Frederick S. B. 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.

Nucleotide sequence analysis of genome segment A of infectious bursal disease virus

The nucleotide sequence of genome segment A cDNA of the STC strain of infectious bursal disease virus (IBDV) was determined and compared with sequences of the homologous genome segment of the 002-73 strain of IBDV and the Jasper strain of infectious pancreatic necrosis virus (IPNV). The STC-IBDV genome segment A was determined to be 3262 base pairs (bp), which is close to the estimated total length of 3300 bp for genome segment A in IBDV, although there is no proof that it is the real length of this genome segment. The STC-IBDV genome segment A contains two major overlapping open reading frames (ORFs). The large ORF of 3036 bp predicts a polyprotein of Mr 109,358, whereas the small ORF is 435 bp and predicts a protein of Mr 16,550 in STC-IBDV. STC-IBDV and 002-73-IBDV polyproteins are closely related (97.4% amino acid homology). Most of the amino acid mismatches are in VP2 sequences, mainly within the area of the conformation-dependent epitope. Comparison with the Jasper-IPNV polyprotein reveals levels of amino acid sequence homology of about 40% in VP2, 32% in VP3 and 21% in VP4. Within the VP2 molecule the conformation-dependent epitope area is again the least homologous, but the heterogeneity is more conspicuous than between the two IBDV strains, which is not surprising since IBDV and IPNV are serologically unrelated. The small ORF proteins have about 88% amino acid sequence homology between STC-IBDV and 002-73-IBDV, and 30% between each IBDV strain and Jasper-IPNV. There is no homology at all in the non-coding regions of IBDV and IPNV. These comparative sequence data will be useful for subgrouping the Birnaviridae family.

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) 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

BackgroundInfectious 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, comparision 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.ResultsRT-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.ConclusionTaken 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.

Characterization of Salmonella isolates from beef cattle, broiler chickens and human sources on Prince Edward Island

Non-typhoid Salmonella serovars remain a potential threat to human health, and beef cattle and broiler chickens are possible sources of these organisms on Prince Edward Island (PEI). In this study, the ceca of beef cattle belonging to fasted and non-fasted groups, and broiler chickens were examined for Salmonella at the time of slaughter. The characteristics of the isolates, including antimicrobial resistance patterns and virulence genes, were studied along with the isolates obtained from cases of human salmonellosis on PEI during the study period (1996-97). The prevalence of Salmonella in beef cattle was 4.6% (11/240). The rate was significantly higher in fasted cattle (7.46%), than in non-fasted cattle (0.94%). The prevalence rate in chickens was 32.5% (39/120). In beef cattle, Salmonella typhimurium phage type (PT) or definitive type (DT) 104 which was resistant to ampicillin, chloramphenicol, streptomycin, sulfisoxazole and tetracycline, was the most predominant type (64%). In chickens, S. heidelberg, with resistance to gentamicin, streptomycin and sulfisoxazole, predominated. Of 26 isolates from humans, the most common serovar was S. typhimurium, including a multidrug-resistant strain of DT104. Examination by PCR revealed presence of the virulence gene invA in all serovars, and the spvC gene in all S. typhimurium isolates, of both beef cattle and human origin. Among the other serovars the latter gene was found in 7 human isolates, but in none of the chicken or beef isolates. All but 3 of the spvC-positive isolates possessed a 90 kilobasepair (kbp) plasmid suggesting that the 3 isolates had the spvC gene on their chromosome. These findings were confirmed by plasmid DNA isolation using 3 different protocols and by sequence analysis of the spvC-PCR product.

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.

Constitutive expression of Atlantic salmon Mx1 protein in CHSE-214 cells confers resistance to Infectious Salmon Anaemia virus

Infectious salmon anaemia (ISA) is a highly fatal viral disease affecting marine-farmed Atlantic salmon which is caused by ISA virus (ISAV), a fish orthomyxovirus that has recently been assigned to the new genus Isavirus within the family Orthomyxoviridae. Mx proteins are among the interferon (IFN)-induced proteins responsible for the development of an antiviral state in vertebrate cells. We used real-time reverse transcription-polymerase chain reaction (RT-PCR) and Chinook salmon embryo (CHSE-214) cells constitutively expressing Atlantic salmon Mx1 protein (ASMx1) to examine the antiviral properties of ASMx1 against two ISAV strains, NBISA01 and HKS-36, having phenotypically different growth properties (cytopathic vs non-cytopathic) in the CHSE-214 cell line. We present evidence that ISAV is sensitive to ASMx1. CHSE-214 cells constitutively expressing ASMx1 showed increased resistance to infection with the cytopathic ISAV strain NBISA01, manifested as delayed development of cytopathic effects (CPE) and significant reduction in the severity of CPE, as well as a 10-fold reduction in virus yield. However, by real-time RT-PCR we observed no significant difference in the mean threshold cycle (Ct) values of ISAV RNA levels, suggesting that the ASMx1 activity on ISAV occurs at the post-transcription steps of virus replication, possibly in the cytoplasm.