Molly J. T. Kibenge

The need for transparency and good practices in the qPCR literature

Two surveys of over 1,700 publications whose authors use quantitative real-time PCR (qPCR) reveal a lack of transparent and comprehensive reporting of essential technical information. Reporting standards are significantly improved in publications that cite the Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE) guidelines, although such publications are still vastly outnumbered by those that do not.

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

BackgroundInfectious 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.ResultsIn 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.ConclusionIn 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.

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.

Antigenic variation among isolates of infectious salmon anaemia virus correlates with genetic variation of the viral haemagglutinin gene.

Infectious salmon anaemia virus (ISAV), an orthomyxovirus-like virus, is an important fish pathogen in marine aquaculture. Virus neutralization of 24 ISAV isolates in the TO cell line using rabbit antisera to the whole virus and comparative sequence analysis of their haemagglutinin (HA) genes have allowed elaboration on the variation of ISAV isolates. The 24 viruses were neutralized to varying degrees, revealing two major antigenic groups, one American and one European. Sequence analysis of the HA gene also revealed two groups of viruses (genotypes) that correlated with the antigenic groupings. The two HA subtypes had nucleotide sequence identity of only < or =79.4% and amino acid sequence identity of < or =84.5% whereas, within each subtype, the sequence identities were 90.7% or higher. This grouping was also evident upon phylogenetic analysis, which revealed two distinct phylogenetic families. Between the two groups, the amino acid sequence was most variable in the C-terminal region and included deletions of 4-16 amino acids in all isolates relative to ISAV isolate RPC/NB-980 280-2. In order to view the relationships among these sequences and the HA sequences of the established orthomyxoviruses, a second phylogenetic tree was constructed which showed the ISAV sequences to be more closely related to sequences from Influenzavirus A and Influenzavirus B than to sequences from Influenzavirus C and Thogotovirus. The extensive deletions in the gene of European ISAV isolates lead us to speculate that the archetypal ISAV was probably of Canadian origin.

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 anemia virus: causative agent, pathogenesis and immunity

Abstract Infectious salmon anemia (ISA) virus (ISAV), an economically important new pathogen in marine aquaculture, is classified in the family Orthomyxoviridae, genus Isavirus. The main structural properties of this genus include enveloped virions 90–140 nm in diameter with surface projections of a combined receptor-binding hemagglutinin and receptor-destroying enzyme activity demonstrated to be an esterase, hence recently designated HE, and a genome composed of eight segments of linear, single-stranded, negative sense RNA ranging in length from 1.0 to 2.4 kb, with a total size of approximately 14.3 kb. The viral genome encodes at least ten proteins, of which nine are structural and one is non-structural. Examination of more than 160 ISAV isolates has led to the identification of two hemagglutinin subtypes of ISAV, one North American and one European. The immune response against ISAV after infection or vaccination does not provide full protection against the infection. The recent discovery of antibody-mediated uptake and replication of ISAV in macrophage-like fish cell lines suggests that Fc receptor-mediated antibody-dependent enhancement of the ISA virus infection might also occur in vivo, as the virus in Atlantic salmon (Salmo salar) targets endothelial cells lining blood vessels and macrophage-like cells. Cumulative mortalities in Atlantic salmon during natural ISA outbreaks and experimental infections range from 0 to 100%. ISAV causes fatal systemic infections in marine-farmed Atlantic salmon and asymptomatic infections in feral fish. Experimentally induced fatal clinical disease in rainbow trout (Oncorhynchus mykiss) has identified a correlate of virulence of ISAV that may explain its emergence as a fish pathogen.

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

AbstactInfectious 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∆.

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

BackgroundHeart 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.MethodsSampling 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.ResultsThe 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.ConclusionsTo 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.

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

Abstract 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.

Infectious salmon anemia virus RNA in fish cell cultures and in tissue sections of Atlantic salmon experimentally infected with infectious salmon anemia virus

Current understanding of the etiopathogenesis of infectious salmon anemia (ISA) virus (ISAV) infection in fish comes mostly from virus detection in homogenized tissues taken from ISA-suspected mortalities. This study combined in situ hybridization (ISH) and histology to demonstrate viral RNA transcripts in different fish cell lines infected with ISAV and in tissues collected during the clinical phase of ISAV infection in Atlantic salmon. For this, a riboprobe to mRNA transcripts of ISAV RNA segment 8 was shown to detect viral mRNA in ISAV-infected TO, CHSE-214, and SHK-1 cell cultures. Specific hybridization was initially detected exclusively in the nuclei of infected cells, which is consistent with the nuclear transcription of orthomyxoviruses. For use of the riboprobe on fish tissues fixed in paraformaldehyde or formalin, the conditions used to permeabilize tissues before ISH (Proteinase K or Tween 20) were first optimized. Tissues were collected 15—20 days after challenge from 7 fresh mortalities of Atlantic salmon parr (∼20 g) showing severe gross and microscopic lesions, consistent with ISAV infection. Reverse transcription-polymerase chain reaction on tissue pools confirmed the presence of ISAV in each of the 7 fish. Of the tissues examined in each fish, the heart and liver consistently showed the strongest hybridization signal and, therefore, the most in situ virus, which was located in the endothelium of small blood vessels and in macrophage-like cells.