Leesa D. Bruggink

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.

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.

Evaluation of the Dako IDEIA norovirus EIA assay for detection of norovirus using faecal specimens from Australian gastroenteritis outbreaks

Aims: New techniques for detection of norovirus, a major cause of gastroenteritis, require ongoing evaluation. The aim of this study was to use material from gastroenteritis outbreaks in Victoria, Australia, to evaluate the sensitivity and specificity of the Dako IDEIA norovirus EIA assay, using both photometric and visual analysis. Methods: A total of 130 faecal specimens from 41 gastroenteritis outbreaks were tested for norovirus by electron microscopy (EM), a two‐round multiplex reverse transcription‐polymerase chain reaction (RT‐PCR) method and the IDEIA norovirus assay. All specimens with sufficient amplified product were sequenced to determine their norovirus genotype. In addition, six well‐established RT‐PCR protocols were used to test four EIA‐positive, multiplex RT‐PCR/EM‐negative specimens. Also, a range of RT‐PCR protocols was used to test a specimen positive for GII only by the multiplex RT‐PCR but positive for GI and GII by the EIA. The effect of multiple freezing‐thawing cycles on EIA positivity was tested on seven additional specimens. A further seven specimens, known to contain the gastroenteritis viruses sapovirus, adenovirus, astrovirus and rotavirus were also tested by the IDEIA norovirus assay. Results: The IDEIA norovirus assay gave a single‐specimen sensitivity and specificity of 66% and 85%, respectively (visual analysis compared with the multiplex RT‐PCR), 63% and 88% (photometric analysis compared with the multiplex RT‐PCR), 65% and 87% (visual analysis compared with the multiplex RT‐PCR and/or EM) and 62% and 90% (photometric analysis compared with the multiplex RT‐PCR and/or EM). None of the four EIA‐positive specimens negative by the multiplex RT‐PCR and/or EM was positive by any of the six alternative RT‐PCR protocols. The specimen positive for GI and GII by EIA but for GII only by the multiplex RT‐PCR was not positive for GI by any of the alternative RT‐PCR protocols. A minimum of three specimens per outbreak had to be tested by the EIA to ensure that norovirus‐positive outbreaks (multiplex RT‐PCR and/or EM) were classified as positive for norovirus by the IDEIA norovirus assay (visual or photometric analysis). However, one specimen from a norovirus‐negative outbreak (multiplex RT‐PCR and/or EM) for which four specimens were provided was positive for norovirus by the IDEIA norovirus assay. Seven norovirus genotypes were identified by open reading frame 1 sequencing analysis and specimens from all seven norovirus genotypes (as well as an EM‐positive/multiplex RT‐PCR‐negative specimen) were detected by the IDEIA norovirus assay by both visual and photometric analysis. Repeated freezing‐thawing cycles (up to six) for faecal specimens did not reduce the sensitivity of the EIA assay but could render an EIA‐negative specimen EIA‐positive. The specimens positive for sapovirus, adenovirus, astrovirus and rotavirus were EIA‐negative. Conclusions: The IDEIA norovirus assay lacks the sensitivity and specificity to ascribe a particular result to a particular specimen, but could be useful for detecting norovirus in a gastroenteritis outbreak where specimens are plentiful, although it is difficult to avoid a risk of false positives. Since visual analysis can be used for result assessment almost as reliably as photometric analysis, the test kit would be useful for laboratories lacking specialist equipment such as a photometric microplate reader.

Molecular and epidemiological features of gastroenteritis outbreaks involving genogroup I norovirus in Victoria, Australia, 2002–2010

GI noroviruses are relatively rare and systematic studies of the molecular epidemiology of GI norovirus outbreaks are lacking. The current study examined the molecular virology of GI norovirus outbreaks in Victoria, Australia (2002–2010). Of 1,617 norovirus outbreaks identified, 69 (4.3%) were associated with GI norovirus alone, 1,540 (95.2%) with GII norovirus alone and 8 (0.5%) with GI + GII. Some differences between GI and GII outbreak epidemiology were found. GI outbreaks peaked in the 2‐month period November/December whereas GII outbreaks peaked in the 2‐month period September/October and GI norovirus outbreaks were significantly more common in non‐healthcare settings (37.7%) than GII outbreaks (9.5%). ORF 1/ORF 2 genotypes found in the 69 outbreaks involving GI norovirus alone were: GI.2/GI.2, 7 outbreaks; GI.2/GI.6, 18 outbreaks; GI.3b/GI.3, 14 outbreaks; GI.4/GI.4, 21 outbreaks; GI.8/GI.8, one outbreak; GI.d/GI.3, four outbreaks; and GI.e/GI.13, one outbreak. The current study appears to be the first to have identified the recombinant form, GI.2/GI.6. Whereas GI.2/GI.6 and GI.3b/GI.3 outbreaks occurred with equal frequency in both healthcare and non‐healthcare settings, GI.4/GI.4 occurred predominantly in healthcare settings. GI ORF 1/ORF 2 genotypes found in the eight outbreaks involving GI + GII norovirus were GI.2/GI.6, GI.3b/GI.3, and GI.4/GI.4, indicating GI genotypes in GI + GII outbreaks were similar to those found in outbreaks involving GI alone. Apparent differences in the evolution of different GI genotypes were noted. GI.2/GI.2, GI.2/GI.6, and GI.4/GI.4 strains tended to undergo periodic shifts in nucleotide sequence whereas various GI.3b/GI.3 strains tended to circulate simultaneously. J. Med. Virol. 84:1437–1448, 2012.

The incidence of norovirus-associated gastroenteritis outbreaks in Victoria, Australia (2002-2007) and their relationship with rainfall.

The relationship between the incidence of norovirus-associated gastroenteritis outbreaks (NAGOs) in Victoria, Australia for the period 2002–2007 and rainfall was examined. Statistical analysis involving the correlation between time series indicated that there was a statistically significant (p < 0.05) correlation between monthly NAGO incidence and average monthly rainfall. There was a lag of an average of about three months between peak average rainfall and a NAGO epidemic. The findings thus indicate rainfall can influence NAGO incidence. In an era where there is concern about the potential effects of global warming on weather patterns, it should be borne in mind that future changes in NAGO incidence may reflect altered world weather patterns.

The effect of fecal turbidity on norovirus detection by reverse transcriptase polymerase chain reaction.

A 10-min fecal preparation results in greater specimen turbidity than a 45-min protocol, but reverse transcriptase polymerase chain reaction (RT-PCR) norovirus test sensitivity is essentially the same. Feces processed so that particle size does not exceed approximately 560 nm do not display greater norovirus RT-PCR inhibitory effects than those that have undergone greater purification.

Molecular and Epidemiological Characteristics of Norovirus Associated with Community-Based Sporadic Gastroenteritis Incidents and Norovirus Outbreaks in Victoria, Australia, 2002–2007

Objectives: The molecular and epidemiological features of community-based norovirus-associated sporadic gastroenteritis incidents (NASGIs) are poorly understood. This study examined these features and compared the findings with studies of community-based and institutional norovirus-associated gastroenteritis outbreaks (NAGOs). Methods: Fecal specimens from NASGIs and NAGOs that occurred in Victoria, Australia (2002–2007) were tested for norovirus by reverse transcription-polymerase chain reaction methodology. Norovirus genotype was determined by nucleotide sequence analysis. Results: 106 community-based NASGIs, 116 community-based NAGOs and 902 institutional NAGOs were identified. The mean age and gender ratio of individuals associated with community-based NASGIs and community-based NAGOs were similar but differed from that found for institutional NAGOs. Although GII.4 was the predominant genotype associated with all three incident types, the mix of genotypes was similar for community-based NASGIs and community-based NAGOs but that for institutional NAGOs was different. All three incident types had a similar seasonal periodicity due to the pronounced seasonal periodicity of GII.4 incidents. Conclusions: The molecular and epidemiological features of noroviruses associated with community-based NASGIs and community-based NAGOs are similar but are different from those found for institutional NAGOs.

The relationship between health care and nonhealth care norovirus outbreak settings and norovirus genotype in Victoria, Australia, 2002–2005

BACKGROUND There is evidence that norovirus genotype is an important factor in determining norovirus epidemiology, but detailed information is lacking. This report examined this question by studying whether the mix of norovirus genotypes associated with norovirus outbreaks in health care settings was different to that in nonhealth care settings. METHODS Norovirus outbreaks tested in Victoria, Australia, 2002-2005 were classified as either health care or nonhealth care. Open reading frame 1 nucleotide sequencing analysis was then used to determine the mix of norovirus genotypes in health care and nonhealth care norovirus outbreaks. RESULTS For the three most common genotypes detected (GI.2, GII.4, and GIIb), the differences between health care and nonhealth care settings were significant. GII.4 was significantly more common in health care settings than in nonhealth care settings, whereas the genotypes GI.2 and GIIb were significantly more common in nonhealth care settings than in health care settings. CONCLUSION Norovirus genotype was found to be an important factor associated with norovirus outbreak setting.

Molecular changes in the norovirus polymerase gene and their association with incidence of GII.4 norovirus-associated gastroenteritis outbreaks in Victoria, Australia, 2001-2005.

This study examined whether a specific (six-nucleotide) sequence in the norovirus polymerase (POL) gene could act as a marker of GII.4 epidemics in Australia in 2001–2005. The predominant variant (pv) in an epidemic year (2002, 2004) changed relative to the pv in the previous year (AACTTG in 2001 to AATCTG in 2002, AATCTG in 2003 to AACTTG in 2004). The pv in a non-epidemic year (2003, 2005) was the same as in the preceding year (AATCTG in 2002 and 2003, AACTTG in 2004 and 2005). Thus, specific short nucleotide sequences could act as markers of major GII.4 norovirus epidemics.