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This study describes the characterisation of G8 rotavirus strains isolated from humans with acute gastroenteritis. Six G8 strains were detected in Australia between 2002 and 2008. Four were G8P[14] strains, one was G8P[8]+[14] and one was G8 P non-typeable. By polyacrylamide gel electrophoresis and enzyme immunoassay analysis, four G8 strains with visible RNA exhibited a long electropherotype and five G8 strains displayed subgroup I specificity. Sequence analysis of the VP7 gene indicated that the G8 strains exhibited the highest nucleotide and amino acid identity with a G8P[11] bovine rotavirus strain detected in Japan. VP4 sequence data of one G8P[14] strain revealed that the closest identity was to another human-bovine-like strain detected in Australia, MG6, a G6P[14] strain. The identification of G8 strains causing disease further extends the number of G8P[14] strains detected in Australian children, and indicates that there is a rare but ongoing presence of uncommon human strains within the community in Australia., (Copyright 2009 Elsevier B.V. All rights reserved.)

A limited number of human G6P[14] rotavirus strains that cause gastroenteritis in humans have been isolated in Europe and Australia. The complete genome sequences were determined for five of these human strains--B10925-97 (isolated in Belgium in 1997), 111/05-27 (Italy, 2005), PA169 (Italy, 1987), MG6 (Australia, 1993), and Hun5 (Hungary, 1997)--and their genetic relatedness to animal rotavirus strains was evaluated by sequencing the complete genome of the sheep rotavirus OVR762 (G8P[14]; Spain, 2002), the guanaco (Lama guanicoe) rotavirus strains Arg/Chubut/99 and Arg/Río Negro/98 (G8P[14] and G8P[1], respectively; Argentina, 1999 and 1998), the sable antelope strain RC-18/08 (G6P[14]; South Africa, 2008), and the bovine rotavirus strain Arg/B383/98 (G15P[11]; Argentina, 1998). These analyses revealed an overall consensus genomic constellation (G6/G8)-P[14]-I2-(R2/R5)-C2-M2-(A3/A11)-N2-T6-(E2/E12)-H3, together with a few gene reassortments, and the phylogenetic analyses confirmed that the P[14] human strains evaluated in this study were closely related to rotavirus strains isolated from sheep, cattle, goats, guanacos, and antelopes and to rabbits (albeit to a lesser extent), suggesting that one (or more) of these animal species might be the source of the human G6P[14] strains. The main feature of the genotype and phylogenetic analyses was the close overall genomic relatedness between the five human G6P[14] rotavirus strains and the ovine and antelope rotavirus strains. Taken together, these data strongly suggest a common origin for the human P[14] strains and those of the even-toed ungulates belonging to the mammalian order Artiodactyla, with sheep probably playing a key role in the interspecies transmission responsible for the introduction of P[14] rotavirus strains into the human population.

Serotype G6 rotaviruses are common pathogens of cattle but are rarely found in humans. In Australia, human G6 isolates have previously been detected in two major southern population centres. A new isolate, ASG6.02, was detected in central Australia (Alice Springs) in 1997. Comparison of the deduced amino acid sequence of the major neutralizing antigen, VP7, indicated that ASG6.02 was related to human G6 viruses isolated from children in Italy and Australia. Phylogenetic analysis supported the close relationship between ASG6.02 and other Australian isolates and indicated that G6 VP7 sequences generally clustered according to the species of origin (human, bovine or porcine). The VP4 type of ASG6.02 was determined as P-type [14], in common with other isolates from Australia and Italy. The detection of ASG6.02 indicates that the distribution of this serotype is increasing in this country and may have implications for successful vaccine development.

The incidence of astrovirus infection in children less than 5 years of age hospitalized with acute gastroenteritis in Melbourne, Australia, from 1995 to 1998 was determined. Astrovirus was detected in 40 of 449 specimens tested by Northern hybridization, and astrovirus infection was confirmed by reverse transcription-PCR with or without culture in CaCO-2 cells. This represented 3.0% (40 of 1, 327) of all children tested for enteric pathogens, including viral, bacterial, and parasitic pathogens, over the survey period. The incidences of astrovirus infection in each year were 4.4% (1995), 2. 2% (1996), 3.9% (1997), and 1.4% (1998). In 1995 and 1997, the incidences of astrovirus infection were greater than the incidence of infection with all individual bacterial pathogens and were either greater than or equal to the incidence of adenovirus infection. Astrovirus exhibited an unusual biennial winter peak of incidence that correlated with a greater incidence of serotype 1 virus and an increased rate of hospitalization of children aged 6 to 12 months. Uncommon (serotype 2 and 4) and rare (serotype 8) serotypes were detected during the survey period. Genetic analysis of ORF2 (which encodes the astrovirus capsid precursor) of Melbourne isolates showed nucleotide sequence variation from year to year. This was not accompanied by significant amino acid substitutions. However, geographical variation was apparent by comparison of Melbourne astrovirus isolates with prototype strains identified in the United Kingdom.

A commercial enzyme immunoassay (EIA) for the detection of astrovirus antigen was used to detect the virus during a 12-month survey of enteric pathogens in children in outpatient (n = 238) and hospital (n = 176) settings. It was found to have a 100% sensitivity and 98.6% specificity. Nineteen astrovirus isolates were detected and confirmed by northern hybridization, cell culture, and RT-PCR. The virus was detected mainly amongst outpatients although a comparison of the detection rate with that in hospitalised children did not demonstrate a statistically significant difference (p = 0.1347). In contrast, there was a strong association between hospitalization and rotavirus infection (p = 0.0371), and a strong association between infection detected in outpatients and adenovirus infection (p = 0.0193). Strains of astrovirus were sequenced, genotyped and shown to be: type 1 (n = 11), type 3 (n = 1), and type 4 (n = 7). Maximum genetic variation in type 1 isolates was 8.6% and type 4 was 7.8%. Changes did not result in amino acid substitutions.

An atypical human rotavirus strain, DG8, was isolated from a 13-month-old child hospitalised with acute gastro-enteritis in Australia. The virus could not be serotyped by enzyme immunoassay (EIA) using standard reagents specific for common Group A human rotavirus G serotypes. The deduced amino acid sequence of the outer capsid glycoprotein, VP7, indicated that this strain belonged to the uncommon human serotype G8. This was confirmed by EIA incorporating a G8-specific neutralising monoclonal antibody (NMAb). The VP4 genotype of DG8 was determined as P[14], equivalent to P serotype P3B, by sequence analysis and confirmed by EIA incorporating a P3B-specific NMAb. Electrophoretic analysis of DG8 genomic dsRNA indicated that the virus exhibited a "long" electropherotype. Northern hybridisation analysis (using a whole-genome probe derived from DG8) indicated that DG8 shared overall homology with the European serotype G8 strain, HAL1166 (11 of 11 genes). In contrast, only 9 of 11 genes of DG8 hybridised with the Asian serotype G8 strain, B37, and with the bovine G8 strain, A5. Hence, DG8 displayed features reminiscent of the human serotype G8 rotaviruses isolated in Europe in the mid-1980s rather than the geographically local G8 Asian strains isolated a decade earlier. It is possible that DG8 arose through reassortment between human and bovine rotaviruses., (Copyright 2000 Wiley-Liss, Inc.)

Background: During an epidemiological study on the incidence of astrovirus infection in children hospitalized with acute gastroenteritis, a Northern hybridization method was used to screen stool samples for astrovirus RNA. Positive results were confirmed using reverse transcriptase-polymerase chain reaction (RT-PCR), which showed surprisingly low sensitivity. The low sensitivity of the RT-PCR method was considered likely to be due to the presence of non-specific inhibitors., Objective: To develop and use a simple culture method to improve the sensitivity of diagnosis of astrovirus in clinical stool samples using RT-PCR., Study Design: Stool samples from children hospitalized with acute gastroenteritis were screened for astrovirus using Northern hybridization. The presence of astrovirus RNA was then confirmed using an astrovirus-specific RT-PCR. Hybridization positive samples that failed to generate an RT-PCR product were cultured in CaCO-2 cells for 48 h. RNA was isolated from cultures and re-tested using the same RT-PCR method., Results: Using Northern hybridization, human astroviruses were detected in the stools of 31 patients and confirmed by RT-PCR in 16 samples. RNA extracted directly from 15 faecal specimens could not be amplified by RT-PCR. After culture for 48 h in CaCO-2 cells, RNA extracted from these samples could be amplified and confirmed the presence of astrovirus in all 15 specimens., Conclusions: Development of a simplified culture method for astrovirus positive faecal specimens improved the sensitivity of astrovirus-specific RT-PCR from 52 to 100%. The technique should be of value as a confirmatory test in surveys of human astrovirus infection.

An atypical human rotavirus strain Z10262, isolated from a chronically infected immunodeficient child, displayed an unusual genomic RNA electrophoretic pattern. Besides, Northern blot analysis indicated that this strain contained an abnormally migrating gene 11 equivalent. Sequencing of this gene showed that it was derived from a genetic rearrangement which involved a partial duplication of the open reading frame (ORF) encoding the non-structural protein NSP5. However, the duplicated region contained a deletion and several point mutations relative to the first copy of the ORF. Phylogenetic analysis of human and animal NSP5 amino acid sequences including Z10262 revealed two groups of human proteins related to different animal proteins. The isolation and analysis of Z10262 strain provides further evidence for the genetic complexity of naturally occurring human rotaviruses.

The sequences of the genes encoding the outer capsid glycoprotein, VP7, of twelve serotype G2 rotavirus isolates from Australia were determined. The deduced amino acid sequences were compared by phylogenetic analysis to published sequences from strains collected from various geographical locations worldwide between 1976 an 1993. This analysis showed that geographical clustering of strains was apparent and that most strains were distantly related to strain DS1 whose VP7 is included in the rhesus rotavirus tetravalent vaccine formulation. Comparisons of the neutralization epitope regions of VP7 also indicated that most strains might exhibit antigenic differences to DS1. These results have implications for the development of future vaccine strategies.

The incidence of astrovirus infection in children under 5 years of age hospitalized for acute gastroenteritis in Melbourne, Australia, during 1995 was determined. Astrovirus was detected in 16 fecal specimens by Northern (RNA) dot blot analysis of RNA isolated from feces with an astrovirus-specific cDNA probe. The incidence of astrovirus infection was determined as 4.2% (16 of 378 total samples) compared with rates of 63.2, 3.7, and 4.2% for rotavirus, adenovirus, and all bacterial pathogens, respectively. Astrovirus was detected during the winter season and mainly in infants between 6 and 12 months of age. Serotyping of samples was carried out by reverse transcriptase PCR and direct sequencing of a 348-bp region of the capsid protein gene. Type 1 strains predominated (11 of 13 typeable samples), although type 4 isolates were also detected. Astrovirus was retrospectively identified in 13 fecal samples collected from hospitalized infants between 1980 and 1985 and shown to contain small viruses by electron microscopy. Type 1 isolates were again the most common, although a type 5 strain was also found. Comparative sequence analysis indicated that type 1 astroviruses exhibited up to 7% sequence divergence over a 15-year period; however, all mutations were silent. The incidence of astrovirus reported here indicates that the virus is a significant cause of severe diarrhea in young children. The genetic analysis also provides important molecular epidemiological information relevant to the development of preventative therapies.

Two rotavirus strains, E210 and E212, implicated in epidemics of gastroenteritis in children in central and northern Australia during 1993-1994, exhibited the unusual combination of a 'short' RNA electrophoretic pattern and subgroup II specificity. The outer capsid protein VP7 was found by PCR typing and sequence analysis to be related to that of serotype G2 viruses. Both strains displayed a novel pattern of reactivity to G2-specific monoclonal antibodies that correlated with sequence variation in the antigenic regions of VP7. The VP4 serotype of E210 and E212 was determined as P1B in an enzyme immunoassay, consistent with other G2 viruses. Analysis of the VP6 gene indicated significant identity (98-99%) with other human subgroup II viruses. Northern hybridization analysis of E210 RNA using total genome probes derived from the prototype strains RV4 and RV5 indicated that E210 was derived from multiple gene reassortment between rotaviruses belonging to different genetic types.

An unusual rotavirus strain, MG6, was isolated from a 16-month-old child admitted to hospital with acute gastroenteritis. The virus could not be serotyped (G-typed) by enzyme immunoassay using standard reagents specific for common serotypes of human Group A rotaviruses. Nucleotide sequencing of cDNA derived from the gene encoding the outer capsid protein, VP7, and deduction of the VP7 amino acid sequence indicated that this strain belonged to serotype G6, a serotype normally associated with viruses causing disease in cattle. This was confirmed by polymerase chain reaction typing and enzyme immunoassay using a G6-specific monoclonal antibody. The VP4 genotype of MG6 was determined by hybridization of its VP4 cDNA to genomic RNA isolated from standard strains of defined P-types. This analysis, confirmed by deduced amino acid sequence analysis, classified MG6 into the novel genotype P13. MG6, therefore, is related to the previously described G6P13 human strain PA169, isolated in Italy. The emergence of strain MG6, the first human G6 rotavirus identified in Australia, provides further evidence of reassortment between human and animal rotaviruses.

Two cases of rotavirus gastroenteritis associated with neurological involvement, one with encephalitis (defined by abnormal neurological signs, cerebrospinal fluid (CSF) pleocytosis and detection of rotavirus genomic nucleic acid in the CSF) and one with a non-inflammatory encephalopathy (defined by abnormal neurological signs, an entirely normal CSF and detection of rotavirus genomic nucleic acid in the CSF), are presented and used as a basis to review and explore potential pathogenetic mechanisms, including direct viral replication within neurons and indirect effects of the newly described rotavirus 'enterotoxin'. [ABSTRACT FROM AUTHOR]