Segmentation of virus particle candidates in transmission electron microscopy images.

In this paper, we present an automatic segmentation method that detects virus particles of various shapes in transmission electron microscopy images. The method is based on a statistical analysis of local neighbourhoods of all the pixels in the image followed by an object width discrimination and finally, for elongated objects, a border refinement step. It requires only one input parameter, the approximate width of the virus particles searched for. The proposed method is evaluated on a large number of viruses. It successfully segments viruses regardless of shape, from polyhedral to highly pleomorphic.

Waterborne norovirus outbreak in a municipal drinking-water supply in Sweden.

During Easter 2009, almost 200 people resident in a small Swedish village fell ill with gastrointestinal symptoms. We conducted a retrospective cohort study and a molecular investigation in order to identify the source of the outbreak. Residents living in households connected to the public water network were at an increased risk of developing disease (relative risk 4·80, 95% confidence interval 1·68-13·73) compared to those with no connection to the public network. Norovirus genotype GI.3 was identified in stool samples from six patients and in a sample from the public water network. Contamination of one of the wells supplying the public water network was thought to be the source of the outbreak. This is a description of a norovirus outbreak linked to a municipal drinking-water supply in Sweden. Information from epidemiological and molecular investigations is of utmost importance to guide outbreak control measures and to prevent future outbreaks.

A foodborne norovirus outbreak at a manufacturing company.

Over 400 office workers from the same unit of a manufacturing company in Stockholm County, Sweden, fell ill with gastroenteritis. A retrospective cohort study of office workers in the affected unit demonstrated that canteen visitors on one day had an increased risk of illness [risk ratio (RR) 27.1, 95% confidence interval (CI) 15.7-46.8] compared to non-visitors. A second study, investigating canteen visitors' consumption of particular food items, showed that both tomatoes from the salad buffet (RR 5.6, 95% CI 3.2-9.6) and hamburgers (RR 4.9, 95% CI 2.4-9.8) were the most likely vehicles of infection. Norovirus GI.3 (Desert Shield) was identified in stool samples from three office workers and from a food handler who prepared the tomatoes for the salad buffet and hamburger ingredients before vomiting at the workplace on 12 November. The outbreak could have been prevented if the food items prepared by the food handler some hours before vomiting had not been served.

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.

Ex vivo stability of the rodent-borne Hantaan virus in comparison to that of arthropod-borne members of the Bunyaviridae family.

The possible effect of virus adaptation to different transmission routes on virus stability in the environment is not well known. In this study we have compared the stabilities of three viruses within the Bunyaviridae family: the rodent-borne Hantavirus Hantaan virus (HTNV), the sand fly-borne Phlebovirus sandfly fever Sicilian virus (SFSV), and the tick-borne Nairovirus Crimean-Congo hemorrhagic fever virus (CCHFV). These viruses differ in their transmission routes: SFSV and CCHFV are vector borne, whereas HTNV is spread directly between its hosts, and to humans, via the environment. We studied whether these viruses differed regarding stability when kept outside of the host. Viral survival was analyzed at different time points upon exposure to different temperatures (4 degrees C, 20 degrees C, and 37 degrees C) and drying at 20 degrees C. We observed clearly different stabilities under wet conditions, particularly at 4 degrees C, where infectious SFSV, HTNV, and CCHFV were detectable after 528, 96, and 15 days, respectively. All three viruses were equally sensitive to drying, as shown by drying on aluminum discs. Furthermore, HTNV and SFSV partially survived for 2 min in 30% ethanol, whereas CCHFV did not. Electron microscopy images of HTNV, SSFSV, and CCHFV stored at 37 degrees C until infectivity was lost still showed the occurrence of virions, but with abnormal shapes and densities compared to those of the nonincubated samples. In conclusion, our study points out important differences in ex vivo stability among viruses within the Bunyaviridae family.

Murine pneumotropic virus VP1 virus-like particles (VLPs) bind to several cell types independent of sialic acid residues and do not serologically cross react with murine polyomavirus VP1 VLPs.

The ability of murine pneumotropic virus (MPtV) major capsid protein VP1 to form virus-like particles (VLPs) was examined. MPtV-VLPs obtained were used to estimate the potential of MPtV to attach to different cells and to assess some characteristics of the MPtV cell receptor. Furthermore, to evaluate if MPtV-VLPs could potentially complement murine polyomavirus (MPyV) VP1 VLPs (MPyV-VLPs) as vectors for prime-boost gene therapy, the capability of MPtV-VLPs to serologically cross react with MPyV-VLPs and to transduce DNA into cells was examined. MPtV VP1 obtained in a recombinant baculovirus system formed MPtV-VLPs readily. MPtV-VLPs were shown by FACS analysis to bind to different cells, independent of MHC class I antigen expression. In addition, MPtV-VLPs did not cause haemagglutination of red blood cells and MPtV-VLP binding to cells was neuraminidase resistant but mostly trypsin and papain sensitive, indicating that the MPtV receptor lacks sialic acid components. When tested by ELISA and in vivo neutralization assays, MPtV-VLPs did not serologically cross react with MPyV-VLPs, suggesting that MPtV-VLPs and MPyV-VLPs could potentially be interchanged as carriers of DNA in repeated gene therapy. Finally, MPtV-VLPs were shown to transduce foreign DNA in vitro and in vivo. In conclusion, the data suggest that MPtV-VLPs, and possibly also MPtV, bind to several different cell types, that binding is neuraminidase resistant and that MPtV-VLPs should potentially be able to complement MPyV-VLPs for prime-boost gene transfer in vivo.

A norwalk-like virus waterborne community outbreak in a Swedish village during peak holiday season.

An outbreak of gastroenteritis due to Norwalk-like virus (NLV) affecting approximately 500 people occurred in a Swedish ski resort during February-March 2002. Epidemiological investigations were performed on cohorts of schoolchildren, permanent residents and skiers visiting the area. Attack rates were respectively 39.7, 29.9 and 38.5%. Drinking un-boiled water originating from one of the three communal water systems was a significant risk factor for all groups. For schoolchildren, the risk of illness increased with increasing amount of water consumed. Nine of 12 stool samples of patients analysed tested positive for NLV. The water tested negative for indicator bacteria and results of NLV tests were inconclusive. In the absence of microbiological findings, the environmental authorities were reluctant to act based on the epidemiological analysis alone, and intervention was delayed until mid-April, following the discovery of a crack in a sewage pipe 10 m from the well.

Epidemiology of Norwalk-like human caliciviruses in hospital outbreaks of acute gastroenteritis in the Stockholm area in 1996.

OBJECTIVES: Outbreaks of acute gastroenteritis associated with 'Norwalk-like viruses' (NLVs) cause significant health problems in hospitals. Hospital outbreaks in the Stockholm area in 1996 were investigated, in order to identify the magnitude of the problem, the mode of transmission, the effect of control measures and the genetic variability of outbreak strains. Determining the epidemiological and clinical significance involves a broad range of possibilities. METHODS: Ten hospitals, representing 66% of the hospitals in the Stockholm area, participated in the study, which included 211 wards. Of these, 18 were selected as control. A standardized protocol that included personal contacts was administered. Outbreak wards were visited between 5 and 10 times. Wards that had reported outbreaks in 1996 were prospectively followed through 1999 by personal contacts, and the available data from 1991 on outbreak reports were collected. A total of 253 stool samples from outbreaks in 1996 were analyzed by electron microscopy (EM) for the presence of NLVs. Positive samples were confirmed by the reverse transcriptase-polymerase chain reaction (RT-PCR). RESULTS: In total, 4 326 patients and 1 119 staff were exposed on the 43 wards that reported 54 outbreaks. The mean attack rate was 13% for patients and 21% for staff. The number of outbreaks in 1996 outnumbered the reported outbreaks in the preceding years (4-70%) and later years (35-40%). Admission to 24 (56%) of the outbreak wards was stopped. The mean duration of illness for patients was 35 hours and for staff, 30 hours. The main symptoms were diarrhoea (80%) and vomiting (68%). Genotyping revealed that the majority of the hospital outbreaks in the Stockholm area in 1996 were caused by a single NLV strain. CONCLUSIONS: The study confirmed that outbreaks of NLV are an increasing public-health problem in hospitals. The risk of being affected by an outbreak was significantly greater on wards that had reported outbreaks in the previous year. It was not obvious which measures had helped to shorten the outbreaks to any appreciable extent. Different managements must therefore be carefully interpreted and adapted to the prevailing circumstances. Genotyping of strains is an important tool of getting a better insight into transmission routes and the mechanism behind the appearance of epidemic strains.

Astrovirus epidemiologically linked to pre-weaning diarrhoea in mink.

Diarrhoea and excessive secretion from the cervical apocrine glands in young, suckling mink kits is a well-known, but poorly defined, syndrome often referred to as "sticky", "greasy", or "wet" kits. We have performed a case-control study, at farm level as well as at mink kit level, in Denmark and Sweden to investigate whether enteric virus infections may be a risk factor in the development of pre-weaning diarrhoea. Tissue samples from the enteric tract of 180 sacrificed mink kits were analysed histologically. Faecal contents were examined by electron microscopy (EM). Astrovirus was detected in abundance and found to be a significant risk factor both at farm level (OR=21.60, p<0.001) and at mink kit level (OR=7.95, p<0.001). Other factors, i.e. low body weight, coccoid bacteria adherent to the enteric villi, and presence of calicivirus were also shown to increase the risk of pre-weaning diarrhoea, although with less impact than astrovirus.

A one-year study of foodborne illnesses in the municipality of Uppsala, Sweden.

Surveillance was enhanced and a retrospective interview study performed in 1998-99 to determine incidence, causes, and costs of foodborne illnesses in Uppsala, Sweden. Sixty-eight percent of the detected foodborne illness incidents were single cases, and 32% were outbreaks. Most (85%) of the incidents came to the attention of the municipal authorities through telephone calls from affected persons. Calicivirus, Campylobacter spp., and Staphyloccocus aureus were the most common etiological agents; meat, meat products, and mixed dishes were the most implicated food categories. The incidence of foodborne illness was estimated to be 38 cases per 1,000 inhabitants per year. The estimated average costs per illness were 2,164 Swedish Krona (SEK) ($246) to society and 500 SEK ($57) to the patient. The annual cost of foodborne illnesses in Sweden was estimated to be 1,082 million SEK ($123 million).

Clinical spectrum and transmission characteristics of infection with Norwalk-like virus: findings from a large community outbreak in Sweden.

A large foodborne outbreak caused by Norwalk-like virus (NLV) among children and staff at 30 day care centers provided an opportunity to study symptomatology and attack rates among patients in different age groups, as well as secondary transmission rates in centers and households. A retrospective cohort study of 775 subjects from 13 randomly chosen centers was performed. Diarrhea was more common in adults than in children (P=.001), whereas the reverse was noted with regard to vomiting (P=.003). The primary attack rate was 27% (142 of 524 subjects): 54% of adults versus 19% of children (P<.001). The mean incubation time for foodborne cases of infection was 34 hours. The secondary attack rate was 17%. Risk factors for spread into households were the primary case being a child (relative risk [RR], 3.8; 95% confidence interval [CI], 1.9-7.6) and vomiting (RR, 2.4; 95% CI, 1.0-5.5). The incubation time for person-to-person transmission was approximated by a mean serial interval of 52 hours. This is the first reported outbreak of NLV infection in which secondary transmission into households by individuals has been studied.

Experimental reproduction of winter dysentery in lactating cows using BCV -- comparison with BCV infection in milk-fed calves.

Infection models were developed for adult cows and for young calves using the same strain of bovine coronavirus (BCV), which for the first time allows experimental reproduction of winter dysentery (WD) in seronegative lactating cows. The cattle were infected through direct contact with an experimentally inoculated calf. All experimental cattle shed faecal BCV with development of diarrhoea, being profusely watery with small amounts of blood in the most severely affected animals, including both cows and calves. The cows, in contrast to the calves, showed depressed general condition and appetite leading to a marked decrease in milk yield. Further age-associated differences were a shorter incubation period in the two youngest calves, but with milder fever and milder decrease in white blood cell counts. These findings shed light on the apparent epidemiological differences between WD and calf BCV diarrhoea suggesting that, (1) the same strains of BCV cause natural outbreaks of calf diarrhoea and WD, (2) seronegative cows are more severely affected by the infection than seronegative conventionally reared calves, and (3) unaffected general condition in diarrhoeic calves may lead to underestimation of the occurrence of calf diarrhoea in WD outbreaks. In response to infection, all cattle produced early interferon type 1 in serum and, except for one calf, in nasal secretions. A finding not previously reported is the detection of interferon type 1 responses in bovine milk. All cattle developed high IgM antibody responses and long-lasting IgA antibody responses both systemically and locally. The serum IgM antibody responses came earlier in most of the calves than in the cows. Prolonged IgM antibody responses were detected in serum and milk, while those in nasal secretions were much shorter. BCV-specific IgA was present in nasal secretions from all cattle throughout the 6 months follow-up. The IgA antibody response in serum was detected up to 17 months post-infection and the duration showed an age-related variation indicating a more prominent IgA memory in the adult cattle and in the older calves than in the younger ones. BCV-specific IgG was detected in all cattle during the experimental period of up to 22 months. In conclusion, WD was reproduced in seronegative lactating cows. The cows showed a more severe general diseases than seronegative calves infected concurrently. Very long-lasting IgA antibody responses were detected both systemically and locally.

Incidence and genetic diversity of group C rotavirus among adults.

Fecal samples from a 1-year prospective study were investigated to establish the role of group C rotavirus infections in acute diarrhea in Swedish adults (>15 years old). Rotaviruses were found in samples from 3% of the patients, and, in 35% of these, group C rotavirus was detected. Clinical symptoms of group C rotavirus infection were generally milder than those of group A rotavirus infection. Gene 8 (vp7) from 12 group C isolates, including strains from the prospective study, a military outbreak, and a sporadic case, was sequenced. The gene was found to be extremely conserved, with identities of 99.1%-100% at the amino acid level. This study has systematically investigated the prevalence and genetic diversity of group C rotavirus in adults. The data confirm the extreme sequence conservation within human group C rotavirus strains and suggest that symptomatic group C rotavirus infections occur more frequently in adults than has been previously recognized.

Enteropathogens in adult patients with diarrhea and healthy control subjects: a 1-year prospective study in a Swedish clinic for infectious diseases.

A 1-year prospective study was conducted to identify enteropathogens in adults with diarrhea (n=851) and in healthy control subjects (n=203) by use of conventional laboratory methods. Virulence factor genes for diarrheagenic Escherichia coli were detected by polymerase chain reaction. Enteropathogens were identified in 56% of patients and 16% of control subjects. The isolation rate was 65% for patients with symptoms for <1 week and for travelers; >1 pathogen was found in 11% of patients. The most frequent enteropathogens were Campylobacter (13% of patients), Clostridium difficile (13%), enterotoxigenic Escherichia coli (8%), Salmonella (7%), Shigella (4%), Blastocystis hominis (4%), calicivirus (3%), rotavirus (3%), enteroaggregative E. coli (2%), Aeromonas (2%), Giardia intestinalis (2%), Cryptosporidium (2%), and astrovirus (2%). Less frequently isolated (< or =1% of patients) were verotoxigenic E. coli, enteropathogenic E. coli, enteroinvasive E. coli, Entamoeba histolytica/Entamoeba dispar, microsporidia, and adenovirus. Fifty percent of the patients were hospitalized, and 43% needed intravenous fluids. The median duration of diarrhea was 14 days. Clinical features were not helpful for predicting the etiology of diarrhea.

Epidemiology of calicivirus infections in Sweden, 1994-1998.

Outbreaks of acute gastroenteritis are frequently caused by caliciviruses. Electron microscopy was used to search for these viruses in fecal samples from patients with acute gastroenteritis. Of 5800 samples collected and analyzed from November 1994 to June 1998, 3700 were associated with outbreaks. A total of 676 outbreaks were analyzed, and viruses were found in 67%. Caliciviruses, usually Norwalk-like viruses (NLVs), were found in 407 (89%) of 455 outbreaks, while Sapporo-like viruses were identified in nine outbreaks, including six that were suspected to include foodborne transmission. Sixty percent of the 1041 patients with calicivirus infections were between 70 and 90 years of age. Food- and waterborne infections were associated with 66 calicivirus outbreaks. Virus-positive outbreaks were documented mainly during winter and spring. The longitudinal survey showed that caliciviruses, and especially the NLVs, cause most nosocomial and community-associated outbreaks in Sweden.

Molecular detection and epidemiology of Sapporo-like viruses.

Sapporo-like viruses (SLVs) are associated with acute gastroenteritis in humans. Due to a limited supply of available reagents for diagnosis, little is known about the incidence and pathogenicity of these viruses. We have developed a first-generation generic reverse transcriptase (RT) PCR assay based on a single primer pair targeting the RNA polymerase gene. With this assay, 55 (93%) of the 59 stool specimens collected in a 10-year period of time (1988 to 1998) and containing typical caliciviruses by electron microscopy tested positive and could be confirmed by Southern hybridization. By phylogenetic analysis, most SLV strains could be classified into one of the three recently described genotypes. However, three samples clustered separately, forming a potential new genotype. We sequenced the complete capsid gene of one of the strains in this cluster: Hu/SLV/Stockholm/97/SE. Alignment of the capsid sequences showed 40 to 74% amino acid identity among strains of the different clusters. Phylogenetic analysis of the aligned sequences confirmed the placing of Hu/SLV/Stockholm/97/SE into a new distinct genetic cluster. This is the first report on the development of a broadly reactive RT-PCR assay for the detection of SLVs.

Viral diarrhea in children in Beijing, China.

A study was undertaken from November 1994 to August 1996 to determine the role of viruses in children (< or =5 years of age) hospitalized at Beijing Children Hospital, Beijing China, for acute diarrhea. Stool samples from diarrheal patients were investigated by ELISA, electron microscopy, and RT-PCR for the presence of rotavirus, calicivirus, astrovirus, and adenovirus. Group A rotavirus was detected in 55.9% of all diarrheal patients and comprised 82.5% of all viruses detected. Group A rotavirus samples were further characterized for their G-type specificity by RT-PCR. Four major G types (1-4) were identified. G1 to G4 accounted for 58.9%, 15.7%, 16.8%, and 6.3%, respectively, of the serotyped samples. Almost all rotavirus infections occurred in children less than 1 year of age, with a significant clustering during the winter months. Group C rotavirus was detected in one 18-month-old child. Astroviruses, caliciviruses, and adenoviruses were detected in 8.5%, 7.6%, and 2.5% of the hospitalized children, respectively. This, the first viral etiological study of childhood diarrhea in China, concludes that rotavirus G1-4 strains play an important role in severe diarrhea in Beijing children.