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.

Astroviruses as a cause of nosocomial outbreaks of infant diarrhea.

During a 16-month study period at a children's hospital, 32 children developed nosocomial gastroenteritis caused by astroviruses. Twenty-five of these occurred during 2 epidemic outbreaks in medical and surgical infants' wards. From the community, 13 confirmed cases were admitted during the study period. Both community-acquired and nosocomial cases occurred during autumn, winter and early spring. The attack rates during outbreaks ranged between 7 and 62% and were highest among children with underlying gastrointestinal diseases. Diarrhea and vomiting were the most common clinical manifestations. The median duration of symptoms was 4 days and that of virus excretion was 5 days. Hospital infection with astroviruses is common and usually affects children less than 2 years of age. The probable mode of transmission is spread via contaminated hands.

Nosocomial calicivirus gastroenteritis in a pediatric hospital.

At St. Göran's Children's Hospital (a tertiary center), we perform electron microscopy of feces in most cases of nosocomial gastroenteritis. From September 1987 through April 1992 we identified 32 episodes of calicivirus infection, 25 of which were nosocomial and, except for one outbreak, sporadic. Systematic study of the nosocomial outbreak of calicivirus gastroenteritis from November 1991 to January 1992, revealed calicivirus in the stools of 8 of 23 children with diarrhea and 0 of 10 without diarrhea. In 3 of 7 sampled after cessation of diarrhea, calicivirus excretion continued for 3 to 6 days. We found no calicivirus in 42 staff members or 9 members of infected patients' families. Nosocomial transmission of calicivirus can occur among infants.

Respiratory infection and iatrogenic diarrhea in Honduras and El Salvador during the 1991-1992 season.

The etiology of acute respiratory tract infection and its association with diarrhea was analyzed in 135 hospitalized children less than three years of age with mainly respiratory symptoms in two pediatric hospitals in Honduras and El Salvador. Etiologic diagnoses were performed on nasopharyngeal samples by tissue culture and immunofluorescence, including a search for the presence of respiratory virus-specific immunoglobulin A antibodies. Fecal samples were subjected to electron microscopy and tissue culture. The majority (83%) of the children (65% boys and 35% girls) were less than 12 months old and 45% were less than four months old. In 63 (47%) patients, at least one viral infection of the respiratory tract was identified. Respiratory syncytial virus (RSV) or the respective IgA antibodies were found in 43 (32%) patients., influenza A and B viruses in 29 (21%) patients, adenovirus in four patients, and enterovirus in two patients. Twenty (15%) patients had two or more viral agents. An association between RSV cases and environmental temperature was established. In 124 fecal samples, we identified four cases with astrovirus, seven with nonpolio enterovirus, and eight with adenovirus, but only three cases with rotavirus. Diarrhea was present in 55 (45%) of the patients. There was a statistically significant association between diarrhea and cases with RSV. This was shown to be associated with antibiotic treatment.

Effects of nerve growth factor on sensory neurons in the chick embryo: a stereological study.

Chicken embryos on days 6-13 of incubation received injections of purified beta NGF (80 micrograms/day) for 3 or 4 days and were then killed. Sensory ganglia were fixed and taken for embedding and sectioning. A stereological method based on unfolding of cell-diameter frequencies was used to determine the number of neurons of different size in the spinal, trigeminal and nodose ganglia. The total volume of the ganglia was also determined. NGF induced increases in diameter of the neural crest-derived dorsomedial (DM) neurons in spinal and trigeminal ganglia. Injected NGF did not influence ventrolateral (VL) neurons of neural crest origin in the spinal ganglia nor the ventrolateral neurons of placodal origin in the trigeminal ganglion. The volumes of spinal and trigeminal ganglia increased by 50 and 100%, respectively. The volume of the nodose ganglion and the total number and size of the placodal nodose neurons were unaffected by NGF. The results demonstrate a clear difference in the response to NGF in vivo between smaller and larger sensory neurons.

Changes of CD2 receptor density determined by electron microscopy on blood lymphocytes following radiation treatment for breast cancer.

Changes of relative CD2 receptor on lymphocytes were examined in 11 women following radiation treatment for breast cancer by electron microscopy using antibody-coated gold particles. Proportions of blood lymphocytes with a high density of CD2 receptors were reduced, whereas those with no or a low density of such receptors were increased after radiation treatment.

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.

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 estimates of the disease burden of rotavirus in Sweden.

Laboratory and hospitalization data from two children's hospitals with large primary catchment areas and national laboratory and hospitalization data for children under 4 y of age with acute diarrhoea were compiled to estimate the number of hospitalizations and the cost burden associated with rotavirus diarrhoea in Sweden. According to our estimates 1500-1700 rotavirus-associated hospitalizations occur annually in Sweden in children under 4 y of age (3.7 hospitalizations/1000 children/y). This number represents 2.3% of admissions for all diagnoses in children of this age group. The cost of these hospitalizations is 13.5-15 million Swedish crowns (US$1.8-2 million). Serotyping by PCR for two years revealed that serotype 1 (G1) was the most common (49% and 58%, respectively) identified. Serotypes 2-4 were identified in the following proportions G2 (23% and 5%), G3 (21% and 0%) and G4 (7% and 16%). The national laboratory report data for 1993-96 show that as much as 7-13% of rotavirus infections occur in elderly people.

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.

Locomotion and cell surface movements of fibroblasts in fibrillar collagen gels.

We have used light microscopy and scanning, scanning transmission, transmission, and high voltage electron microscopy to study the shape and cell surface movements of chick embryo fibroblasts cultured in Vitrogen collagen gels. Cells in gels are less well spread than their counterparts on glass but they retain a dorso-ventral polarity, and move by extending long, narrow lamellar processes. We show here for the first time that cells moving in 3D substrata form perinuclear caps of cross-linked cell surface receptors.

The chick embryo nodose ganglion: effects of nerve growth factor in culture.

Nodose ganglia from 8-day-old chick embryos were cultured in collagen gels for 2 days, with or without added nerve growth factor (NGF), in order to discover whether the nodose neurons, derived from an epidermal placode, are susceptible to this trophic factor. Neuronal survival and neurite outgrowth were stimulated only after addition of NGF, and the enhancing effects could be blocked by introducing antibodies to NGF. Stereological analysis of ganglia sectioned for light microscopy showed that the NGF-treated neurons increased their volume by about 50%, as did the nodose neurons in ovo from the eighth to the tenth day of incubation. The volume density, however, was significantly lower in vitro indicating a limited cell death during culture despite the presence of exogenously supplied NGF. The number of neurofilaments and microtubules increased in the cell centre of neurons treated with NGF; this region also showed numerous dense bodies and an extensive Golgi complex by electron microscopy. Ultrastructural similarities between neurons responding to NGF and neurons undergoing the axon reaction which follows axotomy are indicated. A role for a trophic factor resembling NGF in the normal development of placode-derived neurons of the sensory cranial ganglia is suggested.