Nucleocapsid protein-specific monoclonal antibodies protect mice against Crimean-Congo hemorrhagic fever virus.

Crimean-Congo hemorrhagic fever virus (CCHFV) is a WHO priority pathogen. Antibody-based medical countermeasures offer an important strategy to mitigate severe disease caused by CCHFV. Most efforts have focused on targeting the viral glycoproteins. However, glycoproteins are poorly conserved among viral strains. The CCHFV nucleocapsid protein (NP) is highly conserved between CCHFV strains. Here, we investigate the protective efficacy of a CCHFV monoclonal antibody targeting the NP. We find that an anti-NP monoclonal antibody (mAb-9D5) protected female mice against lethal CCHFV infection or resulted in a significant delay in mean time-to-death in mice that succumbed to disease compared to isotype control animals. Antibody protection is independent of Fc-receptor functionality and complement activity. The antibody bound NP from several CCHFV strains and exhibited robust cross-protection against the heterologous CCHFV strain Afg09-2990. Our work demonstrates that the NP is a viable target for antibody-based therapeutics, providing another direction for developing immunotherapeutics against CCHFV.

Binding, recovery, and infectiousness of enveloped and non-enveloped viruses associated with plastic pollution in surface water.

Microplastics in wastewater and surface water rapidly become colonised by microbial biofilm. Such 'plastisphere' communities are hypothesised to persist longer and be disseminated further in the environment and may act as a vector for human pathogens, particularly as microplastics entering wastewater treatment plants are exposed to high concentrations of pathogenic bacteria. However, the potential for human viral pathogens to become associated with the plastisphere has never before been quantified. Here, we have used rotavirus (RV) SA11 (a non-enveloped enteric virus) and the enveloped bacteriophage Phi6 as model viruses to quantify binding and recovery from biofilm-colonised microplastic pellets in three different water treatments (filtered and non-filtered surface water, and surface water with added nutrients). Viruses associated with biofilm-colonised pellets were more stable compared to those remaining in the water. While infectious particles and genome copies of RV remained stable over the 48 h sampling period, Phi6 stability was highly impacted, with a reduction ranging from 2.18 to 3.94 log10. Virus particles were protected against inactivation factors when associated with the biofilm on microplastic surfaces, and when there was a high concentration of particulate matter in the liquid phase. Although our results suggest that the presence of an envelope may limit virus interaction with the plastisphere, the ability to recover both enveloped and non-enveloped infectious viruses from colonised microplastic pellets highlights an additional potential public health risk of surface waters becoming contaminated with microplastics, and subsequent human exposure to microplastics in the environment.

Sewage-associated plastic waste washed up on beaches can act as a reservoir for faecal bacteria, potential human pathogens, and genes for antimicrobial resistance.

Sewage-associated plastic wastes, such as wet wipes and cotton bud sticks, commonly wash up on beaches; however, it is unclear whether this represents a public health risk. In this study, sewage-associated plastic waste, and naturally occurring substrates (seaweed and sand), were collected from ten beaches along the Firth of Forth estuary (Scotland, UK) and analysed using selective media for the faecal indicator organisms (FIOs) E. coli and intestinal enterococci (IE), and potential human pathogens (Vibrio spp.). Minimum inhibitory concentration (MIC) analysis was used to determine antibiotic resistance in selected strains. FIOs and Vibrio were more often associated with wet wipes and cotton bud sticks than with seaweed, and there was evidence of resistance to several antibiotics. This work demonstrates that plastics associated with sewage pollution can facilitate the survival and dissemination of FIOs and Vibrio and thus, could present an as yet unquantified potential risk to human health at the beach.

Prevalence, distribution and environmental effects on faecal indicator bacteria and pathogens of concern in commercial shellfish production areas in a subtropical region of a developing country (Santa Catarina, Brazil).

This paper reviews recent literature on the abundance and distribution of faecal indicator bacteria and pathogens in shellfish production areas in the state of Santa Catarina, on the subtropical coast of Brazil. This state supplies > 95% of the national production of shellfish. Microbiological monitoring data were mapped using GIS and the results compared with those from other countries. Coastal human population is the main predictive parameter for faecal bacteria in the production areas. Temporal variations of the bacteria can also be predicted by solar radiation and rainfall. The prevalence of pathogens such as hepatitis A virus, human norovirus, Salmonella spp. and Vibrio spp. does not differ substantially from that in developed countries. The information reported here can be used to inform development of microbiological risk profiles for shellfish production areas.

Quantifying the importance of plastic pollution for the dissemination of human pathogens: The challenges of choosing an appropriate 'control' material.

Discarded plastic wastes in the environment are serious challenges for sustainable waste management and for the delivery of environmental and public health. Plastics in the environment become rapidly colonised by microbial biofilm, and importantly this so-called 'plastisphere' can also support, or even enrich human pathogens. The plastisphere provides a protective environment and could facilitate the increased survival, transport and dissemination of human pathogens and thus increase the likelihood of pathogens coming into contact with humans, e.g., through direct exposure at beaches or bathing waters. However, much of our understanding about the relative risks associated with human pathogens colonising environmental plastic pollution has been inferred from taxonomic identification of pathogens in the plastisphere, or laboratory experiments on the relative behaviour of plastics colonised by human pathogens. There is, therefore, a pressing need to understand whether plastics play a greater role in promoting the survival and dispersal of human pathogens within the environment compared to other substrates (either natural materials or other pollutants). In this paper, we consider all published studies that have detected human pathogenic bacteria on the surfaces of environmental plastic pollution and critically discuss the challenges of selecting an appropriate control material for plastisphere experiments. Whilst it is clear there is no 'perfect' control material for all plastisphere studies, understanding the context-specific role plastics play compared to other substrates for transferring human pathogens through the environment is important for quantifying the potential risk that colonised plastic pollution may have for environmental and public health.

Survival of human enteric and respiratory viruses on plastics in soil, freshwater, and marine environments.

The public health significance of plastics and microplastics in different environmental matrices has mainly focused on the toxicological effects of human ingestion. But these pollutants can also harbour pathogenic bacteria as the surfaces of plastics in the environment quickly become colonised by microbial biofilm. This novel microbial habitat has been termed the 'plastisphere' and could facilitate the survival and dissemination of important bacterial and fungal pathogens. Importantly, however, the role of plastic pollution as a secondary pathway for the transmission of human pathogenic viruses has never been addressed. Due to the high prevalence of both enteric and respiratory viruses in the population and in the environment, there is significant potential for human viruses to become associated with the plastisphere. In this review we critically evaluate current knowledge on the interaction of human enteric and respiratory viruses with plastic surfaces and identify the main environmental conditions and plastic characteristics that could affect virus survival and persistence in the environment. Our hypothesis is that the plastisphere can enhance the adhesion, survival and dissemination of human pathogenic viruses and potentially lead to more effective transfer and transmission of viral diseases within the environment. We identify key research questions needed to more fully assess the potential human health risks associated with viruses on plastic surfaces. These include understanding, (1) the mechanisms of viral attachment to either naked or biofilm-colonised plastic (2) how the structural characteristics of viruses (e.g., enveloped, or non-enveloped), affect their persistence in the plastisphere, (3) whether the plastisphere offers protection and increases the persistence of infectious viruses in soil, freshwater, and marine environments.

Presence of enteric viruses, bioaccumulation and stability in Anomalocardia brasiliana clams (Gmelin, 1791).

Bivalve mollusks are filter feeders and may accumulate human pathogens in their tissues. Many studies demonstrated human diseases associated with bivalve consumption, especially oysters. Anomalocardia brasiliana clams are distributed along the Brazilian coastal area and are an exotic ingredient for some typical dishes in Brazil. Even though there are several reports describing the contamination of oysters and mussels with human pathogens, there is a lack of studies reporting contamination of A. brasiliana with human pathogens. An evaluation of natural microbiological contamination in A. brasiliana samples over a period of 18months (November 2014 to April 2016) showed that the bacteria indices were in accordance with Brazilian regulations (E. coli<230MPN and Salmonella sp. absent in 25g of meat). However, the enteric viruses evaluated were detected throughout the analysis period, with the highest result for the hepatitis A virus (HAV); followed by Rotavirus-A (RVA); Human Adenovirus (HAdV) and Norovirus GI (NoV GI). The bioaccumulation of enteric viruses by A. brasiliana during a period of 24h was performed using NoV GI and GII, HAV, RVA and HAdV as models. Interestingly the mollusk demonstrated different uptake behaviors in relation to these viruses throughout the time period. NoV GI was the most adsorbed virus after 24h. HAV concentration was <1% at 3h, but it increased to <10% at 8h, remaining unchanged until 12h, and decreasing to <3% at 24h; HAdV reached its highest concentration at 12h, being released by the animals and lowering to <3% at 24h. RVA bioaccumulation was unstable over time, reaching its highest values after 24h (<5%); NoV GII bioaccumulation remained <1%. Thermal inactivation of HAdV-2 in A. brasiliana was also evaluated. After the usual gentle cooking procedure using different times (0, 1, 1.5, 3 and 5mins), viral infectivity was evaluated using ICC-et-RT-qPCR. The temperature inside the DT remained <80°C over time and after 5min of cooking the HAdV reached a decay of 90% (1 log10). The results showed a real warn to the consumers that can be exposed to infectious human viruses if they eat these clams improperly cooked. HAV was the most detected virus in these animals, which may lead to outbreaks. A. brasiliana exhibited distinct behavior in NoV GI bioaccumulation and persistence, pointing to the need for further studies about the cellular ligands used by these viruses to become attached to these clams.

Adenovirus and Norovirus Contaminants in Commercially Distributed Shellfish.

Shellfish complying with European Regulations based on quantification of fecal bacterial indicators (FIB) are introduced into markets; however, information on viruses, more stable than FIB, is not available in the literature. To assess the presence of noroviruses (NoVs) GI and GII and human adenoviruses (HAdV) in domestic and imported mussels and clams (n = 151) their presence was analyzed during winter seasons (2004-2008) in north-west Spanish markets through a routine surveillance system. All samples tested negative for NoV GI and 13 % were positive for NoV GII. The role of HAdV as viral indicator was evaluated in 20 negative and 10 positive NoV GII samples showing an estimated sensitivity and specificity of HAdV to predict the presence of NoV GII of 100 and 74 % (cut-off 0.5). The levels of HAdV and NoVs and the efficiency of decontamination in shellfish depuration plants (SDP) were evaluated analyzing pre- and post-depurated mussels collected in May-June 2010 from three different SDP. There were no statistically significant differences in the prevalence and quantification of HAdV between pre- and post-depurated shellfish and between seawater entering and leaving the depuration systems. Moreover, infectious HAdV were detected in depurated mussels. These results confirm previous studies showing that current controls and depuration treatments limiting the number of FIB do not guarantee the absence of viruses in shellfish.

Virus, protozoa and organic compounds decay in depurated oysters.

AIMS: (1) Evaluate the dynamic of the depuration process of Crassostrea gigas oysters using different ultraviolet doses with different amounts of contaminants (virus, protozoa and organic contaminants) and (2) investigate the morphological changes in the oysters' tissues produced by the depuration procedures. METHODS: The oysters were allocated in sites with different degrees of contamination and analyzed after 14 days. Some animals were used as positive controls by artificial bioaccumulation with HAdV2 and MNV1 and subjected to depuration assays using UV lamps (18 or 36 W) for 168 h. The following pollutants were researched in the naturally contaminated oysters, oysters after 14 days in sites and oysters during the depuration processes: virus (HAdV, HAV, HuNoV GI/GII and JCPyV), by (RT) qPCR; protozoa (Cryptosporidium and Giardia species), by immunomagnetic separation and immunofluorescence; and organic compounds (AHs, PAHs, LABs, PCBs and organochlorine pesticides-OCs), by chromatography. Changes in the oysters' tissues produced by the depuration processes were also evaluated using histochemical analysis by light microscopy. In the artificially bioaccumulated oysters, only HAdV2 and MNV1 were investigated by (RT) qPCR before the depuration procedures and after 96 and 168 h of these procedures. RESULTS: At 14 days post-allocation, HAdV was found in all the sites (6.2 × 105 to 4.4 × 107 GC g(-1)), and Giardia species in only one site. Levels of PCBs and OCs in the oyster's tissues were below the detection limit for all samples. AHs (3.5 to 4.4 µg g(-1)), PAHs (11 to 191 ng g(-1)) and LABs (57 to 751 ng g(-1)) were detected in the samples from 3 sites. During the depuration assays, we found HAdV, Giardia and Cryptosporidium species until 168 h, independent of UV treatment. AHs, PAHs and LABs were found also after 168 h of depuration (36 W and without UV lamp). The depuration procedures did not produce changes in the oysters' tissues. In the artificially contaminated and depurated oysters, we detected HAdV until 168 h and MNV1 until 96 h of depuration. CONCLUSION: The applied depuration treatments were unable to eliminate the protozoa or to degrade the HAdV genomes but were able to degrade the MNV1 genomes. Similarly, the UV water treatment was not efficient for aliphatic hydrocarbons, PAHs and LABs, as their concentrations were equivalent or higher to the concentrations of the control samples and samples from depuration tanks without UV treatment.

The depuration dynamics of oysters (Crassostrea gigas) artificially contaminated with hepatitis A virus and human adenovirus.

Within the country of Brazil, Santa Catarina is a major shellfish producer. Detection of viral contamination is an important step to ensure production quality and consumer safety during this process. In this study, we used a depuration system and ultraviolet (UV) disinfection to eliminate viral pathogens from artificially infected oysters and analysed the results. Specifically, the oysters were contaminated with hepatitis A virus (HAV) or human adenovirus type 5 (HAdV5). After viral infection, the oysters were placed into a depuration tank and harvested after 48, 72 and 96 h. After sampling, various oyster tissues were dissected and homogenised and the viruses were eluted with alkaline conditions and precipitated with polyethylene glycol. The oyster samples were evaluated by cell culture methods, as well as polymerase chain reaction (PCR) and quantitative-PCR. Moreover, at the end of the depuration period, the disinfected seawater was collected and analysed by PCR. The molecular assays showed that the HAdV5 genome was present in all of the depuration time samples, while the HAV genome was undetectable after 72 h of depuration. However, viral viability tests (integrated cell culture-PCR and immunofluorescence assay) indicated that both viruses were inactivated with 96 h of seawater recirculation. In conclusion, after 96 h of UV treatment, the depuration system studied in this work purified oysters that were artificially contaminated with HAdV5 and HAV.

The depuration dynamics of oysters (Crassostrea gigas) artificially contaminated with hepatitis A virus and human adenovirus

Within the country of Brazil, Santa Catarina is a major shellfish producer. Detection of viral contamination is an important step to ensure production quality and consumer safety during this process. In this study, we used a depuration system and ultraviolet (UV) disinfection to eliminate viral pathogens from artificially infected oysters and analysed the results. Specifically, the oysters were contaminated with hepatitis A virus (HAV) or human adenovirus type 5 (HAdV5). After viral infection, the oysters were placed into a depuration tank and harvested after 48, 72 and 96 h. After sampling, various oyster tissues were dissected and homogenised and the viruses were eluted with alkaline conditions and precipitated with polyethylene glycol. The oyster samples were evaluated by cell culture methods, as well as polymerase chain reaction (PCR) and quantitative-PCR. Moreover, at the end of the depuration period, the disinfected seawater was collected and analysed by PCR. The molecular assays showed that the HAdV5 genome was present in all of the depuration time samples, while the HAV genome was undetectable after 72 h of depuration. However, viral viability tests (integrated cell culture-PCR and immunofluorescence assay) indicated that both viruses were inactivated with 96 h of seawater recirculation. In conclusion, after 96 h of UV treatment, the depuration system studied in this work purified oysters that were artificially contaminated with HAdV5 and HAV.

Evaluation of tropical water sources and mollusks in southern Brazil using microbiological, biochemical, and chemical parameters.

Florianópolis, a city located in the Santa Catarina State in southern Brazil, is the national leading producer of bivalve mollusks. The quality of bivalve mollusks is closely related to the sanitary conditions of surrounding waters where they are cultivated. Presently, cultivation areas receive large amounts of effluents derived mainly from treated and non-treated domestic, rural, and urban sewage. This contributes to the contamination of mollusks with trace metals, pesticides, other organic compounds, and human pathogens such as viruses, bacteria, and protozoan. The aim of this study was to perform a thorough diagnosis of the shellfish growing areas in Florianópolis, on the coast of Santa Catarina. The contamination levels of seawater, sediments, and oysters were evaluated for their microbiological, biochemical, and chemical parameters at five sea sites in Florianópolis, namely three regular oyster cultivation areas (Sites 1, 2, and oyster supplier), a polluted site (Site 3), and a heavily polluted site (Site 4). Samples were evaluated at day zero and after 14 days. Seawater and sediment samples were collected just once, at the end of the experiment. Antioxidant defenses, which may occur in contaminated environments in response to the increased production of reactive oxygen species (ROS) by organisms, were analyzed in oysters, as well as organic compounds (in oysters and sediment samples) and microbiological contamination (in oysters and seawater samples). The results showed the presence of the following contaminants: fecal coliforms in seawater samples (four sites), human adenovirus (all sites), human noroviruses GI and GII (two sites), Hepatitis A viruses (one site), JC Polyomavirus in an oyster sample from the oyster supplier, Giardia duodenalis cysts, and Cryptosporidium sp oocysts (one site). Among organochlorine pesticides, only DDT (dichlorodiphenyltrichloroethane) and HCH (hexachlorocyclohexane) were detected in some sediment and oysters samples in very low levels; site 4 had the highest concentrations of total aliphatic hydrocarbons, PAHs, and linear alkylbenzenes (LABs) found either in oysters or in sediment samples. The major concentration of fecal sterol coprostanol was found at site 4, followed by site 3. After 14 days of allocation in the four selected sites, there was a significant difference in the enzymes analyzed at the monitored spots. The detection of different contaminants in oysters, seawater, and sediment samples in the present study shows the impact untreated or inadequately treated effluents have on coastal areas. These results highlight the need for public investment in adequate wastewater treatment and adequate treatment of oysters, ensuring safe areas for shellfish production as well as healthier bivalve mollusks for consumption.

Depuration dynamics of oysters (Crassostrea gigas) artificially contaminated by Salmonella enterica serovar Typhimurium.

The State of Santa Catarina produces the greatest quantity of edible mollusks in Brazil. To guarantee sanitary qualify, mollusk cultures should be monitored for contamination by pathogenic microorganisms. A self-purification or "depuration" system that eliminates Salmonella enterica serovar Typhimurium contamination from oysters has been developed and evaluated. The depuration process occurred within a closed system, in which 1000 L of water was recirculated for 24 h. The water was sterilized with ultraviolet (UV) light, chlorine, or both together. Oysters (Crassostrea gigas) artificially contaminated with S. typhimurium were harvested every 6 h. Samples of oyster tissue were excised and both the presence and numbers of bacteria were determined. Combined UV light and chlorine treatments resulted in total elimination of bacteria within 12 h. Polymerase chain reaction detected bacteria in water exposed to the three treatments. This pioneering study is the first of its kind in Brazil and represents a major contribution to commercial mollusk culture in this country.