Prevalence of hepatitis E virus antibodies in workers occupationally exposed to swine in Portugal.

The concept of zoonotic hepatitis E in industrialized countries has emerged with the discovery of swine strains of hepatitis E virus (HEV) genotype 3, closely related to human HEV. Different routes of zoonotic HEV transmission have been recognized, including contact with infected pigs. Workers occupationally exposed to swine (WOES) have been considered a risk group for HEV infection, but contradictory results have been reported. In the present study, we searched for anti-HEV IgG in WOES (butchers, slaughterhouse workers, veterinarians and pig farmers; n = 114) and in the general population (n = 804) in order to investigate the potential occupational risk of zoonotic HEV infection in this work group. A significantly higher (p = 0.008) anti-HEV IgG seroprevalence was found in WOES (30.7 %) when compared with the general population (19.9 %). Multivariate analysis showed that having professions with exposure to pigs for more than 16.5 years was a risk factor for being positive for anti-HEV IgG (aOR of 5.4, 95 % CI 1.9-15.6, p = 0.002). To our knowledge, this is the first study on the prevalence of anti-HEV IgG in WOES in Portugal, also showing increased probability for infection in this group.

Large and prolonged food-borne multistate hepatitis A outbreak in Europe associated with consumption of frozen berries, 2013 to 2014.

In May 2013, Italy declared a national outbreak of hepatitis A, which also affected several foreign tourists who had recently visited the country. Molecular investigations identified some cases as infected with an identical strain of hepatitis A virus subgenotype IA. After additional European Union/European Economic Area (EU/EEA) countries reported locally acquired and travel-related cases associated with the same outbreak, an international outbreak investigation team was convened, a European outbreak case definition was issued and harmonisation of the national epidemiological and microbiological investigations was encouraged. From January 2013 to August 2014, 1,589 hepatitis A cases were reported associated with the multistate outbreak; 1,102 (70%) of the cases were hospitalised for a median time of six days; two related deaths were reported. Epidemiological and microbiological investigations implicated mixed frozen berries as the vehicle of infection of the outbreak. In order to control the spread of the outbreak, suspected or contaminated food batches were recalled, the public was recommended to heat-treat berries, and post-exposure prophylaxis of contacts was performed. The outbreak highlighted how large food-borne hepatitis A outbreaks may affect the increasingly susceptible EU/EEA general population and how, with the growing international food trade, frozen berries are a potential high-risk food.

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.

Infectious pancreatic necrosis virus in fish by-products is inactivated with inorganic acid (pH 1) and base (pH 12).

The aquaculture industry needs a simple, inexpensive and safe method for the treatment of fish waste without heat. Microbial inactivation by inorganic acid (HCl) or base (KOH) was determined using infectious pancreatic necrosis virus (IPNV) as a model organism for fish pathogens. Salmonella and spores of Clostridium perfringens were general hygiene indicators in supplementary examinations. IPNV, which is considered to be among the most chemical- and heat-resistant fish pathogens, was reduced by more than 3 log in 4 h at pH 1.0 and pH 12.0. Salmonella was rapidly inactivated by the same treatment, whereas spores of C. perfringens were hardly affected. The results indicate that low and high pH treatment could be particularly suitable for fish waste destined for biogas production. pH treatment at aquaculture production sites could reduce the spread of fish pathogens during storage and transportation without disturbing the anaerobic digestion process. The treatment could also be an alternative to the current energy-intensive steam pressure sterilization of fish waste to be used by the bioenergy, fertilizer and soil improver industries.

The impact of temperature on the inactivation of enteric viruses in food and water: a review.

Temperature is considered as the major factor determining virus inactivation in the environment. Food industries, therefore, widely apply temperature as virus inactivating parameter. This review encompasses an overview of viral inactivation and virus genome degradation data from published literature as well as a statistical analysis and the development of empirical formulae to predict virus inactivation. A total of 658 data (time to obtain a first log(10) reduction) were collected from 76 published studies with 563 data on virus infectivity and 95 data on genome degradation. Linear model fitting was applied to analyse the effects of temperature, virus species, detection method (cell culture or molecular methods), matrix (simple or complex) and temperature category (<50 and ≥50°C). As expected, virus inactivation was found to be faster at temperatures ≥50°C than at temperatures <50°C, but there was also a significant temperature-matrix effect. Virus inactivation appeared to occur faster in complex than in simple matrices. In general, bacteriophages PRD1 and PhiX174 appeared to be highly persistent whatever the matrix or the temperature, which makes them useful indicators for virus inactivation studies. The virus genome was shown to be more resistant than infectious virus. Simple empirical formulas were developed that can be used to predict virus inactivation and genome degradation for untested temperatures, time points or even virus strains.

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.

Enteric viruses in inlet and outlet samples from sewage treatment plants.

Samples collected every two weeks from the inlet and outlet of three sewage treatment plants were screened for the presence of noro-, rota-, astro-, adeno-, hepatitis A- and circoviruses by (RT)-nested PCR, and for F-specific bacteriophages by isolation in Escherichia coli Famp. Plants A and B were secondary treatment plants and plant C used primary treatment. Noroviruses were detected in 43%, 53% and 24% of the inlet samples and 26%, 40% and 21% of the outlet samples from plants A, B and C, respectively. Astroviruses, rotaviruses and adenoviruses were more prevalent. Adenoviruses were detected in 96% of inlet and 94% of outlet samples, supporting the potential of these viruses as indicators of viral contamination from sewage. Hepatitis A virus and circoviruses were found only rarely. Reduction of infective viral particles during sewage treatment was evaluated using F-specific bacteriophages. The phages were reduced by, respectively, 99%, 87% and 0% in plants A, B and C, which corresponded to the observed differences in reduction of norovirus positive samples between the same plants. The study shows that the high viral load in sewage results in a discharge to the environment of a large amount of virus despite sewage treatment. On the other hand, the advantage of a more advanced treatment is demonstrated.

Detection of enteric viruses in shellfish from the Norwegian coast.

Common blue mussels (Mytilus edulis), horse mussels (Modiolus modiolus), and flat oysters (Ostrea edulis) obtained from various harvesting and commercial production sites along the Norwegian coast were screened for the presence of norovirus by a real-time reverse transcription (RT)-nested PCR assay and for possible indicators of fecal contamination, i.e., for F-specific RNA bacteriophages (F-RNA phages) by plaque assay and for human adenoviruses and human circoviruses by nested PCR assay. The aims were to obtain relevant information for assessing the risk of transmission of enteric viruses by shellfish and to investigate the potential of various indicator viruses in routine screening. Noroviruses were detected in 6.8% of the samples, and the indicators were detected in 23.8% (F-RNA phages), 18.6% (adenoviruses), and 8.0% (circoviruses) of the samples. A seasonal variation was observed, with the exception of circoviruses, with more positive samples in the winter. A positive correlation was found between F-RNA phages and noroviruses. However, F-RNA phages were present in only 43% of the norovirus-positive samples. The results show that mussels from the Norwegian coast can constitute a risk of infection with enteric viruses and that routine testing of samples may be justified. Advantages and disadvantages of various options for screening are discussed.

Antigenic diversity of Norwalk-like viruses: expression of the capsid protein of a genogroup I virus, distantly related to Norwalk virus.

The gene encoding the capsid protein of a genogroup I Norwalk-like virus (NLV) (Hu/NLV/Stav/95/Nor) was cloned and expressed in insect cells using a baculovirus vector. The His-tagged recombinant capsid protein (rStav) was antigenic and immunogenic, showed an apparent molecular weight of approximately 68 kD in protein gels, and was only soluble under denaturing conditions. The amino acid sequence of the rStav protein showed 65-88% similarity to capsid protein sequences from other genogroup I NLV and was most closely related to Desert Shield virus. Norwegian recruit sera were tested for antibodies against rStav by Western blotting (rStav WB). The sera had previously been tested for antibodies against a recombinant Norwalk virus capsid protein in an ELISA (rNV ELISA). Several rNV ELISA-negative sera showed a positive response in the rStav WB, indicating that the use of antigens representing different stains may be necessary when screening sera for antibodies against genogroup I NLV.

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.

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.

Prevalence of serum antibodies to Norwalk virus among Norwegian military recruits.

The prevalence of serum antibodies against Norwalk virus among military recruits in Norway was investigated using an enzyme-linked immunosorbent assay (ELISA). A total of 1017 sera were assayed for anti-Norwalk virus total antibodies (ig), of which 300 (29.5%) were positive. Of 227 positive sera, 10.6 and 15.4% were positive for IgA and IgM, respectively, while 2.2% were positive for both. The prevalence of antibodies against Norwalk virus in south-east Norway was significantly higher than that in northern Norway.