I. Di Bartolo

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

ABSTRACT 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.

Massive outbreak of viral gastroenteritis associated with consumption of municipal drinking water in a European capital city

On 24 August 2008, an outbreak alert regarding cases of acute gastroenteritis in Podgorica triggered investigations to guide control measures. From 23 August to 7 September, 1699 cases were reported in Podgorica (population 136 000) and we estimated the total size of the outbreak to be 10 000-15 000 corresponding to an attack rate of approximately 10%. We conducted an age- and neighbourhood-matched case-control study, microbiologically analysed faecal and municipal water samples and assessed the water distribution system. All cases (83/83) and 90% (80/90) [corrected] of controls drank unboiled chlorinated municipal water [matched odds ratio (mOR) 11.2, 95% confidence interval (CI), 1.6-infinity]. Consumption of bottled water was inversely associated with illness (mOR 0.3, 95% CI 0.1-0.8). Analyses of faecal samples identified six norovirus genotypes (21/38 samples) and occasionally other viruses. Multiple defects in the water distribution system were noted. These results suggest that the outbreak was caused by faecally contaminated municipal water. It is unusual to have such a large outbreak in a European city especially when the municipal water supply is chlorinated. Therefore, it is important to establish effective multiple-barrier water-treatment systems whenever possible, but even with an established chlorinated supply, sustained vigilance is central to public health.

Detection of Hepatitis E Virus (HEV) in Italian pigs displaying different pathological lesions

In this study we investigated the HEV prevalence in Italian pigs displaying different pathological lesions, possible risk factors related to the infection, and the possible relations occurring between HEV and other concomitant pig pathogens. Genetic characterization of some of the identified strains was also performed. Detection of HEV RNA was accomplished using a nested reverse-transcription polymerase chain reaction on bile samples from 137 pigs of 2-4months of age submitted for diagnostic purposes. Forty-one of the 137 examined pigs (29.9%) tested positive for HEV RNA. Animals of 80-120days of age showed a higher prevalence of HEV infection (46.9% against 20% of younger animals). No statistically significant correlations between HEV positivity and the presence of other pathological conditions detected at necropsy, or concomitant coinfections with PCV2 and/or PRRSV were detected. All identified strains belonged to genotype 3, and were similar to other HEV subtypes 3e, 3f, 3c circulating in Europe.

Presence of Hepatitis E Virus in a RED Deer (Cervus elaphus) Population in Central Italy.

&NA; Hepatitis E is an acute human disease caused by the hepatitis E virus (HEV). In addition to humans, HEV has been detected in several animal species and is recognized as a zoonotic pathogen. Pigs, wild boar and deer can be reservoir. In this study, we evaluated HEV prevalence in a free‐living red deer (Cervus elaphus) population in central Italy by detecting virus‐specific antibodies and RNA in sera. A total of 35 of 251 red deer sera were positive for anti‐HEV IgG. HEV RNA was detected in 10 of 91 sera examined. Two genomic fragments targeted by diagnostic PCRs in the capsid region were sequenced, both matching with genotype 3 HEV. Overall results confirmed the occurrence of HEV infection in deer also in Italy.

Detection of hepatitis E virus in Italian pig herds

HEPATITIS E is a public health concern in many developing countries, where it is primarily transmitted by the faecal-oral route through contaminated water and food (Emerson and Purcell 2003). The disease is caused by a small, non-enveloped single-stranded RNA virus classified as the separate genus Hepevirus. Although hepatitis E disease occurs only sporadically in countries with good health care systems, the seroprevalence in healthy individuals can be high (Emerson and Purcell 2003). Porcine hepatitis E virus (HEV) is not pathogenic to general pig populations, but there is evidence that the virus may be a zoonotic agent and that animal reservoirs may exist. Experimental interspecies transmission of HEV between non-human primates and pigs has been demonstrated (Meng and others 1998), and seroepidemiological studies have shown that pig handlers are at higher risk of HEV infection than the general population (Meng and others 2002). In Japan, studies have supported the possibility of zoonotic transmission, as consumption of undercooked pig organs or meat and, in one case, of deer meat, was closely linked to cases of hepatitis E in human beings (Tei and others 2003, Yazaki and others 2003). The first porcine strain of HEV was characterised in the USA in 1997 (Meng and others 1997). Since then, several other porcine strains have been described worldwide. In the past few years, sporadic cases of autochthonous hepatitis E in human beings have been reported in several European countries, including Italy (Zanetti and others 1999). In many of these cases, the infecting HEV strain showed a high degree of homology with porcine strains of HEV detected in the same country (van der Poel and others 2001, Clemente-Casares and others 2003, Banks and others 2004a). In recent years in Europe, HEV in pig herds has been reported only in Spain (Clemente-Casares and others 2003), the UK (Banks and others 2004b) and the Netherlands (van der Poel and others 2001). This short communication describes the detection, by a nested reverse transcriptase-PCR (nested RT-PCR), of HEV in two Italian pig farms, and the phylogenetic analysis of the viral strains. Thirty-four faecal and 22 serum samples were collected from five different farrow-to-finish farms located in northcentral Italy. Samples were collected from healthy pigs between two and five months of age. Faecal samples represented pools of faeces from animals of the same age group. Total RNA was extracted from 140 μl of faecal suspension or serum using a QIAmp viral RNA kit (Qiagen), according to the manufacturer’s instructions. Template cDNA was reverse transcribed using random hexamers, according to standard protocols. A 145 base pair (bp) fragment of the open reading frame 2 of HEV was amplified from the prepared cDNA by nested PCR, as described by Erker and others (1999). A faecal suspension from a human patient with hepatitis E was used as a positive control. Nested RT-PCR products were visualised on 2 per cent agarose gel, and bands of the correct size were excised and purified with the QIAquick Gel Extraction Kit (Qiagen). Nucleotide sequencing was performed using the ABI PRISM BigDye Terminator kit, version 2·0 (Applied Biosystems). Sequences were assembled with SEQMAN (DNASTAR), and alignment was performed using the ClustalX algorithm. The HEV genome was detected in two faecal pools (5·9 per cent) collected at two different farms, but all the serum samples were negative. The positive faecal pools were obtained from groups of pigs aged 4·5 and 2·5 months, respectively. Phylogenetic analysis of the two viral sequences (113 bp), termed HEVBO/01 and HEVPI/01, was performed by the neighbour-joining method using PHYLIP 3·6. Bootstrap confidence values (500 replicates) were calculated by using the Seqboot and Consense programs. A phylogenetic tree (Fig 1) was created with the Treeview software using an avian HEV isolate as out group. Phylogenetic analysis of the viral sequences showed that the two Italian strains, HEVBO/01 and HEVPI/01, belonged to genotype 3, as did other porcine and human HEV strains indigenous to Europe. However, they differed significantly from each other, being only 84 per cent identical (18 nucleotide changes). The two Italian strains clustered with strains from countries where HEV is considered non-endemic. In particular, HEVPI/01 was related (with 90 per cent identity) to a human strain (AY540113) detected in a sporadic case of acute autochthonous hepatitis E in Spain (Buti and others 2004). In conclusion, this report represents the first description of HEV in Italian pig herds, and confirms the presence of the virus in apparently healthy pigs. These findings are important, because of the potential risk of transmission of porcine HEV to human beings, either by contact with infected pigs or by ingestion of contaminated undercooked meat. However, further studies are needed to address the true zoonotic potential of HEV in pigs. Studies are in progress to evaluate the prevalence of the infection in Italian pigs. The study of a high number of viral strains will be necessary to assess intraspecies and interspecies HEV homologies and to understand whether zoonotic transmission of HEV may occur in Italy.

A pilot survey of bovine norovirus in northern Italy.

NOROVIRUS has a positive-sense single-stranded RNA genome and belongs to the family Caliciviridae. Members of the family are assigned to four genera, named Vesivirus , Lagovirus , Norovirus and Sapovirus ,on the basis of their genomic organisation and phylogenetic pattern (Mayo 2002). Two virus strains detected in faecal samples of cattle called Newbury agent-1 (Bridger and others 1984) and Nebraska (Smiley and others 2002) cluster together in a separate group for which the genus names Becovirus or Nabovirus have been proposed (Oliver and others 2006). Norovirus and sapovirus are an important cause of acute gastroenteritis in human beings (Koo and others 2010) and may also infect herds of pigs and cattle (van der Poel and others 2000, Reuter and others 2010) and pet animals (Martella and others 2008). Norovirus infection in animals has been either associated with diarrhoea or found to be asymptomatic (van der Poel and others 2000, Mijovski and others 2010). Bovine norovirus prototype strains identified so far are the Newbury2 strain, previously known as Newbury agent 2, and the Jena virus. All bovine norovirus strains fall into genogroup III (GIII) of Norovirus , which includes two genotypes. Viruses genetically related to Jena virus were assigned to genotype 1 (GIII.1) and viruses genetically related to Newbury2 were assigned to genotype 2 (GIII.2) (Ando and others 2000). This short communication describes the identification of bovine norovirus strains belonging to either GIII.1 or GIII.2 in a sample of calves from Italy presenting …

Retrospective Study Evaluating Seroprevalence of Hepatitis E Virus in Blood Donors and in Swine Veterinarians in Italy (2004)

Hepatitis E is an emerging viral disease in developed countries, with sporadic cases occasionally linked to the consumption of raw or undercooked pork, wild boar or deer meat. Cases due to transfusion or transplantation have also been reported. In developed countries, hepatitis E is considered a zoonosis and pig is the main reservoir. In the last few years, several studies conducted in Europe reported variable seroprevalence rates among the general population, ranging between 0.26% and 52.5%. A higher seroprevalence was described among workers who come in contact with pigs. The aim of this retrospective study was to evaluate the seroprevalence of anti‐HEV IgG and IgM antibodies in blood donors (170) and in pig veterinarians (83). Archival sera were collected in Italy in 2004. The observed seroprevalence was 9.64% and 8.82% in veterinarians and blood donors, respectively. Overall, only three sera from blood donors were positive for IgM, but no HEV‐RNA was detected.

Molecular detection and phylogenetic analysis of hepatitis E virus strains circulating in wild boars in south-central Italy

Hepatitis E virus (HEV) is a zoonotic pathogen with a worldwide distribution, and infects several mammalian species, including pigs and wild boars, which are recognized as its natural reservoirs. The virus causes a usually self-limiting liver disease with a mortality rate generally below 1%, although mortality rates of 15%-25% have been recorded in pregnant woman. Chronic infections can also occur. The prevalence of HEV has been extensively studied in wild boars and pigs in northern Italy, where intensive pig herds are predominantly located. In contrast, few data have been collected in south-central Italy, where small pig herds are surrounded by large regional parks populated with heterogeneous wild fauna. In this study, 291 liver samples from wild boars caught in south-central Italy were analysed with the molecular detection of viral RNA. Our results confirm the circulation of HEV in these animals, with a mean prevalence of 13.7% (40 of 291). A nucleotide sequence analysis showed that the HEV strains were highly conserved within the same geographic areas. The wild boar HEV strains belonged to the HEV-3c subtype, which is frequently described in wild boars, and to an uncommon undefined subtype (HEV-3j-like).The viral prevalence detected is concerning because it could represent a potential risk to hunters, meat workers and consumers of wild boar liver and derivative products. The hypothesized inter-species transmission of HEV to pigs and the possibility that the virus maintains its virulence in the environment and the meat chain also present potential risks to human health, and warrant further investigations in the near future.

Pathogenic Escherichia coli and enteric viruses in biosolids and related top soil improvers in Italy

To investigate the presence of genomic traits associated with a set of enteric viruses as well as pathogenic Escherichia coli in top soil improvers (TSI) from Italy.

Pilot survey of norovirus in Northern Italy: an example of surveillance of norovirus gastroenteritis

In this study, we describe the results of virological investigations carried out on cases of gastroenteritis reported in different communities within a 2-year pilot surveillance programme (January 2012 to December 2013) in the autonomous province of Bolzano (Northern Italy). Among the 162 norovirus (NoV)-positive cases out of 702 cases investigated, 76 were grouped in nine suspected outbreaks, 37 were hospital-acquired and 49 were community-acquired sporadic cases. NoV infections were found in all age groups in outbreak and community-acquired cases, while the highest peak of hospital-acquired infections occurred in the elderly. Sequence analyses helped to identify suspected outbreaks both in the community and in hospital wards. Although GII.4 is the predominant genotype, sequence data confirmed that at least seven genotypes circulate causing sporadic cases. Findings in this study confirmed the relevance of NoV infections as a cause of outbreaks, and impact of NoV infections in community-acquired sporadic cases in adults that are rarely described because of a lack of reporting.