Sensitive and specific quantitative detection of rotavirus A by one-step real-time reverse transcription-PCR assay without antecedent double-stranded-RNA denaturation.

A real-time quantitative reverse transcription-PCR (qRT-PCR) assay using the recombinant thermostable Thermus thermophilus (rTth) enzyme was developed to detect and quantify rotavirus A (RVA). By using rTth polymerase, significant improvement was achieved over the existing real-time RT-PCR assays, which require denaturation of the RVA double-stranded RNA (dsRNA) prior to assay setup. Using a dsRNA transcript for segment 7, which encodes the assay target NSP3 gene, the limit of detection for the improved assay was calculated to be approximately 1 genome copy per reaction. The NSP3 qRT-PCR assay was validated using a panel of 1,906 stool samples, 23 reference RVA strains, and 14 nontarget enteric virus samples. The assay detected a diverse number of RVA genotypes and did not detect other enteric viruses, demonstrating analytical sensitivity and specificity for RVA in testing stool samples. A XenoRNA internal process control was introduced and detected in a multiplexed qRT-PCR format. Because it does not require an antecedent dsRNA denaturation step, this assay reduces the possibility of sample cross-contamination and requires less hands-on time than other published qRT-PCR protocols for RVA detection.

Virus detection and duration of illness among patients with 2009 pandemic influenza A (H1N1) virus infection in Texas.

Knowledge from early outbreaks is limited regarding the virus detection and illness duration of the 2009 pandemic influenza A (H1N1) infections. During the period from April to May 2009 in Texas, we collected serial nasopharyngeal (NP) and stool specimens from 35 participants, testing by real-time reverse transcriptase-polymerase chain reaction (rRT-PCR) and culture. The participants were aged 2 months to 71 years; 25 (71%) were under 18. The median duration of measured fever was 3.0 days and of virus detection in NP specimens was 4.2 days; however, few specimens were collected between days 5-9. The duration of virus detection (4.2 days) was similar to the duration of fever (3.5 days) (RR, 1.14; 95% CI, .66-1.95; P = .8), but was shorter than the duration of cough (11.0 days) (RR, .41; 95% CI, .24-.68; P < .001). We detected viral RNA in two participants' stools. All cultures were negative. This investigation suggests that the duration of virus detection was likely similar to the seasonal influenza virus.

G and P types of circulating rotavirus strains in the United States during 1996-2005: nine years of prevaccine data.

BACKGROUND: Rotavirus vaccine was recommended for routine use among US infants in 2006. To provide prevaccine data, we conducted strain surveillance for 9 consecutive seasons during 1996-2005. METHODS: Using reverse-transcriptase polymerase chain reaction genotyping and nucleotide sequencing, we determined P/G genotypes of >3100 rotavirus strains collected in up to 12 cities each year from different US regions. RESULTS: The most prevalent strain globally, P[8] G1, was the most prevalent each year in the United States (overall, 78.5% of strains; range, 60.0%-93.9%), and 9.2% of the samples were P[4] G2, 3.6% were P[8] G9, 1.7% were P[8] G3, and 0.8% were P[8] G4. Genotype P[6] G9, which emerged in 1995, was detected continuously for several seasons (from 1996-1997 to 2000-2001, 0.2%-5.4%) but was not identified in the subsequent 4 seasons. Single or a few detections of rare genotypes (eg, P[6] G12, P[9] G6, and P[9] G3) were observed during several rotavirus seasons at frequencies of 0.5%-1.7% and, overall, comprised 0.6% of all the samples from the entire surveillance period. Several globally common strains in addition to G1, especially G2 and G9, circulated at high prevalence (33%-62%) in some cities during certain years. CONCLUSIONS: Almost 85% of strains during 1996-2005 had either a G or P antigen that is present in both RotaTeq (Merck) and Rotarix (GlaxoSmithKline). Monitoring of strains after introduction of rotavirus vaccines is important.

Molecular characterization of a rare, human-porcine reassortant rotavirus strain, G11P[6], from Ecuador.

The Pan-American Health Organization established a rotavirus pre-vaccination disease burden and strain surveillance network in Latin America and the Caribbean in 2004. During strain surveillance in Ecuador in 2005-2006, a rare rotavirus genotype, G11P[6], was detected among common strains. Sequencing and phylogenetic analysis of this strain identified a novel lineage of the G11 VP7 gene, most closely related to A253 (91.8% nt identity), a porcine rotavirus strain identified in Venezuela. Most genes of this strain clustered with porcine, human-porcine or bovine-porcine reassortant strains; only VP6 and perhaps NSP2 genes were more closely related to cognate genes of human rotaviruses. Thus, this strain was likely generated by gene reassortment between porcine and human parental strains. Our study provides further evidence that animal rotaviruses play an important role in genetic and antigenic diversity of rotaviruses pathogenic for humans.

Anogenital distance measured at weaning is correlated with measures of blood chemistry and behaviors in 450-day-old female mice.

In female mice, anogenital distance (AGD), measured at weaning, provides an estimate of uterine exposure to testosterone from flanking male mouse littermates. A variant of the anogenital distance index (AGDI) that uses the residual value of AGD after accounting for the effect of weight by regression (AGDWTRES) was measured at weaning in F(2) female mice from a C57BL/6J x DBA2/J cross. AGDWTRES was used to examine the relationship between intrauterine environment and blood chemistry variables and activity-related behaviors when the females were 450 days old. Longer AGDWTRES values correlated with lower levels of calcium, cholesterol, phosphorus, iron, and protein, which is opposite to the expected direction, based on underlying sex differences for blood chemistry. A positive correlation was found between AGDWTRES and two activity-related measures (the number of rears in a test of exploration, and the number of sectors of a rod that are entered by the mouse). These findings suggest that in utero proximity to males, as indexed by AGDWTRES, may have effects on fundamental aspects of blood chemistry and behavior that extend well into mouse middle age, and could play an important role in health.

Rotavirus genotypes in Belarus, 2008-2012.

This study describes group A rotavirus (RVA) genotype prevalence in Belarus from 2008 to 2012. In 2008, data from 3 sites in Belarus (Brest, Mogilev, Minsk) indicated that G4P[8] was the predominant genotype. Data from Minsk (2008-2012) showed that G4P[8] was the predominant RVA genotype in all years except in 2011 when G3P[8] was most frequently detected. Other RVA genotypes common in Europe (G1P[8], G2P[4]) were detected each year of the study. This study reveals the dominance of genotype G4P[8] in Belarus and helps to establish the baseline genotype prevalence prior to RVA vaccine introduction in the country.

Genetic analysis of G12P[8] rotaviruses detected in the largest U.S. G12 genotype outbreak on record.

In 2006-07, 77 cases of gastroenteritis in Rochester, NY, USA were associated with rotavirus genotype G12P[8]. Sequence analysis identified a high degree of genetic relatedness among the VP7 and VP4 genes of the Rochester G12P[8] strains and between these strains and currently circulating human G12P[8] strains. Out of 77 samples, two and seven unique nucleotide sequences were identified for VP7 and VP4 genes, respectively. Rochester strain VP7 genes were found to occupy the G12-III lineage and VP4 genes clustered within the P[8]-3 lineage. Six strains contained non-synonymous nucleotide substitutions that produced amino acid changes at 6 sites in the VP8(∗) region of the VP4 gene. Two sites (amino acids 242 and 246) were located in or near a described trypsin cleavage site. Selection analyses identified one positively selected VP7 site (107) and strong purifying selection at 58 sites within the VP7 gene as well as 2 of the 6 variant sites (79 and 218) in VP4.

United States rotavirus strain surveillance from 2005 to 2008: genotype prevalence before and after vaccine introduction.

BACKGROUND: A live, attenuated rotavirus vaccine, RotaTeq®, was approved in 2006 for immunization of infants in the United States. To monitor the distribution of rotavirus genotypes before and after vaccine introduction, the Centers for Disease Control and Prevention conducted strain surveillance with the National Rotavirus Strain Surveillance System. METHODS: Over 3 rotavirus seasons, 2005-2006, 2006-2007, and 2007-2008, National Rotavirus Strain Surveillance System laboratories collected rotavirus-positive stool specimens and submitted them to the Centers for Disease Control and Prevention. Rotavirus strains were G- and P-genotyped by multiplex reverse transcription-polymerase chain reaction or nucleotide sequencing. RESULTS: During 2005-2006 and 2006-2007 seasons, G1 was the dominant G-type but in the 2007-2008 season, G3 replaced G1 as the most frequently detected strain. Four genotypes, G1P[8], G2P[4], G3P[8], and G9P[8] were detected in every season. Uncommon strains observed during the study period were G2P[8], G1P[6], G2P[6], G4P[6], G1P[4], G3P[9], G12P [6], and G12P[8]. The mean age of rotavirus cases in the 2007-2008 season increased significantly in patients less than 3 years old compared with the 2 previous seasons. CONCLUSIONS: : The increased overall prevalence of G3P [8] strains in 2007-2008, the first rotavirus season with reasonable rotavirus vaccine coverage, was consistent with Australian reports of G3 dominance following RotaTeq introduction. However, these strain changes in both countries have occurred in the context of large declines in severe rotavirus disease and we cannot rule out that they are simply the result of naturally occurring changes in rotavirus strain prevalence. These findings underscore the need for careful monitoring of strains to assess possible vaccine pressure-induced changes and vaccine effectiveness against various rotavirus genotypes.

Characterization of VP6 genes from rotavirus strains collected in the United States from 1996-2002.

We sequenced 22 VP6 genes from common rotavirus strains P[8], G1; P[4], G2; P[8], G3; P[8], G4 and P[8], G9 and uncommon type P[6], G9 collected in the US over a 6-year period. All strains defined as members of VP6 antigenic subgroup (SG) I according to reactivity patterns with monoclonal antibodies formed a genetic cluster (Genogroup I) with SG I reference strains. Similarly, all strains in antigenic SGII formed a group (Genogroup II) with corresponding standard strains of the same SG. Most US strains of each genogroup had diverged by 10-15% from the VP6 gene sequence of reference strains collected >20 years earlier and some recent isolates from other countries. Evolutionary analysis demonstrated that recently isolated US strains of both genogroups have diverged into 2-3 related clusters consistent with other recent findings. Unexpectedly, some recent isolates from other countries have diverged greatly from both older reference isolates and from the recent US isolates characterized here. This finding suggests that genetic diversity in human rotavirus VP6 genes may be greater than previously recognized. These sequences will help in the construction of a VP6 gene database to aid in the development of broadly reactive molecular assays and permit identification of regions where primers and probes for existing assays may need to be redesigned.