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Hepeviruses of aquatic organisms

Originally reported in California, the cutthroat trout virus (CTV) has now been isolated from nine species of salmonids in North America. Early work focused on the replication and physical characteristics of the virus, but 20 years later was determined to be most closely related to the hepatitis E virus. The small genome is positive-sense, single-stranded RNA similar to other members of the family Hepeviridae , which now contains its own genus Piscihepevirus with two distinct genotypes, CTV-1 and CTV-2. While CTV has not been associated with acute disease in fish, the virus could form persistently infected cell cultures that may aid research in treating hepatitis E-like viruses affecting humans or other animals. Interestingly, trout exposed to CTV were protected for about a month against subsequent exposure to the infectious hematopoietic necrosis virus. Replicating agents suspected to be CTV can be confirmed by polymerase chain reaction (PCR), quantitative PCR, and sequencing. Other unclassified hepeviruses detected in fish using viral metagenomics include Wenling fish hepevirus, Wenling moray eel hepevirus, Murray–Darling carp hepevirus, and eastern mosquitofish hepevirus. The family Hepeviridae has been placed in the order Hepevirales together with the family Matonaviridae (rubella virus), with member viruses having amino acid homology in the helicase and replicase regions of the nonstructural proteins. In addition, using next-generation sequencing, a hepe-like sequence was characterized in diseased giant freshwater prawn Macrobrachium rosenbergii and named Crustacea hepe-like virus 1. Thus the family Hepeviridae continues to expand among aquatic animal hosts.

Book chapter↗

Ammocoetes of Pacific lamprey are not susceptible to common fish rhabdoviruses of the U.S. Pacific Northwest

Pacific Lampreys Entosphenus tridentatus have experienced severe population declines in recent years and efforts to develop captive rearing programs are under consideration. However, there is limited knowledge of their life history, ecology, and potential to harbor or transmit pathogens that may cause infectious disease. As a measure of the possible risks associated with introducing wild lampreys into existing fish culture facilities, larval lampreys (ammocoetes) were tested for susceptibility to infection and mortality caused by experimental exposures to the fish rhabdovirus pathogens: infectious hematopoietic necrosis virus (IHNV) and viral haemorrhagic septicaemia virus (VHSV). Two IHNV isolates, representing the U and M genogroups, and one VHSV isolate from the IVa genotype were each delivered to groups of ammocoetes by immersion at moderate and high viral doses, and by intraperitoneal injection. Ammocoetes were then held in triplicate tanks with no substrate or sediment. During 41 d of observation postchallenge there was low or no mortality in all groups, and no virus was detected in the small number of fish that died. Ammocoetes sampled for incidence of infection at 6 and 12 d after immersion challenges also had no detectable virus, and no virus was detected in surviving fish from any group. A small number of ammocoetes sampled 6 d after the injection challenge had detectable virus, but at levels below the original quantity of virus injected. Overall there was no evidence of infection, replication, or persistence of any of the viruses in any of the treatment groups. Our results suggest that Pacific Lampreys are highly unlikely to serve as hosts that maintain or transmit these viruses.

Journal of Aquatic Animal Health↗

Cyclic avian mass mortality in the northeastern United States is associated with a novel orthomyxovirus

Since 1998, cyclic mortality events in common eiders ( Somateria mollissima ), numbering in the hundreds to thousands of dead birds, have been documented along the coast of Cape Cod, Massachusetts, USA. Although longitudinal disease investigations have uncovered potential contributing factors responsible for these outbreaks, detecting a primary etiological agent has proven enigmatic. Here we identify a novel orthomyxovirus, tentatively named Wellfleet Bay virus (WFBV), as a potential causative agent of these outbreaks. Genomic analysis of WFBV revealed that it is most closely related to members of the Quaranjavirus genus within the family Orthomyxoviridae . Similar to other members of the genus, WFBV contains an alphabaculovirus gp64-like glycoprotein, which was demonstrated to have fusion activity, and also tentatively suggests that ticks (and/or insects) may vector the virus in nature. However, in addition to the six RNA segments encoding the prototypical structural proteins identified in other quaranjaviruses, a previously unknown RNA segment (segment 7) encoding a novel protein designated as VP7 was discovered in WFBV. Although WFBV shows low to moderate levels of sequence similarity to Quaranfil virus and Johnston Atoll virus , the original members of the Quaranjavirus genus, additional antigenic and genetic analyses demonstrated that it is closely related to the recently identified Cygnet River virus (CyRV) from South Australia, suggesting that WFBV and CyRV may be geographic variants of the same virus. Although the identification of WFBV in part may resolve the enigma of these mass mortality events, the details of the ecology and epidemiology of the virus remain to be determined. Importance The emergence or reemergence of viral pathogens resulting in large-scale outbreaks of disease in humans and/or animals is one of the most important challenges facing biomedicine. For example, understanding how orthomyxoviruses such as novel influenza A virus reassortants and/or mutants emerge to cause epidemic or pandemic disease is at the forefront of current global health concerns. Here we describe the emergence of a novel orthomyxovirus, Wellfleet Bay virus (WFBV), which has been associated with cyclic large-scale bird die-offs in the northeastern United States. This initial characterization study provides a foundation for further research into the evolution, epidemiology, and ecology of newly emerging orthomyxoviruses, such as WFBV, and their potential impacts on animal and/or human health.

Massachusets↗

Structural proteins of two salmonid rhabdoviruses

Purified infectious hematopoietic necrosis (IHN) virus and the virus of haemorrhagic septicaemia (VHS) (Egtved virus) each contain five structural proteins which were designated L, G, N, M-1, and M-2. The IHN viral polypeptides have molecular weights estimated to be 157,000, 72,000, 40,000, 25,000 and 20,000, respectively, whereas those of VHS viral polypeptides are estimated to be 157,000 74,000, 41,000, 21,500, and 19,000, respectively. The carbohydrate composition of the glycoprotein (G) was confirmed by demonstrating selective incorporation of [3H]glucosamine into the designated G protein of both viruses. Phosphoproteins were identified by incorporation of [32P]orthophosphate into the N and M-1 proteins of IHN virus and into the N protein of VHS virus. The glycoprotein of each virus was selectively solubilized by treatment with Triton X-100 in low salt buffer, whereas the M-1, and M-2 proteins along with the G protein were solubilized by Ttition X-100 in 0.43 M NaCl. The protein composition of the salmonid rhabdoviruses resembles that of the rabies virus group more closely than the vesicular stomatitis virus group.

Journal of Virology↗

Rabies outbreak in captive big brown bats (Eptesicus fuscus) used in white-nose syndrome vaccine trial

An outbreak of rabies occurred in a captive colony of wild-caught big brown bats ( Eptesicus fuscus ). Five of 27 bats exhibited signs of rabies virus infection 22–51 d after capture or 18–22 d after contact with the index case. Rabid bats showed weight loss, aggression, increased vocalization, hypersalivation, and refusal of food. Antigenic typing and virus sequencing confirmed that all five bats were infected with an identical rabies virus variant that circulates in E. fuscus in the United States. Two bats with no signs of rabies virus infection were seropositive for rabies virus-neutralizing antibodies; the brains of these bats had no detectable viral proteins by the direct fluorescence antibody test. We suspect bat-to-bat transmission of rabies virus occurred among our bats because all rabies-infected bats were confined to the cage housing the index case and were infected with viruses having identical sequences of the entire rabies nucleoprotein gene. This outbreak illustrated the risk of rabies virus infection in captive bats and highlights the need for researchers using bats to assume that all wild bats could be infected with rabies virus.

Journal of Wildlife Diseases↗

Efficacy of commercial canarypox vaccine for protecting Hawai'i 'Amakihi from field isolates of Avipoxvirus

At least three variants of avian pox virus are present in Hawai‘i - Fowlpox from domestic poultry and a group of genetically distinct viruses that cluster within two clades (Pox Variant 1 and Pox Variant 2) that are most similar to Canarypox based on DNA sequence of the virus 4b core protein gene. We tested whether Hawai‘i ‘Amakihi can be protected from wild virus isolates with an attenuated live Canarypox vaccine that is closely related to isolates that cluster within clade 1 (Pox Variant 1) based on sequence of the attenuated Canarypox virus 4b core protein. Thirty-one (31) Hawai`i ‘Amakihi ( Hemignathus virens ) with no prior physical evidence of pox infection were collected on Mauna Kea from xeric, high elevation habitats with low pox prevalence and randomly divided into two groups. One group of 16 was vaccinated with Poximmune C® while the other group received a sham vaccination with virus diluent. Four of 15 (27%) vaccinated birds developed potentially life-threatening disseminated lesions or lesions of unusually long duration, while one bird never developed a vaccine-associated lesion or "take". After vaccine-associated lesions healed, vaccinated birds were randomly divided into three groups of five and challenged with either a wild isolate of Fowlpox, a Hawai`i `Amakihi isolate of a Canarypox-like virus from clade 1 (Pox Variant 1) or a Hawai`i `Amakihi isolate of a Canarypox-like virus from clade 2 (Pox Variant 2). Similarly, three random groups of five unvaccinated ‘Amakihi were challenged with the same virus isolates. Vaccinated and unvaccinated ‘Amakihi challenged with Fowlpox had transient infections with no clinical signs of infection. Mortality in vaccinated ‘Amakihi that were challenged with Pox Variant 1 and Pox Variant 2 ranged from 0% (0/5) for Pox Variant 1 to 60% (3/5) for Pox Variant 2. Mortality in unvaccinated ‘Amakihi ranged from 40% (2/5) for Pox Variant 1 to 100% (5/5) for Pox Variant 2. While the vaccine provided some protection against Pox Variant 1, serious side effects and low efficacy against Pox Variant 2 make it risky to use in captive or wild honeycreepers.

Hawai'i↗

Efficacy of a commercial canarypox vaccine for protecting Hawai'i 'Amakihi from field isolates of avipoxvirus

At least three variants of avian pox virus are present in Hawai’i - Fowlpox from domestic poultry and a group of genetically distinct viruses that cluster within two clades (Pox Variant 1 and Pox Variant 2) that are most similar to Canarypox based on DNA sequence of the virus 4b core protein gene. We tested whether Hawai’i ‘Amakihi can be protected from wild virus isolates with an attenuated live Canarypox vaccine that is closely related to isolates that cluster within clade 1 (Pox Variant 1) based on sequence of the attenuated Canarypox virus 4b core protein. Thirty-one (31) Hawai`i ‘Amakihi ( Hemignathus virens ) with no prior physical evidence of pox infection were collected on Mauna Kea from xeric, high elevation habitats with low pox prevalence and randomly divided into two groups. One group of 16 was vaccinated with Poximmune C® while the other group received a sham vaccination with virus diluent. Four of 15 (27%) vaccinated birds developed potentially life-threatening disseminated lesions or lesions of unusually long duration, while one bird never developed a vaccine-associated lesion or “take”. After vaccine-associated lesions healed, vaccinated birds were randomly divided into three groups of five and challenged with either a wild isolate of Fowlpox, a Hawai`i `Amakihi isolate of a Canarypox-like virus from clade 1 (Pox Variant 1) or a Hawai`i `Amakihi isolate of a Canarypox-like virus from clade 2 (Pox Variant 2). Similarly, three random groups of five unvaccinated ‘Amakihi were challenged with the same virus isolates. Vaccinated and unvaccinated ‘Amakihi challenged with Fowlpox had transient infections with no clinical signs of infection. Mortality in vaccinated ‘Amakihi that were challenged with Pox Variant 1 and Pox Variant 2 ranged from 0% (0/5) for Pox Variant 1 to 60% (3/5) for Pox Variant 2. Mortality in unvaccinated ‘Amakihi ranged from 40% (2/5) for Pox Variant 1 to 100% (5/5) for Pox Variant 2. While the vaccine provided some protection against Pox Variant 1, serious side effects and low efficacy against Pox Variant 2 make it risky to use in captive or wild honeycreepers.

Hawaii↗

Virion RNA polymerases of two salmonid rhabdoviruses

RNA-dependent RNA polymerases were found to be associated with two salmonid rhabdoviruses: infectious hematopoietic necrosis (IHN) virus and the virus of hemorrhagic septicemia (VHS). The protein composition of these rhabdoviruses closely resembles that of rabies virus rather than that of vesicular stomatitis virus (McAllister and Wagner, 1975). The optimal temperature for in vitro transcription was found to be approximately 18 degrees C for IHN virus and approximately 15 degrees for VHS, closely approximating optimal temperatures for growth of these viruses in salmonid cells. Unlike vesicular stomatitis virus, manganese ion (1 mM) could be used as a divalent cation substitute for magnesium ion (5 mM). The in vitro transcription products of IHN and VHS viruses hybridized completely to the homologous genome but not at all to the heterologous genome.

Journal of Virology↗

A point mutation in the polymerase protein PB2 allows a reassortant H9N2 influenza isolate of wild-bird origin to replicate in human cells.

H9N2 influenza A viruses are on the list of potentially pandemic subtypes. Therefore, it is important to understand how genomic reassortment and genetic polymorphisms affect phenotypes of H9N2 viruses circulating in the wild bird reservoir. A comparative genetic analysis of North American H9N2 isolates of wild bird origin identified a naturally occurring reassortant virus containing gene segments derived from both North American and Eurasian lineage ancestors. The PB2 segment of this virus encodes 10 amino acid changes that distinguish it from other H9 strains circulating in North America. G590S, one of the 10 amino acid substitutions observed, was present in ~ 12% of H9 viruses worldwide. This mutation combined with R591 has been reported as a marker of pathogenicity for human pandemic 2009 H1N1 viruses. Screening by polymerase reporter assay of all the natural polymorphisms at these two positions identified G590/K591 and S590/K591 as the most active, with the highest polymerase activity recorded for the SK polymorphism. Rescued viruses containing these two polymorphic combinations replicated more efficiently in MDCK cells and they were the only ones tested that were capable of establishing productive infection in NHBE cells. A global analysis of all PB2 sequences identified the K591 signature in six viral HA/NA subtypes isolated from several hosts in seven geographic locations. Interestingly, introducing the K591 mutation into the PB2 of a human-adapted H3N2 virus did not affect its polymerase activity. Our findings demonstrate that a single point mutation in the PB2 of a low pathogenic H9N2 isolate could have a significant effect on viral phenotype and increase its propensity to infect mammals. However, this effect is not universal, warranting caution in interpreting point mutations without considering protein sequence context.

Infection, Genetics and Evolution↗

Hydrologic, land cover, and seasonal patterns of waterborne pathogens in Great Lakes tributaries

Great Lakes tributaries are known to deliver waterborne pathogens from a host of sources. To examine the hydrologic, land cover, and seasonal patterns of waterborne pathogens (i.e. protozoa (2), pathogenic bacteria (4) human viruses, (8) and bovine viruses (8)) eight rivers were monitored in the Great Lakes Basin over 29 months from February 2011 to June 2013. Sampling locations represented a wide variety of land cover classes from urban to agriculture to forest. A custom automated pathogen sampler was deployed at eight sampling locations which provided unattended, flow-weighted, large-volume (120–1630 L) sampling. Human and bovine viruses and pathogenic bacteria were detected by real-time qPCR in 16%, 14%, and 1.4% of 290 samples collected while protozoa were never detected. The most frequently detected pathogens were: bovine polyomavirus (11%), and human adenovirus C, D, F (9%). Human and bovine viruses were present in 16.9% and 14.8% of runoff-event samples (n = 189) resulting from precipitation and snowmelt, and 13.9% and 12.9% of low-flow samples (n = 101), respectively, indicating multiple delivery mechanisms could be influential. Data indicated human and bovine virus prevalence was different depending on land cover within the watershed. Occurrence, concentration, and flux of human viruses were greatest in samples from the three sampling locations with greater than 25% urban influence than those with less than 25% urban influence. Similarly, occurrence, concentration, and flux of bovine viruses were greatest in samples from the two sampling locations with greater than 50 cattle/km 2 than those with less than 50 cattle/km 2 . In seasonal analysis, human and bovine viruses occurred more frequently in spring and winter seasons than during the fall and summer. Concentration, occurrence, and flux in the context of hydrologic condition, seasonality, and land use must be considered for each watershed individually to develop effective watershed management strategies for pathogen reduction.

Great Lakes tributaries↗

Spring viremia of carp

pring viremia of carp (SVC) is an important disease affecting cyprinids, mainly common carp Cyprinus carpio . The disease is widespread in European carp culture, where it causes significant morbidity and mortality. Designated a notifiable disease by the Office International des Epizooties, SVC is caused by a rhabdovirus, spring viremia of carp virus (SVCV). Affected fish show destruction of tissues in the kidney, spleen and liver, leading to hemorrhage, loss of water-salt balance and impairment of immune response. High mortality occurs at water temperatures of 10 to 17°C, typically in spring. At higher temperatures, infected carp develop humoral antibodies that can neutralize the spread of virus and such carp are protected against re-infection by solid immunity. The virus is shed mostly with the feces and urine of clinically infected fish and by carriers. Waterborne transmission is believed to be the primary route of infection, but bloodsucking parasites like leeches and the carp louse may serve as mechanical vectors of SVCV. The genome of SVCV is composed of a single molecule of linear, negative-sense, single-stranded RNA containing 5 genes in the order 3¹-NPMGL-5¹ coding for the viral nucleoprotein, phosphoprotein, matrix protein, glycoprotein, and polymerase, respectively. Polyacrylamide gel electrophoresis of the viral proteins, and sequence homologies between the genes and gene junctions of SVCV and vesicular stomatitis viruses, have led to the placement of the virus as a tentative member of the genus Vesiculovirus in the family Rhabdoviridae . These methods also revealed that SVCV is not related to fish rhabdoviruses of the genus Novirhabdovirus . In vitro replication of SVCV takes place in the cytoplasm of cultured cells of fish, bird and mammalian origin at temperatures of 4 to 31°C, with an optimum of about 20°C. Spring viremia of carp can be diagnosed by clinical signs, isolation of virus in cell culture and molecular methods. Antibodies directed against SVCV react with the homologous virus in serum neutralization, immunofluorescence, immunoperoxidase, or enzyme-linked immunosorbent assays, but they cross-react to various degrees with the pike fry rhabdovirus (PFR), suggesting the 2 viruses are closely related. However, SVCV and PFR can be distinguished by certain serological tests and molecular methods such as the ribonuclease protection assay.

Diseases of Aquatic Organisms↗

Potential spread of highly pathogenic avian influenza H5N1 by wildfowl: dispersal ranges and rates determined from large-scale satellite telemetry

1. Migratory birds are major candidates for long-distance dispersal of zoonotic pathogens. In recent years, wildfowl have been suspected of contributing to the rapid geographic spread of the highly pathogenic avian influenza (HPAI) H5N1 virus. Experimental infection studies reveal that some wild ducks, geese and swans shed this virus asymptomatically and hence have the potential to spread it as they move. 2. We evaluate the dispersive potential of HPAI H5N1 viruses by wildfowl through an analysis of the movement range and movement rate of birds monitored by satellite telemetry in relation to the apparent asymptomatic infection duration (AID) measured in experimental studies. We analysed the first large-scale data set of wildfowl movements, including 228 birds from 19 species monitored by satellite telemetry in 2006–2009, over HPAI H5N1 affected regions of Asia, Europe and Africa. 3. Our results indicate that individual migratory wildfowl have the potential to disperse HPAI H5N1 over extensive distances, being able to perform movements of up to 2900 km within timeframes compatible with the duration of asymptomatic infection. 4. However, the likelihood of such virus dispersal over long distances by individual wildfowl is low: we estimate that for an individual migratory bird there are, on average, only 5–15 days per year when infection could result in the dispersal of HPAI H5N1 virus over 500 km. 5. Staging at stopover sites during migration is typically longer than the period of infection and viral shedding, preventing birds from dispersing a virus over several consecutive but interrupted long-distance movements. Intercontinental virus dispersion would therefore probably require relay transmission between a series of successively infected migratory birds. 6. Synthesis and applications . Our results provide a detailed quantitative assessment of the dispersive potential of HPAI H5N1 virus by selected migratory birds. Such dispersive potential rests on the assumption that free-living wildfowl will respond analogously to captive, experimentally-infected birds, and that asymptomatic infection will not alter their movement abilities. Our approach of combining experimental exposure data and telemetry information provides an analytical framework for quantifying the risk of spread of avian-borne diseases.

Journal of Applied Ecology↗

Molecular characterization of a novel orthomyxovirus from rainbow and steelhead trout ( Oncorhynchus mykiss )

A novel virus, rainbow trout orthomyxovirus (RbtOV), was isolated in 1997 and again in 2000 from commercially-reared rainbow trout ( Oncorhynchus mykiss ) in Idaho, USA. The virus grew optimally in the CHSE-214 cell line at 15°C producing a diffuse cytopathic effect; however, juvenile rainbow trout exposed to cell culture-grown virus showed no mortality or gross pathology. Electron microscopy of preparations from infected cell cultures revealed the presence of typical orthomyxovirus particles. The complete genome of RbtOV is comprised of eight linear segments of single-stranded, negative-sense RNA having highly conserved 5′ and 3′-terminal nucleotide sequences. Another virus isolated in 2014 from steelhead trout (also O. mykiss ) in Wisconsin, USA, and designated SttOV was found to have eight genome segments with high amino acid sequence identities (89–99%) to the corresponding genes of RbtOV, suggesting these new viruses are isolates of the same virus species and may be more widespread than currently realized. The new isolates had the same genome segment order and the closest pairwise amino acid sequence identities of 16–42% with Infectious salmon anemia virus (ISAV), the type species and currently only member of the genus Isavirus in the family Orthomyxoviridae . However, pairwise comparisons of the predicted amino acid sequences of the 10 RbtOV and SttOV proteins with orthologs from representatives of the established orthomyxoviral genera and a phylogenetic analysis using the PB1 protein showed that while RbtOV and SttOV clustered most closely with ISAV, they diverged sufficiently to merit consideration as representatives of a novel genus. A set of PCR primers was designed using conserved regions of the PB1 gene to produce amplicons that may be sequenced for identification of similar fish orthomyxoviruses in the future.

Virus Research↗

Host jump of an exotic fish rhabdovirus into a new class of animals poses a disease threat to amphibians

Spring viremia of carp virus (SVCV) is a rhabdovirus that primarily infects cyprinid finfishes and causes a disease notifiable to the World Organization for Animal Health. Amphibians, which are sympatric with cyprinids in freshwater ecosystems, are considered non-permissive hosts of rhabdoviruses. The potential host range expansion of SVCV in an atypical host species was evaluated by testing the susceptibility of amphibians native to the Pacific Northwest. Larval long-toed salamanders Ambystoma macrodactylum and Pacific tree frog Pseudacris regilla tadpoles were exposed to SVCV strains from genotypes Ia, Ib, Ic, or Id by either intraperitoneal injection, immersion, or cohabitation with virus-infected koi Cyprinus rubrofuscus . Cumulative mortality was 100% for salamanders injected with SVCV, 98–100% for tadpoles exposed to virus via immersion, and 0–100% for tadpoles cohabited with SVCV-infected koi. Many of the animals that died exhibited clinical signs of disease and SVCV RNA was found by in situ hybridization in tissue sections of immersion-exposed tadpoles, particularly in the cells of the gastrointestinal tract and liver. SVCV was also detected by plaque assay and RT-qPCR testing in both amphibian species regardless of the virus exposure method, and viable virus was detected up to 28 days after initial exposure. Recovery of infectious virus from naïve tadpoles cohabited with SVCV-infected koi further demonstrated that SVCV transmission can occur between classes of ectothermic vertebrates. Collectively, these results indicated that SVCV, a fish rhabdovirus, can be transmitted to and cause lethal disease in two amphibian species. Therefore, members of all five of the major vertebrate groups (mammals, birds, reptiles, fish, and amphibians) appear to be vulnerable to rhabdovirus infections. Future research studying potential spillover and spillback infections of aquatic rhabdoviruses between foreign and domestic amphibian and fish species will provide insights into the stressors driving novel interclass virus transmission events.

Viruses↗

Experimental challenge of a North American bat species, big brown bat (Eptesicus fuscus), with SARS-CoV-2

The recently emerged novel coronavirus, SARS‐CoV‐2, is phylogenetically related to bat coronaviruses (CoVs), specifically SARS‐related CoVs from the Eurasian bat family Rhinolophidae. As this human pandemic virus has spread across the world, the potential impacts of SARS‐CoV‐2 on native North American bat populations are unknown, as is the ability of North American bats to serve as reservoirs or intermediate hosts able to transmit the virus to humans or to other animal species. To help determine the impacts of the pandemic virus on North American bat populations, we experimentally challenged big brown bats ( Eptesicus fuscus ) with SARS‐CoV‐2 under BSL‐3 conditions. We inoculated the bats both oropharyngeally and nasally, and over the ensuing three weeks, we measured infectivity, pathology, virus concentrations in tissues, oral and rectal virus excretion, virus transmission, and clinical signs of disease. We found no evidence of SARS‐CoV‐2 infection in any examined bat, including no viral excretion, no transmission, no detectable virus in tissues, and no signs of disease or pathology. Based on our findings, it appears that big brown bats are resistant to infection with the SARS‐CoV‐2. The potential susceptibility of other North American bat species to SARS‐CoV‐2 remains to be investigated.

Transboundary and Emerging Diseases↗

Survey of selected pathogens and blood parameters of northern yellowstone elk: Wolf sanitation effect implications

The restoration or conservation of predators could reduce seroprevalences of certain diseases in prey if predation selectively removes animals exhibiting clinical signs. We assessed disease seroprevalences and blood parameters of 115 adult female elk (Cervus elaphus) wintering on the northern range of Yellowstone National Park [YNP] during 2000-2005 and compared them to data collected prior to wolf (Canis lupus) restoration (WR) in 1995 and to two other herds in Montana to assess this prediction. Blood parameters were generally within two standard deviations of the means observed in other Montana herds (Gravelly-Snowcrest [GS] and Garnet Mountain [GM]), but Yellowstone elk had higher seroprevalences of parainfluenza-3 virus (95% CI YNP = 61.1-78.6, GS = 30.3-46.5) and bovine-virus-diarrhea virus type 1 (95% CI YNP = 15.9-31.9, GM = 0). In comparisons between pre-wolf restoration [pre-WR] (i.e., prior to 1995) seroprevalences with those post-wolf restoration [post-WR] in Yellowstone, we found lower seroprevalences for some disease-causing agents post-wolf restoration (e.g., bovine-virus-diarrhea virus type-1 [95% CI pre-WR = 73.1-86.3, post-WR = 15.9-31.9] and bovine-respiratory syncytial virus [95% CI pre-WR = 70.0-83.8, post-WR = 0]), but similar (e.g., Brucella abortus [95% CI pre-WR = 0-4.45, post-WR = 0-4.74] and epizootic hemorrhagic disease virus [95% CI pre-WR = 0, post-WR = 0]) or higher for others (e.g., Anaplasma marginale [95% CI pre-WR = 0, post-WR = 18.5-38.7] and Leptospira spp. [95% CI pre-WR = 0.5-6.5, post-WR = 9.5-23.5]). Though we did not detect an overall strong predation effect through reduced disease seroprevalence using retrospective comparisons with sparse data, our reference values will facilitate future assessments of this issue.

American Midland Naturalist↗

Evaluating microbial purification during soil treatment of wastewater with multicomponent tracer and surrogate tests

Soil treatment of wastewater has the potential to achieve high purification efficiency, yet the understanding and predictability of purification with respect to removal of viruses and other pathogens is limited. Research has been completed to quantify the removal of virus and bacteria through the use of microbial surrogates and conservative tracers during controlled experiments with three-dimensional pilot-scale soil treatment systems in the laboratory and during the testing of full-scale systems under field conditions. The surrogates and tracers employed included two viruses (MS-2 and PRID-1 bacteriophages), one bacterium (ice-nucleating active Pseudomonas ), and one conservative tracer (bromide ion). Efforts have also been made to determine the relationship between viruses and fecal coliform bacteria in soil samples below the wastewater infiltrative surface, and the correlation between Escherichia coli concentrations measured in percolating soil solution as compared with those estimated from analyses of soil solids. The results suggest episodic breakthrough of virus and bacteria during soil treatment of wastewater and a 2 to 3 log (99-99.9%) removal of virus and near complete removal of fecal coliform bacteria during unsaturated flow through 60 to 90 cm of sandy medium. Results also suggest that the fate of fecal coliform bacteria may be indicative of that of viruses in soil media near the infiltrative surface receiving wastewater effluent. Concentrations of fecal coliform in percolating soil solution may be conservatively estimated from analysis of extracted soil solids.

Journal of Environmental Quality↗

Microbiological Water Quality in Relation to Water-Contact Recreation, Cuyahoga River, Cuyahoga Valley National Park, Ohio, 2000 and 2002

The microbiological water quality of a 23-mile segment of the Cuyahoga River within the Cuyahoga Valley National Park was examined in this study. This segment of the river receives discharges of contaminated water from stormwater, combined-sewer overflows, and incompletely disinfected wastewater. Frequent exceedances of Ohio microbiological water-quality standards result in a health risk to the public who use the river for water-contact recreation. Water samples were collected during the recreational season of May through October at four sites on the Cuyahoga River in 2000, at three sites on the river in 2002, and from the effluent of the Akron Water Pollution Control Station (WPCS) both years. The samples were collected over a similar range in streamflow in 2000 and 2002. Samples were analyzed for physical and chemical constituents, as well as the following microbiological indicators and pathogenic organisms: Escherichia coli (E. coli), Salmonella, F-specific and somatic coliphage, enterovirus, infectious enterovirus, hepatitis A virus, Clostridium perfringens (C. perfringens), Cryptosporidium, and Giardia. The relations of the microorganisms to each other and to selected water-quality measures were examined. All microorganisms analyzed for, except Cryptosporidium, were detected at least once at each sampling site. Concentrations of E. coli exceeded the Ohio primary-contact recreational standard (298 colonies per 100 milliliters) in approximately 87 percent of the river samples and generally were higher in the river samples than in the effluent samples. C. perfringens concentrations were positively and significantly correlated with E. coli concentrations in the river samples and generally were higher in the effluent samples than in the river samples. Several of the river samples that met the Ohio E. coli secondary-contact recreational standard (576 colonies per 100 milliliters) had detections of enterovirus, infectious enterovirus, hepatitis A virus, and Salmonella, indicating that there are still risks even when the E. coli standard is not exceeded. River samples in which the secondary-contact recreational standard for E. coli was exceeded showed a higher percentage of the co-occurrence of pathogenic organisms than samples that met the standard. This indicates that in this study area, E. coli is a useful indicator of human health risk. Detections of hepatitis A virus tended to be associated with higher median concentrations of somatic coliphage, F-specific coliphage, and infectious enterovirus. In addition, geometric mean C. perfringens concentrations tended to be higher in samples where hepatitis A virus was present than in samples where hepatitis A virus was absent. Hepatitis A virus was not detected in samples collected upstream from the Akron WPCS; all downstream detections had coincident detections in the Akron WPCS effluent, suggesting that Akron WPCS was a principal source of hepatitis A virus at the downstream sites. Geometric mean concentrations of E. coli were calculated on the basis of analytical results from at least five samples collected at each river site during May, July, and September of 2000. In each case, the Ohio geometric-mean primary-contact recreational standard of 126 col/100 mL was exceeded. E. coli concentrations were significantly correlated with streamflow and increased with streamflow at sites upstream and downstream from the Akron WPCS. This indicates that E. coli loads from sources upstream from the Akron WPCS have the potential to appreciably influence the frequency of attainment of recreational water-quality standards at downstream locations.

Water-Resources Investigations Report↗