Mette Myrmel

Molecular Epidemiology of Norovirus Outbreaks in Norway during 2000 to 2005 and Comparison of Four Norovirus Real-Time Reverse Transcriptase PCR Assays

ABSTRACT During the period from January 2000 to August 2005 a total of 204 outbreaks of norovirus gastroenteritis were diagnosed at the Norwegian Institute of Public Health. A clear increase in the norovirus activity was seen in healthcare institutions during the winter seasons. Polymerase sequence analysis of norovirus strains from 122 outbreaks showed that 112 were caused by GII strains (91.8%). Two norovirus variants seen during the study period—GIIb and GII.4—were predominant between January 2000 and September 2002, whereas GII.4 was predominant from September 2002 onward. The highest norovirus activity was seen during the 2002-2003 and 2004-2005 seasons with the emergence of new GII.4 variants. This study describes the molecular epidemiology of norovirus strains circulating in Norway during the five previous seasons and compares four norovirus real-time reverse transcriptase PCR assays. A suitable assay for routine diagnostics is suggested.

Immunomagnetic separation of a Norwalk-like virus (genogroup I) in artificially contaminated environmental water samples

Rabbit polyclonal antibodies were raised against a recombinant capsid protein from a genogroup I Norwalk-like virus (NLV). Magnetic beads coated with these antibodies were used in immunomagnetic separation (IMS) of the NLV. After capture of the NLV and washing of the beads, viral RNA was heat released and detected by RT-PCR. This IMS procedure was shown to have high sensitivity for detection of homologous NLV, while capture of a genogroup II NLV was less efficient. Antigen capture was not influenced by the content of humic acids in the samples. The combination of IMS and heat release was found to be more efficient than organic extraction of RNA from water contaminated with humic acids. The efficacy and simplicity of IMS/heat release render this combination a feasible tool for the preparation of NLV RNA from environmental samples, although the antigenic diversity of NLV may be a complicating factor.

Potential internalisation of caliciviruses in lettuce.

Fresh produce such as lettuce (Lactuca sativa) has often been linked to epidemic viral gastroenteritis. In these cases, it is unknown whether the viral contamination has occurred during the growing or the processing of the implicated product. In this study lettuce was grown in the presence of enteric viruses, and the uptake of viruses via the roots into the edible parts (leaves and stem) of the lettuce plants was investigated, for plants with both intact and damaged roots. The roots of lettuce, growing either in hydroponic culture or in soil, were exposed to canine calicivirus (CaCV) and a human genogroup 2 norovirus (HuNoV) by these being added into the water or soil in which the lettuce was growing. Leaves from lettuce plants and seedlings were examined for viruses by real-time RT-PCR. When the lettuce plants were exposed to very high concentrations of CaCV, the virus was detected in lettuce leaves, indicating contamination via the roots, but the frequency of positive results was low. Internalisation occurred in both seedlings and grown plants, in both hydroponic and soil cultures, and occurred whether the roots were intact or damaged. However, internalisation of HuNoV was not detected in any of the experimental set ups, although the concentrations to which the plants were exposed were relatively high. Based on these results, viral contamination of lettuce plants via roots cannot be excluded, but is apparently not an important transmission route for viruses.

Detection of norovirus genotype I.3b and II.4 in bioaccumulated blue mussels using different virus recovery methods

Noroviruses (NoVs) are the most common non bacterial human pathogens associated with shellfish borne gastroenteritis. Norovirus detection is based on molecular procedures such as reverse transcriptase (RT)-PCR. A variety of methods have been developed to extract viral RNA from complex shellfish matrixes and to reduce the level of RT-PCR inhibitors. The present study had three objectives: 1) Determine the most appropriate sample treatment protocol for detection of NoVs in mussels, 2) Examine whether there is a variation of the binding affinity between a NoV GI and a GII strain to mussel digestive tissue and how this influences the detection sensitivity, 3) Establish an internal control for sample processing and virus detection. Three RNA extraction methods were evaluated on extracts from blue mussels (Mytilus edulis) spiked with NoV GII.4. The most efficient RNA extraction method was subsequently used for evaluation of three virus recovery methods of blue mussels bio accumulated with NoV GI.3b and GII.4. Mengovirus was evaluated as an internal process control and TaqMan RT-PCRs were used for virus detection. Elution of the two viruses from shellfish tissue differed, indicating a difference in binding affinities. Only a method based upon Proteinase K digestion followed by NucliSenseasyMAG was able to detect both NoV GI.3b and GII.4 (3.0% and 3.5% recovery respectively). The results show that the processing method influences the possibility to detect different variants of NoV.

Evaluation of a rapid method for recovery of norovirus and hepatitis A virus from oysters and blue mussels

Foodborne outbreaks caused by noroviruses (NoVs) and hepatitis A virus (HAV) are often linked to consumption of contaminated shellfish. The objective of this study was to identify an appropriate virus recovery method for real-time reverse transcriptase (RT)-PCR detection and subsequently to evaluate this method on shellfish bioaccumulated with virus in a collaborative study. Five methods were compared for recovery of NoV GII.7 and feline calicivirus from spiked digestive tissue of oysters and mussels. A method based on proteinase K digestion followed by NucliSENS miniMAG extraction was found to be the most efficient with a 50% limit of detection (LOD(50)) of 62 and 12 RT-PCR U/1.5 g digestive tissue for NoV GII.7 in oysters and mussels, respectively. Evaluation of the method in four laboratories found the percentage of sensitivity, based on low/high levels of virus bioaccumulated in oysters, to be 33/80 for NoV GI.3b, 13/92 for NoV GII.4 and 50/42 for HAV. A specificity of 100% was found for all three viruses in non-bioaccumulated oysters. As process control Mengovirus (vMC(0)) showed an average recovery of 1.8% from oysters and 1.2% from mussels. The study demonstrates that this recovery method can be useful for harmonized data generation and routine viral analyses of shellfish.

Bluetongue: a historical and epidemiological perspective with the emphasis on South Africa

Bluetongue (BT) is a non-contagious, infectious, arthropod transmitted viral disease of domestic and wild ruminants that is caused by the bluetongue virus (BTV), the prototype member of the Orbivirus genus in the family Reoviridae. Bluetongue was first described in South Africa, where it has probably been endemic in wild ruminants since antiquity. Since its discovery BT has had a major impact on sheep breeders in the country and has therefore been a key focus of research at the Onderstepoort Veterinary Research Institute in Pretoria, South Africa. Several key discoveries were made at this Institute, including the demonstration that the aetiological agent of BT was a dsRNA virus that is transmitted by Culicoides midges and that multiple BTV serotypes circulate in nature. It is currently recognized that BT is endemic throughout most of South Africa and 22 of the 26 known serotypes have been detected in the region. Multiple serotypes circulate each vector season with the occurrence of different serotypes depending largely on herd-immunity. Indigenous sheep breeds, cattle and wild ruminants are frequently infected but rarely demonstrate clinical signs, whereas improved European sheep breeds are most susceptible. The immunization of susceptible sheep remains the most effective and practical control measure against BT. In order to protect sheep against multiple circulating serotypes, three pentavalent attenuated vaccines have been developed. Despite the proven efficacy of these vaccines in protecting sheep against the disease, several disadvantages are associated with their use in the field.

A 1-Year Quantitative Survey of Noro-, Adeno-, Human Boca-, and Hepatitis E Viruses in Raw and Secondarily Treated Sewage from Two Plants in Norway

A study of enteric viruses in raw and treated sewage from two secondary treatment plants, which received sewage from Oslo city (plant A) and small municipalities in Hedmark county in Norway (plant B), showed high levels of noro-, adeno-, and bocavirus throughout the year. A seasonal variation was observed for adeno- and GII norovirus with higher levels during winter and bocavirus that had more positive samples during winter. The virus concentrations in raw sewage were comparable in the two plants, with medians (log10 genome copies per liter) of 6.1, 6.3, 6.0, and 4.5 for noro GI, noro GII, adeno-, and bocavirus, respectively. The level of hepatitis E virus was not determined as it was below the limit of quantification. The mean log10 virus reduction was 0.55 (plant A) and 1.44 (plant B) with the highest reduction found in the plant with longer hydraulic retention time. The adenoviruses were dominantly serotype 41, while serotype 12 appeared sporadically. Of the 102 raw and treated sewage samples that were tested, eight were positive for hepatitis E virus of which four were from treated sewage. Two of the four obtained gene sequences from hepatitis E virus originated from the rural sewage samples and showed high similarity with a genotype 3 strain of hepatitis E virus detected in local piglets. Two other hepatitis E virus sequences obtained from urban sewage samples showed high similarities with genotype 3 strains isolated from urban sewage in Spain and a human genotype 1 isolate from India. The study gives information on the levels of noroviruses in raw and treated sewage, which is valuable to risk assessment, information indicating that some infections with hepatitis E viruses in Norway have a regional origin and that human bocavirus 2 and 3 are prevalent in the Norwegian population.

SYBR Green based real-time RT-PCR assay for detection and genotype prediction of bovine noroviruses and assessment of clinical significance in Norway.

Abstract A novel SYBR Green based real-time RT-PCR assay for detection of genogroup III bovine noroviruses (BoNoV) was developed and the assay applied to 419 faecal samples from calves with and without diarrhoea. The samples were obtained from 190 Norwegian dairy and beef herds. BoNoV was detected in 49.6% of the samples from 61.1% of the herds indicating that BoNoV is ubiquitous in Norway. The overall prevalence was not significantly different in diarrhoea and non-diarrhoea samples. Analyses of polymerase gene sequences revealed both genotype III/1 and III/2 with genotype III/2 (Newbury2-like) being the most prevalent. Detected capsid sequences were restricted to Newbury2-like and the chimeric Bo/Thirsk10/00/UK strain. The RNA polymerase genotypes of the circulating BoNoVs in Norway were predicted by melting temperature analysis. Additional data from a challenge experiment suggest that a high proportion of young calves are shedding low levels of BoNoV for a prolonged time after recovering from the associated diarrhoea. The findings may explain some of the discrepancies in detection rates from previous studies and explain why some studies have failed to detect significant prevalence differences between calves with and without diarrhoea. It may also shed new light on some epidemiological aspects of norovirus infections.

Quantitative microbial risk assessment combined with hydrodynamic modelling to estimate the public health risk associated with bathing after rainfall events

This study investigated the public health risk from exposure to infectious microorganisms at Sandvika recreational beaches, Norway and dose-response relationships by combining hydrodynamic modelling with Quantitative Microbial Risk Assessment (QMRA). Meteorological and hydrological data were collected to produce a calibrated hydrodynamic model using Escherichia coli as an indicator of faecal contamination. Based on average concentrations of reference pathogens (norovirus, Campylobacter, Salmonella, Giardia and Cryptosporidium) relative to E. coli in Norwegian sewage from previous studies, the hydrodynamic model was used for simulating the concentrations of pathogens at the local beaches during and after a heavy rainfall event, using three different decay rates. The simulated concentrations were used as input for QMRA and the public health risk was estimated as probability of infection from a single exposure of bathers during the three consecutive days after the rainfall event. The level of risk on the first day after the rainfall event was acceptable for the bacterial and parasitic reference pathogens, but high for the viral reference pathogen at all beaches, and severe at Kalvøya-small and Kalvøya-big beaches, supporting the advice of avoiding swimming in the day(s) after heavy rainfall. The study demonstrates the potential of combining discharge-based hydrodynamic modelling with QMRA in the context of bathing water as a tool to evaluate public health risk and support beach management decisions.

Detection of small round structured viruses in artificially contaminated water using filter adsorption and reverse transcription polymerase chain reaction

The small round structured viruses (SRSV) are common causes of gastroenteritis worldwide. Fecally contaminated water is an important vehicle for transmission, but detection of SRSV in environmental samples has been hampered by the lack of sensitive detection methods. The present work describes the detection of SRSV in artificially contaminated deionized water and raw drinking water. SRSV-containing fecal extracts were added to water and virus was recovered by filter adsorption-elution, followed by flocculation. RNA was extracted and SRSV were detected by the use of reverse transcription polymerase chain reaction. The sensitivity of the method corresponded to a positive SRSV detection in 500 ml deionized water with an estimated concentration of 0.5-5 virus particles per ml.