The complete nucleotide sequence of the fourth gene of symptomatic (Wa, DS-1, P, and VA70) and asymptomatic (M37, 1076, McN13, and ST3) rotaviruses of serotype 1, 2, 3, or 4 was determined by the dideoxy chain termination method. In each strain, the fourth gene, which encodes the outer capsid protein VP3, is 2,359 base pairs in length and has 5'and 3'-noncoding regions of 9 and 25 nucleotides, respectively. The gene has a single long open reading frame of 2,325 base pairs that is capable of coding for a protein of 775 amino acids. A total of 14 N-terminal and 12 C-terminal amino acids are completely conserved or almost completely conserved, respectively, among nine human rotavirus VP3 genes that have been sequenced. In addition, there is conservation of arginine at the two trypsin cleavage sites as well as conservation of clusters of amino acids in different regions of the two VP3 cleavage products, VP8 and VP5. Three distinct forms of VP3 were identified among the nine human rotavirus strains analyzed. Three symptomatic rotaviruses (serotypes 1, 3, and 4) possess highly related VP3 genes (92.2 to 97% nucleotide identity). Two symptomatic serotype 2 rotaviruses possess VP3 genes which are even more closely related to each other (98.6% nucleotide identity) and only moderately related to the aforementioned VP3 genes of serotypes 1, 3, and 4 (87.4 to 88.2% nucleotide identity). The four asymptomatic rotaviruses, which constitute the third group, possess highly related VP3 genes (95.5 to 97.5% nucleotide identity) which are distinct from those of the virulent rotaviruses (73 to 74.8% nucleotide identity). At 91 positions in the protein sequence of VP3, an amino acid is conserved among the asymptomatic rotaviruses, while a different amino acid is conserved among the symptomatic rotaviruses. Notably, five regions are conserved among the symptomatic rotaviruses, while a different set of sequences are conserved among the asymptomatic rotaviruses. It is possible that some or all of these regions of sequence dimorphism may be responsible for the difference in virulence of these two groups of human rotaviruses. There are 13 regions in the VP3 protein sequence which exhibit the greatest variability; the majority of these variable regions are observed between amino acids 106 to 192. These regions may represent potential antigenic sites related to heterotypic rotavirus neutralization.
RNA-RNA hybridization was performed to assess the extent of genetic relatedness among human rotaviruses isolated from children with gastroenteritis and from asymptomatic newborn infants. 32P-labeled single-stranded RNAs produced by in vitro transcription from viral cores of the different strains tested were used as probes in two different hybridization assays: undenatured genomic RNAs were resolved by polyacrylamide gel electrophoresis, denatured in situ, electrophoretically transferred to diazobenzyloxymethyl-paper (Northern blots), and then hybridized to the probes under two different conditions of stringency; and denatured genomic double-stranded RNAs were hybridized to the probes in solution and the hybrids which formed were identified by polyacrylamide gel electrophoresis. When analyzed by Northern blot hybridization at a low level of stringency, all genes from the strains tested cross-hybridized, providing evidence for some sequence homology in each of the corresponding genes. However, when hybridization stringency was increased, a difference in gene 4 sequence was detected between strains recovered from asymptomatic newborn infants ("nursery strains") and strains recovered from infants and young children with diarrhea. Although the nursery strains exhibited serotypic diversity (i.e., each of the four strains tested belonged to a different serotype), the fourth gene appeared to be highly conserved. Similarly, each of the virulent strains tested belonged to a different serotype; nonetheless, there was significant conservation of sequence among the fourth genes of three of these viruses. Significantly, the conserved fourth genes of the nursery strains were distinct from the fourth gene of each of the virulent viruses. These results were confirmed and extended during experiments in which the RNA-RNA hybridization was carried out in solution and the resulting hybrids were analyzed by polyacrylamide gel electrophoresis. Under these conditions, the fourth genes of the nursery strains were closely related to each other but not to the fourth genes of the virulent viruses. Full-length hybrids did not form between the fourth genes from the nursery strains and the corresponding genes from the strains recovered from symptomatic infants and young children.
We have used a recently developed monoclonal antibody enzyme-linked immunosorbent assay (K. Taniguchi, T. Urasawa, Y. Morita, H. B. Greenberg, and S. Urasawa, J. Infect. Dis. 155:1159-1166, 1987) for serotyping rotaviruses recovered from 134 Venezuelan infants over a period of 15 months. One hundred and nine of the specimens were typed with the following distribution: serotype 1, 48%; serotype 2, 16%; serotype 3, 22%; and serotype 4, 14%. Three specimens reacted with two different monoclonal antibodies. In addition, 6 specimens (5%) containing enough outer capsid antigen could not be typed; partial RNA sequence analysis of the glycoprotein gene from three of these six strains failed to reveal sequence differences with prototype strains that could be serotyped with the monoclonal antibodies. Variations in the recovery rates of the different serotypes were observed. Serotypes 2, 3, and 4 predominated at the beginning of the study, and serotype 1 predominated at the end of the study. Diarrheal illness appeared to be more prolonged in infants shedding rotavirus serotypes 1 and 3 than in those shedding serotypes 2 and 4.
The gene encoding outer capsid protein VP3 of subpopulations of two animal rotaviruses, simian SAil and Nebraska calf diarrhea virus (NCDV), was analyzed. Two laboratory strains of simian SA1l rotavirus (SAll-SEM and SAll-FEM) differed with respect to VP3. This dimorphism was indicated by a difference in electrophoretic mobility and a difference in reactivity with anti-VP3 monoclonal antibodies. The overall VP3 amino acid homology between the two SA1l VP3 proteins was 82.7%, whereas the VP3 protein of SAll-FEM was 98.5% homologous in amino acid sequence to NCDV VP3, suggesting that SAll-FEM VP3 was derived by gene reassortment in the laboratory during contamination with a bovine rotavirus. A comparison of the deduced amino acid sequence of the VP3 of two virulent NCDV strains and an attenuated NCDV strain (RIT 4237), revealed only five amino acid differences which were scattered throughout the protein but did not involve the trypsin cleavage sites. Of interest, the VP3 of the standard strain of NCDV which is virulent for cows differed in only one amino acid (position 23, Gln to Lys) from the VP3 of an NCDV mutant which was attenuated both for cows and for children.
BackgroundNorovirus, astrovirus, and sapovirus are known to cause acute gastroenteritis and are associated with chronic viral excretion in stool among immunocompromised patients. Because molecular tools for their detection only recently became widely available, the prevalence and chronic excretion of these viruses has not been well defined. We describe features of these viral infections among patients receiving care at the Clinical Center of the National Institutes of Health.MethodsWe identified patients with a positive BioFire FilmArray® gastrointestinal panel result for norovirus, astrovirus, or sapovirus from September 15, 2015 through November 30, 2016. We reviewed patient medical records to abstract clinical and microbiologic information. Chronic excretion was defined as more than one positive test for a given virus with more than 30 days between tests.ResultsOf 932 samples tested, 102 (11%) samples from 48 patients tested positive for norovirus, 15 (2%) samples from 11 patients tested positive for sapovirus, and 16 (2%) samples from 7 patients tested positive for astrovirus. One of these patients had a sample that tested positive for both sapovirus and norovirus, and one tested positive for astrovirus and sapovirus at separate points during the study period. Of the 48 patients with norovirus, 16 (33%) had evidence of chronic excretion, with a median duration of 189 days (range 72–372). Of these 16, 14 were known or suspected to be immunodeficient, and 4 had hematologic malignancies. Of 7 patients with astrovirus, 1 (14%) had evidence of chronic excretion (132 days). This patient had a hematologic malignancy and was taking immunosuppressive medication. No patients with sapovirus had evidence of chronic excretion. Overall, 20 (31%) patients additionally tested positive for another gastrointestinal pathogen, most commonly enteropathogenic E. coli and C. difficile.ConclusionNorovirus remains common in this immunocompromised patient population, and both sapovirus and astrovirus are present. Additional follow-up in this and other cohorts with new molecular tools will enable more complete description of the prevalence, excretion duration, and clinical features of infection with these enteric viruses.This research was supported by the Intramural Research Program of the NIH, NIAID, and the NIH CC.Disclosures All authors: No reported disclosures.
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