Atypical Rotavirus Research Articles

Unusual BA222-like strains of Rotavirus A (Sedoreoviridae: Rotavirus: Rotavirus A): molecular and genetic analysis based on all genome segments.

Rotavirus infection is the major cause of severe dehydrating diarrhea requiring hospitalization in young children worldwide. Due to their segmented genome, rotaviruses are capable of gene reassortment, which makes the emergence and spread of genetically novel strains possible. The purpose of this study was to search for unusual rotaviruses circulating in Nizhny Novgorod in 2021‒2023 and their molecular genetic characterization based on all genome segments. Rotavirus-positive stool samples of children were examined by PCR genotyping and electrophoresis in PAAG. cDNA fragments of each of the 11 genes (VP1‒VP4, VP6, VP7, NSP1‒NSP5), 570 to 850 nucleotide pairs in length were sequenced for the selected strains. The phylogenetic analysis was performed in the MEGA X program. In the study period 2021‒2023, 11 G[P] combinations with a predominance of G3P[8] (59.5%) were identified. Six atypical Rotavirus А (RVA) strains were identified: 2 strains of the G2P[4] genotype (G2-P[4]-I2-R2-C2-M2-A3-N2-T3-E2-H3, G2-P[4]-I2-R2-C2-M2-A3-N2-T3-E3-H2) and 4 G3P[9] strains (all strains had the genotype G3-P[9]-I2-R2-C2-M2-A3-N2-T3-E3-H3). Phylogenetic analysis based on all genes showed an evolutionary relationship between rotaviruses similar to rotaviruses of cats and dogs (BA222-like) and unusual strains of the G2P[4] genotype, for which a mixed combination of genotypes was identified and characterized for the first time. The results obtained expand the understanding of the diversity of reassortant RVAs, as well as complement the data on the genotypic structure of the rotavirus population in Nizhny Novgorod. The wide genetic diversity of reassortant RVA can help rotaviruses overcome the immunological pressure provided by natural and vaccine-induced immunity. In this regard, to control the emergence of new variants and assess changes in the virulence of rotaviruses after reassortment processes, continuous molecular monitoring for circulating RVA is necessary.

The Emergence of New Rotavirus Strains in America

Rotavirus infections are the most common causes of infectious diarrhea in young children and animal worldwide. In some countries in Latin American specifically in Central American and Caribbean countries, rotavirus infections are not subject to specific surveillance. This review is about the unusually strains detected and potential zoonotic of rotavirus in Latin American. Although, interspecies transmission has not been documented to occur directly, an increase of the number of reports of atypical rotavirus genotypes; apparently derived from transmission between animal of farm, domestic and wild with humans, has been reported in some Latin American countries and the world. We consider that the rapid increase in the detection of new unusual strains with genetic heterogeneity, raises interesting questions about the evolution of rotavirus in The Latin American region. The emergence of novel strains derived from interspecies transmission has implications for the design and implementation of successful human rotavirus vaccine strategies.

Rotavirus strain diversity in Eastern and Southern African countries before and after vaccine introduction

BackgroundThe African Rotavirus Surveillance Network has been detecting and documenting rotavirus genotypes in the African sub-continent since 1998 in anticipation of the rollout of rotavirus vaccination in routine Expanded Programme on Immunisation. This paper reports distribution of the rotavirus strains circulating in 15 Eastern and Southern African (ESA) countries from 2010-2015 as part of active World Health Organization (WHO) rotavirus surveillance, and investigates possibility of emergence of non-vaccine or unusual strains in six selected countries post-vaccine introduction. Material and methodsStool samples were collected from children <5 years of age presenting with acute gastroenteritis at sentinel hospitals pre- and post-rotavirus vaccine introduction. Samples were tested for group A rotavirus using an enzyme immunoassay by the national and sentinel laboratories. At the WHO Rotavirus Regional Reference Laboratory in South Africa, molecular characterisation was determined by PAGE (n = 4186), G and P genotyping (n = 6447) and DNA sequencing for both G and P types (n = 400). ResultsThe six-year surveillance period demonstrated that 23.8% of the strains were G1P[8], followed by G2P[4] (11.8%), G9P[8] (10.4%), G12P[8] (4.9%), G2P[6] (4.2%) and G3P[6] (3.7%) in 15 ESA countries. There was no difference in circulating strains pre- and post-rotavirus vaccine introduction with yearly fluctuation of strains observed over time. Atypical rotavirus G and P combinations (such as G1P[4], G2P[8], G9P[4] and G12P[4]) that might have arisen through inter-genogroup or inter-genotypes reassortment were detected at low frequency (2%). Close genetic relationship of African strains were reflected on the phylogenetic analysis, strains segregated together to form an African cluster in the same lineages/sub-lineage or monophyletic branch. ConclusionThere has been considerable concern about strain replacement post-vaccine introduction, it was not clear at this early stage whether observed cyclical changes of rotavirus strains were due to vaccine pressure or this was just part of natural annual fluctuations in the six ESA countries, long-term surveillance is required.

Emerging rotavirus genotype G9P[6] in an outbreak on a neonatal medium care unit

We observed two consecutive episodes of an outbreak of rotavirus (RV) on our neonatal medium care unit in May-June 2009. We investigated the genotype of the RV; describe the spread of the virus among neonates and the measures taken to control the outbreak. Stool samples of symptomatic neonates, and during the second episode stool samples of all neonates, were tested for RV antigens. Reverse transcriptase polymerase chain reaction was performed on ten samples positive for RV, followed by genotyping. Staff members and samples of the environment were also tested for RV. An infection control advisor attended shifts on the ward to observe the daily routines. Eighteen of 44 neonates were tested positive for RV antigen and RNA. Ten samples were genotyped and revealed the G9P[6] strain. One male premature neonate developed a serious neurologic complication. None of the staff members were positive for RV. It is concluded that the RV strain G9P[6] can present as a hard to eradicate nosocomial pathogen. Since atypical RV strains can cause severe illness among neonates, surveillance and genotyping during an outbreak is recommended.

Porcine rotavirus groups A, B, and C identified by polymerase chain reaction in a fecal sample collection with inconclusive results by polyacrylamide gel electrophoresis

Objective: To evaluate the frequency of groups A, B, and C rotaviruses in diarrheic pigs from Brazilian pig herds. Materials and methods: Fecal samples with inconclusive results when tested by the silver-stained polyacrylamide gel electrophoresis (ss-PAGE) technique were selected for this study (n = 144). Rotavirus A (VP4 and VP7 genes), rotavirus B (NSP2 gene), and rotavirus C (VP6 gene) were identified by reverse transcription-polymerase chain reaction (RT-PCR). Results: Of the 144 fecal specimens analyzed by RT-PCR assays, 103 (71.5%) were positive for rotavirus infection. Single infections were detected in 58 samples (40.3%), with 34 (23.6%), 19 (13.2%), and 5 (3.5%) identified as rotavirus groups A, B, and C, respectively. Mixed infections with two and even three rotavirus groups were identified in 45 fecal samples (31.2%). Rotaviruses B and C were more frequently identified in mixed (65.2%) than in single infections. Implications: The inclusion criteria for sample selection and use of RT-PCR assays for diagnosis in this study contributed to the higher frequencies of rotaviruses B and C, which are sporadically implicated in porcine neonatal diarrhea. The high rate of diagnosis of atypical rotavirus showed that rotaviruses B and C, as well as rotavirus A, are disseminated in Brazilian pig herds. These results suggest that failure to identify porcine rotaviruses B and C in diarrheic fecal samples is primarily due to use of diagnostic methods of low sensitivity, and not to low prevalence of infection.

Novel Nonstructural Protein 4 Genetic Group in Rotavirus of Porcine Origin

Novel Nonstructural Protein 4 Genetic Group in Rotavirus of Porcine Origin

Atypical rotavirus identified from young children with diarrhoea in South Africa.

The study was undertaken for antigenic characterization of rotavirus strains isolated from South African children. During July 1996-July 1997, an epidemiological surveillance of rotavirus-associated gastroenteritis was carried out in Pretoria and Gauteng provinces. In total, 1,229 diarrhoeal faecal samples were collected from a pathology laboratory. Of 389 (32%) rotavirus strains detected, most (97%) were group A rotaviruses by serological assay. However, 12 rotavirus strains identified did not apparently carry the common group A-specific antigen as determined by both commercial and monoclonal antibody-based ELISAs. Electrophoretic analysis of the viral RNA genome revealed that these strains shared a common electropherotype and that this resembled the group A rotavirus constellation of RNA segments. Furthermore, the conserved terminal sequences of the group A VP6 and VP7 genes could not be targeted by standard reverse transcriptase-polymerase chain reaction with a routine set of primers to the VP6 or VP7 genes. These strains present an interesting phenotypic variation of the recognized rotaviruses and warrant further characterization.

Enteric virus infections and diarrhea in healthy and human immunodeficiency virus-infected children.

Forty-three stool samples from 27 human immunodeficiency virus (HIV)-seropositive children and 38 samples from 38 HIV-negative children, collected during a 15-month period, were examined for enteric viruses. Diagnostic assays included enzyme immunoassays for rotavirus, adenovirus, and Norwalk virus; polyacrylamide gel electrophoresis for picobirnavirus and atypical rotavirus; and PCR for astrovirus and enterovirus. Specimens from HIV-positive children were more likely than those of HIV-negative children to have enterovirus (56 versus 21%; P < 0.0002) and astrovirus (12 versus 0%; P < 0.02), but not rotavirus (5 versus 8%; P > 0.5). No adenoviruses, picobirnaviruses, or Norwalk viruses were found. The rates of virus-associated diarrhea were similar among HIV-positive and HIV-negative children. Enteroviruses were excreted for up to 6 months in HIV-positive children; however, no evidence for prolonged excretion of poliovirus vaccine was observed. These results suggest that although infection with enterovirus and astrovirus may be frequent in HIV-infected children, enteric viruses are not associated with the diarrhea frequently suffered by these children.

Sequence of the VP7 gene of an atypical human rotavirus: evidence for genetic and antigenic drift.

The nucleotide sequence of the gene encoding the outer capsid glycoprotein, VP7, isolated from a reassortant human rotavirus, M3014, was determined. The deduced amino acid sequence exhibited significant identity to the VP7 from a standard strain belonging to serotype G4, although the antigenic regions of the M3014 VP7 resembled sequences from both serotype G4 and G9 viruses. However, reactivity with G4 or G9 serotype-specific monoclonal antibodies was not observed. We suggest that the M3014 VP7 was derived from sequential mutation of a G4-like progenitor gene resulting in a protein with novel antigenic properties.

Multinucleate Enterocytes Associated with Experimental Group A Porcine Rotavirus Infection

It has been recently demonstrated that atypical (non-group A) porcine rotaviruses are capable of producing villous enterocyte syncytia both in experimental and spontaneous cases of porcine Epithelial syncytia may also occur in atypical rotavirus infections of lambs,3 calves,8 and rats.I0 Since group A rotavirus infection is not known to be associated with small intestinal syncytia, it has been suggested that in the pig, this phenomenon is specific enough to allow its use as a histologic diagnostic ~r i ter ion.~ We present evidence indicating that enterocyte syncytia occasionally occur in group A rotavirus infection of piglets; therefore, the presence of these cells does not denote a non-group A infection. For this study, cesarian-derived piglets were maintained in gnotobiotic isolators. One ml of group A rotavirus (type OSU)6 was further passaged in seven newborn gnotobiotic piglets. Feces and intestinal contents from these pigs were collected 24 hours after infection, pooled, clarified, and filtered (0.45 pm). Electropherotyping6 performed on RNA extracted from this material yielded a banding pattern identical to reference group A OSU porcine rotavirus. No adventitial agents were detected. Two ml of filtered feces ( lo7 plaqueforming units titrated in MA-104 cells) were used to orally infect six gnotobiotic pigs varying in age from 2 to 4 days. One pig, infected at 2 days of age and euthanatized 24 hours after infection, forms the basis for this report. Tissue sections from seven equidistant regions of small intestine7 were fixed in 10% neutral buffered formalin, processed in low-melt paraffin (Peel-A-Way, American Scientific Products, McGaw Park, IL), and sectioned 5 pm thick. They were then processed for immunohistochemical localization of group A specific rotavirus antigen. The glass slides containing the paraffin sections were incubated for 30 minutes at 56 C and then deparaffinized in xylene. After hydration through a series of graded ethanols, endogenous peroxidase activity was blocked by 0.3% hydrogen peroxide for 30 minutes. After washing the slides in 0.0 1 M phosphate buffered saline (pH 7.6, wash buffer and diluent), diluted normal goat serum was added to the sections and the slides were incubated at room temperature for 30 minutes. Excess serum was shaken from the slides, and a 1 : 200 dilution of rabbit antihuman group A rotavirus antibody (DAKO Corp., Santa Barbara, CA) was added to the tissue sections followed by overnight incubation in a humid chamber at 4 C. After washing, a secondary antibody, biotinylated goat antirabbit IgG (Vector Labs, Burlingame, CA), was incubated on the tissue sections for 30 minutes at room temperature. Following washing, horseradish peroxidase-conjugated avidin biotin complex (Vector Labs, Burlingame, CA) was incubated on the sections for 60 minutes at room temperature, after which the slides were washed. A peroxidase substrate solution (0.05% 3,3’diaminobenzidine in 0.05 M Tris buffer [pH 7.21 with 0.0 1% hydrogen peroxide) was added to the tissue sections. After incubation for 5 minutes at room temperature to allow for color development, the slides were rinsed in tap water, counterstained in Mayer’s hematoxylin, dehydrated through a series of graded alcohols, cleared in xylene, and mounted with a coverslip. Paired control sections of porcine intestine, known to be immunohistochemically positive or negative for group A rotavirus antigen, were stained as described above and also with substitution of the primary antibody with normal rabbit serum diluted to the same protein concentration as the pnmary antibody. In addition, a positive control section was reacted with the primary rabbit antibody that had been incubated overnight at 4 C with OSU rotavirus. Enterocytes containing group A rotavirus antigen had variable numbers of cytoplasmic brown granules as opposed to negative tissues and negative controls that lacked such granules. Preincubation of virus with antibody completely blocked specific staining of rotavirus antigen in tissue sections. Examination of the paraffin-embedded, avidin-biotin stained tissues from the experimental pigs demonstrated the presence of brown granules in the cytoplasm of large numbers of villous enterocytes from the mid-jejunum through the ileum. In addition, many cells were vacuolated, many were exfoliating from the villous tips, and there were areas where the villi were denuded of epithelium. These results were similar to previous reports of the pathogenesis of rotavirus infection in Several multinucleated enterocytes were present along the villous sides and tips of one pig. For the most part, these cells lacked the specific cytoplasmic staining indicative of group A rotavirus infection. An occasional sloughed multinucleate cell was seen, however, that had vacuolated cytoplasm and virus-specific immunoperoxidase positive cytoplasmic granules (Fig. 1) indicating replication of group A rotavirus within the cell. We were unable to ascertain from this study whether multinucleate enterocytes are occasionally found in the intestines

Group C Rotavirus Infection and Infiltration

We recently found many cases of gastroenteritis involving older children and sometimes other members of their families. We suspected epidemiological infiltration of an atypical rotavirus (RV). PAGE analysis of 130 cases in 1985 and 1986 revealed that the RNA profiles were similar to that of group C RV in 12 cases. We performed IEM analysis on one of the strains and found it to be group C. The group C RV was detected for 2 months toward the end of the outbreak of group A RV infection. The ages of the group C RV infected patients were 4 years or older. We also studied the prevalence of antibody to group C in the stored sera that had been collected since 1971. Antibody was seldom found in the sera of patients under 3 years of age but it was found in 20-40% of those who were 4 years or older. The clinical manifestations of the group C RV infection were similar to those of group A, except that body temperature was slightly lower and other symptoms were generally milder.

C群ロタウイルスの疫学的研究

By electron microscopy survey of acute gastroenteritis of children in Matsuyama, rotaviruses were detected in 561 of 2479 fecal samples obtained between October, 1984 and September, 1988, in which 60 atypical and 259 typical rotaviruses, so far tested, were detected by polyacrylamide gel electrophoresis (PAGE) of viral RNA. Out of 60 atypical rotaviruses, 5 were observed in 1985, 7 in 1986, none in 1987 and 48 in 1988. These atypical rotaviruses were morphologically indistinguishable from typical ones and showed very similar RNA migration patterns of PAGE to those of group C rotavirus. Furthermore, one atypical virus (86-542) reacted with antiserum against a porcine group C rotavirus in immune electron microscopy, while it did not react with anti-group A rotavirus serum. On the other hand, hyperimmune guinea pig antiserum against 86-542 reacted with a couple of atypical rotavirus including viruses isolated in 1988, so far tested, but did not react with any of the typical viruses. These findings showed that atypical rotaviruses which were endemic in Matsuyama city in 1988 were defined as group C rotavirus. Moreover, these group C rotaviruses showed two kind of RNA migration patterns in PAGE, which clearly discriminated the virus were isolated before and after 1987. Epidemiological features of group C rotavirus were as follows. Children from whom group C rotavirus was isolated were older than those from of whom group A rotavirus was isolated. The epidemic season was in February through April, compared to December through March in group A virus. Retrospective seroepidemiological study by immune adherence hemagglutination test (IAHA) using the purified 86-542 virus as antigen indicated that 15 of 78 sera of children obtained in 1971 already possessed IAHA antibody against group C rotavirus.

Epidemiology of typical and atypical rotavirus infections in New Zealand pigs

Polyacrylamide gel electrophoresis (PAGE) and enzyme-linked immunosorbent assay (ELISA) were employed to investigate the epidemiology of typical and atypical rotavirus infections in five piggeries. Of 152 faecal samples examined, 46 (30 per cent) were positive by ELISA for group A rotavirus. Rotaviruses with electrophoretic patterns resembling groups A, B and C were detected. At least two and up to five different rotavirus electrophoretypes (typical and/or atypical) were detected in each of the five piggeries. Out of 152 faecal samples examined, 28 (18 per cent) contained rotaviruses with group A electrophoretypes, 9 (6 per cent) with group C but only 1 with Group B. Six samples contained both group A and group C rotaviruses. No common electrophoretypes of group A or C rotaviruses were detected in these five piggeries. The PAGE technique was also used to analyze group A rotavirus isolated sequentially from another piggery over a three year period. A single electrophoretype was found during the first two years, but in the third year a different electrophoretype was detected.

Group C Rotavirus Infections in Patients with Diarrhea in Thailand, Nepal, and England

Atypical rotavirus obtained from fecal specimens of six patients with diarrhea from Thailand, Nepal, and England were characterized by using polyacrylamide gel electrophoresis and immune electron microscopy. The electropherotypes were characteristic of the porcine reference group C rotavirus strain but demonstrated considerable strain-to-strain variation. Human convalescent group C sera had a high titer (1:320) when tested against the human isolates and a low titer (1:40) when tested against a porcine reference strain (Cowden). When porcine antiserum (Cowden) was tested against the human isolates, the titers ranged from 1:40 to 1:320, indicating significant antigenic diversity between strains. Group C rotavirus appears to have a worldwide distribution as an agent associated with diarrhea in children and adults.

Atypical rotavirus among diarrhoeic children living in Belém, Brazil

Atypical rotaviruses were detected in faeces from two diarrhoeic children living in Belém, Pará, Brazil. Rotavirus particles were detected by electron microscopy and the RNA electrophoresis showed patterns which were compatible with group C rotaviruses. Tests for the presence of group A antigen by enzyme-linked-immunosorbent assay (ELISA) were negative. The two children had three successive rotavirus infections and in both cases the atypical strains were excreted at the time of the third infection, causing a mild and short-lasting disease.

Atypical rotavirus and villous epithelial cell syncytia in piglets

Histopathological examination of small and large intestine from piglets with enteritis has shown the presence of epithelial multinucleate syncytia. Syncytia were associated with a specific type of atypical rotavirus infection, determined by electron microscopy and polyacrylamide gel electrophoresis analysis of viral RNA. The observations are consistent with similar previously described natural or experimental infections in other animals.

Heterogeneity of genome rearrangements in rotaviruses isolated from a chronically infected immunodeficient child.

Rotaviruses with genome rearrangements, isolated from a chronically infected immunodeficient child, were adapted to growth in BSC-1 cells. Preparations of viral RNA from fecal extracts showed a mixed atypical rotavirus RNA profile, which was due to the presence of at least 12 subpopulations of viruses grossly differing in genotype. Besides various forms of genome rearrangements involving segment 8-, 10-, and 11-specific sequences, reassortment in vivo was likely to have occurred during the emergence of these viruses. The protein products of viral genomes with various forms of segmental rearrangements seemed to be largely unaltered. Genome rearrangement is proposed to be a third mechanism directing the evolution of rotaviruses.

Effects of two negative staining methods on the Chinese atypical rotavirus

The Chinese atypical (Group B) rotavirus, previously called the adult diarrhoea rotavirus (ADRV), was examined by transmission electron microscopy using either uranyl acetate or potassium phosphotungstate (PTA) as negative stains. Complete rotavirus particles were seen using uranyl acetate which were indistinguishable morphologically from typical rotaviruses. In the same preparations virus particles with differing degrees of degradation were seen after staining with PTA. This effect was not related to pH of the PTA and could be prevented by fixation of the specimen by 0.1 per cent glutaraldehyde. It is concluded that the use of PTA can give rise to falsely negative results for specimens containing this virus.

Atypical rotavirus in chickens in Argentina

In Argentina the presence of rotavirus was investigated in a chicken flock experiencing periodic episodes of diarrhoea during the winter of 1986. All the samples analysed were negative by the enzyme-linked immunosorbent assay (ELISA). However, when samples were observed by electron microscopy, particles which were indistinguishable from standard rotaviruses were detected in some samples. Ten of the 36 samples were positive after polyacrylamide gel electrophoresis (PAGE) analysis, all of them showing the same electropherotype. Based on these results these viruses were classified as rotavirus-like or atypical rotaviruses.

Detection of an atypical (possibly Group C) rotavirus in New Zealand pigs

Abstract Extract Madam:— I wish to report the first detection of an atypical (possible Group C) rotavirus in New Zealand pigs.