Simultaneous detection of multiple bacterial and viral aquatic pathogens using a fluorogenic loop-mediated isothermal amplification-based dual-sample microfluidic chip.

Rapid and user-friendly diagnostic tests are necessary for early diagnosis and immediate detection of diseases, particularly for on-site screening of pathogenic microorganisms in aquaculture. In this study, we developed a dual-sample microfluidic chip integrated with a real-time fluorogenic loop-mediated isothermal amplification assay (dual-sample on-chip LAMP) to simultaneously detect 10 pathogenic microorganisms, that is Aeromonas hydrophila, Edwardsiella tarda, Vibrio harveyi, V. alginolyticus, V. anguillarum, V. parahaemolyticus, V. vulnificus, infectious hypodermal and haematopoietic necrosis virus, infectious spleen and kidney necrosis virus, and white spot syndrome virus. This on-chip LAMP provided a nearly automated protocol that can analyse two samples simultaneously, and the tests achieved limits of detection (LOD) ranging from 100 to 10-1  pg/µl for genomic DNA of tested bacteria and 10-4 to 10-5  pg/µl for recombinant plasmid DNA of tested viruses, with run times averaging less than 30 min. The coefficient of variation for the time-to-positive value was less than 10%, reflecting a robust reproducibility. The clinical sensitivity and specificity were 93.52% and 85.53%, respectively, compared to conventional microbiological or clinical methods. The on-chip LAMP assay provides an effective dual-sample and multiple pathogen analysis, and thus would be applicable to on-site detection and routine monitoring of multiple pathogens in aquaculture.

A-to-I editing of Malacoherpesviridae RNAs supports the antiviral role of ADAR1 in mollusks.

BACKGROUND: Adenosine deaminase enzymes of the ADAR family are conserved in metazoans. They convert adenine into inosine in dsRNAs and thus alter both structural properties and the coding potential of their substrates. Acting on exogenous dsRNAs, ADAR1 exerts a pro- or anti-viral role in vertebrates and Drosophila. RESULTS: We traced 4 ADAR homologs in 14 lophotrochozoan genomes and we classified them into ADAD, ADAR1 or ADAR2, based on phylogenetic and structural analyses of the enzymatic domain. Using RNA-seq and quantitative real time PCR we demonstrated the upregulation of one ADAR1 homolog in the bivalve Crassostrea gigas and in the gastropod Haliotis diversicolor supertexta during Ostreid herpesvirus-1 or Haliotid herpesvirus-1 infection. Accordingly, we demonstrated an extensive ADAR-mediated editing of viral RNAs. Single nucleotide variation (SNV) profiles obtained by pairing RNA- and DNA-seq data from the viral infected individuals resulted to be mostly compatible with ADAR-mediated A-to-I editing (up to 97%). SNVs occurred at low frequency in genomic hotspots, denoted by the overlapping of viral genes encoded on opposite DNA strands. The SNV sites and their upstream neighbor nucleotide indicated the targeting of selected adenosines. The analysis of viral sequences suggested that, under the pressure of the ADAR editing, the two Malacoherpesviridae genomes have evolved to reduce the number of deamination targets. CONCLUSIONS: We report, for the first time, evidence of an extensive editing of Malacoherpesviridae RNAs attributable to host ADAR1 enzymes. The analysis of base neighbor preferences, structural features and expression profiles of molluscan ADAR1 supports the conservation of the enzyme function among metazoans and further suggested that ADAR1 exerts an antiviral role in mollusks.

L'impact des microvariants du virus herpétique ostreid herpesvirus 1 sur la culture d'huîtres creuses dans les hémisphères Nord et Sud depuis 2008.

Mollusc farming is the third most productive aquaculture activity in the world, and the Pacific oyster (Crassostrea gigas) is one of the most important farmed species. Since 2008, mass mortalities in C. gigas due to ostreid herpesvirus 1 microvariants have challenged the viability of this industry in Europe, New Zealand and Australia. Ten years after the emergence of this disease, there is evidence that the industry has become consolidated into fewer, larger companies, with the displacement of small farming enterprises and loss of employment in coastal communities. Rather than seeking technical solutions, the industry has turned to compensatory production strategies, such as increasing the number of spat placed on farms, higher market prices for table oysters and direct marketing, which appear to have allowed profitability. Biosecurity policies and responses to outbreaks, including those from within the industry, have had unintended consequences for hatcheries and farmers in areas free of disease, mainly caused by restrictions on animal movements, and have not prevented global spread. There may be opportunities for better coordination of industry and government responses to epizootic disease emergence in aquaculture. There is certainly a need for increased adoption of technical advances from research, once these solutions have been adequately verified.

L'élevage de mollusques occupe le troisième rang mondial parmi les activités de l'aquaculture en termes de production ; l'une des principales espèces élevées est l'huître creuse (Crassostrea gigas). Depuis 2008, la rentabilité des élevages de C. gigas en Europe, en Nouvelle-Zélande et en Australie a été fortement compromise par une mortalité massive due à des microvariants du virus herpétique Ostreid herpesvirus 1. Dix ans après l'émergence de cette maladie, on observe une forte concentration du secteur autour d'entreprises moins nombreuses mais de plus grande envergure qui ont remplacé l'ancien tissu d'exploitations artisanales et occasionné un déclin de l'emploi dans les communautés littorales. Au lieu de rechercher des solutions techniques, le secteur a eu recours à des stratégies de compensation axées sur la production, par exemple en augmentant le nombre de naissains mis en place dans les fermes, en augmentant le prix des huîtres de consommation ou en développant la vente directe, stratégies dont l'impact sur la rentabilité semble avoir été positif. En revanche, les mesures de biosécurité mises en place et les réponses apportées aux foyers, y compris celles introduites par le secteur lui-même ont eu des conséquences imprévues pour les écloseries et les éleveurs des zones indemnes de maladie, principalement en raison des restrictions imposées aux transferts d'animaux, sans pour autant prévenir la propagation de la maladie à l'échelle mondiale. Une meilleure coordination des réponses sectorielles et publiques face à l'émergence des maladies épizootiques affectant l'aquaculture devrait être possible. Il sera également indispensable de recourir davantage aux avancées techniques mises au point par la recherche dès que ces solutions auront été dûment validées.

La producción de moluscos es la tercera actividad acuícola más productiva del mundo, y la ostra japonesa (o del Pacífico) (Crassostrea gigas) ocupa un lugar destacado entre las principales especies cultivadas. Desde 2008, la viabilidad de esta industria en Europa, Nueva Zelanda y Australia está amenazada por episodios de mortandad masiva de C. gigas causados por microvariantes del herpesvirus de los ostreidos 1 (ostreid herpesvirus 1). Diez años después de la aparición de la enfermedad, lo que se observa es que la industria se ha ido concentrando en unas pocas empresas de grandes dimensiones, que han desplazado a las pequeñas empresas ostrícolas y causado la pérdida de numerosos empleos en las comunidades costeras. En lugar de buscar soluciones técnicas, la industria ha optado más bien por estrategias de producción compensatorias (como aumentar el número de semillas de ostra por explotación, subir los precios de mercado de las ostras de mesa o recurrir a la comercialización directa) que parecen haber deparado rentabilidad. Las políticas de seguridad biológica y la respuesta a los brotes, incluida la del propio sector, han tenido consecuencias imprevistas para los viveros y acuicultores situados en zonas libres de la enfermedad, debido sobre todo a las restricciones impuestas a los desplazamientos de animales, sin que ello haya servido para impedir la diseminación mundial de esta patología. Puede haber margen para coordinar más eficazmente las respectivas respuestas de la industria y de los poderes públicos ante la aparición de enfermedades epizoóticas en la acuicultura. Lo que sin ninguna duda es necesario es incorporar en mayor medida los adelantos técnicos resultantes de la investigación, una vez contrastada debidamente cada solución.

Low prevalence of Aichi virus in molluscan shellfish samples from Galicia (NW Spain).

AIMS: The aim of this study was to detect and quantify Aichi virus (AiV) in shellfish from three estuaries in Galicia, the main producer of molluscs in Europe. METHODS AND RESULTS: A total of 249 shellfish samples were analysed using a reverse transcription-quantitative PCR procedure. AiV was detected in 15 of 249 (6·02%) samples. Ría de Ares-Betanzos showed the highest prevalence (11·1%), followed by Ría do Burgo (3·7%) and Ría de Vigo, (2·56%). AiV quantifications ranged from nonquantifiable (under the limit of quantification of the method) to 6·9 × 103 RNAc per g DT, with a mean value of 1·9 × 102 RNAc per g DT. CONCLUSION: Results obtained indicated that the prevalence of this enteric virus in the studied area is considerably lower than those of other enteric viruses, such as Norovirus, Sapovirus, HAV or HEV. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first study that detects the presence of AiV in shellfish from authorized harvesting areas in Spain. Further studies with clinical samples are needed to determine the potential risk of AiV for human health in Galicia.

Viruses infecting marine molluscs.

Although a wide range of viruses have been reported in marine molluscs, most of these reports rely on ultrastructural examination and few of these viruses have been fully characterized. The lack of marine mollusc cell lines restricts virus isolation capacities and subsequent characterization works. Our current knowledge is mostly restricted to viruses affecting farmed species such as oysters Crassostrea gigas, abalone Haliotis diversicolor supertexta or the scallop Chlamys farreri. Molecular approaches which are needed to identify virus affiliation have been carried out for a small number of viruses, most of them belonging to the Herpesviridae and birnaviridae families. These last years, the use of New Generation Sequencing approach has allowed increasing the number of sequenced viral genomes and has improved our capacity to investigate the diversity of viruses infecting marine molluscs. This new information has in turn allowed designing more efficient diagnostic tools. Moreover, the development of experimental infection protocols has answered some questions regarding the pathogenesis of these viruses and their interactions with their hosts. Control and management of viral diseases in molluscs mostly involve active surveillance, implementation of effective bio security measures and development of breeding programs. However factors triggering pathogen development and the life cycle and status of the viruses outside their mollusc hosts still need further investigations.

Australian abalone (Haliotis laevigata, H. rubra and H. conicopora) are susceptible to infection by multiple abalone herpesvirus genotypes.

From 2006 to 2012, acute mortalities occurred in farmed and wild abalone (Haliotis spp.) along the coast of Victoria, Australia. The disease (abalone viral ganglioneuritis; AVG) is associated with infection by an abalone herpesvirus (AbHV). The relative pathogenicity of 5 known variants of AbHV was evaluated on abalone stocks from different states in Australia. Results indicated that all virus variants (Vic1, Tas1, Tas2, Tas3 and Tas4) cause disease and mortality in all abalone stocks tested (greenlip, blacklip and brownlip). In order to avoid further AVG outbreaks in Australian wild abalone, strict regulations on the transfer of abalone stocks must be implemented.

Antiviral immunity in marine molluscs.

Marine molluscs, like all living organisms, are constantly exposed to viruses and have evolved efficient antiviral defences. We review here recent developments in molluscan antiviral immunity against viruses belonging to the order Herpesvirales. Emerging results suggest an interferon-like response and autophagy are involved in the antiviral defence of bivalves to viral infection. Multi-functional plasma proteins from gastropods and bivalves have been identified to have broad-spectrum antiviral activity against mammalian viruses. The antiviral defences present in molluscs can be enhanced by genetic selection, as shown by the presence of oyster strains specifically resistant to ostreid herpesvirus type 1. Whether varying amounts or different isoforms of these antiviral plasma proteins contributes to genetic resistance is worthy of further research. Other evolutionarily conserved antiviral mechanisms, such as RNA interference and apoptosis, still need further characterization.

Epidemiology of foodborne norovirus outbreaks, United States, 2001-2008.

Noroviruses are the leading cause of foodborne illness in the United States. To better guide interventions, we analyzed 2,922 foodborne disease outbreaks for which norovirus was the suspected or confirmed cause, which had been reported to the Foodborne Disease Outbreak Surveillance System of the Centers for Disease Control and Prevention during 2001-2008. On average, 365 foodborne norovirus outbreaks were reported annually, resulting in an estimated 10,324 illnesses, 1,247 health care provider visits, 156 hospitalizations, and 1 death. In 364 outbreaks attributed to a single commodity, leafy vegetables (33%), fruits/nuts (16%), and mollusks (13%) were implicated most commonly. Infected food handlers were the source of 53% of outbreaks and may have contributed to 82% of outbreaks. Most foods were likely contaminated during preparation and service, except for mollusks, and occasionally, produce was contaminated during production and processing. Interventions to reduce the frequency of foodborne norovirus outbreaks should focus on food workers and production of produce and shellfish.

Passive immune-protection of small abalone against Vibrio alginolyticus infection by anti-Vibrio IgY-encapsulated feed.

Small abalone (Haliotis diversicolor supertexta) is a high value-added shellfish. It however has been suffering Vibrio alginolyticus infections, which cause mass death of small abalone and thus great economic losses, particularly in artificial aquaculture. In this study, we attempted to treat small abalone with anti-Vibrio IgY to elicit a passive immunity directly against V. alginolyticus infections. Anti-Vibrio IgY was alginate encapsulated in egg powders as feed, which may avoid antibody inactivation in the gastrointestinal tract of small abalone. The feed was tested for the stability of anti-Vibrio IgY in a gastrointestinal mimic environment. The result showed anti-Vibrio IgY retained activity as high as 90% after 4 h exposure to pancreatic enzymes. Addition of 0, 5 or 10% anti-Vibrio IgY-encapsulated egg powders into a basal diet to form abalone diet formulae. Small abalones fed with the anti-Vibrio IgY formulae showed a relatively high respiratory burst activity than those without anti-Vibrio IgY treatments. The survival rates of small abalones fed with 5 or 10% anti-Vibrio IgY egg powders were in the range of 65-70% 14 days post-V. alginolyticus challenge (1 x 106 c.f.u.), which was significantly higher than 0% of those fed without anti-Vibrio IgY. The anti-Vibrio IgY-encapsulated formulae were thus concluded to be an effective means to prevent small abalone from V. alginolyticus infection, and may be practical in use in abalone aquaculture.

Norovirus, hepatitis A virus and enterovirus presence in shellfish from high quality harvesting areas in Portugal.

This is the first report on the screening of shellfish from Portugal for the presence of human enteropathogenic viruses. Approximately 2000 shellfish (Curbicula fluminea, Ruditapes decussatus, Tellina crassa, Spisula solida, Dosinia exoleta, Ensis spp., Mytilus spp., Ostrea edulis and Cerastoderma edule), organized in 49 batches, were collected between March 2008 and February 2009. They were tested for norovirus (NoV), hepatitis A virus (HAV) and enterovirus (EV) by RT-PCR followed by nucleotide sequencing. Bacterial contamination was also evaluated by Escherichia coli counts. Viral contamination was detected throughout the year in all shellfish species and in all collection areas, independently of their harvesting areas classification. Overall, 67% of all analyzed batches were contaminated by at least one of the studied viruses while the simultaneous presence of two and three viruses was detected in 22% and 6% batches, respectively. Of the three viruses, NoV was detected in 37% of the batches, followed by EV in 35%, and HAV in 33%. Nucleotide sequencing of the NoV and HAV RT-PCR products demonstrated that all strains belonged to NoV genotype GII.4 and HAV subgenotype 1B. The presence of NoV and HAV in shellfish from "A class" harvesting areas of Portugal can represent a potential health risk.

[Distribution and detection of pathogens in shellfish--a review].

Shellfish is one of important vehicles for dissemination of food-borne pathogens. The incidence of food-borne diseases increases every year. Therefore, monitoring and control on the food safety of shellfish is a significant public health concern worldwide. In recent years, our group has studied the pathogens in molecular detection, bioaccumulation and control in shellfish. Based on the our previous studies, the purpose of this article was to provide a review on the pathogens in shellfish in four aspects: the detection methods, distribution, depuration and epidemiology. The molecular methods were widely used in detection of pathogens in shellfish. In addition, the pathogens were bio-accumulated in the gills and digestive glands, including stomach and digestive diverticula, which are good candidate sites for detection of pathogens.

Special Issue "Emerging Viruses in Aquaculture".

According to the 2018 FAO report on aquaculture, there are 598 species of finfish, molluscs, crustaceans, and other organisms used in aquafarming around the world [...].

Susceptibility of Four Abalone Species, Haliotis gigantea, Haliotis discus discus, Haliotis discus hannai and Haliotis diversicolor, to Abalone asfa-like Virus.

Abalone amyotrophia is a viral disease that causes mass mortality of juvenile Haliotis discus and H. madaka. Although the cause of this disease has yet to be identified, we had previously postulated a novel virus with partial genome sequence similarity to that of African swine fever virus is the causative agent and proposed abalone asfa-like virus (AbALV) as a provisional name. In this study, three species of juvenile abalone (H. gigantea, H. discus discus, and H. diversicolor) and four species of adult abalone (the above three species plus H. discus hannai) were experimentally infected, and their susceptibility to AbALV was investigated by recording mortality, quantitatively determining viral load by PCR, and conducting immunohistological studies. In the infection test using 7-month-old animals, H. gigantea, which was previously reported to be insusceptible to the disease, showed multiplication of the virus to the same extent as in H. discus discus, resulting in mass mortality. H. discus discus at 7 months old showed abnormal cell masses, notches in the edge of the shell and brown pigmentation inside of the shell, which are histopathological and external features of this disease, while H. gigantea did not show any of these characteristics despite suffering high mortality. Adult abalones had low mortality and viral replication in all species; however, all three species, except H. diversicolor, became carriers of the virus. In immunohistological observations, cells positive for viral antigens were detected predominantly in the gills of juvenile H. discus discus and H. gigantea, and mass mortality was observed in these species. In H. diversicolor, neither juvenile nor adult mortality from infection occurred, and the AbALV genome was not increased by experimental infection through cohabitation or injection. Our results suggest that H. gigantea, H. discus discus and H. discus hannai are susceptible to AbALV, while H. diversicolor is not. These results confirmed that AbALV is the etiological agent of abalone amyotrophia.

The primitive interferon-like system and its antiviral function in molluscs.

The phylum mollusca is a very important group in the animal kingdom for the large number and diversified species. Recently, interest in molluscan immunity has increased due to their phylogenetic position and importance in worldwide aquaculture and aquatic environment. As the main aquaculture animal, most molluscs live in the water environment and they have to cope with many pathogen challenges, in which virus is one of the primary causes for the mass mortality. In vertebrates, interferon (IFN) system is generally recognized as the first line of defence against viral infection, while the antiviral mechanisms in molluscs remain to be clearly illuminated. Recently, some IFN-like proteins and IFN-related components have been characterized from molluscs, such as pattern recognition receptors (PRRs), interferon regulatory factors (IRFs), IFN-like receptors, JAK/STAT and IFN-stimulated genes (ISGs), which reinforce the existence of IFN-like system in molluscs. This system can be activated by virus or poly (I:C) challenges and further regulate the antiviral response of haemocytes in molluscs. This review summarizes the research progresses of IFN-like system in molluscs with the emphases on the uniformity and heterogeneity of IFN-like system of molluscs compared to that of other animals, which will be helpful for elucidating the antiviral modulation in molluscs and understanding the origin and evolution of IFN system.

Development and validation of a TaqMan PCR assay for the Australian abalone herpes-like virus.

The recent emergence of a herpes-like virus in both farmed and wild populations of abalone in Victoria, Australia, has been associated with high mortality rates in animals of all ages. Based on viral genome sequence information, a virus-specific real-time TaqMan assay was developed for detection and identification of the abalone herpes-like virus (AbHV). The assay was shown to be specific as it did not detect other viruses from either the Herpesvirales or the Iridovirales orders which have genome sequence similarities. However, the TaqMan assay was able to detect DNA from the Taiwanese abalone herpes-like virus, suggesting a relationship between the Taiwanese and Australian viruses. In addition, the assay detected < 300 copies of recombinant plasmid DNA per reaction. Performance characteristics for the AbHV TaqMan assay were established using 1673 samples from different abalone populations in Victoria and Tasmania. The highest diagnostic sensitivity and specificity were 96.7 (95% CI: 82.7 to 99.4) and 99.7 (95% CI: 99.3 to 99.9), respectively, at a threshold cycle (C(T)) value of 35.8. The results from 2 separate laboratories indicated good repeatability and reproducibility. This molecular assay has already proven useful in confirming presumptive diagnosis (based on the presence of ganglioneuritis) of diseased abalone in Victorian waters as well as being a tool for surveillance of wild abalone stocks in other parts of Australia.

Norovirus in retail shellfish.

Norovirus is a common cause of gastroenteritis outbreaks associated with consumption of raw shellfish. The majority of norovirus infections worldwide are due to genogroup II noroviruses. Bivalve molluscs (mussels, clams and oysters) at the end of the commercial chain, the points of purchase, were sampled between 2005 and 2008 in several retail points in Apulia, Italy, and screened by a semi-nested RT-PCR specific for genogroup II noroviruses. Noroviral RNA was detected in 12.1% of the samples, with lower frequency being observed in samples obtained from hypermarkets (8.1%) rather than in samples from open-air markets and fish shops (17.6% and 16.2%, respectively). By sequence analysis, the strains were characterized as norovirus variants GII.4/2004 and GII.b/Hilversum, which were both circulating in Italy in the same time-span.

Assessment of human enteric viruses in cultured and wild bivalve molluscs.

Standard and real-time reverse transcription-PCR (rRT-PCR) procedures were used to monitor cultured and wild bivalve molluscs from the Ría de Vigo (NW Spain) for the main human enteric RNA viruses, specifically, norovirus (NoV), hepatitis A virus (HAV), astrovirus (AsV), rotavirus (RT), enterovirus (EV), and Aichi virus (AiV). The results showed the presence of at least one enteric virus in 63.4% of the 41 samples analyzed. NoV GII was the most prevalent virus, detected in 53.7% of the samples, while NoV GI, AsV, EV, and RV were found at lower percentages (7.3, 12.2, 12.2, and 4.9%, respectively). In general, samples obtained in the wild were more frequently contaminated than those from cultured (70.6 vs. 58.3%) molluscs and were more readily contaminated with more than one virus. However, NoV GI was detected in similar amounts in cultured and wild samples (6.4 x 10(2) to 3.3 x 10(3) RNA copies per gram of digestive tissue) while the concentrations of NoV GII were higher in cultured (from 5.6 x 10(1) to 1.5 x 10(4) RNA copies per gram of digestive tissue) than in wild (from 1.3 x 10(2) to 3.4 x 10(4) RNA copies per gram of digestive tissue) samples.

Characterization of a nucleus located mollusc mitoferrin and its response to OsHV-1 infection.

Mitoferrin genes as members of SLC25 family are conservatively existed across species, mainly locate on mitochondria and serve an important role in the regulation of whole cellular iron metabolism. Available iron withholding from pathogens presents an important host defense strategy, while the regulation role of mitoferrin against invading pathogens is largely unknown. In this study, a unique mollusc mitoferrin gene was identified in ark clams, named SbmiFn, that showed conserved three-dimensional structure with other mitoferrins, and its iron binding activity was verified by iron chelating assay. Besides cytoplasmic distribution, colocalization between SbmiFn and nuclei was observed by immunohistochemistry assay. Moreover, the response of SbmiFn to viral pathogen OsHV-1 was investigated. The results showed that nucleus located signal of SbmiFn was enhanced, the expressions of SbmiFn and ferritin were coordinately decreased, which might assist host against OsHV-1 replication as the increase of OsHV-1 copies were hardly detected after that. These results refreshed our knowledge on the sequence, structure and functional characteristics of mitoferrin subfamily, and would contribute to further comparative studies on iron metabolism.

Prevalence of human pathogenic enteric viruses in bivalve molluscan shellfish and cultured shrimp in south west coast of India.

The prevalence of human enteric viruses in bivalve molluscan shellfish and shrimp collected off the south west coast of India was studied to assess the extent of fecal pollution of coastal environment. Out of 194 samples analyzed, 37% of oyster, 46% of clam and 15% of shrimp samples were positive for enteroviruses (EV). Adenoviruses (ADV) were detected in 17% of oyster and 27% of clam samples. However, other enteric viruses such as noroviruses (NoV) and hepatitis A virus (HAV) were not detected in any of the samples. High prevalence of EV and ADV was noticed between May to December. Thirty four percent of oyster and 49% of clam samples showed fecal coliform values higher than the limit. MS-2 phage was detected in 57% of oyster and 73% of clam samples. The presence of MS-2 phage and human enteric viruses showed association while fecal coliforms and enteric viruses showed no association. However, 17 samples, which were positive for enteric viruses (EV and ADV), were negative for MS-2 phage.

Evaluation of RT-PCR and reverse line blot hybridization for detection and genotyping F+ RNA coliphages from estuarine waters and molluscan shellfish.

AIMS: To evaluate a PCR-based detection and typing method for faecal indicator viruses (F+ RNA coliphages) in water and shellfish, and apply the method for better understanding of the ecology and microbial source tracking potential of these viruses. METHODS AND RESULTS: Water and shellfish samples were collected over 3 years at nine estuaries in the East, West and Gulf Coasts of the USA, providing 1033 F+ RNA coliphage isolates. F+ RNA coliphage genotyping rates by reverse transcriptase-PCR-reverse line blot (RLB) hybridization ranged from 94.7% to 100% among estuaries, and were not significantly different in oysters, clams, mussels or water (P = 0.8427). Twenty samples negative by RLB were nucleotide sequenced for confirmation, and to refine RLB probes. More F+ RNA coliphages were genotyped from colder water than warmer waters, while the water salinity did not affect F+ RNA coliphage levels. CONCLUSIONS: RT-PCR-RLB was a robust method for detecting and genotyping F+ RNA coliphages from diverse coastal areas, which provided new information on the ecology of F+ RNA coliphages. SIGNIFICANCE AND IMPACT OF THE STUDY: This performance-validated F+ RNA coliphage method can be used for faecal indicator monitoring and microbial source tracking, to protect recreational bathers and shellfish consumers from exposure to pathogenic virus and their disease risks.