, the lack of need of a buffering agent, low dose volume (0.5 mL), non-interference with other concomitantly administered vaccines, and conformance with WHO-prequalification requirements provide ROTAVAC® the potential for widespread global usage. Post completion of this study, ROTAVAC® is now a WHO-prequalified vaccine. Clinical Trials Registration: (CTRI Number: CTRI/2015/12/006428).

Rotavirus G (RVG) strains have been detected in a variety of avian species, but RVG genomes have been published from only a single pigeon and two chicken strains. Two turkey RVG strains were identified and characterized, one in a hatchery with no reported health issues and the other in a hatchery with high embryo/poult mortality. The two turkey RVG strains shared only an 85.3 % nucleotide sequence identity in the VP7 gene while the other genes possessed high nucleotide identity among them (96.3–99.9 %). Low nucleotide percentage identities (31.6–87.3 %) occurred among the pigeon and chicken RVG strains. Interestingly, potential recombination events were detected between our RVG strains and a human RVB strain, in the VP6 and NSP3 segments. The epidemiology of RVG in avian flocks and the pathogenicity of the two different RVG strains should be further investigated to understand the ecology and impact of RVG in commercial poultry flocks.

Surveillance of Rotavirus A (RVA) infections in North America swine populations are limited and not performed over a significant time period to properly assess the diversity of RVA strains in swine. The VP7 (G) and VP4 (P) genes of 32 Canadian RVA strains, circulating between 2009 and 2015 were sequenced, identifying the G3P[13], G5P[7], G9P[7], G9[13], and G9[19] genotype combinations. The Canadian RVA strains were compared to the RVA strains present in the swine ProSystems RCE rotavirus vaccine. The comparison revealed multiple amino acid differences in the G and P antigenic epitopes, regardless of the G and P genotypes but specifically in the Canadian G3, P[13] and P[19] genotypes. Our study further contributes to the characterization of RVA's evolution and disease mitigation among swine, which may optimize target vaccine design, thereby minimizing RVA disease in this economically important animal population.

Objective To assess the safety and immunogenicity of the MF59®-adjuvanted trivalent influenza vaccine (aTIV; Fluad®) compared with modified aTIV formulations. Methods A total of 196 subjects ≥ 65 years were randomized to receive 7 different formulations of vaccine containing a range of adjuvant and antigen doses by single injection, or divided into two injections at a single time point. The primary study objective was to compare the serologic response of different formulations of aTIV containing increased amounts of adjuvant and antigen 21 days after vaccination. Subjects were followed for immunogenicity and safety for one year. Results The highest immune response, as measured by hemagglutination inhibition (HI) assay, 3 weeks after vaccination was observed in subjects in Group 6 with GMT 382.2 (95% confidence interval [CI] 237.5 to 615.0), 552.3 (364.8 to 836.1), and 54.1 (36.9 to 79.4) against A/H1N1, A/H3N2, and B respectively. Rates of seroconversion were also generally highest in this treatment group: 75% (95% CI 55.1 to 89.3), 75% (55.1 to 89.3), and 42.9% (24.5 to 62.8), respectively, against A/H1N1, A/H3N2, and B strains. The highest incidence of solicited adverse events (AEs) was reported by subjects who received both the highest dosage of antigen in combination with the highest dosage of adjuvant at the same site: 67.9% and 57.1% in Groups 4 and 6, respectively. The majority of solicited AEs were mild to moderate in severity. The number of unsolicited AEs was similar across the different dosages. Conclusion In this phase I trial of adults ≥ 65 years of age who received increased adjuvant and antigen dosages relative to the licensed aTIV, increased dosage of MF59 resulted in increased immunogenicity against all 3 components of seasonal influenza vaccine. The increase in immunogenicity was accompanied by an increase in the incidence of local reactogenicity.

Rotavirus B (RVB) has been associated with enteric disease in many animal species. An RVB strain was identified in pooled intestinal samples from Alpine caprine kids (between 2 and 3 days of age) experiencing high (>90 %) morbidity, and the complete caprine RVB genome was characterized. Histology revealed villus atrophy, the samples tested positive for RVB by real-time RT-PCR and metagenomic next-generation sequencing identified only RVB and orf virus. In the VP4 gene segment, the caprine RVB strain had a higher percentage nucleotide identity to the Indian bovine RVB strains than to the Japanese bovine RVB strains, but the VP7, VP6, VP2, NSP1, NSP2 and NSP5 gene segments of the American caprine RVB strain were genetically related to the Japanese bovine RVB strains. The results indicate a lack of RVB sequences to understand reassortment or the evolutionary relationship of RVB strains from cattle and goats.

Rotaviruses are important cause of diarrhea in animals, including humans. Currently, rotavirus species A, B, C, E, and H (RVA-RVC, RVE, and RVH) have been identified in pigs. Traditionally, RVA has been considered the primary cause of diarrhea in pigs, and RVB and RVC had been described sporadically in pigs until recently. Qualitative porcine RVA, RVB, and RVC RT-PCR (RT-qPCR) assays were designed and 7508 porcine diarrheic samples, submitted to University of Minnesota, were tested to estimate the percentage of RVA, RVB, and RVC over a period of approximately 2 years (from 2009 to 2011). The individual RVA and RVC RT-qPCR assays were multiplex into a single RT-qPCR while the RVB RT-qPCR assay remained as an individual RT-qPCR. In total, 83% of the samples were positive for RVA, RVB, or RVC. As expected, RVA was detected at the highest overall percentage (62%). However, 33% and 53% of the samples were positive for RVB and RVC, respectively, indicating that both RVB and RVC are also epidemiologically important in the swine population. RVC was most predominant in young pigs (1-20 days of age), while RVA and RVB were most predominant in ≥21 day old pigs. As diagnostic tools, the developed RT-qPCR assays could successfully discriminate among infecting RV species, which could lead to better surveillance and epidemiological studies for ultimately better prevention and control strategies.

During the vaccination phase of the Rotavirus Efficacy and Safety Trial (REST), the period between the administration of dose 1 through 13 days after the administration of dose 3, there were more wild-type rotavirus gastroenteritis (RVGE) cases among vaccine recipients compared with placebo recipients using the protocol-specified microbiological plaque assay in the clinical-efficacy cohort, a subset of subjects where vaccine efficacy against RVGE of any severity was assessed. In this study, a rotavirus genome segment 6-based reverse transcriptase–polymerase chain reaction assay was applied post hoc to clarify the accuracy of type categorization of all these RVGE cases in vaccine recipients during the vaccination phase of REST. The assay characterized 147 (90%) of 163 re-assayed RVGE cases or rotavirus-associated health care contacts as type-determinable: either wild-type or vaccine-type rotavirus strains. In the clinical-efficacy cohort (N = 5673), 19 (18.8%) of 101 samples from RVGE cases contained wild-type rotavirus, 70 (69.3%) vaccine virus, and 12 (11.9%) were indeterminable. In the large-scale cohort (N = 68,038), 10 (34.5%) of 29 samples from RVGE-related health care contacts contained wild-type rotavirus strains, 15 (51.7%) vaccine-type rotavirus strains, and 4 (13.8%) were indeterminable. Of the 33 samples from RVGE cases in placebo recipients, all were confirmed to contain wild-type rotaviruses. Altogether, this post-hoc re-evaluation showed that the majority (75%) of type-determinable RVGE cases or health care contacts that occurred during the vaccination phase of REST in vaccine recipients were associated with vaccine-type rotavirus strains rather than wild-type rotavirus strains.

Rotavirus may be an important causative agent of acute gastroenteritis (AGE) in the elderly, a population that is particularly vulnerable due to waning immunity. It is estimated that rotavirus may account for 2-5% of adult gastroenteritis hospitalizations in the United States. This is the first study to assess the safety and immunogenicity of the live pentavalent rotavirus vaccine (RV5) in an elderly population. In this study, healthy, independently living adults aged 65-80 years were randomized in a 2:1 ratio to receive three 2-mL oral doses of RV5 or placebo administered 28-42 days apart. All subjects were followed for safety for 42 days post any vaccination and up to 180 days after the final vaccination for clinical adverse events. Immunogenicity of RV5 was measured by serum anti-rotavirus IgA enzyme immunoassay and serum neutralizing antibody responses to human rotavirus serotypes prior to and after each dose. Results of this study demonstrated that RV5 was generally safe and well tolerated in healthy elderly adults, where 9% of placebo and 27% of RV5 recipients experienced a vaccine-related adverse event of mild or moderate intensity. Immune responses (serum anti-rotavirus immunoglobulin A [IgA] and serum neutralizing antibodies against human rotavirus serotypes in the vaccine) were augmented in this population after a single dose of RV5, despite the factors of older age and preexisting antibodies to the virus. Therefore, if vaccination in the elderly is needed, further evaluation of RV5 as a candidate vaccine in this age group may be warranted.

We investigated the presence in US pigs of rotavirus H (RVH), identified in pigs in Japan and Brazil. From 204 samples collected during 2006-2009, we identified RVH in 15% of fecal samples from 10 US states, suggesting that RVH has circulated in the United States since 2002, but probably longer. ispartof: Emerging Infectious Diseases vol:20 issue:7 pages:1195-1198 ispartof: location:United States status: published

Group A rotavirus (RVA) infections cause severe economic losses in intensively reared livestock animals, particularly in herds of swine and cattle. RVA strains are antigenically heterogeneous, and are classified in multiple G and P types defined by the two outer capsid proteins, VP7 and VP4, respectively. This study summarizes published literature on the genetic and antigenic diversity of porcine and bovine RVA strains published over the last 3 decades. The single most prevalent genotype combination among porcine RVA strains was G5P[7], whereas the predominant genotype combination among bovine RVA strains was G6P[5], although spatiotemporal differences in RVA strain distribution were observed. These data provide important baseline data on epidemiologically important RVA strains in swine and cattle and may guide the development of more effective vaccines for veterinary use.

During the 2008–2009 rotavirus season, 10 G3P[6] rotavirus strains were isolated for the first time in Belgium, while an outbreak of G2P[6] strains occurred in the USA in 2005–2006. Partial sequencing of the 11 genome segments of the 10 Belgian G3P[6] strains revealed a clonal origin. Two of these strains, and a G2P[6] strain representative of the American outbreak, were selected and sequenced completely to analyze their evolutionary relationships. Genetic analysis revealed that all strains possessed a DS-1-like genotype constellation. The 2 Belgian G3P[6] strains showed >99% sequence identity at the nucleotide level and the American G2P[6] strain was phylogenetically closely related to the Belgian P[6] strains. These data suggest that reassortment(s) involving VP7 occurred recently, and that the prevalence of DS-1-like P[6] rotavirus strains need to be closely monitored because the currently licensed RVA vaccines contain neither the P[6] genotype nor strains with a complete human DS-1 genotype constellation.

BACKGROUND P[6] rotaviruses have been circulating with a high prevalence in African and, to a more limited extent, Asian countries, but they have not been highly prevalent in other parts of the world. METHODS To investigate the genomic relationship between African and Asian human P[6] rotaviruses and P[4] and P[8] rotaviruses circulating worldwide, we sequenced 39 P[6] strains, collected in Ghana, Mali, Kenya and Bangladesh, providing the largest data set of P[6] rotavirus genomes isolated in low-income countries or anywhere else in the world that has been published thus far. RESULTS Overall, the data indicate that the genetic backbone of human P[6] strains from the low-income countries are similar to those of P[4] or P[8] strains circulating worldwide. CONCLUSIONS The observation that gene segment 4 is the main differentiator between human P[6] and non-P[6] strains suggests that the VP4 spike protein is most likely one of the main reasons preventing the rapid spread of P[6] strains to the rest of the world despite multiple introductions. These observations reinforce previous findings about the receptor specificity of P[6] rotavirus strains.

Background Efficacy of the pentavalent rotavirus vaccine (PRV), RotaTeq®, against severe rotavirus gastroenteritis (RVGE) was evaluated in two double-blind, placebo-controlled, multicenter Phase III clinical trials conducted in GAVI-eligible countries in Africa (Ghana, Kenya, and Mali) and in Asia (Bangladesh and Vietnam) from March 2007 through March 2009. The findings from each continent have been analyzed and presented separately, according to a single identical protocol. Ad hoc analyses combining data from the five sites were performed to further assess the impact of PRV. Methods 6674 infants (4705 infants from Africa and 1969 infants from Asia), randomized 1:1 to receive 3 doses of PRV/placebo at approximately 6-, 10-, and 14-weeks of age according to each country's EPI schedule, were included in the per protocol efficacy analysis. Breastfeeding and concomitant administration of EPI vaccines, including OPV, were allowed. Episodes of gastroenteritis (GE) in infants who presented to study facilities were captured and scored using the 20-point Vesikari scale. Stool samples were analyzed by rotavirus-specific EIA to detect presence of rotavirus antigen and RT-PCR to determine the G/P genotypes. We assessed efficacy to prevent all-cause GE and RVGE at a variety of cut-off points (score ≥ 11, severe; score ≥ 15, very severe). Results Vaccine efficacy (VE) against RVGE, regardless of serotype, through the entire follow-up period for any severity, severe (score ≥ 11), and very severe (score ≥ 15) was 33.9%, 95% CI (22.7, 43.5), 42.5%, 95% CI (27.4, 54.6), and 51.2%, 95% CI (26.3, 68.2), respectively. Through the first year of life, VE against severe RVGE was 58.9%, 95% CI (40.0, 72.3) and against all-cause severe GE was 23.0%, 95% CI (5.4, 37.3). VE against severe RVGE caused by non-vaccine G serotypes, G8 and G9, through the entire follow-up period was 87.5%, 95% CI (6.8, 99.7) and 48.0%, 95% CI (−5.5, 75.6), respectively. All G8 strains were associated with P2A[6] (a P-type not contained in PRV), while the majority of the G9 strains were associated with P1A[8] (a P-type contained in PRV). Conclusions Combining data from the 5 sites strengthens the precision of VE estimates and reveals rising VE with increased RVGE severity. Extrapolating data from VE against severe GE and RVGE suggest that 39% of severe GE episodes during the first year of life were due to rotavirus, highlighting substantial, potentially preventable, public health burden of RVGE. PRV provides protection against non-vaccine serotypes (G8P2A[6]).

Background We evaluated the immunogenicity of the pentavalent rotavirus vaccine (PRV) in two GAVI-eligible Asian countries, Bangladesh and Vietnam, nested in a larger randomized, double-blind, placebo-controlled efficacy trial conducted over a two-year period from 2007 through 2009. Methods 2036 infants were randomly assigned, in a 1:1 ratio, to receive three oral doses of PRV or placebo approximately at 6, 10, and 14 weeks of age. Concomitant use of EPI vaccines, including oral poliovirus vaccine (OPV) and diphtheria-tetanus-whole cell pertussis (DTwP) vaccine, was encouraged in accordance to the local EPI schedule. A total of 303 infants were evaluated for immunogenicity and blood samples were collected before the first dose (pD1) and approximately 14 days following the third dose (PD3). The seroresponse rates (≥3-fold rise from pD1 to PD3) and geometric mean titers (GMTs) were measured for anti-rotavirus immunoglobulin A (IgA) and serum neutralizing antibody (SNA) to human rotavirus serotypes G1, G2, G3, G4, and P1A[8], respectively. Results Nearly 88% of the subjects showed a ≥3-fold increase in serum anti-rotavirus IgA response in the analysis of the two countries combined. When analyzed separately, the IgA response was lower in Bangladeshi children (78.1% [95% CI: 66.0, 87.5]) than in Vietnamese children (97.0% [95% CI: 89.6, 99.6]), with a PD3 GMT of 29.1 (units/mL) and 158.5 (units/mL), respectively. In the combined population, the SNA responses to the individual serotypes tested ranged from 10 (G3) to 50 (G1) percentage points lower than the responses shown in the developed countries. However, the SNA response to G3 in Vietnamese subjects was 37.3% (95% CI: 25.8, 50.0), which was similar to the G3 response rate in developed countries. Conclusions Three oral doses of PRV were immunogenic in two GAVI-eligible Asian countries: Bangladesh and Vietnam. The GMTs of both the serum anti-rotavirus IgA and SNA responses were generally higher in Vietnamese than in Bangladeshi children. The SNA responses varied by individual serotypes and were lower than the results from developed countries. The clinical significance of these observations is not understood because an immune correlate of protection has not been established.

An efficacy clinical trial with pentavalent rotavirus vaccine (PRV), RotaTeq(®), was conducted at Matlab field site of ICDDR,B, Bangladesh from March 2007 to March 2009. The methodology, including operation logistics, and lessons-learned are described in this report. Vaccination was organized at 41 fixed-site clinics twice/month. A total of 1136 infants were randomized 1:1 to receive 3 doses of PRV/placebo at approximately 6-, 10-, and 14-weeks of age with routine vaccines of the Expanded Programme on Immunization (EPI) schedule. Twelve field-workers routinely visited study participants for safety and efficacy follow-up. The study was conducted following good clinical practices and maintaining cold-chain requirements. There were no temperature deviations of clinical vaccine supplies. Data entry was done using the source documents to a central database developed by the sponsor which was linked to web. Among enrolled infants, 1128 (99.3%) received 3 doses of PRV/placebo and efficacy follow-up was conducted for a median of 554 days. For the evaluation of immunogenicity, blood samples were collected from 150 participants predose 1 and from 147 (98%) of the same participants post dose 3. Stool samples were collected from 778 (99.9%) acute gastroenteritis episodes among children who reported to diarrhoea treatment centres. Thirty-nine serious adverse events, including 6 deaths, occurred among study participants. The efficacy of PRV against severe rotavirus gastroenteritis was 42.7% through the entire follow-up period; serum anti-rotavirus IgA response was 78.1%. Inclement weather, difficult transportation, and movement of study participants were some of the challenges identified. This is the first vaccine trial in rural Bangladesh with online data entry. The study was well accepted in the community and was completed successfully.

The oral, pentavalent rotavirus vaccine (PRV), RotaTeq was assessed for prevention of severe rotavirus gastroenteritis (RVGE) in young children in two multi-site, randomized, placebo-controlled field trials; one in Asia (Vietnam and Bangladesh) and the other in sub-Saharan Africa (Ghana, Kenya and Mali). The efficacy results for the Mali site of the multi-country trial are presented here. We randomly assigned infants in a 1:1 ratio to receive 3 doses of PRV/placebo at approximately 6, 10, and 14 weeks of age. Gastroenteritis episodes were captured passively at the local health centers and by home visits. The primary study outcome was severe RVGE, as defined by a score of ≥ 11 using the Vesikari Clinical Scoring System occurring ≥ 14 days after the third dose until the end of the study. Other efficacy analyses included efficacy against severe RVGE through the first year and during the second years of life, as well as efficacy after receiving at least one dose of vaccine. In total, 1960 infants were enrolled in the trial at the Mali site and sera were collected on a subset of infants (approximately 150) for immunogenicity testing. In the first year of follow-up, largely due to cultural practices to visit traditional healers as the first point of care, the point estimate of efficacy was unreliable: the per protocol vaccine efficacy against severe RVGE was 1% (95% confidence interval [CI]: -431.7, 81.6); the intention-to-treat vaccine efficacy was 42.9% (95% CI: -125.7, 87.7). During the second year of follow-up, after the surveillance system was modified to adapt to local customs and health care seeking practices, the point estimate of per-protocol vaccine efficacy was 19.2% (95% CI: -23.1,47.3%). 82.5% of Malian infants (95% CI: 70.1,91.3%) who received PRV mounted a seroresponse (≥ 3-fold rise from baseline (prevaccination) to post-dose 3 vaccination) of anti-rotavirus immunoglobulin A antibody, with a post third-dose geometric mean titer (GMT) of 31.3 units/mL. By contrast, only 20.0% of placebo recipients (95% CI: 10.0, 33.7%) developed a seroresponse and the post-third dose GMT was 3.2 units/mL. None of the serious clinical adverse events observed were considered to be vaccine-related.

Two multicenter Phase III trials were conducted in five countries from March 2007 to March 2009 to evaluate the safety and efficacy of the pentavalent rotavirus vaccine (PRV), RotaTeq(®), in Africa and Asia. In this report, we evaluate the safety of this vaccine, including among HIV-infected and HIV-exposed infants, in Kenya. 1308 Infants were randomized 1:1 to receive 3 doses of PRV/placebo at approximately 6, 10, and 14 weeks of age. HIV counseling and testing were offered to all participants. A positive PCR result indicated HIV infection; the presence of HIV antibody in PCR-negative children indicated HIV exposure without HIV infection. All serious adverse events (SAE) within 14 days of any dose, and vaccine-related SAEs, intussusception, and deaths occurring at any time during the study, were reported ("SAE surveillance"). In addition, 297 participants were followed for 42 days after any dose for any adverse event (AE), regardless of severity ("intensive safety surveillance"). The safety evaluation was stratified by HIV status. SAEs were reported in 20/649 vaccine recipients (3.1%) and 21/643 placebo recipients (3.3%) within 14 days following vaccination (p = 0.9). The most common SAE in the vaccinated group was pneumonia (1.7%). No individual SAE was significantly more common among vaccine vs. placebo recipients. Seventy-two deaths were reported, 38 (5.9%) and 34 (5.3%) among vaccine and placebo recipients, respectively (p = 0.66). No cases of intussusception were reported. During intensive safety surveillance, 137/147 (93.2%) vaccine recipients and 147/150 (98.0%) placebo recipients experienced one or more AEs (risk ratio = 0.95; 95% CI: 0.91-1.0; p = 0.05). 88.5% of the infants were tested for HIV infection; 21/581 (3.6%) children in the vaccine group and 17/577 (2.9%) in the placebo group were HIV-infected. Among the 37 HIV-infected infants with full safety follow-up, 5/21 (23.8%) vaccine recipients and 2/16 (12.5%) placebo recipients reported an SAE (p = 0.67). In total, 12 deaths occurred among identified HIV-infected infants: 8 (38%) receiving vaccine vs. 4 (23.5%) receiving placebo (RR = 1.6, 95% CI: 0.59-4.5). Among the 21 HIV-infected infants in the vaccine group, 2 of 8 deaths were gastroenteritis-related; among the 17 HIV-infected infants in the placebo group, 3 of 4 deaths were gastroenteritis-related. There were no significant differences in serious or non-serious AEs, including vaccine-related SAEs, between the 88 HIV-exposed vaccine recipients vs. the 89 HIV-exposed placebo recipients. PRV appears to be a safe intervention against rotavirus gastroenteritis among infants in Kenya. AEs, including serious AEs, were not associated with receipt of vaccine. Further, SAEs were not significantly more common among HIV-infected or HIV-exposed participants; however, the low number of HIV-infected infants did not provide sufficient power to fully assess safety in HIV-infected vaccine recipients.

Two live-attenuated rotavirus group A (RVA) vaccines, Rotarix (G1P[8]) and RotaTeq (G1-G4, P[8]), have been successfully introduced in many countries worldwide, including Belgium. The parental RVA strains used to generate the vaccines were isolated more than 20 years ago in France (G4 parental strain in RotaTeq) and the United States (all other parental strains). At present, little is known about the relationship between currently circulating human RVAs and the vaccine strains. In this study, we determined sequences for the VP7 and VP4 outer capsid proteins of representative G1P[8], G2P[4], G3P[8], G4P[8], G9P[8], and G12P[8] RVAs circulating in Belgium during 2007 to 2009. The analyses showed that multiple amino acid differences existed between the VP7 and VP4 antigenic epitopes of the vaccine viruses and the Belgian isolates, regardless of their G and P genotypes. However, the highest variability was observed among the circulating G1P[8] RVA strains and the G1 and P[8] components of both RVA vaccines. In particular, RVA strains of the P[8] lineage 4 (OP354-like) showed a significant number of amino acid differences with the P[8] VP4 of both vaccines. In addition, the circulating Belgian G3 RVA strains were found to possibly possess an extra N-linked glycosylation site compared to the G3 RVA vaccine strain of RotaTeq. These results indicate that the antigenic epitopes of RVA strains contained in the vaccines differ substantially from those of the currently circulating RVA strains in Belgium. Over time, these differences might result in selection for strains that escape the RVA neutralizing-antibody pressure induced by vaccines.

Rotavirus gastroenteritis (RVGE) is the leading cause of severe diarrhea in children worldwide. This paper provides an overview of the development, clinical evaluation, and postlicensure impact of RotaTeq™(Rotavirus Vaccine, Live, Oral, Pentavalent, Merck & Co., Inc.). RotaTeq, an oral vaccine, is uniquely designed to contain five human-bovine reassortant rotavirus strains expressing the human serotypes G1, G2, G3, G4, and P1A[8], which represent the most common human rotavirus serotypes responsible for ∼85% of RVGE worldwide. The development required novel solutions for manufacturing, testing, and formulation for each of the reassortants. In one of the largest vaccine clinical trials conducted, the vaccine was shown to be well tolerated and highly efficacious against severe RVGE. Efficacy has also been demonstrated in lower-income countries. In large U.S. postlicensure studies, there have been no safety signals identified, and RotaTeq has had a significant impact on reducing RVGE and its associated medical costs since licensure in 2006.

Background Influenza virus infection in the elderly remains one of the ten leading causes of death. One successful strategy to enhance the magnitude of their influenza vaccine immune response has been the addition of the adjuvant MF59®. Methods 196 subjects ≥ 65 years of age were enrolled in a dose ranging study with seven treatment arms to assess the safety and immunogenicity of the current formulation of aTIV compared with aTIV-modified formulations in which the dosage of MF59 was doubled or tripled and/or the dosage of the three influenza virus strains (A/H1N1, A/H3N2, and B) was doubled. Vaccine was administered by single or bilateral deltoid inoculations. The antibody responses to all three influenza virus vaccine strains were compared 21 days after a dose or doses of aTIV or aTIV-modified formulations, as measured by hemagglutination inhibition (HI) assay and microneutralization (MN) assay. Results In general, HI and MN titers at Day 22 increased to a greater degree with the dosage of MF59 compared with that of HA (HI presented in Figure 1). This was evident when comparing the HI and MN titers where antigen content was a constant 45 μg, but MF59 dose ranged from 9.75, 19.5 to 29.25 mg in a single vaccine dose (Group 1, 2 and 6, respectively). Generally, the highest titers against all strains were evident with the highest MF59 dose (29.25 mg). The relationship of antigen content and immunogenicity of the vaccine was less apparent when comparing titers between groups in which HA antigen content doubled from 45 to 90 μg. Administering the dose of MF59 (19.5 mg) and TIV (90 μg) into either a single arm or dividing between two arms resulted in comparable titers. The incidence of solicited AEs tended to increase with the dose of MF59 and to a lesser degree, antigen. The majority of solicited AEs were mild to moderate in severity. The number of unsolicited AEs were similar across the different dosages used in this trial. Conclusion In elderly subjects ≥65 years of age, increase in MF59 dose is associated with increased immunogenicity against all 3 components of seasonal influenza vaccine. Figure 1. Geometric mean titer (HI) against influenza A/H3N2/Texas/2012 according to dose of MF59 and HA. Disclosures G. Otten, Seqirus: Employee, Salary. V. Matassa, Seqirus: Employee, Salary. M. Ciarlet, Novartis Vaccines: Employee, Salary. B. Leav, Seqirus: Employee and Shareholder, Salary.

Cell culture derived rotavirus preparations contain a mixture of double-layered particles (DLPs) and triple-layered particles (TLPs). Characterization of rotavirus vaccine products is important to demonstrate a consistent manufacturing process. A capillary zone electrophoresis (CZE) method was developed to separate and quantitate rotavirus DLPs and TLPs in cell lysate samples and CsCl-purified vaccine preparations of each of the five reassortant rotavirus vaccine strains (G1, G2, G3, G4 and P1) contained in the pentavalent rotavirus vaccine, RotaTeq. The CZE electropherograms showed that migration of DLPs and TLPs from both CsCl-purified and cell lysates resulted in a separation distance of approximately 3 min between the two rotavirus particle types. The identification of the peak(s) containing TLPs was confirmed for both CsCl-purified and cell lysate samples by treatment of the samples with 50mM EDTA, which converted TLPs to DLPs. The migration pattern of the DLPs was consistent (23-24 min) among all reassortant strains tested, whether the DLPs were CsCl-purified or from cell lysates. However, the migration pattern of the TLP electropherograms of the reassortant rotavirus strains in cell lysates differed from those of the CsCl-purified reassortant rotavirus strains. In the cell lysate samples, the TLPs of the G1 and G2 reassortant rotavirus strains migrated slower that the corresponding TLPs from the CsCl-purified samples, while the migration time of the TLPs of the G3, G4 and P1 reassortants strains from the cell lysate and CsCl-purified samples appeared similar. Also, the TLPs from the CsCl-purified samples appeared as a defined single peak, while most of the TLPs from the cell lysate samples appeared as a broad peak or as multiple peaks. All the migration patterns were reproducible and consistent. Taking into account reproducibility, objective quantitation, and minimal sample manipulation as well as volume, CZE allowed consistent and quantitative characterization of rotavirus vaccine preparations, which is required for evaluation of vaccine products, including process validation.

Phase III studies of an oral, live, pentavalent, human-bovine reassortant rotavirus vaccine (RotaTeq; Merck) in developed countries have demonstrated that it is well tolerated with regard to intussusception and other adverse events and is efficacious in preventing rotavirus gastroenteritis and associated healthcare encounters. However, it cannot be assumed that rotavirus vaccines will be equally efficacious in infants and young children in the developing world. Differences in host populations, associated health conditions, and the epidemiology of rotavirus disease could affect vaccine performance. Concern about the potential for differences in efficacy stems from studies of previous candidate rotavirus vaccines, including bovine and rhesus rotaviruses, which showed no or variable efficacy in developing regions. Given this history, the World Health Organization (WHO) recommended that the efficacy of "new" rotavirus vaccines should be demonstrated in diverse geo- graphic areas, including developing countries, before widespread implementation. Successful implementation of any rotavirus vaccine in the developing world requires additional clinical research and sharing of early introduction experiences. We discuss efforts to bring RotaTeq vaccine to the developing world. Critical steps to achieve this goal include the clinical evaluation of vaccine safety and efficacy in a multisite trial in Asia and Africa, evaluation of concomitant use with other pediatric vaccines routinely used, and vaccine assessment in special populations (premature, human immunodeficiency virus-infected, and malnourished infants). Com- pletion of WHO prequalification of RotaTeq and affordability are also key requirements to routine vaccine introduction. The RotaTeq Partnership with the Nicaraguan Ministry of Health provides an example of the successful introduction of this vaccine into a developing world country.

Summary Background Rotavirus vaccine has proved effective for prevention of severe rotavirus gastroenteritis in infants in developed countries, but no efficacy studies have been done in developing countries in Asia. We assessed the clinical efficacy of live oral pentavalent rotavirus vaccine for prevention of severe rotavirus gastroenteritis in infants in Bangladesh and Vietnam. Methods In this multicentre, double-blind, placebo-controlled trial, undertaken in rural Matlab, Bangladesh, and urban and periurban Nha Trang, Vietnam, infants aged 4–12 weeks without symptoms of gastrointestinal disorders were randomly assigned (1:1) to receive three oral doses of pentavalent rotavirus vaccine 2 mL or placebo at around 6 weeks, 10 weeks, and 14 weeks of age, in conjunction with routine infant vaccines including oral poliovirus vaccine. Randomisation was done by computer-generated randomisation sequence in blocks of six. Episodes of gastroenteritis in infants who presented to study medical facilities were reported by clinical staff and from parent recollection. The primary endpoint was severe rotavirus gastroenteritis (Vesikari score ≥11) arising 14 days or more after the third dose of placebo or vaccine to end of study (March 31, 2009; around 21 months of age). Analysis was per protocol; infants who received scheduled doses of vaccine or placebo without intervening laboratory-confirmed naturally occurring rotavirus disease earlier than 14 days after the third dose and had complete clinical and laboratory results were included in the analysis. This study is registered with ClinicalTrials.gov, number NCT00362648. Findings 2036 infants were randomly assigned to receive pentavalent rotavirus vaccine (n=1018) or placebo (n=1018). 991 infants assigned to pentavalent rotavirus vaccine and 978 assigned to placebo were included in the per-protocol analysis. Median follow up from 14 days after the third dose of placebo or vaccine until final disposition was 498 days (IQR 480–575). 38 cases of severe rotavirus gastroenteritis (Vesikari score ≥11) were reported during more than 1197 person-years of follow up in the vaccine group, compared with 71 cases in more than 1156 person years in the placebo group, resulting in a vaccine efficacy of 48·3% (95% CI 22·3–66·1) against severe disease (p=0·0005 for efficacy >0%) during nearly 2 years of follow-up. 25 (2·5%) of 1017 infants assigned to receive vaccine and 20 (2·0%) of 1018 assigned to receive placebo had a serious adverse event within 14 days of any dose. The most frequent serious adverse event was pneumonia (vaccine 12 [1·2%]; placebo 15 [1·5%]). Interpretation In infants in developing countries in Asia, pentavalent rotavirus vaccine is safe and efficacious against severe rotavirus gastroenteritis, and our results support expanded WHO recommendations to promote its global use. Funding PATH (GAVI Alliance grant) and Merck.

These viruses may become human pathogens., G11 rotaviruses are believed to be of porcine origin. However, a limited number of G11 rotaviruses have been recently isolated from humans in combination with P[25], P[8], P[6], and P[4]. To investigate the evolutionary relationships of these strains, we analyzed the complete genomes of 2 human G11P[25] strains, 2 human G11P[8] strains, and 3 porcine reference strains. Most of the 11 gene segments of these 7 strains belonged to genotype 1 (Wa-like). However, phylogenetic clustering patterns suggested that an unknown G11P[25] strain with a new I12 VP6 genotype was transmitted to the human population, in which it acquired human genotype 1 gene segments through reassortment, resulting in a human G11P[8] rotavirus strain with an entire human Wa-genogroup backbone. This Wa-like backbone is believed to have caused the worldwide spread of human G9 and G12 rotaviruses. G11 human rotavirus strains should be monitored because they may also become major human pathogens.

Rotavirus nonstructural protein 4 (NSP4) is a protein with pleiotropic properties. It functions in rotavirus morphogenesis, pathogenesis, and is the first described viral enterotoxin. Since many bacterial toxins function as potent mucosal adjuvants, we evaluated whether baculovirus-expressed recombinant simian rotavirus SA11 NSP4 possesses adjuvant activity by co-administering NSP4 with keyhole limpet hemocyanin (KLH), tetanus toxoid (TT) or ovalbumin (OVA) as model antigens in mice. Following intranasal immunization, NSP4 significantly enhanced both systemic and mucosal immune responses to model immunogens, as compared to the control group, in an antigen-specific manner. Both full-length and a cleavage product of SA11 NSP4 had adjuvant activity, localizing this activity to the C-terminus of the protein. NSP4 forms from virulent and avirulent porcine rotavirus OSU strain, and SA11 NSP4 localized within a 2/6-virus-like particle (VLP) also exhibited adjuvant effects. These studies suggest that the rotavirus enterotoxin NSP4 can function as an adjuvant to enhance immune responses for a co-administered antigen.

Rotaviruses are important enteric pathogens of humans and animals. Group A rotaviruses (GARVs) account for up to 1 million children deaths each year, chiefly in developing countries and human vaccines are now available in many countries. Rotavirus-associated enteritis is a major problem in livestock animals, notably in young calves and piglets. Early in the epidemiological GARV studies in humans, either sporadic cases or epidemics by atypical, animal-like GARV strains were described. Complete genome sequencing of human and animal GARV strains has revealed a striking genetic heterogeneity in the 11 double stranded RNA segments across different rotavirus strains and has provided evidence for frequent intersections between the evolution of human and animal rotaviruses, as a result of multiple, repeated events of interspecies transmission and subsequent adaptation.

Background: The pentavalent rotavirus vaccine (PRV), RotaTeq, can be concomitantly administered with most routine childhood vaccines. This study evaluated the immunogenicity and reactogenicity of PRV when used concomitantly with a hexavalent vaccine containing diphtheria, tetanus, acellular pertussis, hepatitis B, inactivated poliovirus, and Haemophilus influenzae type b. Methods: Healthy infants (N = 403) received hexavalent vaccine concomitantly with either PRV or placebo at 2, 3, and 4 months of age. Antibody responses were measured immediately before and 42 +/- 3 days after vaccination. Parents/legal guardians recorded all adverse events for 14 days after vaccination. Results: Seroprotective titers for hepatitis B (hepatitis B surface antigen >=10 mIU/mL) were achieved by 97.8% of subjects in both vaccine treatment groups. Seroprotective titers to H. influenzae type b (polyribosylribitol phosphate >=0.15 [mu]g/mL) were achieved by 91.4% of subjects receiving both vaccines and 95.1% of subjects receiving only hexavalent vaccine. Seroprotective titers to diphtheria, tetanus, and poliovirus were also comparable between the vaccine treatment groups, as were geometric mean antibody titers to the pertussis antigens. Among PRV recipients, 92% had a >=3-fold rise in serum antirotavirus immunoglobulin A levels. Concomitant administration was well tolerated. The incidence of adverse events was similar for both groups, with no statistically significant increases in fever, vomiting, diarrhea, or irritability. Conclusions: In this study, concomitant administration of PRV with hexavalent vaccine was well tolerated and the immune responses to the antigens of the hexavalent vaccine were noninferior when compared with those of the control group. In addition, PRV was immunogenic when administered concomitantly with hexavalent vaccine.

Recently, a classification system was proposed for rotaviruses in which all the 11 genomic RNA segments are used (Matthijnssens et al., 2008; J. Virol. 82:3204-3219). Based on nucleotide identity cut-off percentages, different genotypes were defined for each genome segment. A nomenclature for the comparison of complete rotavirus genomes was considered in which the notations Gx-P[x]-Ix-Rx-Cx-Mx-Ax-Nx-Tx-Ex-Hx are used for the VP7-VP4-VP6-VP1-VP2-VP3-NSP1-NSP2-NSP3-NSP4-NSP5/6 encoding genes, respectively. This classification system is an extension of the previously applied, genotype-based system which made use of the rotavirus gene segments encoding VP4, VP7, VP6, and NSP4. In order to assign rotavirus strains to one of the established genotypes or a new genotype, a standard procedure is proposed in this report. As more human and animal rotavirus genomes will be completely sequenced, new genotypes for each of the eleven gene segments may be identified. A Rotavirus Classification Working Group (RCWG) including specialists in molecular virology, infectious diseases, epidemiology, and public health was formed, which can assist in the appropriate delineation of new genotypes, thus avoiding duplications and helping minimize errors. Scientists discovering a potentially new rotavirus genotype for any of the 11 gene segments are invited to send the novel sequence to the RCWG, where the sequence will be analyzed, and a new nomenclature will be advised as appropriate. The RCWG will update the list of classified strains regularly and make this accessible on a website. Close collaboration with the Study Group Reoviridae of the International Committee on the Taxonomy of Viruses will be maintained.

Group A rotavirus classification is currently based on the molecular properties of the two outer layer proteins, VP7 and VP4, and the middle layer protein, VP6. As reassortment of all the 11 rotavirus gene segments plays a key role in generating rotavirus diversity in nature, a classification system that is based on all the rotavirus gene segments is desirable for determining which genes influence rotavirus host range restriction, replication, and virulence, as well as for studying rotavirus epidemiology and evolution. Toward establishing such a classification system, gene sequences encoding VP1 to VP3, VP6, and NSP1 to NSP5 were determined for human and animal rotavirus strains belonging to different G and P genotypes in addition to those available in databases, and they were used to define phylogenetic relationships among all rotavirus genes. Based on these phylogenetic analyses, appropriate identity cutoff values were determined for each gene. For the VP4 gene, a nucleotide identity cutoff value of 80% completely correlated with the 27 established P genotypes. For the VP7 gene, a nucleotide identity cutoff value of 80% largely coincided with the established G genotypes but identified four additional distinct genotypes comprised of murine or avian rotavirus strains. Phylogenetic analyses of the VP1 to VP3, VP6, and NSP1 to NSP5 genes showed the existence of 4, 5, 6, 11, 14, 5, 7, 11, and 6 genotypes, respectively, based on nucleotide identity cutoff values of 83%, 84%, 81%, 85%, 79%, 85%, 85%, 85%, and 91%, respectively. In accordance with these data, a revised nomenclature of rotavirus strains is proposed. The novel classification system allows the identification of (i) distinct genotypes, which probably followed separate evolutionary paths; (ii) interspecies transmissions and a plethora of reassortment events; and (iii) certain gene constellations that revealed (a) a common origin between human Wa-like rotavirus strains and porcine rotavirus strains and (b) a common origin between human DS-1-like rotavirus strains and bovine rotaviruses. These close evolutionary links between human and animal rotaviruses emphasize the need for close simultaneous monitoring of rotaviruses in animals and humans.

Twenty-five years passed between the discovery of the parent strain (WC3) of the pentavalent human-bovine reassortant rotavirus vaccine (PRV) in 1981 and the licensure of PRV in 2006. This orally administered liquid vaccine, which is given as a 3-dose series, is indicated for the prevention of rotavirus gastroenteritis, caused by the G serotypes contained in the vaccine, in infants and children. PRV is recommended by the Advisory Committee on Immunization Practices and the American Academy of Pediatrics for administration to all infants in the United States. These recommendations are supported by the results of the phase III studies, which demonstrated that PRV is well tolerated and efficacious. PRV reduced rotavirus-related hospitalizations by 96% and was not associated with an increased incidence of serious adverse events, including intussusception. This report focuses on the safety and efficacy data from the late-phase studies of PRV and discusses plans for providing this vaccine to the developing world.

RotaTeqTM is a pentavalent rotavirus vaccine based on a bovine rotavirus genetic backbone in vitro reassorted with human outer capsid genes. During clinical trials of RotaTeqTM in Sub-Saharan Africa, the vaccine efficacy over a 2-year follow-up was lower against the genotypes contained in the vaccine than against the heterotypic G8P[6] and G8P[1] rotavirus strains of which the former is highly prevalent in Africa. Complete genome analyses of 43 complete rotavirus genomes collected during phase III clinical trials of RotaTeqTM in Sub-Saharan Africa, were conducted to gain insight into the high level of cross-protection afforded by RotaTeqTM against these G8 strains. Phylogenetic analysis revealed the presence of a high number of bovine rotavirus gene segments in these human G8 strains. In addition, we performed an in depth analysis on the individual amino acid level which showed that G8 rotaviruses were more similar to the RotaTeqTM vaccine than non-G8 strains. Because RotaTeqTM possesses a bovine genetic backbone, the high vaccine efficacy against G8 strains might be partially explained by the fact that all these strains contain a complete or partial bovine-like backbone. Altogether, this study supports the hypothesis that gene segments other than VP7 and VP4 play a role in vaccine-induced immunity. ispartof: Scientific Reports vol:5 pages:14658- ispartof: location:England status: published

The immune response elicited by the rotavirus nonstructural protein NSP4 and its potential role in protection against rotavirus disease are not well understood. We investigated the serological response to NSP4 and its correlation with disease protection in sera from 110 children suffering acute diarrhea, associated or not with rotavirus, and from 26 children who were recipients of the rhesus rotavirus tetravalent (RRV-TV) vaccine. We used, as antigens in an enzyme-linked immunosorbent assay (ELISA), affinity-purified recombinant NSP4 (residues 85 to 175) from strains SA11, Wa, and RRV (genotypes A, B, and C, respectively) fused to glutathione S -transferase. Seroconversion to NSP4 was observed in 54% (42/78) of the children who suffered from natural rotavirus infection and in 8% (2/26) of the RRV-TV vaccine recipients. Our findings indicate that NSP4 evokes significantly ( P < 0.05) higher seroconversion rates after natural infection than after RRV-TV vaccination. The serum antibody levels to NSP4 were modest (titers of ≤200) in most of the infected and vaccinated children. A heterotypic NSP4 response was detected in 48% of the naturally rotavirus-infected children with a detectable response to NSP4. Following natural infection or RRV-TV vaccination, NSP4 was significantly less immunogenic than the VP6 protein when these responses were independently measured by ELISA. A significant ( P < 0.05) proportion of children who did not develop diarrhea associated with rotavirus had antibodies to NSP4 in acute-phase serum, suggesting that serum antibodies against NSP4 might correlate with protection from rotavirus diarrhea. In addition, previous exposures to rotavirus did not affect the NSP4 seroconversion rate.

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 (

Summary Background Rotavirus infection is thought to be confined to the intestine. Reports of rotavirus RNA in the cerebral spinal fluid and serum of children infected with rotavirus suggest the possibility that rotavirus escapes the intestine into the circulatory system. We assessed whether rotavirus antigen, RNA, or both, were present in serum samples from immunocompetent rotavirus-infected children and animals. Methods We obtained sera from immunocompetent mice, rats, rabbits, and calves 1–10 days after inoculation with rotavirus or matched vehicle. We obtained sera retrospectively from immunocompetent children diagnosed with rotavirus diarrhoea (n=33), healthy children (n=6) and adults (n=12), children convalescing from rotavirus (n=6), and children with non-rotavirus diarrhoea (n=11). Samples were analysed for the presence of rotavirus antigen or RNA by EIA or RT-PCR, respectively. Findings Rotavirus antigen was present in sera from rotavirus-infected animals, but not in sera from control animals. Infectious rotavirus or rotavirus RNA was detected in sera of mice and calves, respectively. Antigen was present in 22 of 33 serum samples from children with confirmed rotavirus infection but in none of 35 samples from controls. Detection of serum antigen was inversely related to the number of days between symptom onset and sample collection, and directly related to stool antigen concentration. Rotavirus RNA was detected by RT-PCR in three of six rotavirus-positive sera. Interpretation Rotavirus can escape the gastrointestinal tract in children, resulting in antigenaemia and possible viraemia. This finding is important for the understanding of the pathogenesis, immunology, and clinical manifestations of rotavirus infection.

The gene encoding the VP8* trypsin-cleavage product of the VP4 protein of porcine rotavirus strain A34 was sequenced, and the predicted amino acid (aa) sequence was compared to the homologous region of all known P genotypes. The aa sequence of the VP8* of strain A34 shared low identity, ranging from 39% (bovine strain B223, P8[11]) to 76% (human strain 69M, P4[10]), with the homologous sequences of representative strains of the remaining 21 P genotypes. Phylogenetic relationships showed that the VP8* of strain A34 shares a common evolutionary lineage with those of human 69M (P4[10]) and equine H-2 (P4[12]) strains. Hyperimmune sera raised to strain A34 and to a genetic reassortant strain containing the VP4 gene from strain A34, both with high homologous neutralization titer via VP4, failed to neutralize strains representative of 15 different P genotypes. These results indicate that strain A34 should be considered as prototype of a new P genotype and serotype (P14[23]) and provide further evidence for the vast genetic and antigenic diversity of group A rotaviruses.

The genes encoding the glycoprotein VP7, the VP8* trypsin-cleavage product of the protein VP4, a fragment of the protein VP6 associated with subgroup (SG) specificity, and the enterotoxin NSP4 of rotavirus strains identified in diarrheic fecal samples of rabbits in Italy were sequenced. The Italian lapine rotavirus (LRV) strains possessed a G3 VP7, SG I VP6, and KUN-like NSP4, a gene constellation typical of LRVs. One LRV strain (30/96), isolated in 1996, shared the closest amino acid (aa) identity (87–96%) with the P[14] genotype, composed of human and LRV strains. Conversely, three LRV strains (160/01, 229/01, and 308/01), identified in 2001, were highly identical (90–95%) among each other, but showed low aa identity (34–77%) to the VP8* genotype-specific sequences of representative rotavirus strains of all remaining P genotypes. This report confirms the worldwide genetic constellations of LRVs and identifies a novel VP4 genotype in rabbits, tentatively proposed as genotype P[22].

Virus-like particles (VLPs) are being evaluated as a candidate rotavirus vaccine. Rotavirus VLPs composed of simian SA11 strain VP2 and VP6 proteins (homologous 2/6-VLPs) were produced by cloning the rotavirus simian SA11 genes 2 and 6 into a single baculovirus transfer vector (pAcAB4). The overall yield of homologous 2/6-VLPs produced with the dual recombinant baculovirus was at least 30-fold higher than that of VLPs composed of bovine RF strain VP2 and simian SA11 strain VP6 (heterologous 2/6-VLPs), produced with single recombinant baculoviruses. Adult mice were immunized intramuscularly twice with various doses of homologous or heterologous 2/6-VLPs in QS-21, orally with or without cholera toxin (CT), or intranasally with mutant Escherichia coli heat-labile enterotoxin (LT-R192G). Both homologous and heterologous 2/6-VLPs were immunogenic and induced protection from challenge, with those administered parenterally or intranasally affording the highest mean protection from challenge. The 2/6-VLPs did not induce serum neutralizing antibody (N-Ab) responses, but these VLPs primed for a broad heterotypic N-Ab response, which was elicited after rotavirus challenge. Heterotypic N-Ab responses were not observed in 2/6-VLP vaccinated mice that were > or =94% protected from challenge. After challenge, control mice immunized with adjuvant alone developed only homotypic serum N-Ab responses. Similar results were obtained after challenge of rabbits immunized parenterally or intranasally with heterologous 2/6-VLPs. These results suggest that 2/6-VLPs prime the immune system to enhance the production of heterotypic N-Ab responses, but the induction of heterotypic N-Abs requires that virus replication occurs after challenge. The use of 2/6-VLPs expressed from a single recombinant baculovirus simplifies production and would reduce the cost of a VLP-based vaccine.

Group A rotaviruses are major pathogens causing acute gastroenteritis in children and animals. To determine if group A rotavirus replicates and induces disease in rats, antibody-negative Lewis neonatal or adult rats were inoculated orally with tissue culture-adapted human (Wa, WI61, and HAL1166), simian (rhesus rotavirus [RRV] and SA11), bovine (WC3), lapine (ALA), or porcine (OSU) rotavirus strains, wild-type murine (EC wt ) rotavirus strain, or phosphate-buffered saline (PBS). Rotavirus infection in rats was evaluated by (i) clinical findings, (ii) virus antigen shedding or infectious virus titers in the feces or intestinal contents measured by enzyme-linked immunosorbent assay or fluorescent-focus assay, (iii) histopathological changes in the small intestine, (iv) distribution of rotavirus antigen in small-intestine sections by immunofluorescence, and (v) growth rate. Rotavirus infection of 5-day-old but not ≥21-day-old rats resulted in diarrhea that lasted from 1 to 10 days postinoculation. The severity of disease and spread of infection to naÏve littermates differed depending on the virus strain used for inoculation. The duration of virus antigen shedding following infection was considerably prolonged (up to 10 days) in neonatal rats compared to that in 21-day-old rats (1 or 2 days). Based on lack of virus antigen shedding and disease induction, the murine EC wt rotavirus was the only strain tested that did not infect rats. Histopathological changes in the small-intestine mucosa of 5-day-old RRV-inoculated rats but not of PBS-inoculated rats was limited to extensive enterocyte vacuolation in the ileum. In RRV-inoculated neonatal rats, rotavirus antigen was detected in the epithelial cells on the upper half of the intestinal villi of the jejunum and ileum. In addition, infection of neonatal rats with RRV but not with PBS resulted in reduced weight gain. Rats infected with group A rotaviruses provide a new animal model with unique features amenable to investigate rotavirus pathogenesis and the molecular mechanisms of intestinal development, including physiological factors that may regulate age-dependent rotavirus-induced diarrhea.

Infection of epithelial cells by some animal rotaviruses, but not human or most animal rotaviruses, requires the presence ofN-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-μm 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 [3H]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.

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.

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.