Evolution of Animal South American RVA Told by the NSP4 Gene E12 Genotype.

Rotavirus A (RVA) possesses a genome of 11 double-stranded (ds) RNA segments, and each segment encodes one protein, with the exception of segment 11. NSP4 is a non-structural multifunctional protein encoded by segment 10 that defines the E-genotype. From the 31 E-genotypes described, genotype E12 has been described in Argentina, Uruguay, Paraguay, and Brazil in RVA strains infecting different animal species and humans. In this work, we studied the evolutionary relationships of RVA strains carrying the E12 genotype in South America using phylogenetic and phylodynamic approaches. We found that the E12 genotype has a South American origin, with a guanaco (Lama guanicoe) strain as natural host. Interestingly, all the other reported RVA strains carrying the E12 genotype in equine, bovine, caprine, and human strains are related to RVA strains of camelid origin. The evolutionary path and genetic footprint of the E12 genotype were reconstructed starting with the introduction of non-native livestock species into the American continent with the Spanish conquest in the 16th century. The imported animal species were in close contact with South American camelids, and the offspring were exposed to the native RVA strains brought from Europe and the new RVA circulating in guanacos, resulting in the emergence of new RVA strains in the current lineages' strongly species-specific adaption. In conclusion, we proposed the NSP4 E12 genotype as a genetic geographic marker in the RVA strains circulating in different animal species in South America.

Impact of Vaccination on Rotavirus Genotype Diversity: A Nearly Two-Decade-Long Epidemiological Study before and after Rotavirus Vaccine Introduction in Sicily, Italy.

Sicily was the first Italian region to introduce rotavirus (RV) vaccination with the monovalent G1P[8] vaccine Rotarix® in May 2012. In this study, the seasonal distribution and molecular characterization of RV strains detected over 19 years were compared to understand the effect of Rotarix® on the evolutionary dynamics of human RVs. A total of 7846 stool samples collected from children < 5 years of age, hospitalized with acute gastroenteritis, were tested for RV detection and genotyping. Since 2013, vaccine coverage has progressively increased, while the RV prevalence decreased from 36.1% to 13.3% with a loss of seasonality. The local distribution of RV genotypes changed over the time possibly due to vaccine introduction, with a drastic reduction in G1P[8] strains replaced by common and novel emerging RV strains, such as equine-like G3P[8] in the 2018−2019 season. Comparison of VP7 and VP4 amino acid (aa) sequences with the cognate genes of Rotarix® and RotaTeq® vaccine strains showed specific aa changes in the antigenic epitopes of VP7 and of the VP8* portion of VP4 of the Italian RV strains. Molecular epidemiological surveillance data are required to monitor the emergence of novel RV strains and ascertain if these strains may affect the efficacy of RV vaccines.

A phase 1, randomized, observer blind, antigen and adjuvant dosage finding clinical trial to evaluate the safety and immunogenicity of an adjuvanted, trivalent subunit influenza vaccine in adults ≥ 65 years of age.

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 receive7different formulations of vaccine containing a range of adjuvant and antigen dosesby 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 antigen21 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.

A randomized, open-labelled, non-inferiority phase 4 clinical trial to evaluate the immunogenicity and safety of the live, attenuated, oral rotavirus vaccine, ROTAVAC® in comparison with a licensed rotavirus vaccine in healthy infants.

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).

Complete genome characterization of a rotavirus B (RVB) strain identified in Alpine goat kids with enteritis reveals inter-species transmission with RVB bovine strains.

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.

Genome characterization of Turkey Rotavirus G strains from the United States identifies potential recombination events with human Rotavirus B strains.

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.

Genotypic and epitope characteristics of group A porcine rotavirus strains circulating in Canada.

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.

Human P[6] Rotaviruses From Sub-Saharan Africa and Southeast Asia Are Closely Related to Those of Human P[4] and P[8] Rotaviruses Circulating Worldwide.

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.

Three-Level Mixed-Effects Logistic Regression Analysis Reveals Complex Epidemiology of Swine Rotaviruses in Diagnostic Samples from North America.

Rotaviruses (RV) are important causes of diarrhea in animals, especially in domestic animals. Of the 9 RV species, rotavirus A, B, and C (RVA, RVB, and RVC, respectively) had been established as important causes of diarrhea in pigs. The Minnesota Veterinary Diagnostic Laboratory receives swine stool samples from North America to determine the etiologic agents of disease. Between November 2009 and October 2011, 7,508 samples from pigs with diarrhea were submitted to determine if enteric pathogens, including RV, were present in the samples. All samples were tested for RVA, RVB, and RVC by real time RT-PCR. The majority of the samples (82%) were positive for RVA, RVB, and/or RVC. To better understand the risk factors associated with RV infections in swine diagnostic samples, three-level mixed-effects logistic regression models (3L-MLMs) were used to estimate associations among RV species, age, and geographical variability within the major swine production regions in North America. The conditional odds ratios (cORs) for RVA and RVB detection were lower for 1-3 day old pigs when compared to any other age group. However, the cOR of RVC detection in 1-3 day old pigs was significantly higher (p < 0.001) than pigs in the 4-20 days old and >55 day old age groups. Furthermore, pigs in the 21-55 day old age group had statistically higher cORs of RV co-detection compared to 1-3 day old pigs (p < 0.001). The 3L-MLMs indicated that RV status was more similar within states than among states or within each region. Our results indicated that 3L-MLMs are a powerful and adaptable tool to handle and analyze large-hierarchical datasets. In addition, our results indicated that, overall, swine RV epidemiology is complex, and RV species are associated with different age groups and vary by regions in North America.

Comparative analysis of pentavalent rotavirus vaccine strains and G8 rotaviruses identified during vaccine trial in Africa.

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.

Safety and immunogenicity of pentavalent rotavirus vaccine in a randomized, double-blind, placebo-controlled study in healthy elderly subjects.

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.

Analysis by rotavirus gene 6 reverse transcriptase-polymerase chain reaction assay of rotavirus-positive gastroenteritis cases observed during the vaccination phase of the Rotavirus Efficacy and Safety Trial (REST).

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.

Rapid detection and high occurrence of porcine rotavirus A, B, and C by RT-qPCR in diagnostic samples.

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.

Widespread rotavirus H in commercially raised pigs, United States.

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.

VP6 genetic diversity, reassortment, intragenic recombination and classification of rotavirus B in American and Japanese pigs.

Rotavirus B (RVB) has been identified as a causative agent of diarrhea in rats, humans, cattle, lambs, and swine. Recently, 20 RVB VP7 genotypes were determined based on an 80% nucleotide percent cut-off value. In this study, we sequenced the RVB VP6 gene segment from 80 RVB positive swine samples from the United States and Japan. Phylogenetic analyses, using the 30 available RVB VP6 sequences from GenBank and our 80 novel RVB VP6 sequences, revealed a large genetic diversity of RVB strains, mainly in pigs. For classification purposes, pairwise identity frequency analyses suggested an 81% nucleotide percent cut-off value, resulting in 13 RVB VP6 (I) genotypes. In addition, an intragenic recombinant RVB VP6 segment was identified from Japan. Furthermore, the data indicates frequent reassortment events occurred between the porcine RVB VP7 and VP6 gene segments.

Identification, phylogenetic analysis and classification of porcine group C rotavirus VP7 sequences from the United States and Canada.

Rotavirus C (RVC) is a major cause of gastroenteritis in swine. Between December 2009 and October 2011, 7520 porcine samples were analyzed from herds in the US and Canada. RVC RNA was detected in 46% of the tested samples. In very young pigs (≤3 days old) and young piglets (4-20 days old), 78% and 65%, respectively, RVC positive samples were negative for RVA and RVB. RVC RNA was also detected in 10% of tested lung tissues. Additionally, we investigated the porcine RVC molecular diversity by sequencing the VP7 gene segment of 65 specimens, yielding 70 VP7 gene sequences. Based on pairwise identity frequency profiles and phylogenetic analyses, an 85% nucleotide classification cut-off value was calculated using the novel sequence data generated in this study (n=70) and previously published RVC VP7 sequences (n=82), which resulted in the identification of 9 VP7 RVC genotypes, G1 to G9.

Global distribution of group A rotavirus strains in horses: a systematic review.

Group A rotavirus (RVA) is a major cause of diarrhea and diarrhea-related mortality in foals in parts of the world. In addition to careful horse farm management, vaccination is the only known alternative to reduce the RVA associated disease burden on horse farms. The precise evaluation of vaccine effectiveness against circulating strains needs enhanced surveillance of equine RVAs in areas where vaccine is already available or vaccine introduction is anticipated. Therefore, we undertook the overview of relevant information on epidemiology of equine RVA strains through systematic search of public literature databases. Our findings indicated that over 99% of equine RVA strains characterized during the past three decades belonged to two common genotypes, G3P[12] and G14P[12], whereas most of the minority equine RVA strains were probably introduced from a heterologous host by interspecies transmission. These baseline data on RVA strains in horses shall contribute to a better understanding of the spatiotemporal dynamics of strain prevalence in vaccinated and non-vaccinated herds.

The path to an RSV vaccine.

Respiratory syncytial virus (RSV) is the greatest remaining unmet infant vaccine need in developed countries and an important unmet infant vaccine need worldwide. More than 40 years of effort have yet to result in a licensed RSV vaccine for humans. Key challenges to RSV vaccine development include a peak of severe disease at 2-3 months of age, problematic biochemical behavior of key vaccine antigens, a history of vaccine-mediated disease enhancement, and reliance on animal models that may not accurately reflect human disease processes. Potential paths to overcome these challenges include maternal immunization, structure-based engineering of vaccine antigens, the design of a novel platform for safe infant immunization, and the development of improved animal models for vaccine-enhanced disease.

Review of group A rotavirus strains reported in swine and cattle.

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.

Complete genetic characterization of human G2P[6] and G3P[6] rotavirus strains.

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.