Quantitation of human astrovirus by real-time reverse-transcription-polymerase chain reaction to examine correlation with clinical illness.

Human astroviruses (HAstVs) cause gastroenteritis. Real-time, reverse-transcription-polymerase chain reaction (RT2-PCR) was developed to quantitate HAstV RNA. An 88 bp amplicon from the conserved 3' genomic region was detected by binding of SYBR Green. RT2-PCR was reproducible, with a correlation coefficient of 0.998-1.00 and PCR efficiency of 94.4-100% (mean 97%). The coefficient of variation was 0.6-2.5%, dynamic range with RNA standard up to 5 x 10(8) RNA copies (RNACN) and sensitivity 5 RNACN. Of 54 blinded, archived stool samples from children hospitalized because of gastroenteritis tested by RT-PCR, 49 (91%) agreed by RT2-PCR for HAstV-positivity (Cohen kappa=0.81, 95%CI 0.66-0.97). HAstV RNACN in stools ranged from 7.6 x 10(1) to 3.6 x 10(14)copies/0.1g. Children coinfected with rotavirus had lower RNACN (mean log 4.22/standard deviation=2.26) than those without coinfection (7.57/3.06; p=.019). Children taking infant formula also had lower RNACN (5.96/2.98) than breast-fed or weaned children (8.73/2.92; p=.027). Higher RNACN tended to occur with longer duration of diarrhea for the episode (r=0.49, p=.064), but was not associated with change in age, gender, illness day, severity or breast-feeding. RT2-PCR quantitated HAstV RNA and RNACN in stool correlates with features of clinical illness.

Immunogenicity and safety of inactivated influenza virus vaccine in young children in 2003-2004.

This study assessed the safety and immunogenicity of a pediatric formulation of the 2003-2004 inactivated, trivalent, split virion influenza vaccine, administered in a 2-dose schedule in healthy children ages 6-36 months, of whom 94% had protective titers (> or =1/40) to at least 1 antigen. The 2003-2004 split virion influenza vaccine was safe and immunogenic in young children.

Genome prediction of putative genome-linked viral protein (VPg) of astroviruses.

Positive-sense single-stranded RNA (+ssRNA) viruses replicate by uncoating the RNA genome for translation to provide viral proteins essential for genome replication and the production of new viral particles. The viral proteins are synthesized from a polyprotein precursor, which is cleaved nascently. The synthesized proteins include viral RNA-dependent RNA polymerase (RdRP), viral genome-linked protein (VPg), and a helicase. VPg is covalently attached to the genomic form of +ssRNA viruses. Helicases and NTPase unwind the RNA before replication. VPg and helicases have been identified in +ssRNA families, however, the presence of VPg and helicase in the Astroviridae, another +ssRNA family, has not been fully elucidated. Computational tools were utilized to provide sequence analysis evidence for the presence and genomic location of astrovirus VPg and helicase. HMMER program v2.1.1 was used to build Hidden Markov Model (HMM) profile for calicivirus VPg to search for conserved motifs in the astrovirus genome. We performed phylogenetic analysis of two genomic regions of astroviruses and caliciviruses (encoding the RdRP and VPg). We identified a putative VPg coding region in astrovirus. This region was located in open reading frame 1a (ORF1 a) and included sites with high sequence similarity to the VPg coding regions of Caliciviridae, Piconaviridae, and Potyviridae. A region encoding a putative astrovirus helicase identified conserved motifs only with pestivirus helicase sequences. Sequence analysis and comparison to other +ssRNA viruses supports the presence of VPg in the Astroviridae. Further laboratory analysis will be necessary to confirm these findings.

Immunogenicity of a recombinant human cytomegalovirus gB vaccine in seronegative toddlers.

BACKGROUND: Immunization of young children against cytomegalovirus (CMV) might decrease child-to-child and child-to-adult transmission of CMV and thereby reduce maternal infection during pregnancy. We conducted a Phase I trial in CMV-seronegative toddlers to evaluate the reactogenicity and immunogenicity of a CMV gB vaccine administered with MF59, an oil and water adjuvant. METHODS: Eighteen children between 12 and 35 months of age received either 20 microg of CMV gB/MF59 (n = 15) or a control hepatitis A vaccine (n = 3) at 0, 1 and 6 months. The study was open-label for the first six children and then observer-blinded and randomized. Children were monitored for local and systemic reactions and for the development of antibodies to the envelope protein gB and CMV-neutralizing antibodies. RESULTS: Adverse reactions were uncommon and mild. Two children were excluded from the immunogenicity analysis because they had serologic evidence of CMV infection. Reciprocal geometric mean neutralizing titers were: 0 preimmunization (n = 18); 90 (range, 53 to 188) after Dose 2 (n = 6); and 638 (range, 210 to 1645) 1 month after Dose 3 (n = 13). The reciprocal geometric mean neutralizing titers of antibody to gB by EIA were: 0 preimmunization (n = 18); 857 (range, 307 to 2073) after Dose 1 (n = 12); 27 457 (range, 9312 to 55,080) after Dose 2 (n = 6); and 98,264 (range, 35,480 to 228,780) 1 month after Dose 3 (n = 5). After Dose 3 antibody responses of toddlers were greater than those of naturally infected adults and were notably higher than among 149 adults given 3 doses of the same vaccine in other trials. CONCLUSION: The CMV gB vaccine is well-tolerated and highly immunogenic in toddlers.

Molecular characterization and sequence analysis of human astroviruses circulating in Hungary.

Human astroviruses (HAstVs) are major pathogens in viral gastroenteritis worldwide. Twenty-five HAstV strains were detected from stool specimens of children hospitalized for acute gastroenteritis in Budapest, Hungary, between 1995 and 1999. Sequence analysis was performed at the 3' end of the capsid gene to determine genotypic diversity of HAstVs circulating in Hungary. Five different genotypes of HAstVs were identified: HAstV-1 was predominant, followed by types 5, 8, 3 and 4. Two different subtypes of HAstV-1 were detected, but only one at a time in the community. This is the first report on the genetic diversity of HAstVs in Hungary and Central/Eastern Europe.

Prebiotic effect of fructo-oligosaccharide supplemented term infant formula at two concentrations compared with unsupplemented formula and human milk.

BACKGROUND: Human milk components, including oligosaccharides, affect the gastrointestinal flora of infants. Previous studies in adults have demonstrated that fructo-oligosaccharides increase potentially beneficial fecal bacteria, including bifidobacteria. The purpose of this study was to determine the prebiotic effect of infant formula supplemented with fructo-oligosaccharides. METHODS: Healthy term infants 2 to 6 weeks of age were enrolled in a 5-week, prospective, randomized, crossover, single-site study with a nonrandomized human milk comparator group. Washout weeks preceded and followed a week of feeding with fructo-oligosaccharide-supplemented formula (1.5 or 3.0 g/L). Stool specimens were quantitatively cultured weekly for bacteroides, lactobacilli, bifidobacteria, clostridia and enterococci and were tested for Clostridium difficile toxin. RESULTS: Seventy-two of 87 infants completed the trial; 58 were formula fed and 14 were human milk fed. Mean counts of bifidobacteria and lactobacilli were similar in all groups at entry and no group experienced a significant change in counts with fructo-oligosaccharide supplementation. After 7 days of fructo-oligosaccharide supplementation the bifidobacteria counts were greater in the 1.5 g/L fructo-oligosaccharide formula group than in the human milk fed or 3.0 g/L fructo-oligosaccharide formula groups. Formula-fed infants had higher counts of enterococci and bacteroides before fructo-oligosaccharide supplementation, and these counts did not change after supplementation. Clostridium counts increased 7 days after supplementation in the 1.5 g/L fructo-oligosaccharide formula group (P = 0.0356). No human milk fed infants had C. difficile toxin in stools. Fructo-oligosaccharide (3.0 g/L) supplementation resulted in more frequent and significantly softer stools. CONCLUSIONS: Infant formula supplemented with 1.5 or 3.0 g/L fructo-oligosaccharides was safe but had minimal effect on fecal flora and C. difficile toxin.

Comparison of clinical characteristics between astrovirus and rotavirus infections diagnosed in 1997 to 2002 in Hungary.

AIM: To determine the severity and clinical characteristics of human astrovirus (HAstV) infections among hospitalized children and compare them with children infected by rotavirus. METHODS: Retrospective, case-control study of astrovirus-infected and rotavirus-infected children. Astroviruses were detected in stool samples by enzyme immunoassay and/or reverse transcriptase-polymerase chain reaction. All stool samples were tested for rotavirus and bacterial pathogens, and all negative samples were further tested for human astrovirus. Children with astrovirus-positive stool samples and complete clinical data were included in this study. RESULTS: Astrovirus was detected in 29 (1.8%) children, and 63 rotavirus-infected children were included as controls. Astrovirus-infected children had shorter duration of diarrhea than rotavirus-infected children (median 4 and 6 d, respectively; p<0.05), and 79% of the astrovirus infections were associated with a short duration of vomiting (median 1 and 4 d, respectively; p<0.0001). Rotavirus-infected children had longer hospitalization (p<0.050) than astrovirus-infected children. CONCLUSION: HAstV-infected children had similar symptoms to those occurring in rotavirus infection. However, astrovirus-infected patients had a significantly shorter duration of diarrhea and vomiting, and they required a shorter hospitalization. On the basis of the clinical data and severity scores, children with rotavirus infection had more severe illness.

Astrovirus infection in children.

PURPOSE OF REVIEW: Public health concerns related to enteric viral agents, such as astroviruses and caliciviruses, include their ability to cause sporadic diarrhea, large outbreaks of gastroenteritis, and hospitalizations or deaths resulting from vomiting, diarrhea, and dehydration. Improved surveillance and application of sensitive molecular assays has increased awareness of these enteric pathogens and reduced the 'diagnostic gap' or unknown causes of non-bacterial gastroenteritis. RECENT FINDINGS: Molecular assays have been applied to further describe the epidemiology of human astroviruses from a variety of geographic areas. The burden of astrovirus infections compared with other enteric viral agents, including rotaviruses, caliciviruses, and enteric adenoviruses have been reported. New methods for detection of astroviruses such as reverse transcription-polymerase chain reaction and molecular typing methods have advanced the understanding of the epidemiology. Additional molecular studies have described the protein processing mechanisms of this single-stranded RNA virus. SUMMARY: Astroviruses are increasingly recognized as significant gastrointestinal pathogens. The understanding of molecular epidemiology and molecular processing of the virus may lead to specific prevention strategies.