Activated sludge and UV-C254 for Sapovirus, Aichivirus, Astrovirus, and Adenovirus processing.

In this study, the detection rates of four enteric viruses, Human Astrovirus (HAstVs), Aichivirus (AiVs), Human Adenovirus (HAdVs), and Sapovirus (SaVs) are carried out to assess the virological quality of the treated wastewater. A total of 140 samples was collected from wastewater treatment plant WWTP of Tunis-City. Real-time RT-PCR and conventional RT-PCR results showed high frequencies of detection of the four enteric viruses investigated at the entry and exit of the biological activated sludge procedure and a significant reduction in viral titers after tertiary treatment with UV-C254 irradiation. These results revealed the ineffectiveness of the biological activated sludge treatment in removing viruses and the poor quality of the treated wastewater intended for recycling, agricultural reuse, and safe discharge into the natural environment. The UV-C254 irradiation, selected while considering the non-release of known disinfection by-products because of eventual reactions with the large organic and mineral load commonly present in the wastewater.

Detection of Sapoviruses in two biological lines of Tunisian hospital wastewater treatment.

The efficiency of rotating biodisks and natural oxidizing lagoon procedures is investigated at a Tunisian semi-industrial pilot plant, El Menzeh I, where the wastewater is mainly provided by three different neighbouring hospital clinics. Throughout 2011, 102 wastewater samples were collected from the two mentioned wastewater treatment procedures. Results showed that the Sapovirus (SaV) frequency was approximately 29.4% using the real-time reverse transcription polymerase chain reaction (RT-PCR) technique, and about 16.6% using the conventional RT-PCR. Also, the SaV genogroups and genotypes were identified and genotyping revealed that all of the four Tunisian SaV strains obtained belonged to the two genogroups GIV.1 and GGI.3. In addition, two new genotypes, D and C, were detected. A moderate decrease in the SaV frequencies was observed at the exit of the two treatment processes and the SaV removal rate was around 90% in the natural oxidizing lagoons and 94% in the rotating biodisks procedure showing the temperate sensitivity of these viruses to the implemented biological wastewater. Therefore, an urgent disinfection process should be implemented downstream of the two biological treatment procedures for safe release of treated effluent in the different natural environments. Abbreviations: NoV: Noroviruses; SaV: Sapoviruses; EC: Electrical Conductivity; COD: Chemical Oxygen Demand; BOD5: Biological Oxygen Demand; SS: Suspended Solids; NH4-N: Ammonium Nitrogen; P-PO4: Ortho-Phosphate; AlCl3: aluminum chloride.

Molecular prevalence of bovine noroviruses and neboviruses in newborn calves in Iran.

In this study, bovine enteric caliciviruses (BECs) were detected in 49.4% of a total of 253 stool specimens for diarrheic calves collected from 42 industrial dairy farms from March 2010 to February 2012. Genogroup III norovirus (NoVsGIII) were more prevalent (39.5%) than neboviruses (NBs) (15%), and coinfections were observed in 5.1% of the samples tested. Sequence analysis of the partial polymerase gene from 13 NoVsGIII samples indicated the circulation of both genotype 1 and genotype 2 strains. Among the six NB strains sequenced, five were related to the Bo/Nebraska/80/US strain, while one was related to the Bo/Newbury1/76/UK strain.

HS-AFM and SERS Analysis of Murine Norovirus Infection: Involvement of the Lipid Rafts.

Studies on human norovirus are severely hampered by the absence of a cell culture system until the discovery of murine norovirus (MNV). The cell membrane domains called lipid rafts have been defined as a port of entry for viruses. This study is conducted to investigate murine norovirus binding on the mouse leukemic monocyte macrophage cell line. Lipid raft related structures are extracted from cells by detergent treatment resulting detergent-resistant membrane (DRMs) domains. The real-time polymerase chain reaction technique is performed to detect the viral genome, thereby the MNV binding on the DRMs. The interactions between MNV and DRMs are investigated by high-speed atomic force microscopy (HS-AFM) combined with surface-enhanced Raman spectroscopy (SERS). The inoculation of the virus onto cells results in the aggregations of detergent-resistant membrane domains significantly. The characteristic Raman band of MNV is found in inoculated samples. To be sure that these results are originated from specific interactions between DRM and MNV, methyl-ß-cyclo-dextrin (MßCD) is applied to disrupt lipid rafts. The MNV binding on DRMs is precluded by the MßCD treatment. The cholesterols chains are defined as a key factor in the interactions between norovirus and DRMs. The authors conclude that the MNV binding involves the presence of DRMs and cholesterol dependent.

A Biocatalytic Nanomaterial for the Label-Free Detection of Virus-Like Particles.

The design of nanomaterials that are capable of specific and sensitive biomolecular recognition is an on-going challenge in the chemical and biochemical sciences. A number of sophisticated artificial systems have been designed to specifically recognize a variety of targets. However, methods based on natural biomolecular detection systems using antibodies are often superior. Besides greater affinity and selectivity, antibodies can be easily coupled to enzymatic systems that act as signal amplifiers, thus permitting impressively low detection limits. The possibility to translate this concept to artificial recognition systems remains limited due to design incompatibilities. Here we describe the synthesis of a synthetic nanomaterial capable of specific biomolecular detection by using an internal biocatalytic colorimetric detection and amplification system. The design of this nanomaterial relies on the ability to accurately grow hybrid protein-organosilica layers at the surface of silica nanoparticles. The method allows for label-free detection and quantification of targets at picomolar concentrations.

Detection of Aichi virus genotype B in two lines of wastewater treatment processes.

Enteric viruses are released in important quantities into the environment where they can persist for a very long time. At very low doses, they can cause human gastroenteritis, and are responsible for a substantial number of waterborne diseases. The aims of this study were multiple: firstly, to study the circulation of Aichi viruses (AiV) in wastewater sampled at the scale of a pilot wastewater treatment plant; secondly, to evaluate the performance of two wastewater treatment procedures, as natural oxidizing lagoons and rotating Biodisks, concerning the AiV removal; and finally, to determine the different type of AiV genotype found during this study. Hence, the pilot wastewater treatment plant is principally irrigated by the wastewater of three neighbouring clinics. Wastewater samples were collected during 2011 from the two lines of biological treatment procedures. AiV detection in wastewater were achieved using the Reverse Transcription Polymerase Chain Reaction (RT-PCR) technique, and the identification of AiV genotype was realized by the direct sequencing of PCR products. The result revealed that AiV strains were identified in 50% (n = 51) of the wastewater samples. A significant increase of the AiV detection frequency was registered from upstream to downstream of the five ponds constituting the natural oxidizing lagoon process, and at the exit of the rotating Biodisks procedure. All detected AiV strains showed the highest nucleotide sequence identity to genotype B that has been recently observed in patients in Asia. This finding represented the first Tunisian survey that revealed and mentioned the first detection of AiV genotype B in sewage and by the same argued for a noticeable resistance or survival of this type of virus in the two lines of treatment considered.

The molecular epidemiology of bovine rotaviruses circulating in Iran: a two-year study.

Bovine group A rotavirus (bovine RVA) is recognized as a major cause of severe gastroenteritis in newborn calves. The purpose of this study was to estimate the prevalence and identify the genotypes of circulating bovine RVA in newborn diarrheic calves. Two hundred fifty-three stool samples of diarrheic calves up to 1 month old were collected from 42 industrial dairy farms in two Iranian provinces during March 2010 to February 2012. All collected samples were screened for the presence of bovine RVA by RT-PCR, and the G and P genotypes were determined by semi-nested multiplex RT-PCR assay. The results of RT-PCR indicated that 49.4 % (125 out of 253) of the samples were positive for bovine RVA. The G and P genotyping of a subset of positive samples (n = 85) by semi-nested multiplex RT-PCR revealed that G6 (55.3 %) and G10 (43.5 %) and P[5] (51.8 %) and P[11] (27 %) were the most prevalent G and P genotypes, respectively. G6P[5] was the dominant genotype (35.3 %), followed by G10P[5], G10P[11] and G6P[11], with prevalence rates of 16.5 %, 15.3 % and 10.6 %, respectively. Sequence analysis of 20 VP7 and four VP4 genes showed highest nucleotide sequence identity with the corresponding genes of strains RVA/Cow-tc/GBR/UK/1973/G6P7[5] and RVA/Cow-tc/USA/B223/XXXX/G10P[11]. The results of this study reveal the diversity of G and P genotypes in bovine RVA samples from diarrheic Iranian calves and expands our knowledge of bovine RVA infections in the Middle East. These results also highlight the importance of producing of an effective rotavirus vaccine and its inclusion in the national cattle immunization program.

Evaluation of immunochromatographic tests for the rapid detection of the emerging GII.17 norovirus in stool samples, January 2016.

A novel GII.17 norovirus emerged in Asia in the winter of 2014/15. A worldwide spread is conceivable and norovirus diagnostic assays need to be evaluated to investigate if they adequately detect this emerging genotype. Seven immunochromatographic kits commercially available in Europe were evaluated on ten stool samples where GII.17 virus had been quantified by real-time reverse transcription-polymerase chain reaction. All the kits detected GII.17 with various sensitivities, partly depending on the virus titre.

Rotavirus P[8] Infections in Persons with Secretor and Nonsecretor Phenotypes, Tunisia.

To determine whether rotavirus infections are linked to secretor status, we studied samples from children in Tunisia with gastroenteritis. We phenotyped saliva for human blood group antigens and tested feces for rotavirus. Rotavirus was detected in 32/114 patients. Secretor genotyping showed that P[8] rotavirus infected secretors and nonsecretors, and infection correlated with presence of Lewis antigen.

Diagnostic Accuracy of Seven Commercial Assays for Rapid Detection of Group A Rotavirus Antigens.

Seven commercial immunochromatographic assays intended for the detection of group A rotavirus antigens in human stool samples were evaluated. These assays showed similar levels of diagnostic accuracy and were suitable for the detection of rotavirus in patients with acute gastroenteritis but missed some asymptomatic rotavirus shedding identified by real-time reverse transcription-PCR.

A new gyrovirus in human feces.

A novel gyrovirus genome found in the feces of an adult with diarrhea is described. The genome shows the three expected main ORFs encoding a structural protein (VP1), nonstructural protein (VP2), and Apoptin protein (VP3), which shared identities of 41, 42, and 38 % with those of the most closely related gyrovirus proteins, respectively. Given the high divergence in its genome, this gyrovirus may be considered the prototype for a new viral species (GyV9) in the Gyrovirus genus. Because the closest relatives of this gyrovirus infect chicken, a possible dietary origin for the presence of this virus in human feces is discussed.

Intrarectal immunization and IgA antibody-secreting cell homing to the small intestine.

According to the current paradigm, lymphocyte homing to the small intestine requires the expression of two tissue-specific homing receptors, the integrin α4ß7 and the CCL25 receptor CCR9. In this study, we investigated the organ distribution and the homing molecule expression of IgA Ab-secreting cells (ASCs) induced by intrarectal immunization with a particulate Ag, in comparison with other mucosal immunization routes. Intrarectal immunization induces gut-homing IgA ASCs that localize not only in the colon but also in the small intestine, although they are not responsive to CCL25, unlike IgA ASCs induced by oral immunization. The mucosal epithelial chemokine CCL28, known to attract all IgA ASCs, does not compensate for the lack of CCL25 responsiveness, because the number of Ag-specific cells is not decreased in the gut of CCR10-deficient mice immunized by the intrarectal route. However, Ag-specific IgA ASCs induced by intrarectal immunization express the integrin α4ß7, and their number is considerably decreased in the gut of ß7-deficient mice immunized by the intrarectal route, indicating that α4ß7 enables these cells to migrate into the small intestine, even without CCL25 responsiveness. In contrast, IgA ASCs induced by intranasal immunization express low α4ß7 levels and are usually excluded from the gut. Paradoxically, after intranasal immunization, Ag-specific IgA ASCs are significantly increased in the small intestine of ß7-deficient mice, demonstrating that lymphocyte homing is a competitive process and that integrin α4ß7 determines not only the intestinal tropism of IgA ASCs elicited in GALTs but also the intestinal exclusion of lymphocytes primed in other inductive sites.

New parvovirus in child with unexplained diarrhea, Tunisia.

A divergent parvovirus genome was the only eukaryotic viral sequence detected in feces of a Tunisian child with unexplained diarrhea. Tusavirus 1 shared 44% and 39% identity with the nonstructural protein 1 and viral protein 1, respectively, of the closest genome, Kilham rat parvovirus, indicating presence of a new human viral species in the Protoparvovirus genus.

Rotavirus surveillance in urban and rural areas of Niger, April 2010-March 2012.

Knowledge of rotavirus epidemiology is necessary to make informed decisions about vaccine introduction and to evaluate vaccine impact. During April 2010-March 2012, rotavirus surveillance was conducted among 9,745 children <5 years of age in 14 hospitals/health centers in Niger, where rotavirus vaccine has not been introduced. Study participants had acute watery diarrhea and moderate to severe dehydration, and 20% of the children were enrolled in a nutrition program. Of the 9,745 children, 30.6% were rotavirus positive. Genotyping of a subset of positive samples showed a variety of genotypes during the first year, although G2P[4] predominated. G12 genotypes, including G12P[8], which has emerged as a predominant strain in western Africa, represented >80% of isolates during the second year. Hospitalization and death rates and severe dehydration among rotavirus case-patients did not differ during the 2 years. The emergence of G12P[8] warrants close attention to the characteristics of associated epidemics and possible prevention measures.

Absolute Humidity Influences the Seasonal Persistence and Infectivity of Human Norovirus.

Norovirus (NoV) is one of the main causative agents of acute gastroenteritis worldwide. In temperate climates, outbreaks peak during the winter season. The mechanism by which climatic factors influence the occurrence of NoV outbreaks is unknown. We hypothesized that humidity is linked to NoV seasonality. Human NoV is not cultivatable, so we used cultivatable murine norovirus (MNV) as a surrogate to study its persistence when exposed to various levels of relative humidity (RH) from low (10% RH) to saturated (100% RH) conditions at 9 and 25°C. In addition, we conducted similar experiments with virus-like particles (VLPs) from the predominant GII-4 norovirus and studied changes in binding patterns to A, B, and O group carbohydrates that might reflect capsid alterations. The responses of MNV and VLP to humidity were somewhat similar, with 10 and 100% RH exhibiting a strong conserving effect for both models, whereas 50% RH was detrimental for MNV infectivity and VLP binding capacity. The data analysis suggested that absolute humidity (AH) rather than RH is the critical factor for keeping NoV infectious, with an AH below 0.007 kg water/kg air being favorable to NoV survival. Retrospective surveys of the meteorological data in Paris for the last 14 years showed that AH average values have almost always been below 0.007 kg water/kg air during the winter (i.e., 0.0046 ± 0.0014 kg water/kg air), and this finding supports the fact that low AH provides an ideal condition for NoV persistence and transmission during cold months.

Norovirus-related chronic diarrhea in a patient treated with alemtuzumab for chronic lymphocytic leukemia.

BACKGROUND: Norovirus infection is increasingly recognized as an important cause of persistent gastroenteritis in immunocompromised hosts and can be a potential cause of morbidity in these populations. CASE PRESENTATION: Here, we report a case of norovirus-related chronic diarrhea occurring in a 62-year-old immunocompromised patient treated with alemtuzumab for chronic lymphocytic leukemia. Despite different therapeutic strategies including tapering of immunosuppressive therapy and immunoglobulin administration, diarrhea unfortunately did not resolve and lasted for a total of more than twelve weeks with prolonged norovirus fecal excretion. CONCLUSIONS: Norovirus infection can occur in the setting of alemtuzumab treatment, even as a single agent, and should be included in the differential diagnoses of acute and chronic diarrhea in these immunocompromised patients. Although the administration of oral immunoglobulin has been described as a promising efficient therapy, this was not the case in our patient. Clinical trials are thus clearly warranted to better define risk factors and efficient therapies for norovirus infection in immunocompromised populations.

Prevalence and genetic diversity of norovirus infection in Tunisian children (2007-2010).

Viral gastroenteritis can be a life-threatening disease in infants and young children, especially in developing countries. The aim of this study was to continue the epidemiological surveillance of norovirus (NoV) infections in Tunisian children suffering from acute gastroenteritis. Surveillance was initiated in January 2003, to monitor potential variations in strains over time, in terms of frequency and diversity of NoV genotypes, and more particularly the potential emergence of new GII.4 variants following the 2004 Hunter variant. From April 2007 to April 2010, a total of 407 stool specimens were collected from sporadic cases (238 inpatients and 169 outpatients). Furthermore, 28 stool samples were collected from children involved in 3 gastroenteritis outbreaks. Stool specimens were screened for NoV genogroup I (GI) and II (GII) by RT-PCR. NoV strains were genotyped, and variants identified, based on sequence and phylogenetic analyses of the polymerase and capsid genes. NoVs were detected in 38 sporadic cases (9.3%) and 21 epidemic cases (75%). Great diversity was observed throughout the period, with seven distinct NoV genotypes characterized in sporadic cases, and three in outbreaks. GIIb/II.3 and GII.4 were predominant globally, with fluctuations of their prevalence over time. Interestingly, the Hunter variant, which was the unique GII.4 variant observed from 2003 to April 2007 in the region of Monastir, was replaced by the 2006b variant. NoV is an important enteropathogen responsible for viral gastroenteritis among infants and children in Tunisia, and the infecting strains between 2007 and 2010 were different from those in previous years.

Molecular prevalence of bovine noroviruses and neboviruses detected in central-eastern Tunisia.

Two genetically distinct bovine enteric caliciviruses are known: noroviruses of genogroup III (NoVsGIII), which are genetically related to human noroviruses, and neboviruses, which represent a new calicivirus genus. To investigate the presence of NoVsGIII and nebovirus strains in diarrheic calves in Tunisia, a total of 169 faecal specimens were collected from January 2006 to October 2010 from different cattle herds located in the central-east regions. RT-PCRs and sequencing were carried out using primers targeting the 3' end of the polymerase gene of NoVsGIII and neboviruses. This study revealed that NoVsGIII and nebovirus are endemic in diarrheic calves in Tunisia. NoVsGIII infections, all with genotype 2, had an apparent molecular prevalence of 16.6 % and were more frequent than nebovirus infections. NoVsGIII infections showed clear seasonality, with a peak in winter. Nebovirus infections, with a prevalence of 3.0 %, were all related to the reference strain Bo/Nebraska/80/US.

Adhesion of human pathogenic enteric viruses and surrogate viruses to inert and vegetal food surfaces.

Enteric viruses, particularly human Noroviruses (NoV) and hepatitis A virus (HAV), are key food-borne pathogens. The attachment of these pathogens to foodstuff and food-contact surfaces is an important mechanism in the human contamination process. Studies were done to investigate the nature of the physicochemical forces, such as hydrophobic and electrostatic ones, involved in the interaction virus/matrix but, at this day, only few data are available concerning surface properties of viruses and prediction of the adhesion capacity of one specific virus onto matrices is still very difficult. The purpose of this study was to propose a reference system, including a representative virus surrogate, able to predict as close as possible behaviour of pathogenic viruses in term of adhesion on inert (stainless steel and polypropylene) and food surfaces (lettuce leaves, strawberries and raspberries). The adhesion of human pathogenic enteric viruses, cultivable strain of HAV and non-cultivable strains of human NoV (genogroups I and II), have been quantified and compared to these of human enteric viruses surrogates, included the MNV-1 and three F-specific RNA bacteriophages (MS2, GA and Qß). A standardized approach was developed to assess and quantify viral adhesion on tested matrices after a contact time with each virus using real-time RT-PCR. Methods used for virus recovery were in accordance with the CEN recommendations, including a bovine Enterovirus type 1 as control to monitor the efficiency of the extraction process and amplification procedure from directly extracted or eluted samples. The adhesion of human pathogenic viruses, ranging from 0.1 to 2%, could be comparable for all matrices studied, except for NoV GII on soft fruits. Adhesion percentages obtained for the studied surrogate virus and phages were shown to be comparable to those of HAV and NoV on inert and lettuce surfaces. The MNV-1 appeared as the best candidate to simulate adhesion phenomena of all human pathogenic enteric viruses on all studied surfaces, while MS2 and GA bacteriophages could be a good alternative as model of viral adhesion on inert and lettuce surfaces. These results will be usable to design relevant experimental systems integrating adhesion behaviour of enteric viruses in the assessment of the efficiency of a technological or hygienic industrial process.

Possible novel nebovirus genotype in cattle, France.

To determine if bovine caliciviruses circulate in France, we studied 456 fecal samples from diarrheic calves. We found a 20% prevalence of genogroup III noroviruses and a predominance of genotype III.2. Neboviruses, with a prevalence of 7%, were all related to the reference strain Bo/Nebraska/80/US, except for the strain Bo/DijonA216/06/FR, which could represent a novel genotype.