Tamas Berke

Clinical presentations of rotavirus infection among hospitalized children.

Background. Although rotaviruses (RVs) are the most common cause of severe gastroenteritis in children, there is a lack of information detailing the spectrum of clinical manifestations of RV disease resulting in hospitalization. Objective. To characterize the clinical spectrum of RV-associated hospitalizations, including short stay visits in children. Methods. Active RV disease surveillance was conducted at three children’s hospitals Sundays through Thursdays in children 15 days through 4 years of age admitted with diarrhea (D), vomiting (V) and/or unexplained fever (F) between November, 1997, and June, 1998. Stool specimens were collected and tested for RV by enzyme immunoassay. Results. Of the 862 children enrolled, 763 (88%) had a stool specimen tested for RV. Overall 31% of children excreted RV. RV excretion was highest when all 3 symptoms (D, V and F) occurred in the same child (56%), lower when 2 symptoms occurred together (38% DV; 19% DF; 13% VF) and lowest when each symptom occurred alone (3% D; 11% V; 6% F). Nine percent of the children without diarrhea excreted RV. Children admitted without diarrhea were more likely to have rotavirus if they developed diarrhea during their hospitalization. Conclusions. RV detection was greatest when diarrhea, vomiting and fever occurred together and lowest when each symptom occurred alone. The spectrum of symptoms of rotavirus disease in children at the time of admission to the hospital or short stay unit may be broader than previously recognized.

Phylogenetic analysis of the caliciviruses

A phylogenetic portrait of the genus Calicivirus in the family Caliciviridae was developed based upon published sequences and newly characterized calicivirus (CV) strains, including additional Sapporo‐like HuCV strains in pediatric diarrhea stool specimens from South Africa, the United Kingdom, and the United States. Distance and parsimony methods were applied to nucleotide and amino acid sequences of human and animal calicivirus 3D RNA‐dependent RNA polymerase (∼470nt) and capsid hypervariable regions (∼1,200nt) to generate phylogenetic trees. Pairwise amino acid identity in the 3D region among the Sapporo‐like strains ranged from 61%; to 100%. Human and animal caliciviruses (HuCVs and AnCVs) separated into five genogroups: small round‐structured viruses (SRSV), Sapporo‐like, and hepatitis E virus (HEV)‐like HuCVs and rabbit‐, and vesicular exanthema of swine virus (VESV)‐like AnCVs, each with a distinct genome organization. Each genogroup, including the Sapporo‐like HuCVs, subdivided further into subgenogroups. The capsid region trees had higher levels of confidence than the 3D region trees and limited conclusions about genogroups could be drawn from the 3D region analyses. This analysis suggested that CVs include five potential virus subfamilies. J. Med. Virol. 52:419–424, 1997.

Molecular characterization of a novel recombinant strain of human astrovirus associated with gastroenteritis in children.

Summary. We report a naturally occurring human astrovirus (HAstV) strain detected in two different geographic locations. We identified two isolates of this strain in a diarrhea outbreak at a child care center in Houston, Texas; and two isolates in diarrhea stool samples from two children in Mexico City. All four isolates were detected in stool samples by enzyme immunoassay (EIA). One of the Mexican isolates was typed by EIA and all four isolates were HAstV-5 by typing RT-PCR. The four isolates were >97% nucleotide-identical in two different genomic regions: ORF1a (246nt), and the 3′ end of the genome (471nt). One isolate from each geographic location was further sequenced in the transition region from ORF1b to ORF2 (1255nt) and this region of the two isolates showed ≥ 99% nt identity. Phylogenetic analyses of sequences of eight HAstV antigenic types and the novel strain in the transition region demonstrated the new strain being closely related to HAstV-3 in ORF1b, but closest to HAstV-5 in ORF2. These results and high sequence identity among all HAstV antigenic types in the transition region and RNA structural predictions supported a potential recombination site at the ORF1b/ORF2 junction. This is the first evidence that recombination occurs among human astroviruses.

Prevalence and genetic diversity of human caliciviruses (HuCVs) in Mexican children.

Human caliciviruses (HuCVs) contain two genera: “Norwalk‐like viruses” (NLVs) and “Sapporo‐like viruses” (SLVs). The importance of the two genera as a cause of acute gastroenteritis of infants and children remains unknown. Beginning in 1989, a birth cohort of children in Mexico was enrolled and monitored for acute gastroenteritis. A subset of 115 diarrhea stool specimens from 76 children and 66 non‐diarrhea stool specimens from 64 children was examined for HuCVs by RT‐PCR by using a primer pair (p289/290) that detects both NLVs and SLVs. Twenty‐two (19%) of the 115 diarrhea stool specimens and 5 (7%) of 66 non‐diarrhea stool specimens produced RT‐PCR products of expected size (319 bp for NLVs and 331 bp for SLVs). Twenty of the twenty‐seven strains were cloned and sequenced. Pairwise sequence analysis showed that 9 (60%) and 6 (40%) of the 15 strains from the diarrhea stools were NLVs and SLVs, respectively. The same proportions of NLVs (60%) and SLVs (40%) were observed in the non‐diarrhea stools. Strains in the NLV genus could be further divided into four clusters: Lordsdale, MxV, and HV and one potentially new cluster. Strains in the SLV genus could be divided into three clusters: Sapporo/82, Lon/92, and a potentially new cluster. Strains from the Lordsdale cluster were the most common among these children. The findings of both genera and multiple clusters of HuCVs co‐circulating and the identification of new strains of HuCVs in the population justify the need for future studies of HuCVs in infants and children. J. Med. Virol. 62:217–223, 2000.

Reclassification of the Caliciviridae into distinct genera and exclusion of hepatitis E virus from the family on the basis of comparative phylogenetic analysis

Summary. Caliciviridae and Picornaviridae belong to the same subphylum and genera within Picornaviridae are well characterized. Until 1998, Caliciviridae included one genus Calicivirus, containing strains with distinct structural and genomic features. Phylogenetic analyses of capsid genes revealed five clusters within Caliciviridae corresponding to differences in genome organization. In order to determine to what taxonomic level these clusters correspond, genomic sequences of caliciviruses, picornavirus prototypes, and two togavirus strains were analyzed. Distance and maximum likelihood methods were used to estimate the phylogenetic relationships among strains. Analysis of the capsid gene revealed separation of five main clusters (Norwalk-like, and Sapporo-like human caliciviruses, hepatitis E virus, vesicular exanthem of swine-like, and lapine caliciviruses) and distances corresponding to those observed among picornavirus genera. Utilizing more conserved (presumed helicase and polymerase) regions for the analyses, only major groups of caliciviruses were separated with confidence, with distances also comparable to those separating picornavirus genera. Anal- ysis in these regions that included togavirus sequences moved HEV strains out of the calicivirus cluster. Our findings support the reclassification of caliciviruses into four genera. The phylogenetic position of hepatitis E virus, by analysis of non-structural genes, is outside of the caliciviruses, in an uncertain taxonomic position.

Molecular Epidemiology of Childhood Astrovirus Infection in Child Care Centers

This study assessed the role of human astrovirus (HAstV) in outbreaks and sporadic cases of diarrhea among children attending child care centers (CCCs) and determined the infecting astrovirus antigenic types by reverse transcriptase-polymerase chain reaction (RT-PCR) and sequence analysis. Eight astrovirus outbreaks occurred in 6 CCCs. Of 179 children with diarrhea, 36 (20%) had astrovirus-associated diarrhea. Diarrhea stools obtained during diarrhea outbreaks were more likely to contain astrovirus (40/476) than were samples not associated with a diarrhea outbreak (14/452) (P<.001). Type-specific RT-PCR and DNA sequencing identified 5 outbreaks associated with HAstV-1 and 3 outbreaks with HAstV-2. Sequential outbreaks in 2 CCCs occurred with a different type in the same year. Phylogenetic analysis identified 6 clades of HAstV-1 and 2 clades of HAstV-2 during this 1-year surveillance. Astrovirus was a significant cause of diarrhea outbreaks, and 2 antigenic types were present in the community during 1 diarrhea season.

Sapporo-like human caliciviruses are genetically and antigenically diverse.

SummaryThe Sapporo-like human caliciviruses (HuCVs) comprise one of three genogroups of HuCVs associated with acute gastroenteritis. Phylogenetic analysis has shown that Sapporo-like HuCVs are related more closely to animal caliciviruses than to other known HuCVs. We produced 3.2 kb cDNA fragments from the 3′ end to three Sapporo-like HuCVs that were associated with acute gastroenteritis in children (Houston/86, Houston/90, and London/92). Sequence analysis of the 3.2 kb cDNAs showed that two of the three viruses had a genomic organization similar to that of other Sapporo-like strains and the third strain (London/92) lacked an open reading frame overlapping the 5′ end of the capsid gene. Alignment of the capsid sequences of these three strains showed 44–78% amino acid identity among the three strains. Phylogenetic analysis of the aligned sequences indicated the three strains are related but each belongs to a distinct genetic cluster. The genetic differences are associated with antigenic differences in that an enzyme immune assay (EIA) specific for the prototype Sapporo/82 strain detected the Houston/86 strain, but not the Houston/90 and London/92 strains. In vitro transcription and translation of viral cDNA containing the predicted capsid gene of Houston/90 resulted in a protein of 63 K, which is immunoprecipitated by sera from children infected with the strain. Genetically and antigenically distinct strains in the Sapporo-like HuCVs have not been described previously and the occurrence of such diverse strains in the same community likely increases the importance of these strains as a cause of illness in children.

Genomic mapping of a calicivirus VPg

SummaryWe identified a primate calicivirus (Pan-1) VPg in Pan-1-infected cells. The Pan-1 VPg was associated with both genomic and subgenomic RNAs. RNase digestion of Pan-1 RNA yielded a residual protein of 16 kDa. The N-terminal sequence of Pan-1 VPg was determined by direct amino acid sequencing and mapped to a region of the genome equivalent to picornavirus VPgs. Alignment of this protein sequence with similar regions of other calicivirus genomes allowed identification of conserved amino acid motifs and potential boundaries of the calicivirus VPg genes. Proteinase K treatment abolished the infectivity of Pan-1 RNA, suggesting that Pan-1 VPg is required for RNA infectivity.

Nonmedical costs associated with rotavirus disease requiring hospitalization.

Background: As the most common cause of severe diarrhea among children, rotavirus has a significant economic impact. Previous studies focused on the direct medical costs of rotavirus infections; however, nonmedical costs account for the majority of the financial burden from this disease. Herein, we report the results from the largest prospective study in the United States determining the nonmedical costs of severe rotavirus infections. Methods: Prospective, active, gastroenteritis case surveillance was conducted between November 1997 and December 1999 at 3 pediatric medical centers. Rotavirus infection was identified for 548 children admitted between 2 weeks and 5 years of age. Detailed information about nonmedical costs during the prehospitalization, hospitalization and posthospitalization periods was obtained through interviews. Results: The average nonmedical cost per case of rotavirus disease was

Partial characterization of the genome of nine animal caliciviruses.

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