Assessment of norovirus contamination in environmental samples from Florianópolis City,Southern Brazil

Aims: To assess norovirus (NoV) contamination in aquatic ecosystems in the city of Florianópolis, in Southern Brazil, to provide epidemiological data that can support actions for environmental contamination control. Methods and Results: An adsorption–elution method, followed by ultrafiltration, was performed to concentrate the viruses. NoV were detected using semi‐nested PCR and quantified by real‐time PCR. From June 2007 to May 2008, NoV were detected in 23% (22/94) of the samples analysed, including seawater, drinking water, superficial water (creek and brackish lagoon) and treated sewage. The mean viral loads for genogroups (G)I and GII in treated sewage samples were 297 and 440 genomic copies (gc) l^?1, respectively, whereas creek water samples contained 2603 and 1361 gc l^?1, respectively. Six samples were sequenced: two samples were GII.4, two were GII.2 and two were GI.3. Conclusions: NoV were detected in all water types analysed, demonstrating the widespread contamination of this geographical area with several cocirculating strains belonging to GI and GII. Significance and Impact of the Study: This study demonstrates the environmental spread of NoV in environmental waters and highlights the potential hazard for human health following the consumption of or contact with these waters, which could result in waterborne or foodborne acute gastroenteritis.     

Interaction between noroviruses and human histo-blood group antigens

Norovirus (NOV), a member of the family Caliciviridae, is a major cause of water and food-borne acute nonbacterial gastroenteritis, and forms many morphologically similar but antigenically diverse groups of viruses. The virus-like particles (VLPs) derived from the prototype strain of NoV, Norwalk virus (NV/68), bind to histo-blood group antigens (HBGAs). HBGAs are carbohydrates that contain structurally related saccharide moieties, and are found in saliva and mucosal secretions from intestinal epithelial cells of secretor individuals who have FUT2 gene encoding a fucosyltransferase. From volunteer challenge studies, there is strong evidence that the carbohydrate-binding is essential for the NV/68 infection. Non-secretors, who do not express FUT2 fucosyltransferase and consequently do not express H type 1 or Leb in the gut, were not infected after the challenge with NV/68. However, other NoV VLPs display different ABH and Lewis carbohydrate-binding profiles, and indeed epidemiological studies showed that some NoV strains could infect individuals with another ABH phenotypes. GII/4 is known to be global epidemic strain and bound more HBGAs when compared with other strains. The strength of the transmission of GII/4 strains may be linked with their wide recognition of HBGAs. It is obvious that HBGAs are important factors to determine the host specificity, although it is still unclear whether the HBGAs act as the primary receptor or enhance NoV infectivity. Further investigation is needed.     

Continuous detection of norovirus and astrovirus in wastewater in a coastal city of China in 2014–2016

Norovirus (NoV) and human astrovirus (HAstV) are important causative agents of acute gastroenteritis in children and adults. They are comprised of multiple genotypes and attention should be paid to genotype changes or emergence of new genetic variants. To study the prevalence and diversity of NoV GI, GII, and HAstV circulating in eastern China, we conducted a three-year environmental surveillance in a coastal city of Yantai. Thirty-six sewage samples were collected, processed, and examined for the presence of viral genomes by PCR. The results showed that NoV GI, GII, and HAstV were detected in all 36 samples. Six NoV GI genotypes, 11 NoV GII genotypes, and 5 HAstV serotypes were identified; GI.6, GII.17, and HAstV-5 were the most prevalent types, respectively. Persistent existence of NoV GII.17 Kawasaki 308 variant was observed during whole study period. Phylogenetic analysis reflected multiple transmission lineages in local population for both viruses. Our results reflect continuous presence of enteric viruses in sewage, improve our understanding on their molecular epidemiology, and demonstrate surveillance on sewage is an effective approach in understanding the local circulation of enteric viruses.     

Host Genetic Factors Affect Susceptibility to Norovirus Infections in Burkina Faso

Norovirus (NoV) constitutes the second most common viral pathogen causing pediatric diarrhea after rotavirus. In Africa, diarrhea is a major health problem in children, and yet few studies have been performed regarding NoV. The association of histo-blood group antigens (HBGA) and susceptibility to NoV infection is well established in Caucasian populations with non-secretors being resistant to many common NoV strains. No study regarding HBGA and NoV susceptibility has yet been performed in Africa. We collected 309 stool and 208 saliva samples from diarrheal children in Ouagadougou, Burkina Faso; May 2009 to March 2010. NoV was detected using real-time PCR, and genotyped by sequencing. Saliva samples were ABO, Lewis and secretor phenotyped using in house ELISA assays. NoV was detected in 12% (n = 37) of the samples. The genotype diversity was unusually large; overall the 37 positive samples belonged to 14 genotypes. Only children <2 years of age were NoV positive and the GII.4 NoVs were more frequent in the late dry season (Jan-May). NoV infections were observed less in children with the secretor-negative phenotype or blood group A (OR 0.18; p = 0.012 and OR 0.31; p = 0.054; respectively), with two non-secretors infected with genotypes GII.7 and GII.4 respectively. Lewis-negative (Le^a−b−) children, representing 32% of the study population, were susceptible to GII, but were not infected with any NoV GI. GII.4 strains preferentially infected children with blood group B whereas secretor-positive children with blood group O were infected with the largest variety of genotypes. This is the first study identifying host genetic factors associated with susceptibility to NoV in an African population, and suggests that while the non-secretor phenotype provides protection; the Lewis b antigen is not necessary for GII infection.     

Detection of genogroup IV norovirus in wastewater and river water in Japan

Aims:  To test wastewater and river water in Japan for genogroup IV norovirus (GIV NoV).
Methods and Results:  Influent and effluent samples from a wastewater treatment plant and the Tamagawa River water samples were collected monthly for a year. The water samples were concentrated by the adsorption–elution method, using an HA electronegative filter with acid rinse procedure, followed by quantitative detection of GIV NoV using TaqMan-based real-time RT-PCR. Both wastewater and river water samples showed a high positive ratio of GIV NoV during winter and spring. The highest concentration in wastewater and river water was 6·9 × 10⁴ and 1·5 × 10⁴ copies l⁻¹, respectively.
Conclusions:  Presence of GIV NoV in the environments demonstrates that not only GI and GII NoVs but also GIV strains are circulating and that routine monitoring of GIV NoV in water environments is recommended to understand its epidemics, environmental distribution and potential health risks.
Significance and Impact of the Study:  This is the first study providing quantitative data on the occurrence of GIV NoV in environmental water over a 1-year period.     

Detection of Norovirus and Sapovirus from diarrheic dogs and cats in Japan

Norovirus (NoV) and sapovirus (SaV) are important causes of human diarrhea. In this study, between 2007 and 2014 fecal samples were collected from 97 dogs and 83 cats with diarrhea and examined to determine the prevalence of NoV and SaV infections in Japan. To detect caliciviruses, approximately 300 bases targeting the polymerase gene were amplified using RT‐PCR and subjected to phylogenetic and homology analyses. Specific PCR products were obtained from four canine and nine feline samples: two canine and one feline isolate were classified as NoV, two canine isolates as SaV and the remaining eight feline isolates as vesivirus (VeV). The three NoV isolates were classified into the same clade as that of known canine and feline NoVs; their homologies (75.9–92.3%) were higher than those with human genogroup IV (GIV) NoVs (59.1–65.9%). The homology of the feline NoV isolate with previously reported feline NoV isolates was particularly high (91.7–92.3%). Regarding SaV, the two canine isolates were classified into the same clade as known canine SaVs and their homologies (72.5–86.5%) were higher than those with other mammal SaVs (20.7–58.0%). The eight feline VeV isolates were assumed to be feline calicivirus. The present study is the first report of the presence of NoV‐ and SaV‐infected dogs and cats in Japan. The findings suggest there are species‐specific circulations of NoV and SaV among dogs and cats, in Japan.     

Evaluation of nine sets of PCR primers in the RNA dependent RNA polymerase region for detection and differentiation of members of the family Caliciviridae, Norwalk virus and Sapporo virus

Norwalk virus and Sapporo virus were approved as type species of the genus "Norwalk-like viruses" and the genus "Sapporo-like viruses," respectively, in the family Caliciviridae. A total of 116 stool specimens containing Norwalk virus (NV) or Sapporo virus (SV) were tested by RT-PCR and Southern hybridization to evaluate nine sets of PCR primers and seven internal oligonucleotide probes in the RNA dependent RNA polymerase region of NV and SV for detection and differentiation of viruses in the NV and SV. Fifty-five stool samples were collected from 11 outbreaks of NV and/or SV gastroenteritis in an infant home, where residents were infants under 2 years of age, in Sapporo, Japan. Sixty specimens were obtained in Sapporo from sporadic cases in children, mainly under 6 years of age, of acute gastroenteritis due to small round structured viruses detected by EM. There is no single primer pair to detect all NV and SV, and at least three primer pairs, G1 set, G2 set and Sapp35/Sapp36, are required to detect viruses in the NV and SV clades. Many NV and SV strains were successfully classified into one of the NV/genogroup I, NV/genogroup II and SV by single-round RT-PCR and Southern hybridization. The new detection method for SV reported in this study combined with those for NV previously reported may elucidate the importance of Norwalk virus and Sapporo virus as a cause of viral gastroenteritis in all age groups in the world.     

Environmental contamination with SARS-CoV-2 in COVID-19 hospitals in Wuhan,China, 2020

Coronavirus disease 2019 (COVID-19) pandemic has caused high number of infections and deaths of healthcare workers globally. Distribution and possible transmission route of SARS-CoV-2 in hospital environment should be clarified. We herein collected 431 environmental (391 surface and 40 air) samples in the intensive care unit (ICU) and general wards (GWs) of three hospitals in Wuhan, China from February 21 to March 4, 2020, and detected SARS-CoV-2 RNA by real-time quantitative PCR. The viral positive rate in the contaminated areas was 17.8% (28/157), whereas there was no virus detected in the clean areas. Higher positive rate (22/59, 37.3%) was found in ICU than that in GWs (3/63, 4.8%). The surfaces of computer keyboards and mouse in the ICU were the most contaminated (8/10, 80.0%), followed by the ground (6/9, 66.7%) and outer glove (2/5, 40.0%). From 17 air samples in the contaminated areas, only one sample collected at a distance of around 30 cm from the patient was positive. Enhanced surface disinfection and hand hygiene effectively decontaminated the virus from the environment. This finding might help understand the transmission route and contamination risk of SARS-CoV-2 and evaluate the effectiveness of infection prevention and control measures in healthcare facilities.     

Transmission of Rickettsia massiliae in the tick, Rhipicephalus turanicus

Rickettsia massiliae, strain Bar29, was detected in engorged female ticks of the Rhiphicephalus sanguineus group collected in Corsica, a French Mediterranean island. Ticks were identified by molecular analysis as Rhipicephalus turanicus (Pomerantsev) (Acari: Ixodidae). Twenty larvae of the second generation obtained from a R. massiliae-infected, engorged female were tested by polymerase chain reaction (PCR) and all were positive for R. massiliae. Larvae of the same cohort were fed on rabbits and specimens of subsequent stages of the second and third generation of ticks were tested by PCR. Both transovarial and transstadial transmission were demonstrated; the transovarial transmission rate was estimated at 100%. A high filial infection rate was demonstrated; 132 out of 134 larvae obtained from five infected females of the fourth generation were infected. When saliva samples from half-engorged Rh. turanicus of the second generation were tested by PCR, four out of five were positive. Rickettsia massiliae was detected in faeces of infected ticks by PCR and immunofluorescence assay, although no rickettsiae could be maintained in culture. Co-feeding/transsexual transmission of R. massiliae Bar29 was demonstrated by feeding male Rh. turanicus on a rabbit with Rhipicephalus sanguineus (Latreille) (Acari: Ixodidae) females (the latter were the only uninfected ticks available). Infection was subsequently detected in nine out of the thirteen females (69.2%). These results suggest that Rh. turanicus ticks are potential vectors and reservoirs for R. massiliae Bar29.     

Non-sand fly transmission of a North American isolate of Leishmania infantum in experimentally infected BALB/c mice

Leishmania infantum, an etiologic agent of zoonotic visceral leishmaniasis, is endemic in the foxhound population in the United States and Canada. Leishmaniasis is usually transmitted by blood-feeding sand flies; however, epidemiological data do not support a significant role for sand flies in the maintenance of foxhound infections in North America, and an alternate mode of transmission may exist. The present study was conducted to determine if transplacental or direct transmission occurs in pregnant BALB/c mice experimentally infected with L. infantum isolated from a naturally infected foxhound from Virginia as well as to determine if the parasite was directly transmitted to the males used to breed the mice. Female BALB/c mice were intravenously inoculated with 1 x 10(6) promastigotes of the LIVT-1 strain of L. infantum. Mice were bred to uninfected male BALB/c mice 2 mo postinoculation. Pregnant mice were killed between days 13 and 18 of gestation. Pups and placentas were collected at necropsy, divided, and used for parasite culture and polymerase chain reaction (PCR) analyses. Culture and PCR analyses were performed on spleens from the male mice to determine the possibility of sexual transmission. Leishmania sp. DNA was detected in 4 of 88 pups and 3 of 16 placentas from LIVT-1-inoculated mice. One male mouse used to breed infected females was PCR positive. This work provides evidence for a low level of nonvector transmission of North American L. infantum in a mouse model.     

Genetic diversity of genogroup IV noroviruses in wastewater in Japan

Aims: To investigate the prevalence, seasonality and genetic diversity of genogroup IV noroviruses (GIV NoVs) in wastewater in Japan. Methods and Results: Untreated and treated wastewater samples were collected monthly for a year from a wastewater treatment plant in Japan. The concentrated wastewater samples were examined for the presence of GIV NoV genomes with seminested RT‐PCR assay targeting partial capsid gene. Among 12 untreated and 12 treated wastewater samples tested, GIV NoV genomes were detected in three (25%) untreated and two (17%) treated wastewater samples with a high positive ratio in winter season. Genetic analysis revealed that the GIV NoVs in the wastewater samples were genetically diverse and were classified into three different genetic clusters. Conclusions: Frequent detection of GIV NoVs in winter season, which is a common epidemic period of human NoVs in Japan, indicates that GIV NoVs exhibit temporal trends similar to GI and GII NoVs. Based on the partial capsid gene sequences, we identified several unique GIV NoV strains belonging to the novel genetic cluster, demonstrating that GIV NoVs are more genetically diverse than previously appreciated. Significance and Impact of the Study: Our findings provide novel evidence of considerable genetic diversity among the GIV NoV strains.     

Occurrence of enteric viruses in shellfish and relation to climatic-environmental factors

Aims: To investigate the presence of enteric viruses [hepatitis A (HAV) and norovirus (NoV)] in shellfish harvested from the deltaic area of the Po river in relation to environmental factors. Methods and Results: Fortnightly sampling of shellfish was carried out in two lagoon areas (category B production areas) and one sea area (category A). Environmental parameters in the lagoon and hydrometric level of the tributary river were monitored throughout the sampling period. Samples (n = 120) were analysed for bacterial (E. coli and Salmonella) and viral (HAV and NoV) contamination; samples from category B areas were analysed before and after purification treatment. All the samples were negative for HAV whereas 10 samples (8·3%), all harvested in the lagoon areas, were positive for NoV. Sequencing identified the strains as genotypes II.4 and II.b. None of the samples was found to be contaminated after depuration. Conclusions: The monitoring showed a low frequency of NoV presence; viral contamination, detected exclusively in shellfish collected from the deltaic area (category B), could be influenced by the flow of the tributary river. Significance and Impact of the Study: The data collected are useful for the design of targeted prevention strategies and for the modulation of control plans after meteorological events.     

The hygienic efficiency of conventional and inverted lamb dressing systems

R.G. BELL AND S.C. HATHAWAY. 1996. Aerobic plate counts (APC 37°C and APC 25°C) and Escherichia coli enumerations (Petrifilm) were used to determine sources of bacterial contamination during sheep dressing, determine the hygienic efficacy of hand wash and knife 'sterilization'procedures and compare the hygiene efficiency of conventional and inverted sheep dressing systems. The major slaughterline sources of microbial contamination were: fleece > workers' hands > faecal pellets > knife blades. Aerobic plate counts (APC 37°C) exceeding log 4.4 cfu cm^-2 were considered indicative of direct fleece contact, whereas E. coli numbers exceeding log 3.3 cfu cm^-2 were considered indicative of direct faecal contact. A 44°C water hand rinse removed 90% of the microbial contamination from workers' hands, but rinsed hands, particularly those contacting the fleece, still carried a microbial population exceeding log 4.0 cfu cm^-2. A 44°C rinse followed by an 82°C water dip reduced the contamination on knife blades to less than log 3.0 cfu cm^-2. Inverted dressing systems produced carcasses with a lower contamination level than conventional systems. With both systems little increase in contamination occurred after pelt removal. The areas of highest contamination were the forequarter region with inverted dressing and the hindquarter with conventional dressing. In both cases these regions are the sites where cuts are made through the skin. With both systems contamination around these cuts was entirely consistent with direct fleece contact resulting from 'rollback'.