In silico identification of multiple conserved motifs within the control region of Culicidae mitogenomes.

Mosquitoes are important vectors for human and animal diseases. Genetic markers, like the mitochondrial COI gene, can facilitate the taxonomic classification of disease vectors, vector-borne disease surveillance, and prevention. Within the control region (CR) of the mitochondrial genome, there exists a highly variable and poorly studied non-coding AT-rich area that contains the origin of replication. Although the CR hypervariable region has been used for species differentiation of some animals, few studies have investigated the mosquito CR. In this study, we analyze the mosquito mitogenome CR sequences from 125 species and 17 genera. We discovered four conserved motifs located 80 to 230 bp upstream of the 12S rRNA gene. Two of these motifs were found within all 392 Anopheles (An.) CR sequences while the other two motifs were identified in all 37 Culex (Cx.) CR sequences. However, only 3 of the 304 non-Culicidae Dipteran mitogenome CR sequences contained these motifs. Interestingly, the short motif found in all 37 Culex sequences had poly-A and poly-T stretch of similar length that is predicted to form a stable hairpin. We show that supervised learning using the frequency chaos game representation of the CR can be used to differentiate mosquito genera from their dipteran relatives.

Distribution and Pathogenicity of Two Cutthroat Trout Virus (CTV) Genotypes in Canada.

The sole member of the Piscihepevirus genus (family Hepeviridae) is cutthroat trout virus (CTV) but recent metatranscriptomic studies have identified numerous fish hepevirus sequences including CTV-2. In the current study, viruses with sequences resembling both CTV and CTV-2 were isolated from salmonids in eastern and western Canada. Phylogenetic analysis of eight full genomes delineated the Canadian CTV isolates into two genotypes (CTV-1 and CTV-2) within the Piscihepevirus genus. Hepevirus genomes typically have three open reading frames but an ORF3 counterpart was not predicted in the Canadian CTV isolates. In vitro replication of a CTV-2 isolate produced cytopathic effects in the CHSE-214 cell line with similar amplification efficiency as CTV. Likewise, the morphology of the CTV-2 isolate resembled CTV, yet viral replication caused dilation of the endoplasmic reticulum lumen which was not previously observed. Controlled laboratory studies exposing sockeye (Oncorhynchus nerka), pink (O. gorbuscha), and chinook salmon (O. tshawytscha) to CTV-2 resulted in persistent infections without disease and mortality. Infected Atlantic salmon (Salmo salar) and chinook salmon served as hosts and potential reservoirs of CTV-2. The data presented herein provides the first in vitro and in vivo characterization of CTV-2 and reveals greater diversity of piscihepeviruses extending the known host range and geographic distribution of CTV viruses.

Transcriptome Analysis of Atlantic Salmon (Salmo salar) Skin in Response to Sea Lice and Infectious Salmon Anemia Virus Co-Infection Under Different Experimental Functional Diets.

Sea lice (Lepeophtheirus salmonis) are ectoparasitic copepods that cause significant economic loss in marine salmoniculture. In commercial salmon farms, infestation with sea lice can enhance susceptibility to other significant pathogens, such as the highly contagious infectious salmon anemia virus (ISAv). In this study, transcriptomic analysis was used to evaluate the impact of four experimental functional feeds (i.e. 0.3% EPA/DHA+high-ω6, 0.3% EPA/DHA+high-ω6+immunostimulant (IS), 1% EPA/DHA+high-ω6, and 1% EPA/DHA+high-ω3) on Atlantic salmon (Salmo salar) during a single infection with sea lice (L. salmonis) and a co-infection with sea lice and ISAv. The overall objectives were to compare the transcriptomic profiles of skin between lice infection alone with co-infection groups and assess differences in gene expression response among animals with different experimental diets. Atlantic salmon smolts were challenged with L. salmonis following a 28-day feeding trial. Fish were then challenged with ISAv at 18 days post-sea lice infection (dpi), and maintained on individual diets, to establish a co-infection model. Skin tissues sampled at 33 dpi were subjected to RNA-seq analysis. The co-infection's overall survival rates were between 37%-50%, while no mortality was observed in the single infection with lice. With regard to the infection status, 756 and 1303 consensus differentially expressed genes (DEGs) among the four diets were identified in "lice infection vs. pre-infection" and "co-infection vs. pre-infection" groups, respectively, that were shared between the four experimental diets. The co-infection groups (co-infection vs. pre-infection) included up-regulated genes associated with glycolysis, the interferon pathway, complement cascade activity, and heat shock protein family, while the down-regulated genes were related to antigen presentation and processing, T-cell activation, collagen formation, and extracellular matrix. Pathway enrichment analysis conducted between infected groups (lice infection vs. co-infection) resulted in several immune-related significant GO terms and pathways unique to this group, such as "autophagosome", "cytosolic DNA-sensing pathway" and "response to type I interferons". Understanding how experimental functional feeds can impact the host response and the trajectory of co-infections will be an essential step in identifying efficacious intervention strategies that account for the complexities of disease in open cage culture.

The Ecobiomics project: Advancing metagenomics assessment of soil health and freshwater quality in Canada.

Transformative advances in metagenomics are providing an unprecedented ability to characterize the enormous diversity of microorganisms and invertebrates sustaining soil health and water quality. These advances are enabling a better recognition of the ecological linkages between soil and water, and the biodiversity exchanges between these two reservoirs. They are also providing new perspectives for understanding microorganisms and invertebrates as part of interacting communities (i.e. microbiomes and zoobiomes), and considering plants, animals, and humans as holobionts comprised of their own cells as well as diverse microorganisms and invertebrates often acquired from soil and water. The Government of Canada's Genomics Research and Development Initiative (GRDI) launched the Ecobiomics Project to coordinate metagenomics capacity building across federal departments, and to apply metagenomics to better characterize microbial and invertebrate biodiversity for advancing environmental assessment, monitoring, and remediation activities. The Project has adopted standard methods for soil, water, and invertebrate sampling, collection and provenance of metadata, and nucleic acid extraction. High-throughput sequencing is located at a centralized sequencing facility. A centralized Bioinformatics Platform was established to enable a novel government-wide approach to harmonize metagenomics data collection, storage and bioinformatics analyses. Sixteen research projects were initiated under Soil Microbiome, Aquatic Microbiome, and Invertebrate Zoobiome Themes. Genomic observatories were established at long-term environmental monitoring sites for providing more comprehensive biodiversity reference points to assess environmental change.

In vivo virulence and genomic comparison of infectious Salmon Anaemia Virus isolates from Atlantic Canada.

The infectious salmon anaemia virus (ISAV) is capable of causing a significant disease in Atlantic salmon, which has resulted in considerable financial losses for salmon farmers around the world. Since the first detection of ISAV in Canada in 1996, it has been a high priority for aquatic animal health management and surveillance programmes have led to the identification of many genetically distinct ISAV isolates of variable virulence. In this study, we evaluated the virulence of three ISAV isolates detected in Atlantic Canada in 2012 by doing in vivo-controlled disease challenges with two sources of Atlantic salmon. We measured viral loads in fish tissues during the course of infection. Sequences of the full viral RNA genomes of these three ISAV isolates were obtained and compared to a high-virulence and previously characterized isolate detected in the Bay of Fundy in 2004, as well as a newly identified ISAV NA-HPR0 isolate. All three ISAV isolates studied were shown to be of low to mid-virulence with fish from source A having a lower mortality rate than fish from source B. Viral load estimation using an RT-qPCR assay targeting viral segment 8 showed a high degree of similarity between tissues. Through genomic comparison, we identified various amino acid substitutions unique to some isolates, including a stop codon in the segment 8 ORF2 not previously reported in ISAV, present in the isolate with the lowest observed virulence.

Performance characteristics of polymerase chain reaction and histological methods for the detection of Haplosporidium nelsoni in the eastern oyster (Crassostrea virginica).

Fitness for purpose and validation are increasingly becoming a benchmark in the development of test methods for the diagnosis of infectious diseases in aquatic animals. The design of the evaluation and the analysis of data are critical to demonstrate test method performance characteristics and fitness for purpose, as stated in the World Organization for Animal Health pathway for test validation. Three test methods for the detection of the oyster parasite Haplosporidium nelsoni were selected for the validation study described herein: histology, end-point polymerase chain reaction (PCR), and real-time PCR (qPCR). Preliminary work evaluated the analytical sensitivity and specificity of the PCR and qPCR assay in development. The following stage used test results on 100 oysters in 3 different laboratories to assess diagnostic sensitivity (DSe), diagnostic specificity (DSp), repeatability, and reproducibility. Repeatability and reproducibility were within 68-95%. The final part of the project evaluated DSe and DSp using test results on 400 oysters and results from the first 100 oysters tested. In the absence of a 100% gold standard test, latent class modeling methods were explored to characterize the tests (i.e., Bayesian analyses). For both PCR methods, DSe was >90%, and in the 60% range for histology, whereas DSp was >90% for all methods. Based on the results of this validation, a threshold cycle value of 30 for qPCR corresponds to the limit of sensitivity for histology where unreliable detection becomes more frequent, thus providing a threshold helpful in diagnostic settings where both histology and qPCR are used.

A modelling approach to predict the variation of repeatability and reproducibility of a RT-PCR assay for infectious salmon anaemia virus across infection prevalences and infection stages.

Conventional studies on the precision of diagnostic tests with binary outcomes report single descriptive estimates of agreement for a particular pool of samples. However, agreement for binary tests is intrinsically associated with the assay operating characteristics that are influenced by population and laboratory covariate factors. Therefore, reporting agreement estimates under various conditions may be more appropriate for diagnostic test comprehension. In this study, the influence of various submission factors (tissue sample homogenization, prevalence of infection and pathogen level) on agreement was further analyzed using test result information from a previous descriptive report of within and between laboratories agreement (repeatability and reproducibility, respectively) of a Reverse-Transcription Polymerase Chain Reaction (RT-PCR) assay for infectious salmon anaemia virus (ISAV). Multilevel logistic regression models were constructed separately for non-, low- or high-infected salmon (classified using a study pseudogold standard) to predict probabilities of testing positive under different testing conditions. For each of the 3 infection categories, agreement and kappa values within and between laboratories were computed from the models' predicted values using probability formulae. Thereafter, overall estimates were predicted using simple category weighting for various proportions of infection stages. Agreement varied substantially among infection categories and, consequently, overall repeatability and reproducibility varied greatly with prevalence. This confirmed that the report of a single descriptive estimate (corresponding to a set prevalence) may not be appropriate. Low-infected fish had the lowest agreement estimate which was improved by sample homogenization. This supported a heterogeneous distribution of ISAV in early infected salmon kidney. However, tissue homogenization increased the probability to obtain a false-positive test result (cross-contamination suspected) and decreased agreement in non-infected fish. Compared to conventional report of test agreement estimation, the modelling approach identified influencing submission factors and provided predictive intervals of agreement that give a better expectation and understanding of assay repeatability and reproducibility under different circumstances of use.

Use of a third class in latent class modelling for the diagnostic evaluation of five infectious salmon anaemia virus detection tests.

In the absence of a reference standard, a latent class model (LCM) was used in this study to assess diagnostic sensitivity (DSe) and specificity (DSp) of a recently developed reverse-transcription polymerase chain reaction (RT-PCR) for infectious salmon anaemia virus (ISAV). The study included 4 populations of Atlantic salmon, and to ensure the identifiability of the LCM, four additional detection methods were used in parallel including real-time RT-PCR (qRT-PCR), virus isolation (VI), indirect fluorescent antibody test (IFAT), and a lateral flow immunoassay (LFI). While a conventional LCM assumes DSe and DSp to be constant across the populations, Nérette et al. (2008) previously reported concerns about non-constant DSp of RT-PCR, which detects viral RNA from both active and inactive viral particles. It was suspected that some ISAV recovered fish may carry residual RNA and may be more likely to test positive compared to naïve fish. The various mixture distributions of the two sub-classes of non-infected fish would lead to a non-constant combined DSp estimate across populations. Within a Bayesian framework, the conventional two-class LCM was extended to three classes of infection stages (naïve non-infected, recovered non-infected carrying RNA, and infected). The resulting analysis confirmed the existence of three classes of fish with substantially different test performances for ISAV. For infected fish, DSe of RT-PCRs and VI approximated 90%, and antibody based assays were the least sensitive (DSe around 65%). Regardless of the test, the DSp estimates on naïve fish were all above 98% with LFI being in average the most specific. Only RT-PCR and qRT-PCR tested positive with the additional class of recovered fish (DSp around 30%). The true infectious status of this sub-class (i.e. viral RNA carriers) is debatable and requires further knowledge about ISAV infection dynamics at the fish level. Promising applications of multiple class estimates require adjustments of traditional test interpretation and further epidemiological knowledge of the infection dynamics at the population level.

Selection of a cutoff value for real-time polymerase chain reaction results to fit a diagnostic purpose: analytical and epidemiologic approaches.

Diagnostic laboratories frequently select a subjective cutoff value for real-time amplification assays, above which a threshold cycle (Ct) value is deemed false. Commonly, higher Ct values are interpreted as amplification or fluorescence artifacts, or cross contaminations. Although the implementation of Ct cutoff might be reasonable, its justification and selection should be based on evidence. The current article reviewed evidence-based strategies to select Ct cutoffs grouped in analytical and epidemiologic approaches. Analytical strategies use criteria gathered during the assay development and include fluorescence threshold, reaction end-cycle, limit of detection, and artifact investigation. Variability in amplification efficacy across test runs may induce some instability in an intended Ct cutoff and requires some standardization or normalization procedures. Epidemiologic strategies use criteria based on either the probability or the cost of a false test result associated with a specified cutoff. Cutoffs, depending on the intended purpose of the test, can be selected graphically to minimize the probability of either false-positive or false-negative results by using two-graph receiver operating characteristics curves. The assay's diagnostic sensitivity and specificity may vary with the tested population, thus, the estimated two-graph receiver operating characteristics curve is population dependent and should be established for the targeted population. Although the selection of a cutoff based on misclassification cost depends on infection prevalence, the selection based on predictive values does not. To optimize the test average diagnostic performance, the Ct cutoff should be selected when diagnostic odds ratio is maximal. Epidemiologic approaches were illustrated by selecting Ct cutoffs for a real-time assay for Infectious salmon anemia virus.

Traditional descriptive analysis and novel visual representation of diagnostic repeatability and reproducibility: application to an infectious salmon anaemia virus RT-PCR assay.

As a component of diagnostic test evaluation, the estimation of repeatability and reproducibility of an assay is necessary to assess the robustness and the transferability of the method among laboratories. Respectively defined as the agreement within and between laboratories, repeatability and reproducibility of a qualitative diagnostic test are traditionally reported using observed proportion of agreement or Kappa values. Applied to a recently designed RT-PCR assay for the detection of infectious salmon anaemia virus, repeatability only within a national reference laboratory and reproducibility with two additional independent regional laboratories were investigated. Homogenization of fish kidney tissue was conducted to potentially provide more uniform submission material, and to assess the effect of homogenization on laboratory comparability. Comparison of agreement between non-homogenized and homogenized tissue samples revealed different patterns of test results and unexpected alterations of agreement due to homogenization. This observation may be explained by cross-contamination of some samples during the homogenization process. One of the laboratories was in clear disagreement with the two others and impacted the overall reproducibility of the assay. Agreement levels were visually described using a novel tree-shape representation inspired from phylogenetic studies. The resulting phylogram illustrated the proximity of test findings between repeated samples within a laboratory and between laboratories, and facilitated the interpretation of the agreement levels.

Lack of evidence of infectious salmon anemia virus in pollock Pollachius virens cohabitating with infected farmed Atlantic salmon Salmo salar.

The infectious salmon anemia (ISA) virus causes lethargy, anemia, hemorrhage of the internal organs, and death in farmed Atlantic salmon Salmo salar. It has been a cause of disease in Norwegian farmed Atlantic salmon since 1984 and has since been identified in Canada, Scotland, the United States, and the Faroe Islands. Wild fish have been proposed as a viral reservoir because they are capable of close contact with farmed salmon. Laboratory studies have shown that brown trout and sea trout Salmo trutta, rainbow trout Oncorhynchus mykiss, and herring Clupea harengus tested positive for the virus weeks after intra-peritoneal injection of the ISA virus. Pollock Pollachius virens are commonly found in and around salmon cages, and their close association with the salmon makes them an important potential viral reservoir to consider. The objective of this study was to determine the presence or prevalence of ISA virus in pollock cohabitating with ISA-infected farmed Atlantic salmon. Kidney tissue from 93 pollock that were living with ISA-infected salmon in sea cages were tested with reverse transcription-polymerase chain reaction (RT-PCR) test. Results yielded the expected 193 bp product for positive controls, while no product was observed in any of the pollock samples, resulting in an ISA viral prevalence of 0%. This study strengthens the evidence that pollock are unlikely to be an ISA virus reservoir for farmed Atlantic salmon.