Antigenic and genetic properties of viruses linked to hemorrhagic fever with renal syndrome.

Hemorrhagic fever with renal syndrome (HFRS) comprises a variety of clinically similar diseases of viral etiology that are endemic to and sporadically epidemic throughout the Eurasian continent and Japan. Although HFRS has not been reported in North America, viruses that are antigenically similar to HFRS agents were recently isolated from rodents in the United States. Examination and comparison of eight representative isolates from endemic disease areas and from regions with no known associated HFRS indicate that these viruses represent a new and unique group that constitutes a separate genus in the Bunyaviridae family of animal 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.

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

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.

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.

Evolutionary conservation in the hepatitis B virus core structure: comparison of human and duck cores.

BACKGROUND: Hepatitis B virus is a major human pathogen which has been extensively studied, yet its structure is unknown. Cryo-electron microscopy of the viral cores expressed in Escherichia coli or isolated from infected liver provides a means for determining the structure of the hepatitis B nucleocapsid. RESULTS: Using cryo-electron microscopy and three-dimensional image reconstruction, we have determined the structures of duck and human hepatitis B virus cores and find that they have similar dimer-clustered T = 3 and T = 4 icosahedral organizations. The duck virus core protein sequence differs from the human in both length and amino acid content; however, the only significant structural differences observed are the lobes of density on the lateral edges of the projecting (distal) domain of the core protein dimer. The different cores contain varying amounts of nucleic acid, but exhibit similar contacts between the core protein and the nucleic acid. Immunoelectron microscopy of intact cores has localized two epitopes on the core surface corresponding to residues 76-84 and 129-132. CONCLUSIONS: The bacterial expression system faithfully reproduces the native hepatitis B virus core structure even in the absence of the complete viral genome. This confirms that proper assembly of the core is independent of genome packaging. Difference imaging and antibody binding map three sequence positions in the structure: the C terminus and the regions near amino acids 80 and 130. Finally, we suggest that the genome-core interactions and the base (proximal) domain of the core dimer are evolutionarily conserved whereas the projecting domain, which interacts with the envelope proteins, is more variable.

Semlike Forest virus membrane proteins. Preparation and characterization of spike complexes soluble in detergent-free medium.

After Triton X-100 delipidation and subsequent Triton X-100 removal in a sucrose gradient the membrane protein spikes of Semliki Forest virus remained soluble in aqueous buffers. It was shown they were present as octameric complexes with a molecular weight of 95-10(4) and that they contain less than 4% lipid and detergent by weight. In electron microscopy after negative staining they appeared as "rosette"-shaped particles. Part of the protein could also be found associated in ordered paracrystalline arrays.

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.

Redistribution of Mr 75,000 plasma membrane protein, cytovillin, into newly formed microvilli in herpes simplex and Semliki Forest virus infected human embryonal fibroblasts.

We have previously purified an Mr 75,000 protein from cultured human JEG-3 choriocarcinoma cells and showed that this protein is specifically confined to the cytoplasmic side of JEG-3 microvillar membranes. Recently, the Mr 75,000 protein, designated as cytovillin, was found to be expressed also in several other cultured human cell lines and strains, in which it was detected in microvillus-related structures. We now demonstrate the redistribution of cytovillin in herpes simplex type 1 (HSV-1) and Semliki Forest virus (SFV) infected human embryonal fibroblasts. Virus infection induced rapidly numerous microvilli on the apical cell surfaces, and cytovillin was enriched into these newly formed structures as shown by indirect immunofluorescence and immunoferritin electron microscopy. In mock-infected cells treated with the anti-cytovillin antibodies a small amount of ferritin particles and faint fluorescence was detected along the smooth plasma membrane. Only occasional cell surface protrusions were observed in these cells. The enrichment of the cytovillin was first seen 2 h after infection. The isoelectric point (IP) and the mobility of the cytovillin polypeptide in sodium dodecyl sulfate polyacrylamide gel electrophoresis was not altered after this redistribution, suggesting that the protein was not significantly modified during infection. Five RNA+ SFV mutants (ts-1, ts-2, ts-3, ts-5, ts-7) with temperature-sensitive defects in processing and transport of viral envelope glycoproteins to the plasma membrane induced microvilli at the restrictive temperature (39 degrees C) as the wild type virus.(ABSTRACT TRUNCATED AT 250 WORDS)

Ultrastructural study of two patterns in the interaction of Helicobacter pylori with neutrophils.

As evidenced by electron microscopy, unopsonised bacteria of some Helicobacter pylori strains were readily taken into cytoplasmic vacuoles of human neutrophils; those of other strains were only rarely absorbed. The strains engulfed like this also induced strong oxidative burst reactions in neutrophils, as measured by chemiluminescence. This has been associated with peptic ulcer disease, in the past. The ultrastructural interaction patterns agreed with the reaction patterns shown by chemiluminescence.

Ultrastructure of Chlamydia pneumoniae in cell culture.

The electron microscopic appearance of Chlamydia pneumoniae elementary bodies with pear-shaped, loose outer membrane has been suggested as one criterion of its classification as a new chlamydial species. The study of the original strain TW 183 in LCL 929 and HL cells and a low-passage isolate of Kajaani-6 isolate in HL cells revealed spherical compact elementary bodies common to other chlamydia.

Confirmation of Norwalk-like virus amplicons after RT-PCR by microplate hybridization and direct sequencing.

A large number of Norwalk-like viruses (NLVs) have been identified from stool samples by RT-PCR by amplifying part of the polymerase-coding gene. A set of probes were selected based on sequence analysis of the viruses circulating in Finland during the years 1996-97 for confirmation of the findings by hybridization. A microplate hybridization test, which provides a rapid semi-automatic detection for PCR products, was designed and compared with agarose gel electrophoresis. From the material of 210 stool samples, mainly from diarrheal outbreaks during years 1997-1998, three probes, one for NLV genogroup GGI and one for each of the two GGII subgroups (Toronto-like and Lordsdale-like), were sufficient to detect 87.8% (36/41) of GGI and 89.0% (49/55) of GGII samples positive by gel electrophoresis. Amplicon sequencing of the strains not detected by the above probes revealed genetic variability in the sequences. Biotin-streptavidin binding was used both for microplate hybridization assays and for direct sequencing to identify the amplicons. Based on the sequences three more probes for the hybridization panel were added so that all the different NLVs of this study could be recognized.

Solid-phase enzyme immunoassay for rotavirus antigen: faecal protease activity as a reason for false-negative results.

Rabbits and guinea pigs were immunized with purified bovine rotavirus. Immunoglobulin G fractions of the resulting antisera were used in a standard four-layer solid-phase enzyme immunoassay (EIA) for rotavirus antigen in human faecal specimens. Samples negative for rotavirus in electron microscopy, when diluted in standard EIA buffers, regularly gave absorbance values lower than those obtained with buffer blank only. By further diluting of the samples the resulting absorbance values were found to increase to the blank levels. When all dilution buffers were supplemented with 1-5% of bovine serum, negative samples at any dilution gave absorbance values close to those of the buffer blanks. Similar results were obtained if the serum was replaced by 1-5 mM of phenylmethylsulphonyl fluoride, a synthetic broad spectrum serine-type protease inhibitor. Aprotinin, another protease inhibitor, was without effect. A similar inhibition pattern was obtained when faecal specimens were tested in a caseinolytic quantitative protease assay in the presence of the above inhibitors. These observations suggest that protease activity present in human faecal samples may cause false-negative results in solid-phase immunoassay for viral antigens, unless appropriate means are used to avoid this interference.

Polyamine depletion of cells reduces the infectivity of herpes simplex virus but not the infectivity of Sindbis virus.

The effect of polyamines on the viral growth was examined using cell strains that could be effectively depleted of polyamines. In order to avoid the polyamines present in serum we used a polyamine auxotrophic Chinese hamster ovary cell line P22 growing in serum-free medium and Vero cells growing in low serum medium. The final yield of an enveloped RNA virus, Sindbis, in P22 cells was not decreased by depletion of cellular polyamines although the onset of the viral replication was delayed. In contrast the final yield of an enveloped DNA virus, Herpes simplex virus (HSV), was considerably reduced in Vero cells, depleted of polyamines by alpha-difluoromethylornithine, an inhibitor of polyamine synthesis. However, the number of HSV particles detected by electronmicroscopy was not decreased. Southern blot analysis of HSV-DNA from the polyamine depleted and the control cells showed changes in the relative abundance of the DNA fragments suggesting that impairment in DNA synthesis may have caused the decreased infectivity of HSV.

Isolation of an iridovirus from pike-perch Stizostedion lucioperca.

We have isolated a large virus from pike-perch Stizostedion lucioperca fingerlings with no signs of disease. The biochemical structural, and serological properties of this newly isolated virus suggest that it belongs to the family Iridoviridae. The virus multiplied and was cytopathogenic in several cultured fish cell lines. The virus has a DNA-containing genome and is assembled in the cytoplasm. When viewed in electron micrographs, the assembly sites showed a paracrystalline array of hexagonal nucleocapsids. The ultrastructure of the pike-perch virus resembled that of previously isolated fish iridoviruses. It is an enveloped icosahedral DNA virus. The diameter of the nucleocapsid in thin sections was 127 +/- 3 nm; in negatively stained preparates the size of the enveloped virus varied from 147 to 187 nm. In immunofluorescence the virus was stained by rabbit antisera against EHN (epizootic haematopoietic necrosis) virus, sheatfish iridovirus and cod iridovirus. The pathogenicity of the virus isolate was studied by inoculation into juvenile rainbow trout Oncorhyncus mykiss. Experimental infection under aquarium conditions suggested that the virus is apothogenic to rainbow trout. The infective virus could be recovered from the viscera of inoculated fish during the first week post-infection, after which the proportion of virus-positive fish declined over time. A small proportion of the fish still carried the virus 24 d post-inoculation.

Outbreak oof calicivirus gastroenteritis associated with eating frozen raspberries.

Small round structured viruses (SRSVs - for example, calici-, astro-, and entero-viruses) are the commonest causes of outbreaks of non-bacterial gastroenteritis worldwide. Transmission of SRSVs by water and by various foods - including salads, bakery prod

Rotavirus gastroenteritis in Finland: burden of disease and epidemiological features.

The burden of disease attributable to childhood rotavirus infection in Finland was assessed from data on hospital admissions for acute gastroenteritis and from reported virological diagnoses of rotavirus from 1985 to 1995. The mean number of hospitalizations (3584 annually in children under 5 y of age) corresponded to approximately 5.6% of the birth cohort. Rotavirus was estimated to be responsible for 54% of cases; accordingly, 3% of all children in Finland are hospitalized for rotavirus diarrhoea. The monthly distribution of hospitalizations for acute diarrhoea showed a similar pattern as monthly diagnoses of rotavirus, with a long epidemic period starting as early as November or December and lasting until June or even July. The prevalent rotavirus G-type throughout the study period was G1, which was detected in over 60% of the cases; however, in the season 1988-89 G4 was the prominent type. Improved case management has led to a shorter duration of hospital stay (3.3 d in 1985 vs. 2.3 d in 1995), but otherwise these was no significant trend for rotavirus gastroenteritis over the years. These findings underscore the need to control rotavirus gastroenteritis with a specific intervention, notably rotavirus vaccination.

Waterborne epidemics in Finland in 1998-1999.

Fourteen waterborne epidemics occurred in Finland during 1998-1999. About 7,300 illness cases were registered in these outbreaks. All except one of the waterborne epidemics were associated with undisinfected groundwaters. An equal number of waterborne epidemics occurred in public and private water systems, but most cases of illness occurred in public water systems. The three largest epidemics comprised 6,700 illness cases. Insufficient purification treatment unable to remove Norwalk-like viruses caused the only waterborne epidemic in a surface water plant. The main reasons for groundwater outbreaks were floods and surface runoffs which contaminated water. Norwalk-like viruses caused eight and Campylobacter three of the outbreaks. In two cases the epidemic ceased by the exhaustion of susceptible persons in the exposed community but in most cases it was terminated by changing the water source, boiling the drinking water, and starting chlorination.

Evaluation of the purification capacity of nine portable, small-scale water purification devices.

A test was performed to evaluate the microbial and chemical purification capacity of nine portable, small-scale water purification filter devices with production capacity less than 100 L/h. The devices were tested for simultaneous removal capacity of bacteria (cultured Escherichia coli, Clostridium perfringens, Klebsiella pneumoniae and Enterobacter cloacae), enteric protozoans (formalin-stored Cryptosporidium parvum oocysts), viral markers (F-RNA bacteriophages) and microcystins produced by toxic cyanobacterial cultures. In general, the devices tested were able to remove bacterial contaminants by 3.6-6.9 log10 units from raw water. Those devices based only on filtration through pores 0.2-0.4 microm or larger failed in viral and chemical purification. Only one device, based on reverse osmosis, was capable of removing F-RNA phages at concentrations under the detection limit and microcystins by 2.5 log10. The present study emphasised the need for evaluation tests of water purification devices from the public safety and HACCP (Hazard Analysis and Critical Control Point) points of view. Simultaneous testing for various pathogenic/indicator microbes and microcystins was shown to be a useful and practical way to obtain essential data on actual purification capacity of commercial small-scale drinking-water filters.