Human enteric viruses in a wastewater treatment plant: evaluation of activated sludge combined with UV disinfection process reveals different removal performances for viruses with different features.

This study assess the quality of wastewater through the detection and quantification of important viruses causing gastroenteritis at different stages of the wastewater treatment process in an activated-sludge wastewater treatment plant with ultraviolet disinfection. Ten sampling events were carried out in a campaign along a period of 18 months collecting wastewater samples from the influent, after the activated-sludge treatment, and after the final disinfection with UV radiation. Samples were concentrated through ultracentrifugation and analysed using retro-transcription, PCR and real time quantitative PCR protocols, for detection and quantification of Group A Rotavirus (RVA), Human Astrovirus (HAstV), Norovirus Genogroup II (NoV GII) and Human Adenovirus (HAdV). HAdV (100%), NoV GII (90%), RVA (70%) and HAstV (60%) were detected in influent samples with concentration from 1·4 (NoV GII) to 8·0 (RVA) log10  gc l-1 . Activated-sludge treatment reached well quality effluents with low organic material concentration, although nonstatistical significant differences were registered among influent and postactivated sludge treatment samples, regarding the presence and concentration for most viruses. All post-UV samples were negative for NoV GII and HAstV, although RVA and HAdV were detected in 38% and 63% of those samples respectively, with concentration ranging from 2·2 to 5·5 and 3·1 to 3·4 log10  gc l-1 . SIGNIFICANCE AND IMPACT OF THE STUDY: This study demonstrates that an activated-sludge wastewater treatment plant with UV disinfection reduces to levels below the detection limit those single-stranded RNA viruses as noroviruses and astroviruses and reach significant lower levels of rotaviruses and adenoviruses after the complete treatment process.

Norovirus molecular detection in Uruguayan sewage samples reveals a high genetic diversity and GII.4 variant replacement along time.

AIMS: To determine the prevalence and molecular epidemiology of norovirus (NoV) genogroup I (GI) and GII in Uruguay. METHODS AND RESULTS: One hundred and sixteen sewage samples were collected in six cities (Bella Unión, Salto, Paysandú, Fray Bentos, Melo and Treinta y Tres) from March 2011 to April 2013, viruses were concentrated by ultracentrifugation and NoV studies were performed by semi-nested RT-PCR (partial capsid region). NoV were detected in samples from all the cities and detected in 72% (84/116) of the samples with nine of them belonging to GI, 48 to GII and 27 to both genogroups. Remarkably, a high genetic diversity was identified: GII.2 (n = 13), GII.4 (n = 13), GI.1 (n = 5), GI.4 (n = 5), GI.8 (n = 4), GII.13 (n = 4), GII.1 (n = 3), GII.6 (n = 3), GI.3 (n = 1), GI.5 (n = 1), GI.6 (n = 1), GII.3 (n = 1), GII.17 (n = 1). Interestingly, a complete replacement of GII.4 New Orleans 2009 by GII.4 Sydney 2012 variants during 2012 was evidenced. CONCLUSION: This study reveals a high circulation of different NoV GI and GII genotypes in sewage evidencing a replacement of GII.4 variants. SIGNIFICANCE AND IMPACT OF STUDY: This approach can be used as an indicator of the presence of a new GII.4 variant which can originate an increase in acute gastroenteritis outbreaks worldwide.

Sewage surveillance reveals the presence of canine GVII norovirus and canine astrovirus in Uruguay.

Canine norovirus (NoV) and astrovirus (AstV) were studied in 20 domestic sewage samples collected in two cities in Uruguay. Four samples were characterized as canine AstV after phylogenetic analysis clustering with strains detected in Italy and Brazil in 2008 and 2012, respectively. One sample was characterized as canine NoV and clustered with a strain detected in Hong Kong and recently classified as GVII. This study shows the occurrence of a canine NoV GVII strain for the first time in the American continent and also warns about possible zoonotic infection, since canine strains were detected in domestic sewage.

RNAi knock-downs support roles for the mucin-like (AeIMUC1) gene and short-chain dehydrogenase/reductase (SDR) gene in Aedes aegypti susceptibility to Plasmodium gallinaceum.

The mosquito midgut represents the first barrier encountered by the Plasmodium parasite (Haemosporida: Plasmodiidae) when it is ingested in blood from an infected vertebrate. Previous studies identified the Aedes aegypti (L.) (Diptera: Culicidae) mucin-like (AeIMUC1) and short-chain dehydrogenase/reductase (SDR) genes as midgut-expressed candidate genes influencing susceptibility to infection by Plasmodium gallinaceum (Brumpt). We used RNA inference (RNAi) by double-stranded RNA (dsRNA) injections to examine ookinete survival to the oocyst stage following individual gene knock-downs. Double-stranded RNA gene knock-downs were performed 3 days prior to P. gallinaceum infection and oocyst development was evaluated at 7 days post-infection. Mean numbers of parasites developing to the oocyst stage were significantly reduced by 52.3% in dsAeIMUC1-injected females and by 36.5% in dsSDR-injected females compared with females injected with a dsß-gal control. The prevalence of infection was significantly reduced in dsAeIMUC1- and dsSDR-injected females compared with females injected with dsß-gal; these reductions resulted in a two- and three-fold increase in the number of uninfected individuals, respectively. Overall, these results suggest that both AeIMUC1 and SDR play a role in Ae. aegypti vector competence to P. gallinaceum.

Individual rotavirus-like particles containing 120 molecules of fluorescent protein are visible in living cells.

Rotaviruses are large, complex icosahedral particles consisting of three concentric capsid layers. When the innermost capsid protein VP2 is expressed in the baculovirus-insect cell system it assembles as core-like particles. The amino terminus region of VP2 is dispensable for assembly of virus-like particles (VLP). Coexpression of VP2 and VP6 produces double layered VLP. We hypothesized that the amino end of VP2 could be extended without altering the auto assembly properties of VP2. Using the green fluorescent protein (GFP) or the DsRed protein as model inserts we have shown that the chimeric protein GFP (or DsRed)-VP2 auto assembles perfectly well and forms fluorescent VLP (GFP-VLP2/6 or DsRed-VLP2/6) when coexpressed with VP6. The presence of GFP inside the core does not prevent the assembly of the outer capsid layer proteins VP7 and VP4 to give VLP2/6/7/4. Cryo-electron microscopy of purified GFP-VLP2/6 showed that GFP molecules are located at the 5-fold vertices of the core. It is possible to visualize a single fluorescent VLP in living cells by confocal fluorescent microscopy. In vitro VLP2/6 did not enter into permissive cells or in dendritic cells. In contrast, fluorescent VLP2/6/7/4 entered the cells and then the fluorescence signal disappear rapidly. Presented data indicate that fluorescent VLP are interesting tools to follow in real time the entry process of rotavirus and that chimeric VLP could be envisaged as "nanoboxes" carrying macromolecules to living cells.

Antigenic structure of human respiratory syncytial virus fusion glycoprotein.

New series of escape mutants of human respiratory syncytial virus were prepared with monoclonal antibodies specific for the fusion (F) protein. Sequence changes selected in the escape mutants identified two new antigenic sites (V and VI) recognized by neutralizing antibodies and a group-specific site (I) in the F1 chain of the F molecule. The new epitopes, and previously identified antigenic sites, were incorporated into a refined prediction of secondary-structure motifs to generate a detailed antigenic map of the F glycoprotein.