John A. Marshall

Small round-structured (Norwalk-like) viruses and classical human caliciviruses in Southeastern Australia

A total of 6,226 fecal samples collected from 1980 to 1996 in the Australian states of Victoria, New South Wales, and Tasmania from individuals with gastroenteritis were tested for small round‐structured viruses (SRSVs) and classical human caliciviruses (ClHuCVs) by electron microscopy. There were 223 samples positive for SRSVs, and nine positive for ClHuCVs. SRSVs were detected in individuals of all ages and were commonly associated with gastroenteritis outbreaks in nursing homes and hospitals. SRSVs were detected throughout the year, but were more common in the period from late winter to early summer in Australia (August to December). There were peaks of virus activity in the early 1980s and more recently in 1995 and 1996. Analyses by RT‐PCR and sequencing of a segment of ORF1 encoding the putative RNA polymerase for SRSVs and ClHuCVs showed the presence of viruses belonging to several genogroups. Viruses of genogroup 1 (Norwalk/Southampton‐like) and genogroup 3 (ClHuCVs) were relatively rare. Viruses of genogroup 2 (Snow Mountain‐like) were common, and could be divided into two subgroups, one containing Toronto/Mexico‐like viruses, the other Lordsdale/Camberwell‐like viruses. The majority of viruses detected belonged to this latter subgroup. J. Med. Virol. 55:312–320, 1998.

Heminested Multiplex Reverse Transcription-PCR for Detection and Differentiation of Norwalk-Like Virus Genogroups 1 and 2 in Fecal Samples

ABSTRACT The present study describes a heminested multiplex reverse transcription (RT)-PCR assay which enables simultaneous detection and differentiation of Norwalk-like virus (NLV) genogroups from clinical fecal samples without the need to perform sequencing or hybridization. The assay developed was able to detect concentrations of fewer than 100 viral particles per 5 μl of clarified fecal extract and could differentiate the two genogroups with a specificity of 100%. Although the multiplex RT-PCR assay failed to detect NLV in about 3% of the fecal samples which were NLV positive by electron microscopy (EM), the assay was approximately six times more sensitive than EM for NLV detection.

Molecular characterization of Camberwell virus and sequence variation in ORF3 of small round-structured (Norwalk-like) viruses.

Five small round‐structured viruses (SRSVs) associated with gastroenteritis in Victoria, Australia, from January to November 1994 were examined by sequencing cDNA prepared from faecal samples using RT‐PCR. The sequence of the 3′ half (3.8 kb) of the genome of one of these viruses, Camberwell, was determined. Camberwell virus was related most closely to Bristol and Lordsdale viruses, and belonged to the genetic group of SRSVs containing Bristol, Lordsdale, Toronto, OTH‐25, Mexico, and Hawaii viruses. The amino acid identities between Camberwell and Bristol viruses for proteins encoded by ORF1 (partial), ORF2, and ORF3 were 99%, 98%, and 90%, respectively. A highly variable region in ORF3 corresponding to amino acid residues 123 to 169 (Bristol and Camberwell numbering) were identified. Short segments of ORF1 (polymerase region) and the highly variable ORF3 region was analysed for the other four viruses. The results obtained indicated the potential usefulness of the variable region in distinguishing between closely related viruses.

The Dynamics of Norovirus Outbreak Epidemics: Recent Insights

Noroviruses are a major cause of gastroenteritis outbreaks worldwide. Norovirus outbreaks frequently occur as epidemics which appear to be related to both genetic and environmental factors. This review considers recent progress in understanding these factors. The norovirus genome undergoes continuous change and this appears to be important in the persistence of the virus in the community. Studies on the common GII.4 genotype have shown that some norovirus outbreak epidemics involving this genotype are correlated with specific changes in the genome. In contrast to the growing understanding of the role of genetic factors in norovirus outbreak epidemics, the role of environmental factors is less well understood. Topics reviewed here include long term excretion of norovirus in some individuals, long term survivability of norovirus in the environment, the role of meteorological factors in the control of norovirus outbreaks and the possible zoonotic transmission of the virus.

Norovirus mixed infection in an oyster-associated outbreak: an opportunity for recombination

SummaryWe describe an outbreak of gastroenteritis in which the nucleic acid of three distinct noroviruses was amplified from the same fecal sample. To enable the separate amplification of each virus, an inclusion/exclusion RT-PCR primer design strategy was developed. This paired a virus-specific exclusion primer (designed with the exact sequence of one virus in a region displaying low conservation among the three viruses) with a virus-nonspecific inclusion primer (designed in a conserved region). Thus, in each reaction the exclusion primer provided specificity for a single virus, and the inclusion primer increased the sensitivity and allowed hybridization in a region of unknown sequence. Analysis of the partial genomic sequences of the three viruses (3.6–3.8 kb) indicated that each virus belonged to a separate genogroup II cluster, and each displayed evidence of a potential recombination event when the sequences were compared with other published norovirus sequences. Our results, which show a mixed norovirus infection in a single individual, confirm the need to be aware of the possibility of mixed norovirus infections, and of the possibility of genomic recombination causing anomalies in phylogenetic analyses in such instances.

Hospital Outbreak of Norwalk-Like Virus

An outbreak of gastroenteritis caused by Norwalk-like virus occurred in two areas of the hospital: area 1, consisting of three adjacent and interconnected wards, with mostly elderly patients; and area 22, an acute ward in a separate building with elderly patients. In area 1, 40 patients and 20 staff were affected; in area 2, 18 patients and 14 staff were affected. Infection control measures were instituted in consultation with the government health authority. These measures did not appear to affect the course of the outbreak, but may have prevented spreads to the other wards.

Evaluation of the RIDA®QUICK immunochromatographic norovirus detection assay using specimens from Australian gastroenteritis incidents

A range of laboratory methods is now available for the detection of norovirus, a major cause of gastroenteritis. Recently, a commercial immunochromatographic assay for norovirus detection, the RIDA(®)QUICK assay, has become available, but there is still only limited information on its efficacy. This study examined the sensitivity and specificity of the RIDA(®)QUICK assay, using faecal material received for testing in a major diagnostic/reference laboratory in Australia. The sensitivity of the assay was found to be 83% and the specificity was 100%. No false positive norovirus results were found and the assay did not cross-react with common faecal viruses such as rotavirus, astrovirus, sapovirus and adenovirus. The assay was less reliable for genogroup I (GI) noroviruses than for genogroup II (GII) noroviruses. Genotypes detected by the assay included GII.1, GII.2, GII.3, GII.4, GII.6 and GII.7. The assay failed to detect any GI specimens in the test group. Genotypes not detected included GI.4 and GI.6. The assay was simple and quick to perform. It is valuable in a point-of-care situation or as a backup in a laboratory where a rapid initial norovirus result is required.

A comparative evaluation of the sensitivity of two automated and two manual nucleic acid extraction methods for the detection of norovirus by RT-PCR

The aim of the study was to compare the sensitivity of a norovirus RT-PCR method using two manual RNA extraction methods (Qiagen and Roche) and two automated RNA extraction methods (Qiagen and Corbett). All four RNA extraction methods gave similar sensitivities although the automated methods, especially the Corbett, required significantly less labour than the manual methods. The automated methods also enabled RNA extraction of approximately two to three times the number of specimens in a given time period compared to manual methods.

High level excretion of norwalk-like virus following resolution of clinical illness

Summary We report the case of an elderly woman excreting high levels (about 5 2 10 5 virions per gram of faeces) of Norwalk‐like virus (NLV) in the absence of any clinical symptoms of gastroenteritis. Analysis by reverse transcription, polymerase chain reaction and DNA sequencing was carried out on a 342‐nucleotide region of open reading frame 1. This indicated that the NLV belonged to genogroup 2 and was more closely related to the Camberwell subgroup, the most common circulating in southeast Australia at present, than to the Norwalk and Mexico viruses.

Replication complexes associated with the morphogenesis of rubella virus

SummaryThin section electron microscopy was used to investigate cellular changes associated with the replication of rubella virus (RV) in Vero cells and to compare these changes to those of the related alphavirus, Semliki Forest virus (SFV). Conspicuous membrane-bound cytoplasmic vacuoles analogous to the alphavirus replication complexes were observed in RV infected cells but not in mock infected cells. The vacuoles were characterised by membrane-bound vesicles measuring about 60 nm which often displayed an irregular dense core and/or a network of fibres. These vesicles were morphologically distinct from RV particles and were generally located at regular intervals on the inner side of the surrounding membrane of the RV replication complex. Degenerating cellular material was often found in the membrane-bound vacuole of a replication complex. The replication complexes were intimately associated with the rough endoplasmic reticulum (RER), which was localised 45–75 nm from the surrounding membrane of the replication complex. Parallel studies of replication complexes in SFV infected cells did not reveal such an intimate association with the RER. RV replication complexes appeared as early as 8 h post infection (p.i.), before detection of RV particles by electron microscopy, and their peak production at 24 h p.i. coincided with the time of maximum virus titre.