Jon R. Gentsch

Characterization of unusual G8 rotavirus strains isolated from Egyptian children

SummaryWe report the first detection of P[14], G8 rotaviruses isolated in Egypt from the stool of children participating in a 3 year study of rotavirus epidemiology. Two strains, EGY1850 and EGY2295, were characterized by a serotyping enzyme immunoassay (EIA), virus neutralization, and sequence analysis of the genes encoding VP7 and the VP8* portion of the VP4 gene. These two strains shared a high level of homology of their VP7s (87.8% nucleotide [nt], 97.2% amino acid [aa]) and VP4s (89.6% nt, 97.1% aa) and had the highest VP7 identity to serotype G8 (>82% nt, >92% aa) and VP4 identity to genotype P[14] (≥81% nt, >91% aa) strains. Serological results with a VP7 G8-specific and VP4 P[14]-specific neutralizing monoclonal antibodies supported the genetic classification of EGY1850 and EGY2295 as P[14], G8. Genogroup analysis supports earlier findings that human G8 rotaviruses may be genetically related to bovine rotaviruses. These findings demonstrate that our understanding of the geographic distribution of rotavirus strains is incomplete, emphasize the need to monitor rota- virus serotypes, and extend the known distribution of serotype G8 and genotype P[14] strains in Africa.

Characterization of human rotavirus genotype P(8)G5 from Brazil by probe-hybridization and sequence

SummaryWe report the molecular characterization of rotavirus genotype P[8]G5 strains found in fecal specimens collected in four different regions of Brazil, using digoxigenin (dig)-labeled oligonucleotide probes, sequence analysis, and RNA-RNA hybridization. The closest sequence relationships of the neutralization antigens of these strains were to the VP4 protein of P1A[8]G1 strain KU (93.3% identity in amino acids 11 to 282) and to the VP7 protein of G serotype 5 strain OSU (87.6% identity in amino acids 8 to 232). Based on VP7 sequence differences, we designed dig-probes that allowed us to discriminate porcine OSU-like strains from G5 strains isolated from Brazilian infants. The genetic relationships of two P[8]G5 isolates to other rotavirus genogroups were analyzed by RNA-RNA hybridization with [32P]-GTP probes representative of serotypes P1A[8]G1 (Wa), P[8]G3 (AU17), and P9[7]G5 (OSU). The Brazilian P[8]G5 strains showed sequence homology with genes of Wa-like and OSU-like strains, suggesting that these two strains were naturally occurring reassortants between members of the Wa and porcine rotavirus genogroups. The identification of these strains in diverse geographic areas of Brazil underscores their stability and demonstrates the emergence of clinically important rotavirus diarrhea strains by reassortment.

Molecular epidemiology of group A rotavirus in Buenos Aires, Argentina 2004-2007: reemergence of G2P[4] and emergence of G9P[8] strains.

Detection and characterization of group A rotavirus in Buenos Aires, Argentina, was conducted on 710 fecal samples from children 0–15 years old collected between 2004 and 2007. Rotavirus was detected in 140 (19.7%) samples with G9P[8] (30.0%) and G2P[4] (21.4%) as the most common genotypes. Mixed (G and/or P) infections accounted for 17.9% of the samples and the emerging G12 strain was detected during 2004 (3.5%) and 2007 (2.5%). Genotype G2 was the most prevalent during 2004 (43.9%) and 2007 (57.5%) and G9 during 2005 (58.0%) and 2006 (61.5%). Analysis of genotype prevalences from studies performed since 1996 in the same area showed striking natural fluctuations in G and P genotype frequencies. In particular, G2P[4] strains disappeared after 1999 and reemerged in 2004 to become the predominant strain by 2007 with a concomitant major decrease in G1P[8] prevalence. The VP7 genes from Argentinian G9 and G2 strains were sequenced and phylogenetic analysis was conducted in order to compare with sequences from strains isolated in regional countries reported previously. Several changes in the deduced amino acid sequence in antigenic regions of the VP7 protein from Argentinian and Brazilian strains were identified compared to vaccine strains. Overall, this study revealed relationships in the circulation of rotavirus strains in South American countries and major replacements in dominant genotypes, including the virtual disappearance of G1P[8] strains in a non‐vaccinated population. High numbers of mixed infections speeding up evolution, circulation of rare serotypes, and antigenic drift could, eventually, become challenges for new vaccines. J. Med. Virol. 82:1083–1093, 2010.

Sequence Analysis of the Gene Encoding VP4 of a Bovine Group C Rotavirus: Molecular Evidence for a New P Genotype

Nucleotide sequence of the bovine group C rotavirus Shintoku strain gene 3 was determined. Segment 3 is 2253 nucleotides (nt) in length and contains a long open reading frame (ORF) beginning at nt 22 and terminating at nt 2223. This ORF encodes a polypeptide of 733 amino acids with a predicted molecular mass of 83 kDa. The deduced gene 3 amino acid sequence shares 79% and 73% identities with VP4 of the porcine Cowden and human Bristol strains, respectively. Lack of high amino acid sequence homology in VP4 of bovine, porcine, and human group C rotaviruses indicates that the Shintoku strain represents a new P genotype.

Detection of field isolates of human and animal group C rotavirus by reverse transcription-polymerase chain reaction and digoxigenin-labeled oligonucleotide probes.

Rotaviruses (RV) are important etiological agents of acute gastroenteritis in infants and young children, as well as the young of a variety of animals worldwide. These viruses belong to Reoviridae family and contain a genome of 11 segments of double-stranded RNA (dsRNA). Two major proteins, VP4 and VP7, encoded by genome segments 4 and 7, 8 or 9, respectively, evoke a neutralizing antibody response and form the basis for the current classification of group (gp) A rotavirus into P (VP4) and G (VP7) serotypes. Although much recent progress has been made on the molecular biology of gp C RV, routine methods to detect and discriminate human, porcine, and bovine strains are not available widely. In this study, a multiplex reverse transcriptase-polymerase chain reaction (RT-PCR) and digoxigenin-labeled (dig) oligonucleotide probes using chemiluminescence has been developed to detect and discriminate VP7 genes from culture-adapted and field isolates of human, porcine and bovine gp C RV. The multiplex RT-PCR and dig-probes were specific for the VP7 genes of human, porcine and bovine gp C RV and allowed detection and characterization of single and mixed infections of porcine gp C RV with porcine gp A or gp B rotaviruses. Detection rates for gp C RV were more than 50% when compared with polyacrylamide gel electrophoresis. These new diagnostic assays may help determine the epidemiological importance of these viruses in human and animal infections.

Detection and characterization of group C rotavirus in Buenos Aires, Argentina, 1997-2003.

The role of group C rotaviruses as a cause of diarrhea was examined among children <17 years of age admitted to a Hospital in a suburban area of Buenos Aires, Argentina between 1997 and 2003. A total of 1,579 fecal samples were screened for group A (RVA) and C (RVC) rotaviruses by two in‐house ELISA methods at Quilmes University (UNQ‐ELISA). Samples positive, doubtful and negative by RVC specific UNQ‐ELISA (nu2009=u2009246) were examined further for RVC by another in‐house ELISA (CDC‐ELISA), electron microscopy, RT‐PCR, nested PCR, and Southern hybridization. Sensitivity, specificity, and predictive values for each test were determined. While the sensitivity was comparable for the nested PCR and CDC‐ELISA methods (82.5%), the molecular methods were slightly more specific. Poorly preserved particles were often seen in fecal samples, suggesting that degradation of RNA could be a factor influencing the performance of molecular methods. The incidence of RVC was estimated to be 3% without apparent differences among seasons. RVC infected patients had a significantly (Pu2009<u20090.001) higher median age (6 years vs. 1 year) than those with RVA infection. Sequence of the RVC VP7 gene from six Argentinean strains and sequences reported previously in different countries showed high nucleotide (94.4–99.9%) sequence identities, indicating a high degree of conservation for human RVC VP7 genes among strains collected on five continents over a period of 17 years. These findings indicate that RVC is a significant cause of diarrhea and it is necessary to develop simple and sensitive serological methods for its detection. J. Med. Virol. 81:1109–1116, 2009.

Completion of the Four Large Gene Sequences of Porcine Group C Cowden Rotavirus

The terminal nucleotide sequences of group C Cowden rotavirus gene segments 1-4 were determined. When compared with the published sequences, we found 14 to 29 additional nt at the 5′ ends of the four reported gene sequences. For the 3′ ends, we observed an additional 16 nt in gene 2 and 14 fewer nt in gene 4.