LIMITED ACCUMULATION AND PERSISTENCE OF AN INFLUENZA A VIRUS IN TADPOLE SNAILS (PHYSA SPP.).

Waterfowl infected with avian influenza A viruses (IAVs) shed infectious virus into aquatic environments, providing a mechanism for transmission among waterfowl, while also exposing the entire aquatic ecosystem to the virus. Aquatic invertebrates such as freshwater snails are likely exposed to IAVs in the water column and sediment. Freshwater snails comprise a significant portion of some waterfowl species' diets, so this trophic interaction may serve as a novel route of IAV transmission. In these experiments, tadpole snails (Physa spp.) were exposed to a low-pathogenicity IAV (H3N8) to determine whether snails can accumulate the virus and, if so, how long virus persists in snail tissues. Snail tissues were destructively sampled and tested by reverse-transcription quantitative real-time PCR. Our experiments demonstrated that tadpole snails do accumulate IAV RNA in their tissues, although at low titers, for at least 96 h. These results indicate that it may be possible for IAV transmission to occur between waterfowl via ingestion of a natural invertebrate prey item; however, the time frame for transmission may be limited.

In situ hybridization revealed wide distribution of Haliotid herpesvirus 1 in infected small abalone, Haliotis diversicolor supertexta.

Ganglioneuritis was the primary pathologic change in infected abalone associated with Haliotid herpesvirus 1 (HaHV-1) infection, which eventually became known as abalone viral ganglioneuritis (AVG). However, the distribution of HaHV-1 in the other tissues and organs of infected abalone has not been systemically investigated. In the present study, the distribution of HaHV-1-CN2003 variant in different organs of small abalone, Haliotis diversicolor supertexta, collected at seven different time points post experimental infection, was investigated with histopathological examination and in situ hybridization (ISH) of HaHV-1 DNA. ISH signals were first observed in pedal ganglia at 48 h post injection, and were consistently observed in this tissue of challenged abalone. At the same time, increased cellularity accompanied by ISH signals was observed in some peripheral ganglia of mantle and kidney. At the end of infection period, lesions and co-localized ISH signals in infiltrated cells were detected occasionally in the mantle and hepatopancreas. Transmission electron microscope analysis revealed the presence of herpes-like viral particles in haemocyte nuclei of infected abalone. Our results indicated that, although HaHV-1-CN2003 was primarily neurotropic, it could infect other tissues including haemocytes.

RNA virome screening in diverse but ecologically related citrus pests reveals potential virus-host interactions.

As an evergreen ecosystem, citrus orchards have specialized pest species and stable ecological homeostasis; thus, they provide an ideal model for investigating RNA viromes in diverse but ecologically related species. For this purpose, we collected specialized citrus pests from three classes of invertebrates, Insecta, Arachnida, and Gastropoda and we constructed two kinds of libraries (RNA and small RNA) for the pests by deep sequencing. In total, six virus-derived sequences were identified, including four Picornavirales, one Jingchuvirales and one Nidovirales. The picornavirus-derived small RNAs showed significant small RNA peaks and symmetric distribution patterns along the genome, which suggests these viruses infected the hosts and triggered host antiviral immunity RNA interference. Screening of virus-derived sequences in multiple species of citrus pests (n = 10 per species) showed that Eotetranychus kankitus picorna-like virus and Tetranychus urticae mivirus may be present in multiple pests. Our investigation in citrus pests confirmed that RNA viruses revealed by metagenomics could impact host immunity (e.g. RNAi). An approach with parallel deep sequencing of RNAs and small RNAs is useful not only for viral discoveries but also for understanding virus-host interactions of ecologically related but divergent pest species.

Wuhan poty-like virus 1 is a possible member of the genus Macluravirus.

We have analysed the genome sequence of Wuhan poty-like virus 1 (WuPLV1), reported as an unclassified RNA virus in GenBank (Accession no: KX884573.1). Based on the polyprotein sequence identity (ranging from 55.2 to 71.1%), with classifiable members of the Macluravirus genus of the plant virus family Potyviridae, we suggest that WuPLV1 represents a possible new species of Macluravirus, although the virus was isolated from the Chinese land snail Mastigeulota kiangsinensis, which is not known to be a host or vector of macluraviruses.

Early evidence for a virus-like agent infecting the pest snail Theba pisana (Gastropoda: Pulmonata) in Southern Italy.

The Mediterranean land snail Theba pisana (Mollusca: Helicidae) is an introduced agricultural pest in many countries around the world, including Australia, Israel, USA and South Africa. In addition, this snail is an intermediate host of parasites of importance in both human and veterinary medicine. In this study, a natural population of T. pisana snails on the Domitian coast of Italy was surveyed following a mass mortality event. By light microscopy, 30% of the collected individuals showed in the calcium cells of the digestive gland the presence of hypertrophied nuclei containing eosinophilic to weakly basophilic inclusion bodies. Ultrastructural examination by transmission electron microscopy (TEM) showed nuclear inclusions constituted by a reticulated stroma into which unenveloped, roundish virus-like particles (38±4nm in diameter) were present. To the best of our knowledge this could be the first evidence for a virus-like agent infecting the gastropod T. pisana, which may open new biocontrol perspectives of the this pest worldwide.

Human adenovirus in tissues of freshwater snails living in contaminated waters.

Human adenovirus (HAdV) is resistant to environment and can be used as a marker to detect fecal contamination. Considering the importance of freshwater snails in the aquatic environment, their use as concentrators for HAdV is a complementary tool for viral analysis of water. The goal of the study was to detect HAdV in snails and surface water collected from wetlands of the Sinos River (Rio Grande do Sul, Brazil) basin and to compare rates and viral loads found in both samples. HAdV was detected through real-time PCR. Total and fecal coliforms were detected by Colilert® kit, and viral infectivity of positive samples of the DNA genome was performed in A549 human cell line. All wetlands presented bacterial and viral contamination, but no viral particle was considered viable. The wetland that showed lower fecal coliform mean was Campo Bom, and São Leopoldo (both cities in Rio Grande do Sul) was representative of the highest mean. HAdV was detected in water samples (53%), gastropods' hemolymph (31%) and tissues (16%). Wetlands proved to be environments already altered by human action. Water samples exhibited a higher frequency of HAdV detection; however, in some instances, the target viral genomes were only found in gastropod biological samples. This was a pioneer study in the use of freshwater snails for human enteric viral assessment thus demonstrating that the human organism can retain fecal contamination, complementing and assisting in microbiological water analyzes.

Detection and Genetic Analysis of Noroviruses and Sapoviruses in Sea Snail.

An outbreak of acute gastroenteritis occurred at a restaurant in Yokohama in December 2011. Because many of the customers had consumed raw sea snail, sea snail was suspected to be the source of this outbreak. To determine whether sea snail contains Norovirus (NoV) or Sapovirus (SaV), we analyzed 27 sea snail samples collected over 5 months (May, June, August, October, and December 2012) and 59.3% were positive for NoV and/or SaV. The levels of NoV ranged from 1.5 × 10(3) to 1.5 × 10(5) copies/g tissue, and those of SaV from 1.5 × 10(2) to 1.3 × 10(3) copies/g tissue. The highest levels were observed in sea snails collected in December. A phylogenetic analysis of the NoVs showed that the viral strains were NoV genotypes GI.4, GI.6, GII.4, GII.12, GII.13, and GII.14, and the SaV strains were genotypes GI.2 and GI.3. The NoV GII.4 Sydney 2012 variants were only detected in December. This variant was a major source of gastroenteritis in Japan in the winter of 2012/2013. In contrast, the NoV GII.4 strains detected in May and June 2012 were not the Sydney 2012 variant. This study demonstrates that sea snail contains multiple genogroups and genotypes of NoV and SaV strains. We conclude that the sea snail presents a risk of gastroenteritis when consumed raw.

Failure of transmission of low-pathogenic avian influenza virus between Mallards and freshwater snails: an experimental evaluation.

In aquatic bird populations, the ability of avian influenza (AI) viruses to remain infectious in water for extended periods provides a mechanism that allows viral transmission to occur long after shedding birds have left the area. However, this also exposes other aquatic organisms, including freshwater invertebrates, to AI viruses. Previous researchers found that AI viral RNA can be sequestered in snail tissues. Using an experimental approach, we determined whether freshwater snails (Physa acuta and Physa gyrina) can infect waterfowl with AI viruses by serving as a means of transmission between infected and naïve waterfowl via ingestion. In our first experiment, we exposed 20 Physa spp. snails to an AI virus (H3N8) and inoculated embryonated specific pathogen-free (SPF) chicken eggs with the homogenized snail tissues. Sequestered AI viruses remain infectious in snail tissues; 10% of the exposed snail tissues infected SPF eggs. In a second experiment, we exposed snails to water contaminated with feces of AI virus-inoculated Mallards (Anas platyrhynchos) to evaluate whether ingestion of exposed freshwater snails was an alternate route of AI virus transmission to waterfowl. None of the immunologically naïve Mallards developed an infection, indicating that transmission via ingestion likely did not occur. Our results suggest that this particular trophic interaction may not play an important role in the transmission of AI viruses in aquatic habitats.

Influence of elevated temperatures on the immune response of abalone, Haliotis rubra.

Elevated water temperature can act as a stressor impacting the immune responses of molluscs, potentially increasing their susceptibility to microbial infections. Abalone are commercially important marine molluscs that have recently experienced disease outbreaks caused by a herpesvirus and Vibrio bacteria. Sampling of wild-caught Haliotis rubra showed a significant correlation between water temperature and both antiviral and antibacterial activity, with higher activity in summer than in winter months. However, antibacterial activity was compromised in favour of antiviral activity as the water temperatures peaked in summer. A controlled laboratory experiment was then used to investigate several immune responses of H. rubra, including total haemocyte count (THC), stimulated superoxide anion production (SO), antiviral activity against a model herpesvirus, herpes simplex virus type 1 and antibacterial activity against a representative pathogenic bacterium, Vibrio anguillarum, over one week after raising water temperature from 18 to 21 or 24 °C. THC and SO increased at day 1 and then dropped back to control levels by days 3 and 7. By comparison, the humoural immune parameters showed a delayed response with antibacterial and antiviral activity significantly increasing on days 3 and 7, respectively. Consistent with the field study, antibacterial activity became significantly depressed after prolonged exposure to elevated temperatures. A principal components analysis on the combined immune parameters showed a negative correlation between antiviral and antibacterial activity. SO was positively correlated to THC and neither of these cellular parameters were correlated to the humoural antimicrobial activity. Overall, this study indicates that abalone may have more resilience to viruses than bacterial pathogens under conditions of elevated temperature, such as those predicted under future climate change scenarios.

Geographical features of estuaries for neritid gastropods including Clithon retropictus to preserve thermostable direct hemolysin-producing Vibrio parahaemolyticus.

Thermostable direct hemolysin-producing strain of Vibrio parahaemolyticus was not detected from the alimentary tract of 7 neritid gastropods including Clithon retropictus at 9 estuaries of Southwest Islands in Japan in the present study. The strain has been detected from C. retropictus at 2 estuaries facing The Sea of Japan but not at 2 estuaries facing The Seto Inland Sea and The Pacific Ocean in Western Japan in our previous studies. In comparison with geographical features of the estuaries where the strain was detected and not, thick accumulation of muddy sediments at the riverbed and stagnation of brackish water at low tide seem to be essential for the strain to survive in neritid gastropods including C. retropictus.