Human noroviruses in the faeces of wild birds and rodents-new potential transmission routes.

Human noroviruses (HuNoVs) are one of the leading global causes of diarrhoeal diseases and are transmitted mainly from person to person but also through contaminated food, water and fomites. The possible zoonotic nature of NoVs has occasionally been discussed, although the viruses are generally considered to be host-species-specific. We investigated whether wild birds and rodents could serve as carriers of HuNoVs, thereby transmitting the virus to humans directly or indirectly by contaminating foods. All samples, 115 avian and 100 rat faeces collected in springs 2009-2013 from dump sites, and 85 faeces from yellow-necked mice trapped in late autumn 2008 and 2009 after the rodents entered human settlements due to the first night frosts, were screened for HuNoV using real-time reverse transcription PCR. HuNoVs were detected in 31 (27%) faecal samples of wild birds, in two (2%) faecal samples of rats and in no samples of mice. Most (25) of the positive bird samples and both rat samples contained genogroup II, and six positive bird samples contained genogroup I HuNoV. The avian species shedding faeces containing HuNoVs were identified as gulls and crows using DNA barcoding. Our results show that wildlife, birds and rats in particular, is capable of spreading HuNoVs in the environment.

Performance of pre-RT-qPCR treatments to discriminate infectious human rotaviruses and noroviruses from heat-inactivated viruses: applications of PMA/PMAxx, benzonase and RNase.

AIMS: Detection/Quantification of RNA viruses is mostly done by reverse-transcriptase (RT)-(q)PCR, but it does not distinguish between infectious and noninfectious viruses. Our aim was to test, how different pretreatments before RT-qPCR could eliminate positivity originated from external nucleic acids or genomes of damaged particles. METHODS AND RESULTS: Heat-inactivated (80°C for 10 min) rotavirus Wa strain and faecal samples containing rotavirus or norovirus were treated with PMA/PMAxx, benzonase or crude extract RNase prior to RT-qPCR. PMA/PMAxx pretreatments were not consistently efficient for RV, although they seemed to work to some extent for heat-inactivated norovirus. Benzonase and RNase provided consistently 2·2-2·8 log10 reductions in the titre of faecal rotavirus. CONCLUSIONS: All pretreatments need to be further validated for each virus separately, taking into account sample matrix and inactivation conditions. Although none of the pretreatments could completely render inactivated viruses undetectable, RNase worked most consistently for both rota- and norovirus. SIGNIFICANCE AND IMPACT OF THE STUDY: This study sheds light on capacity of the most common pre-RT-qPCR treatments to eliminate damaged, noninfectious rotaviruses and noroviruses after thermal treatment. To our knowledge, this is the first time, when benzonase has been used in this context.

Hepatitis E Virus Antibodies in Finnish Veterinarians.

We investigated hepatitis E virus (HEV) infections in Finnish veterinarians engaged in different practice specialties and evaluated the effect of different background factors on HEV exposure by examining total HEV antibodies in samples collected from the participants of the 2009 National Veterinary Congress in Helsinki, Finland. Finnish veterinarians commonly have total HEV antibodies with seroprevalence of 10.2%. Of the non-veterinarians, 5.8% were seropositive. Increasing age was associated with HEV seropositivity, and, surprisingly, the highest HEV seroprevalence (17.8%) among veterinarians was detected among small animal practitioners. Although no positive correlation between swine contacts and HEV seropositivity was found, 22.7% of veterinarians who had had needle stick by a needle that had previously been injected into a pig versus 9.0% of those who had not were seropositive, even though the finding was statistically non-significant (P = 0.07). Our results suggest that, although contact with swine is a known risk factor for HEV infection, the sources of HEV infections are probably numerous, including travelling abroad and possibly also other reservoirs of HEV than pigs.

Virological Quality of Irrigation Water in Leafy Green Vegetables and Berry Fruits Production Chains.

This study condenses data acquired during investigations of the virological quality of irrigation water used in production of fresh produce. One hundred and eight samples of irrigation water were collected from five berry fruit farms in Finland (1), the Czech Republic (1), Serbia (2), and Poland (1), and sixty-one samples were collected from three leafy green vegetable farms in Poland, Serbia, and Greece. Samples were analyzed for index viruses of human or animal fecal contamination (human and porcine adenoviruses, and bovine polyoma viruses), and human pathogenic viruses (hepatitis A virus, hepatitis E virus, and noroviruses GI/GII). Both index and pathogenic viruses were found in irrigation water samples from the leafy green vegetables production chain. The data on the presence of index viruses indicated that the highest percentage of fecal contamination was of human origin (28.1 %, 18/64), followed by that of porcine (15.4 %, 6/39) and bovine (5.1 %, 2/39) origins. Hepatitis E virus (5 %, 1/20) and noroviruses GII (14.3 %, 4/28) were also detected. Samples from berry fruit production were also positive for both index and pathogenic viruses. The highest percentage of fecal contamination was of human origin (8.3 %, 9/108), followed by that of porcine, 4.5 % (4/89) and bovine, 1.1 % (1/89) origins. Norovirus GII (3.6 %, 2/56) was also detected. These data demonstrate that irrigation water used in primary production is an important vehicle of viral contamination for fresh produce, and thus is a critical control point which should be integrated into food safety management systems for viruses. The recommendations of Codex Alimentarius, as well as regulations on the use of water of appropriate quality for irrigation purposes, should be followed.

Norovirus transmission between hands, gloves, utensils, and fresh produce during simulated food handling.

Human noroviruses (HuNoVs), a leading cause of food-borne gastroenteritis worldwide, are easily transferred via ready-to-eat (RTE) foods, often prepared by infected food handlers. In this study, the transmission of HuNoV and murine norovirus (MuNoV) from virus-contaminated hands to latex gloves during gloving, as well as from virus-contaminated donor surfaces to recipient surfaces after simulated preparation of cucumber sandwiches, was inspected. Virus transfer was investigated by swabbing with polyester swabs, followed by nucleic acid extraction from the swabs with a commercial kit and quantitative reverse transcription-PCR. During gloving, transfer of MuNoV dried on the hand was observed 10/12 times. HuNoV, dried on latex gloves, was disseminated to clean pairs of gloves 10/12 times, whereas HuNoV without drying was disseminated 11/12 times. In the sandwich-preparing simulation, both viruses were transferred repeatedly to the first recipient surface (left hand, cucumber, and knife) during the preparation. Both MuNoV and HuNoV were transferred more efficiently from latex gloves to cucumbers (1.2% ± 0.6% and 1.5% ± 1.9%) than vice versa (0.7% ± 0.5% and 0.5% ± 0.4%). We estimated that transfer of at least one infective HuNoV from contaminated hands to the sandwich prepared was likely to occur if the hands of the food handler contained 3 log10 or more HuNoVs before gloving. Virus-contaminated gloves were estimated to transfer HuNoV to the food servings more efficiently than a single contaminated cucumber during handling. Our results indicate that virus-free food ingredients and good hand hygiene are needed to prevent HuNoV contamination of RTE foods.

Outbreak of gastroenteritis caused by norovirus GII.4 Sydney variant after a wedding reception at a resort/activity centre, Finland, August 2012.

In August 2012, an outbreak of gastroenteritis occurred among 88 persons attending a wedding reception at a resort/activity centre in Ylöjärvi, Finland. Of 39 interviewed guests, 23 met the case definition. Two persons were hospitalized. Epidemiological, laboratory and environmental investigations were conducted to characterize the outbreak and to recommend control measures. Investigation confirmed the presence of a new strain of norovirus GII.4 Sydney variant in stool specimens obtained from two wedding guests and on several environmental surfaces in the centre. In the questionnaire study, none of the foods or beverages served during the reception were significantly associated with the illness. Additional cases of gastroenteritis that occurred at the centre before and after the wedding reception supported the hypothesis of environmental transmission of norovirus. After thorough cleansing and disinfection and 1 week's quarantine, no new cases with symptoms typical for norovirus infection were identified at the centre.

Ultraviolet Light Inactivation of Murine Norovirus and Human Norovirus GII: PCR May Overestimate the Persistence of Noroviruses Even When Combined with Pre-PCR Treatments.

Transmission of gastroenteritis-causing noroviruses may be significant via contaminated surfaces. Measures for control, e.g. disinfection with ultraviolet irradiation (UV), are therefore necessary for interrupting this transmission. Human norovirus (HuNoV) GII.4 and Murine norovirus (MuNoV) were used to study the efficacy of UV for virus inactivation on dry glass surfaces. MuNoV inactivation was measured using viability assay and the reduction in viral RNA levels for both viruses using reverse transcription quantitative PCR (RT-QPCR). For each UV dose, two parallel sample groups were detected using RT-QPCR: one group was enzymatically pre-PCR treated with Pronase and RNAse enzymes, while the other was not treated enzymatically. In the viability assay, loss of infectivity and a 4-log reduction of MuNoV were observed when the viruses on glass slides were treated with a UV dose of 60 mJ/cm(2) or higher. In the RT-QPCR assay, a steady 2-log decline of MuNoV and HuNoV RNA levels was observed when UV doses were raised from 0 to 150 mJ/cm(2). A distinct difference in RNA levels of pretreated and non-pretreated samples was observed with UV doses of 450-1.8 × 10(3) mJ/cm(2): the RNA levels of untreated samples remained over 1.0 × 10(3) PCR units (pcr-u), while the RNA levels of enzyme-treated samples declined below 100 pcr-u. However, the data show a prominent difference between the persistence of MuNoV observed with the infectivity assay and that of viral RNA detected using RT-QPCR. Methods based on genome detection may overestimate norovirus persistence even when samples are pretreated before genome detection.

A longitudinal study revealing hepatitis E virus infection and transmission at a swine test station.

Hepatitis E virus (HEV) is a zoonotic agent that causes acute hepatitis in humans, and infects several animal species, most importantly swine. In the current study, that presents the first evidence of HEV infections in pigs in Finland, genetic divergence and transmission of HEV was investigated among pigs at a swine test station at two occasions. In 2007, HEV RNA was found in 25% of pens, and 35% of 2-3 month-old pigs at the station. Three different isolates, comprising 13 sequences of HEV genotype 3 e that were imported from different farms were detected. In 2010, 39% of pigs were HEV RNA positive on weeks 1, 3, or 5 of a 3-month follow-up, and 11 sequences, all representing one of the isolates that was also present in 2007, were detected. The isolate was considered to be either re-introduced to, or to persist at the station, and it was transmitted between the pigs. The study sheds light on the rate and time of HEV transmission in swine, and describes the epidemiologic variability of HEV isolates over time.

Multiple norovirus outbreaks linked to imported frozen raspberries.

In 2009, the number of foodborne norovirus outbreaks in Finland seemed markedly high, and many outbreaks seemed to be linked to imported frozen raspberries. We reviewed the data regarding all notified foodborne outbreaks in 2009 in Finland in order to assess the magnitude of the problem and to summarize the information on raspberry-linked outbreaks. Between March and August, 13 norovirus outbreaks affecting about 900 people could be linked to imported frozen raspberries. Two raspberry samples corresponding to two batches of raspberries were positive for norovirus. These two batches proved to have been the likely source in six of the 13 outbreaks. Analytical studies had not been conducted for six outbreaks, and virological test results were inconclusive in two. However, combining epidemiological and microbiological methods often enabled finding the source, as exemplified in investigation of a large school outbreak. To ensure prompt control measures in similar situations in the future, both aspects of outbreak investigations should be strengthened.

Rotavirus genotypes co-circulating in Europe between 2006 and 2009 as determined by EuroRotaNet, a pan-European collaborative strain surveillance network.

EuroRotaNet, a laboratory network, was established in order to determine the diversity of co-circulating rotavirus strains in Europe over three or more rotavirus seasons from 2006/2007 and currently includes 16 countries. This report highlights the tremendous diversity of rotavirus strains co-circulating in the European population during three years of surveillance since 2006/2007 and points to the possible origins of these strains including genetic reassortment and interspecies transmission. Furthermore, the ability of the network to identify strains circulating with an incidence of ≥1% allowed the identification of possible emerging strains such as G8 and G12 since the beginning of the study; analysis of recent data indicates their increased incidence. The introduction of universal rotavirus vaccination in at least two of the participating countries, and partial vaccine coverage in some others may provide data on diversity driven by vaccine introduction and possible strain replacement in Europe.

An extensive gastroenteritis outbreak after drinking-water contamination by sewage effluent, Finland.

An inappropriate cross-connection between sewage- and drinking-water pipelines contaminated tap water in a Finnish town, resulting in an extensive waterborne gastroenteritis outbreak in this developed country. According to a database and a line-list, altogether 1222 subjects sought medical care as a result of this exposure. Seven pathogens were found in patient samples of those who sought treatment. To establish the true disease burden from this exposure, we undertook a population-based questionnaire investigation with a control population, infrequently used to study waterborne outbreaks. The study covered three areas, contaminated and uncontaminated parts of the town and a control town. An estimated 8453 residents fell ill during the outbreak, the excess number of illnesses being 6501. Attack rates were 53% [95% confidence interval (CI) 49.5-56.4] in the contaminated area, 15.6% (95% CI 13.1-18.5) in the uncontaminated area and 6.5% (95% CI 4.8-8.8) in the control population. Using a control population allowed us to differentiate baseline morbidity from the observed morbidity caused by the water contamination, thus enabling a more accurate estimate of the disease burden of this outbreak.

Detection of human norovirus from frozen raspberries in a cluster of gastroenteritis outbreaks.

We describe a cluster of norovirus outbreaks affecting about 200 people in Southern Finland in September and October 2009. All outbreaks occurred after consumption of imported raspberries from the same batch intended for the catering sector. Human norovirus genotype GI.4 was found in frozen raspberries. The berries were served in toppings of cakes in separate catering settings or mixed in curd cheese as a snack for children in a daycare center. The relative risk for consumption of the berry dish was 3.0 (p

Rotavirus surveillance in europe, 2005-2008: web-enabled reporting and real-time analysis of genotyping and epidemiological data.

BACKGROUND: The first European rotavirus surveillance network, EuroRotaNet, comprising 16 laboratories in 15 European countries, has been established. METHODS: Fecal samples from gastroenteritis cases positive for group A rotavirus antigen were collected from multiple European countries from 2005 to mid-2008 and were subjected to G and P genotyping. Epidemiological data collected included age, sex, geographical location, setting, dates of onset and sample collection, and clinical symptoms. RESULTS: A total of 8879 rotavirus-positive samples were characterized: 2129 cases were from the 2005-2006 season, 4030 from the 2006-2007 season, and 2720 from the ongoing 2007-2008 season. A total of 30 different G and P type combinations of strains circulated in the region from 2005 through 2008. Of these strains, 90% had genotypes commonly associated with human infections-G1P[8], G2P[4], G3P[8], G4P[8], and G9P[8]-and 1.37% represented potential zoonotic introductions. G1P[8] remained the most prevalent genotype in Europe as a whole, but the incidence of infection with G1P[8] rotavirus strains was <50% overall, and all 3 seasons were characterized by a significant diversity of cocirculating strains. The peak incidence of rotavirus infection occurred from January through May, and 81% of case patients were aged <2.5 years. Conclusions. Data gathered through EuroRotaNet will provide valuable background information on the rotavirus strain diversity in Europe before the introduction of rotavirus vaccines, and the network will provide a robust method for surveillance during vaccine implementation.

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.

Prolonged norovirus outbreak in a Finnish tertiary care hospital caused by GII.4-2006b subvariants.

Norovirus outbreaks are difficult to control in hospitals. Cohorting and contact isolation, disinfective surface cleaning and hand hygiene are key elements in outbreak control. A new norovirus variant, GII.4.-2006b, spreading across many continents, caused an exceptionally long epidemic period in Finland, from November 2006 to June 2007. Here, we describe the clinical and molecular characteristics of a norovirus outbreak in a large tertiary care hospital in Finland. Altogether 240 (18%) patients and 205 (19%) healthcare workers fell ill in the 504 bedded main building of Helsinki University Central Hospital during December 2006 to May 2007. The epidemic curve had three peaks in January, February and April, and different wards were affected each time. During the outbreak, 502 patient stool specimens were tested for norovirus RNA, 181 (36%) of which were positive. Molecular analysis of 48 positive specimens revealed three main subvariants of GII.4.-2006b circulating temporally within distinct wards. Of all microbiologically confirmed cases, 121 (67%) were nosocomial and nine (5%) died within 30 days of diagnosis. Molecular analysis suggested that the three main GII.4-2006b subvariants entered the hospital with gastroenteritis patients, and the nosocomial spread within wards coincided with the epidemic peaks. Active control measures, including temporary closure of the wards, ultimately confined the single-ward outbreaks. A prolonged outbreak in the community was probably the source for the prolonged outbreak period in the hospital.

Multiple norovirus outbreaks among workplace canteen users in Finland, July 2006.

Multiple gastroenteritis outbreaks occurred between 25 and 31 July 2006 in 10 workplace canteens in south-western Finland. One vegetable processing plant provided raw vegetables to all the canteens. We conducted cohort studies in the three most visited canteens and environmental investigations in the kitchens and the plant. Patients' stools, food, water and environmental samples were tested for enteric bacteria and viruses. Of the three canteens, 150/273 respondents (response rate 82%) had gastroenteritis. Consumption of mixed raw vegetables was significantly associated with the illness but no single vegetable explains the outbreak. An identical norovirus GII.1 genotype was detected from all genotyped patient samples. Water, food, and environmental samples were negative for norovirus. The facilities had appropriate hygienic conditions and no staff member had gastroenteritis prior to the outbreak. Tracing back the vegetables to the farm level proved unsuccessful. This was the largest foodborne norovirus outbreak in Finland.

Analysis of integrated virological and epidemiological reports of norovirus outbreaks collected within the Foodborne Viruses in Europe network from 1 July 2001 to 30 June 2006.

The Foodborne Viruses in Europe network has developed integrated epidemiological and virological outbreak reporting with aggregation and sharing of data through a joint database. We analyzed data from reported outbreaks of norovirus (NoV)-caused gastroenteritis from 13 European countries (July 2001 to July 2006) for trends in time and indications of different epidemiology of genotypes and variants. Of the 13 countries participating in this surveillance network, 11 were capable of collecting integrated epidemiological and virological surveillance data and 10 countries reported outbreaks throughout the entire period. Large differences in the numbers and rates of reported outbreaks per country were observed, reflecting the differences in the focus and coverage of national surveillance systems. GII.4 strains predominated throughout the 5-year surveillance period, but the proportion of outbreaks associated with GII.4 rose remarkably during years in which NoV activity was particularly high. Spring and summer peaks indicated the emergence of genetically distinct variants within GII.4 across Europe and were followed by increased NoV activity during the 2002-2003 and 2004-2005 winter seasons. GII.4 viruses predominated in health care settings and in person-to-person transmission. The consecutive emergence of new GII.4 variants is highly indicative of immune-driven selection. Their predominance in health care settings suggests properties that facilitate transmission in settings with a high concentration of people such as higher virus loads in excreta or a higher incidence of vomiting. Understanding the mechanisms driving the changes in epidemiology and clinical impact of these rapidly evolving RNA viruses is essential to design effective intervention and prevention measures.

Data quality of 5 years of central norovirus outbreak reporting in the European Network for food-borne viruses.

BACKGROUND: The food-borne viruses in Europe (FBVE) network database was established in 1999 to monitor trends in outbreaks of gastroenteritis due to noroviruses (NoVs), to identify major transmission routes of NoV infections within and between participating countries and to detect diffuse international food-borne outbreaks. METHODS: We reviewed the total of 9430 NoV outbreak reports from 13 countries with date of onset between 1 January 2002 and 1 January 2007 for representativeness, completeness and timeliness against these objectives. RESULTS: Rates of reporting ranged from a yearly average of 1.8 in 2003 to 11.6 in 2006. Completeness of reporting of an agreed minimum dataset improved over the years, both for epidemiological and virological data. For the 10 countries that provided integrated (epidemiological AND virological) reporting over the 5-year period, the completeness of the minimum dataset rose from 15% in 2003 to 48% in 2006. Two countries have not been able to combine both data types due to the structure of the national surveillance system (England and Wales and Germany). Timeliness of reporting (median days between the onset of an outbreak and the date of reporting to the FBVE database) differed greatly between countries, but gradually improved to 47 days in 2006. CONCLUSION: The outbreaks reported to the FBVE reflect the lack of standardization of surveillance systems across Europe, making direct comparison of data between countries difficult. However, trends in reported outbreaks per country, distribution of NoV genotypes, and detection of diffuse international outbreaks were used as background data in acute questions about NoV illness and the changing genotype distribution during the 5-year period, shown to be of added value. Integrated reporting is essential for these objectives, but could be limited to sentinel countries with surveillance systems that allow this integration. For successful intervention in case of diffuse international outbreaks, completeness and timeliness of reporting would need to be improved and expanded to countries that presently do not participate.

Norovirus genotypes causing gastroenteritis outbreaks in Finland 1998-2002.

BACKGROUND: Outbreak investigation methods for enteric viruses were improved in 1990s when gene amplification techniques were established in viral laboratories. OBJECTIVES: The objective of the study was to determine the causative agents for Finnish viral gastroenteritis outbreaks. Our aim was also to further characterise the norovirus strains, reveal the temporal occurrence of norovirus (NV) genotypes and to study some epidemiological aspects concerning the outbreaks. STUDY DESIGN: A total of 416 Finnish viral gastroenteritis outbreaks that occurred during 5 years (1998-2002), excluding those among hospitalised children, were investigated for enteric viruses. Stool samples were screened by electron microscopy as well as analyzed by specific noro- and astrovirus RT-PCR tests. Amplicon sequence analysis was used to find out norovirus genotypes. RESULTS: Noroviruses caused 252 (60.6%) of the outbreaks; other viruses, astro- or rotavirus, caused four epidemics. Norovirus epidemics occurred in all kinds of settings, most often in hospitals (30.6%) and in restaurants and canteens (14.3%). Both NV genogroups were found every year, but NV GGII outbreaks always outnumbered those of GGI. All but one outbreak at hospitals and nursing homes were of genotype GII. Polymerase sequence analysis revealed a variety of NV genotypes; six GI and at least eight GII genotypes. The GI.3 Birmingham-like and GII.4 Bristol-like genotype appeared every year, whereas the other types were circulating for shorter periods or sporadically. During the study period the genotypes GII.4 (Bristol), GII.1 (Hawaii), an emerging genotype GIIb, and a new variant of GII.4 predominated in that order. Indication for rapid genetic changes in the genotype GII.4 was also noticed. CONCLUSIONS: Noroviruses were the most prevalent causative agents in the outbreaks. Many NV genotypes were circulating, and a shift in the predominant genotypes was evident between epidemic seasons.

Wading pool water contaminated with both noroviruses and astroviruses as the source of a gastroenteritis outbreak.

In July 2001, an outbreak of gastroenteritis occurred in Helsinki among children and adults after bathing in an outdoor wading pool. The epidemiological survey revealed that at least 242 persons were affected. Microbiological testing of both patient stool samples and of the pool water revealed the presence of two different gastroenteritis viruses: a norovirus (NV) and an astrovirus. Amplicon sequencing of the NV samples showed nucleotide sequence identity between the virus from patients and the water. After changing the pool water and the sand at the bottom of the pool followed by shock chlorination, no virus could be detected in the water. However, NV was continuously detected in the water outlet well as much as 8 months after the incident. Here we show how molecular methods aided in tracing the source of the epidemic and in finding the causative pathogens both in patients and in the environment.