Continuous surveillance and viral discovery in animals and humans are a core component of a one-health approach to address recent viral reverse zoonoses.

Contemporary human and animal viruses have a broad or narrow host range-those with a broad host range are potentially transmitted from animals to humans (ie, zoonosis) or humans to animals (ie, reverse zoonosis). This Currents in One Health article reviews the recent reverse zoonoses involving Coronaviridae, Poxviridae, arboviruses, and, for nonhuman primate species, the human respiratory viruses. The prevention and control of reverse zoonoses are also reviewed. Coronaviruses continue to emerge as new zoonotic agents, including a canine coronavirus, CCoV-HuPn-2018, circulating in people at low levels, and a pangolin coronavirus, MjHKU4r-CoV-1, circulating in Malayan pangolins. Moreover, the risk for SARS-CoV-2 variants to mutate in animal reservoirs and reinfect humans is ongoing. In the case of mpox, the risk of reverse zoonosis is low and there are vaccines for use in humans at risk. The situation with arboviruses is as varied as the number of human arboviruses, and only yellow fever virus and dengue virus have licensed vaccines in the Americas. As for reverse zoonoses in endangered species, solutions require changing human behavior and policies at all levels impacting wildlife. Overall, continuous surveillance and viral discovery in humans and animals remain core components of a one-health approach to reduce and, where possible, eliminate zoonotic and reverse zoonotic diseases. Viral zoonosis and viral reverse zoonosis focusing on recent influenza A virus disease events in humans and other species are the subjects of the companion Currents in One Health by Kibenge, AJVR, June 2023.

A One Health approach to mitigate the impact of influenza A virus (IAV) reverse zoonosis is by vaccinating humans and susceptible farmed and pet animals.

The term reverse zoonosis specifically refers to the natural transmission of disease and infection from humans to animals, with humans as the reservoir host replicating the infectious agent. In the last 20 years, reverse zoonosis has increasingly garnered attention because of human disease outbreaks. In this Currents in One Health article, the author will review host range as the main risk factor for reverse zoonosis, with an emphasis on influenza A virus (IAV) disease events in humans and other species in the context of a "One Health" approach to gain a better understanding of their transmission routes to facilitate their control and prevent them from occurring. The human-to-pig transmission of IAV represents the largest reverse zoonosis of a pathogen documented to date. At the same time, the 2022 farmed mink outbreak in Spain is the most sustained mammal-to-mammal transmission of the highly pathogenic avian influenza (HPAI) H5N1 since its re-emergence in humans in 2003. Without any prospect of eradicating IAVs, the best way to mitigate the impact of IAV reverse zoonosis is by vaccinating humans and susceptible farmed and pet animals. The recent major reverse zoonoses involving other virus groups (Coronaviridae, Poxviridae, arboviruses, and the human respiratory viruses transmitted to endangered non-human primate species) and the prevention and control of reverse zoonoses are addressed in the companion Currents in One Health by Kibenge, JAVMA, June 2023.

Isolation of a New Infectious Pancreatic Necrosis Virus (IPNV) Variant from Genetically Resistant Farmed Atlantic Salmon (Salmo salar) during 2021-2022.

Infectious pancreatic necrosis (IPN), caused by IPNV, affects several species of farmed fish, particularly Atlantic salmon, and is responsible for significant economic losses in salmon aquaculture globally. Despite the introduction of genetically resistant farmed Atlantic salmon and vaccination strategies in the Chilean salmon industry since 2019, the number of IPN outbreaks has been increasing in farmed Atlantic salmon in the freshwater phase. This study examined gross and histopathological lesions of IPNV-affected fish, as well as the IPNV nucleotide sequence encoding the VP2 protein in clinical cases. The mortality reached 0.4% per day, and the cumulative mortality was from 0.4 to 3.5%. IPNV was isolated in the CHSE-214 cell line and was confirmed by RT-PCR, and VP2 sequence analysis. The analyzed viruses belong to IPNV genotype 5 and have 11 mutations in their VP2 protein. This is the first report of IPN outbreaks in farmed Atlantic salmon genetically resistant to IPNV in Chile. Similar outbreaks were previously reported in Scotland and Norway during 2018 and 2019, respectively. This study highlights the importance of maintaining a comprehensive surveillance program in conjunction with the use of farmed Atlantic salmon genetically resistant to IPNV.

SARS-CoV-2 transmission via aquatic food animal species or their products: A review.

Outbreaks of COVID-19 (coronavirus disease 2019) have been reported in workers in fish farms and fish processing plants arising from person-to-person transmission, raising concerns about aquatic animal food products' safety. A better understanding of such incidents is important for the aquaculture industry's sustainability, particularly with the global trade in fresh and frozen aquatic animal food products where contaminating virus could survive for some time. Despite a plethora of COVID-19-related scientific publications, there is a lack of reports on the risk of contact with aquatic food animal species or their products. This review aimed to examine the potential for Severe Acute Respiratory Syndrome-Coronavirus-2 (SARS-CoV-2) contamination and the potential transmission via aquatic food animals or their products and wastewater effluents. The extracellular viability of SARS-CoV-2 and how the virus is spread are reviewed, supporting the understanding that contaminated cold-chain food sources may introduce SAR-CoV-2 via food imports although the virus is unlikely to infect humans through consumption of aquatic food animals or their products or drinking water; i.e., SARS-CoV-2 is not a foodborne virus and should not be managed as such but instead through strong, multifaceted public health interventions including physical distancing, rapid contact tracing, and testing, enhanced hand and respiratory hygiene, frequent disinfection of high-touch surfaces, isolation of infected workers and their contacts, as well as enhanced screening protocols for international seafood trade.

Correction to: Piscine orthoreovirus sequences in escaped farmed Atlantic salmon in Washington and British Columbia.

In the original publication of the article [1], as the quotation below was included without specific permission from Dr. Gary Marty, which is against the Virology Journal guidelines for the citation of unpublished data, all authors request to delete it from their article.

Piscine orthoreovirus sequences in escaped farmed Atlantic salmon in Washington and British Columbia.

BACKGROUND: Piscine orthoreovirus (PRV) is an emergent virus in salmon aquaculture belonging to the family Reoviridae. PRV is associated with a growing list of pathological conditions including heart and skeletal inflammation (HSMI) of farmed Atlantic salmon. Despite widespread PRV infection in commercially farmed Atlantic salmon, information on PRV prevalence and on the genetic sequence variation of PRV in Atlantic salmon on the north Pacific Coast is limited. METHODS: Feral Atlantic salmon caught in Washington State and British Columbia following a large containment failure at a farm in northern Puget Sound were sampled. Fish tissues were tested for PRV by RT-qPCR assay for segment L1 and conventional RT-PCR for PRV segment S1. The PCR products were sequenced and their relationship to PRV strains in GenBank was determined using phylogenetic analysis and nucleotide and amino acid homology comparisons. RESULTS: Following the escape of 253,000 Atlantic salmon from a salmon farm in Washington State, USA, 72/73 tissue samples from 27 Atlantic salmon captured shortly after the escape tested PRV-positive. We estimate PRV-prevalence in the source farm population at 95% or greater. The PRV found in the fish was identified as PRV sub-genotype Ia and very similar to PRV from farmed Atlantic salmon in Iceland. This correlates with the source of the fish in the farm. Eggs of infected fish were positive for PRV indicating the possibility of vertical transfer and spread with fish egg transports. CONCLUSIONS: PRV prevalence was close to 100% in farmed Atlantic salmon that were caught in Washington State and British Columbia following a large containment failure at a farm in northern Puget Sound. The PRV strains present in the escaped Atlantic salmon were very similar to the PRV strain reported in farmed Atlantic salmon from the source hatchery in Iceland that was used to stock commercial aquaculture sites in Washington State. This study emphasizes the need to screen Atlantic salmon broodstock for PRV, particularly where used to supply eggs to the global Atlantic salmon farming industry thereby improving our understanding of PRV epidemiology.

First description of clinical presentation of piscine orthoreovirus (PRV) infections in salmonid aquaculture in Chile and identification of a second genotype (Genotype II) of PRV.

BACKGROUND: Heart and skeletal muscle inflammation (HSMI) is an emerging disease of marine-farmed Atlantic salmon Salmo salar, first recognized in 1999 in Norway, and recently associated with piscine orthoreovirus (PRV) infection. To date, HSMI lesions with presence of PRV have only been described in marine-farmed Atlantic salmon in Norway. A new HSMI-like disease in rainbow trout Oncorhynchus mykiss associated with a PRV-related virus has also been reported in Norway. METHODS: Sampling of Atlantic salmon and coho salmon was done during potential disease outbreaks, targeting lethargic/moribund fish. Fish were necropsied and tissues were taken for histopathologic analysis and testing for PRV by RT-qPCR assay for segment L1 and conventional RT-PCR for PRV segment S1. The PCR products were sequenced and their relationship to PRV strains in GenBank was determined using phylogenetic analysis and nucleotide and amino acid homology comparisons. RESULTS: The Atlantic salmon manifested the classical presentation of HSMI with high PRV virus loads (low Ct values) as described in Norway. The coho salmon with low Ct values had myocarditis but only in the spongy layer, the myositis of red muscle in general was mild, and the hepatic necrosis was severe. Upon phylogenetic analysis of PRV segment S1 sequences, all the Chilean PRV strains from Atlantic salmon grouped as sub-genotype Ib, whereas the Chilean PRV strains from coho salmon were more diversified, grouping in both sub-genotypes Ia and Ib and others forming a distinct new phylogenetic cluster, designated Genotype II that included the Norwegian PRV-related virus. CONCLUSIONS: To our knowledge the present work constitutes the first published report of HSMI lesions with presence of PRV in farmed Atlantic salmon outside of Europe, and the first report of HSMI-like lesions with presence of PRV in coho salmon in Chile. The Chilean PRV strains from coho salmon are more genetically diversified than those from Atlantic salmon, and some form a distinct new phylogenetic cluster, designated Genotype II.

Clinical signs, pathology and dose-dependent survival of adult wood frogs, Rana sylvatica, inoculated orally with frog virus 3 Ranavirus sp., Iridoviridae.

Amphibian populations suffer massive mortalities from infection with frog virus 3 FV3, genus Ranavirus, family Iridoviridae, a pathogen also involved in mortalities of fish and reptiles. Experimental oral infection with FV3 in captive-raised adult wood frogs, Rana sylvatica Lithobates sylvaticus, was performed as the first step in establishing a native North American animal model of ranaviral disease to study pathogenesis and host response. Oral dosing was successful LD50 was 10(2.93 2.423.44) p.f.u. for frogs averaging 35mm in length. Onset of clinical signs occurred 614days post-infection p.i. median 11 days p.i. and time to death was 1014 days p.i. median 12 days p.i.. Each tenfold increase in virus dose increased the odds of dying by 23-fold and accelerated onset of clinical signs and death by approximately 15. Ranavirus DNA was demonstrated in skin and liver of all frogs that died or were euthanized because of severe clinical signs. Shedding of virus occurred in faeces 710 days p.i. 34.5days before death and skin sheds 10 days p.i. 01.5days before death of some frogs dead from infection. Most common lesions were dermal erosion and haemorrhages haematopoietic necrosis in bone marrow, kidney, spleen and liver and necrosis in renal glomeruli, tongue, gastrointestinal tract and urinary bladder mucosa. Presence of ranavirus in lesions was confirmed by immunohistochemistry. Intracytoplasmic inclusion bodies probably viral were present in the bone marrow and the epithelia of the oral cavity, gastrointestinal tract, renal tubules and urinary bladder. Our work describes a ranaviruswood frog model and provides estimates that can be incorporated into ranavirus disease ecology models.

Genetic analysis and comparative virulence of infectious salmon anemia virus (ISAV) types HPR7a and HPR7b from recent field outbreaks in Chile.

BACKGROUND: Infectious salmon anemia (ISA) is a serious disease of marine farmed Atlantic salmon, Salmo salar L. caused by ISA virus (ISAV). ISAV genomic segments 5 and 6 encode surface glycoproteins hemagglutinin-esterase (HE) and F protein important for the pathogenicity of ISAV. In this study, we describe the genetic characteristics and relationship between ISAV-HPR7a and ISAV-HPR7b strains that caused the ISA outbreaks in Chile in 2013 and 2014, respectively, and the evolution of the ISAV clades since 2009 based on segment 5 and 6 sequences. METHODS: The study material included samples from six ISA cases in Chile. RNA was extracted from salmon tissues and ISAV isolated from cell culture; segments 5 and 6 were amplified by RT-PCR and compared by alignment with ISAV sequences from the GenBank database. RESULTS: ISAV-HPR7a and ISAV-HPR7b belong to the European Genotype I strains only found in Europe and Chile, and in both cases, show high similarity in segments 5 and 6 with identity between 95-96%. Our data confirm the hypothesis that the original virus was introduced to Chile in 1996. Compared to the 2007 ISAV-HPR7b isolate, the 2014 ISAV-HPR7b does not have an insertion in segment 5 and was associated with low mortality, which suggests that ISAV virulence was attenuated by the absence of the insertion in segment 5. In contrast, the highly virulent ISAV-HPR14 from April 2013 outbreak did not have the insertion in segment 5 either. CONCLUSION: Variability in the ISAV virulence markers supports the quasispecies theory that multiple evolution forces are likely to shape ISAV genetic diversity. Our findings provide evidence of continuing evolution of ISAV in the Chilean aquaculture industry.

Infectious salmon anaemia virus (ISAV) in Chilean Atlantic salmon (Salmo salar) aquaculture: emergence of low pathogenic ISAV-HPR0 and re-emergence of virulent ISAV-HPR∆: HPR3 and HPR14.

ABSTACT: Infectious salmon anaemia (ISA) is a serious disease of marine-farmed Atlantic salmon (Salmo salar) caused by ISA virus (ISAV), which belongs to the genus Isavirus, family Orthomyxoviridae. ISA is caused by virulent ISAV strains with deletions in a highly polymorphic region (HPR) of the hemagglutinin-esterase (HE) protein (designated virulent ISAV-HPR∆). This study shows the historic dynamics of ISAV-HPR∆ and ISAV-HPR0 in Chile, the genetic relationship among ISAV-HPR0 reported worldwide and between ISAV-HPR0 and ISAV-HPR∆ in Chile, and reports the 2013 ISA outbreak in Chile. The first ISA outbreak in Chile occurred from mid-June 2007 to 2010 and involved the virulent ISAV-HPR7b, which was then replaced by a low pathogenic ISAV-HPR0 variant. We analyzed this variant in 66 laboratory-confirmed ISAV-HPR0 cases in Chile in comparison to virulent ISAV-HPR∆ that caused two new ISA outbreaks in April 2013. Multiple alignment and phylogenetic analysis of HE sequences from all ISAV-HPR0 viruses allowed us to identify three genomic clusters, which correlated with three residue patterns of ISAV-HPR0 (360PST362, 360PAN362 and 360PAT362) in HPR. The virus responsible for the 2013 ISAV-HPR∆ cases in Chile belonged to ISAV-HPR3 and ISAV-HPR14, and in phylogenetic analyses, both clustered with the ISAV-HPR0 found in Chile. The ISAV-HPR14 had the ISAV-HPR0 residue pattern 360PAT362, which is the only type of ISAV-HPR0 variant found in Chile. This suggested to us that the 2013 ISAV-HPR∆ re-emerged from ISAV-HPR0 that is enzootic in Chilean salmon aquaculture and were not new introductions of virulent ISAV-HPR∆ to Chile. The clinical presentations and diagnostic evidence of the 2013 ISA cases indicated a mixed infection of ISAV with the ectoparasite Caligus rogercresseyi and the bacterium Piscirickettsia salmonis, which underscores the need for active ISAV surveillance in areas where ISAV-HPR0 is enzootic, to ensure early detection and control of new ISA outbreaks, as it is considered a risk factor. This is the first report of ISA linked directly to the presence of ISAV-HPR0, and provides strong evidence supporting the contention that ISAV-HPR0 shows a strong relationship to virulent ISAV-HPR∆ viruses and the possibility that it could mutate to virulent ISAV-HPR∆.

Whole-genome analysis of piscine reovirus (PRV) shows PRV represents a new genus in family Reoviridae and its genome segment S1 sequences group it into two separate sub-genotypes.

BACKGROUND: Piscine reovirus (PRV) is a newly discovered fish reovirus of anadromous and marine fish ubiquitous among fish in Norwegian salmon farms, and likely the causative agent of heart and skeletal muscle inflammation (HSMI). HSMI is an increasingly economically significant disease in Atlantic salmon (Salmo salar) farms. The nucleotide sequence data available for PRV are limited, and there is no genetic information on this virus outside of Norway and none from wild fish. METHODS: RT-PCR amplification and sequencing were used to obtain the complete viral genome of PRV (10 segments) from western Canada and Chile. The genetic diversity among the PRV strains and their relationship to Norwegian PRV isolates were determined by phylogenetic analyses and sequence identity comparisons. RESULTS: PRV is distantly related to members of the genera Orthoreovirus and Aquareovirus and an unambiguous new genus within the family Reoviridae. The Canadian and Norwegian PRV strains are most divergent in the segment S1 and S4 encoded proteins. Phylogenetic analysis of PRV S1 sequences, for which the largest number of complete sequences from different "isolates" is available, grouped Norwegian PRV strains into a single genotype, Genotype I, with sub-genotypes, Ia and Ib. The Canadian PRV strains matched sub-genotype Ia and Chilean PRV strains matched sub-genotype Ib. CONCLUSIONS: PRV should be considered as a member of a new genus within the family Reoviridae with two major Norwegian sub-genotypes. The Canadian PRV diverged from Norwegian sub-genotype Ia around 2007 ± 1, whereas the Chilean PRV diverged from Norwegian sub-genotype Ib around 2008 ± 1.

Countermeasures against viral diseases of farmed fish.

Farmed fish provide an increasing fraction of the human food supply, and are of major economic importance in many countries. As in the case of terrestrial agriculture, bringing together large numbers of animals of a single species (i.e., monoculture) increases the risk of infectious disease outbreaks, including viral infections. Aquaculture, in which farmed fish are kept at high population densities in close proximity with wild fish reservoirs, is ideal for the emergence of wild-type pathogens that exist benignly in local wild fish and/or the spreading of aquatic pathogens to wild fish that enter into or come into close proximity with net cages and with fish escaping from them. This paper provides a general review for the nonspecialist of viral diseases of farmed fish and how they could be prevented or treated. It has five principal objectives: (1) to provide an update on the most important and emerging viral diseases of salmonid aquaculture; (2) to review general aspects of innate antiviral defense against virus infections in fish, including recent advances in antiviral signaling; (3) to discuss current principles and practices of vaccinating fish; (4) to review antiviral drugs that have activity against viruses of farmed fish, and current barriers to employing them in aquaculture; and (5) to discuss the growing use of "functional feeds" in salmonid aquaculture to mitigate viral diseases. In conclusion, despite the challenging aquatic environment, it is expected that well thought-out combinations of vaccination and immunostimulants and/or antiviral drugs could provide solid protection against viral diseases of farmed fish.

Replication of low pathogenic avian influenza virus in naturally infected Mallard ducks (Anas platyrhynchos) causes no morphologic lesions.

Although the Mallard (Anas platyrhynchos) is considered an important maintenance host for low pathogenic avian influenza (LPAI) viruses, viral cell tropism and pathology in naturally infected birds are largely unknown. In August 2006, we collected 19 free-living hatch-year Mallards that were positive for LPAI virus by real-time reverse-transcriptase polymerase chain reaction (RRT-PCR) in combined oropharyngeal and cloacal swabs. We investigated virus infection and associated lesions in the digestive and respiratory tracts by RRT-PCR, virus culture, immunohistochemistry (IHC), and histology. By RRT-PCR, 15 birds were positive in cloacal bursa, colon/cloaca, or both, and three were positive in lungs. Virus was isolated from eight birds and typed as H2N3 (three birds), H3N3 (two birds), H3N8 (one bird), H4N6 (one bird), and H?N3 (one bird). By IHC, birds were positive in the cloacal bursa (eight birds), colon (three), cecum (two), or ileum (one). Cell types infected were superficial epithelial cells of the bursa and epithelial cells of the intestinal villi and, less commonly, mucosal glands. By histology, there was no evidence of lesions associated with LPAI virus infection. These results show that epithelia of the cloacal bursa and of the lower intestine are important sites of natural LPAI virus infection in free-living hatch-year Mallards. The lack of lesions associated with this infection suggests that there is a strong selection by LPAI virus to cause minimal virulence in this maintenance host species.

MIQE précis: Practical implementation of minimum standard guidelines for fluorescence-based quantitative real-time PCR experiments.

The conclusions of thousands of peer-reviewed publications rely on data obtained using fluorescence-based quantitative real-time PCR technology. However, the inadequate reporting of experimental detail, combined with the frequent use of flawed protocols is leading to the publication of papers that may not be technically appropriate. We take the view that this problem requires the delineation of a more transparent and comprehensive reporting policy from scientific journals. This editorial aims to provide practical guidance for the incorporation of absolute minimum standards encompassing the key assay parameters for accurate design, documentation and reporting of qPCR experiments (MIQE précis) and guidance on the publication of pure 'reference gene' articles.

The fight between the teleost fish immune response and aquatic viruses.

Teleost fish represent a transition point on the phylogenetic spectrum between invertebrates that depend only on innate immunity and mammals that heavily depend on adaptive immunity. The major mechanisms of the teleost fish innate immune response are suggested to be similar to mammals, although fine details of the process require further studies. Within the innate immune response the type I interferon (IFN) system is an essential innate antiviral component that protects fish from some virus infections. The current progress of cloning and functional characterization of fish antiviral genes is promising in further elucidation of the fish antiviral response. The adaptive immune system of fish utilizes cellular components more or less similar to mammals. Teleost fish produce IgM as a primary antibody response and lack isotype switching to mount virus-specific antibodies during the infection process. Despite this, the development of successful fish rhabdoviral vaccines suggest that vaccination may prove to be an effective way of promoting fish adaptive immune responses to viruses. This paper reviews the bony fish antiviral response with specific discussion on the evolutionary mechanisms that allow aquatic viruses to co-exist with their host. Detailed aspects of the teleost type I IFN system are also addressed.

Infectious salmon anaemia virus (ISAV) isolates induce distinct gene expression responses in the Atlantic salmon (Salmo salar) macrophage/dendritic-like cell line TO, assessed using genomic techniques.

Infectious salmon anaemia virus (ISAV) is a marine orthomyxovirus of significant interest not only as a cause of a fatal disease of farmed Atlantic salmon resulting in severe economic losses to the aquaculture industry, but also as the only poikilothermic orthomyxovirus. ISAV targets vascular endothelial cells and macrophages, and is known to influence the expression of both innate and adaptive immune response relevant genes. ISAV isolates from different geographic regions have been shown to vary considerably in their pathogenicity for Atlantic salmon. This study aimed to characterize the Atlantic salmon TO macrophage/dendritic-like cell responses to infection with a selection of ISAV isolates of different genotypes and pathogenicity phenotypes. The first TO infection trial used ISAV isolates NBISA01 and RPC/NB-04-085-1 of high and low pathogenicity, respectively, and global gene expression analyses were carried out using approximately 16,000 gene (16K) salmonid cDNA microarrays to compare RNA samples extracted from TO cells harvested 24 and 72h post-infection versus time-matched uninfected controls. Overall, the microarray experiment showed that RPC/NB-04-085-1-infected cells had a higher total number of reproducibly dysregulated genes (88 genes: the sum of genes greater than 2-fold up- or down-regulated in all four replicate microarrays of a given comparison) than the NBISA01-infected cells (10 genes) for the combined sampling points (i.e. 24 and 72h). This microarray experiment identified several salmon genes that were differentially regulated by NBISA01 and RPC/NB-04-085-1, and which may be useful as molecular biomarkers of ISAV infection. An initial quantitative reverse transcription-polymerase chain reaction (QRT-PCR) study involving 25 microarray-identified genes confirmed the differences in the level of dysregulation of host transcripts between the two ISAV isolates (i.e. NBISA01 and RPC/NB-04-085-1). A second TO infection trial was run using a selection of four clinical ISAV isolates (Norway-810/9/99, a high pathogenicity isolate of European genotype; RPC/NB-04-085-1, a low pathogenicity isolate of European genotype; NBISA01, a high pathogenicity isolate of North American genotype; and RPC/NB-01-0593-1, an intermediate pathogenicity isolate of North American genotype), and UV-inactivated RPC/NB-04-085-1, with sampling at 24, 36, 48, 72, 96, and 120h post-infection. The microarray-identified, QRT-PCR validated suite of 24 molecular biomarkers of response to ISAV were used in a second QRT-PCR experiment to assess the TO cell gene expression responses to the four ISAV isolates at all six time points in the infection. The QRT-PCR data showed that RPC/NB-04-085-1 caused the highest fold changes of most immune-relevant genes [such as interferon-inducible protein Gig1, Mx1 protein, interferon-induced protein with tetratricopeptide repeats 5, Radical S-adenosyl methionine domain-containing protein (viperin), and several genes involved in the ISGylation pathway], followed by Norway-810/9/99. NBISA01 and RPC/NB-01-0593-01 (both of North American genotype) showed low fold up-regulation of transcripts that were highly induced by RPC/NB-04-085-1 isolate. These findings show that ISAV isolates have strain-specific variations in their ability to induce immune response genes.

Infectious salmon anaemia virus (ISAV) isolated from the ISA disease outbreaks in Chile diverged from ISAV isolates from Norway around 1996 and was disseminated around 2005, based on surface glycoprotein gene sequences.

BACKGROUND: Infectious salmon anaemia (ISA) virus (ISAV) is a pathogen of marine-farmed Atlantic salmon (Salmo salar); a disease first diagnosed in Norway in 1984. For over 25 years ISAV has caused major disease outbreaks in the Northern hemisphere, and remains an emerging fish pathogen because of the asymptomatic infections in marine wild fish and the potential for emergence of new epidemic strains. ISAV belongs to the family Orthomyxoviridae, together with influenza viruses but is sufficiently different to be assigned to its own genus, Isavirus. The Isavirus genome consists of eight single-stranded RNA species, and the virions have two surface glycoproteins; fusion (F) protein encoded on segment 5 and haemagglutinin-esterase (HE) protein encoded on segment 6. However, comparison between different ISAV isolates is complicated because there is presently no universally accepted nomenclature system for designation of genetic relatedness between ISAV isolates. The first outbreak of ISA in marine-farmed Atlantic salmon in the Southern hemisphere occurred in Chile starting in June 2007. In order to describe the molecular characteristics of the virus so as to understand its origins, how ISAV isolates are maintained and spread, and their virulence characteristics, we conducted a study where the viral sequences were directly amplified, cloned and sequenced from tissue samples collected from several ISA-affected fish on the different fish farms with confirmed or suspected ISA outbreaks in Chile. This paper describes the genetic characterization of a large number of ISAV strains associated with extensive outbreaks in Chile starting in June 2007, and their phylogenetic relationships with selected European and North American isolates that are representative of the genetic diversity of ISAV. RESULTS: RT-PCR for ISAV F and HE glycoprotein genes was performed directly on tissue samples collected from ISA-affected fish on different farms among 14 fish companies in Chile during the ISA outbreaks that started in June 2007. The genes of the F and HE glycoproteins were cloned and sequenced for 51 and 78 new isolates, respectively. An extensive comparative analysis of ISAV F and HE sequence data, including reference isolates sampled from Norway, Faroe Islands, Scotland, USA, and Canada was performed. Based on phylogenetic analysis of concatenated ISAV F and HE genes of 103 individual isolates, the isolates from the ISA outbreaks in Chile grouped in their own cluster of 7 distinct strains within Genotype I (European genotype) of ISAV, with the closest relatedness to Norwegian ISAVs isolated in 1997. The phylogenetic software program, BACKTRACK, estimated the Chile isolates diverged from Norway isolates about 1996 and, therefore, had been present in Chile for some time before the recent outbreaks. Analysis of the deduced F protein sequence showed 43 of 51 Chile isolates with an 11-amino acid insert between 265N and 266Q, with 100% sequence identity with Genotype I ISAV RNA segment 2. Twenty four different HE-HPRs, including HPR0, were detected, with HPR7b making up 79.7%. This is considered a manifestation of ISAV quasispecies HE protein sequence diversity. CONCLUSION: Taken together, these findings suggest that the ISA outbreaks were caused by virus that was already present in Chile that mutated to new strains. This is the first comprehensive report tracing ISAV from Europe to South America.

Absolute quantitation of infectious salmon anaemia virus using different real-time reverse transcription PCR chemistries.

Routine laboratory diagnosis of infectious salmon anaemia virus (ISAV) infection is primarily by reverse transcription polymerase chain reaction (RT-PCR) because of the high sensitivity and rapid turnaround time of the test. This paper describes methods for highly reproducible absolute viral load measurements using external standard curves generated with either ISAV recombinant plasmid DNA (pDNA) standards or transcribed RNA standards prepared by in vitro transcription with T7 RNA polymerase, and using a two tube real-time or quantitative (q)RT-PCR with SYBR Green I chemistry and a single tube qRT-PCR with TaqMan probe chemistry. When applied to virus samples of known virus titer for the highly pathogenic ISAV strain NBISA01 and the low pathogenic ISAV strain RPC/NB-04-085-1, both methods showed a 100-fold lower detectable titer for RPC/NB-04-085-1 but with a higher number of viral RNA molecules compared to NBISA01. Overall, the SYBR Green I method overestimated copy numbers in samples having equivalent Ct values with the TaqMan probe method. Taken together, the findings suggest that the TaqMan probe method with the in vitro transcribed RNA standard curve is the preferred method for reliable and rapid quantitation of ISAV in samples.

First detection, isolation and molecular characterization of infectious salmon anaemia virus associated with clinical disease in farmed Atlantic salmon (Salmo salar) in Chile.

BACKGROUND: Infectious salmon anaemia (ISA) is a viral disease of marine-farmed Atlantic salmon (Salmo salar) caused by ISA virus (ISAV), which belongs to the genus Isavirus, family Orthomyxoviridae. The virus is considered to be carried by marine wild fish and for over 25 years has caused major disease outbreaks in marine-farmed Atlantic salmon in the Northern hemisphere. In the Southern hemisphere, ISAV was first detected in Chile in 1999 in marine-farmed Coho salmon (Oncorhynchus kisutch). In contrast to the classical presentation of ISA in Atlantic salmon, the presence of ISAV in Chile until now has only been associated with a clinical condition called Icterus Syndrome in Coho salmon and virus isolation has not always been possible. During the winter of 2007, unexplained mortalities were registered in market-size Atlantic salmon in a grow-out site located in Chiloé in Region X of Chile. We report here the diagnostic findings of the first significant clinical outbreak of ISA in marine-farmed Atlantic salmon in Chile and the first characterization of the ISAV isolated from the affected fish. RESULTS: In mid-June 2007, an Atlantic salmon marine farm site located in central Chiloé Island in Region X of Chile registered a sudden increase in mortality following recovery from an outbreak of Pisciricketsiosis, which rose to a cumulative mortality of 13.6% by harvest time. Based on the clinical signs and lesions in the affected fish, and laboratory tests performed on the fish tissues, a confirmatory diagnosis of ISA was made; the first time ISA in its classical presentation and for the first time affecting farmed Atlantic salmon in Chile. Rapid sequencing of the virus-specific RT-PCR products amplified from the fish tissues identified the virus to belong to the European genotype (Genotype I) of the highly polymorphic region (HPR) group HPR 7b, but with an 11-amino acid insert in the fusion glycoprotein, and ability to cause cytopathic effects (CPE) in CHSE-214 cell line, characteristics which make it distinct from common European Genotype ISAV isolates from Europe and North America. CONCLUSION: In conclusion, the present work constitutes the first report of a case of ISA in farmed Atlantic salmon in Chile. The clinical signs and lesions are consistent with the classical descriptions of the disease in marine-farmed Atlantic salmon in the Northern hemisphere. The outbreak was caused by ISAV of European genotype (or Genotype I) of HPR 7b but distinct from common European Genotype ISAV isolates.