Whole-genome assembly of a novel invertebrate herpesvirus from the gastropod Babylonia areolata.

Molluscan herpesviruses cause disease in species of major importance to aquaculture and are the only known herpesviruses to infect invertebrates, which lack an adaptive immune system. Understanding the evolution of malacoherpesviruses in relation to their hosts will likely require comparative genomic studies on multiple phylogenetic scales. Currently, only two malacoherpesvirus species have genomes that have been fully assembled, which limits the ability to perform comparative genomic studies on this family of viruses. In the present study, we fully assemble a herpesvirus from Illumina and Nanopore sequence data that were previously used to assemble the genome of the gastropod Babylonia areolata. We tentatively assign this novel herpesvirus to the genus Aurivirus within the family Malacoherpesviridae based on a phylogenetic analysis of DNA polymerase. While structurally similar to other malacoherpesvirus genomes, a synteny analysis of the novel herpesvirus with another Aurivirus species indicates that genomic rearrangements might be an important process in the evolution of this genus. We anticipate that future complete assemblies of malacoherpesviruses will be a valuable resource in comparative herpesvirus research.

No trade-off between growth and OsHV-1 tolerance in Pacific oysters (Crassostrea gigas) farmed on the West Coast, USA.

An important pathogen of concern for Pacific oyster growers in the USA is ostreid herpesvirus 1 (OsHV-1). Currently, oyster stocks exist that are tolerant to OsHV-1; however, it is uncertain if a trade-off exists between their tolerance to OsHV-1 and their growth in different environments. To investigate any potential trade-offs, Pacific oyster families with varying levels of OsHV-1 tolerance were grown in a bay where OsHV-1 is endemic (Tomales Bay, CA) and in a bay where OsHV-1 is absent (Willapa Bay, WA). In Tomales Bay, we found that oysters from OsHV-1 tolerant families grew faster than oysters from OsHV-1 susceptible families, while in Willapa Bay, no statistically significant difference in growth was found between oyster families with different levels of OsHV-1 tolerance observed in Tomales Bay. These findings indicate that Pacific oysters bred to be tolerant to OsHV-1 would not be expected to have a longer time-to-market regardless of the presence of OsHV-1 in the growing environment.

Ostreid herpesvirus 1 latent infection and reactivation in adult Pacific oysters, Crassostrea gigas.

Ostreid herpesvirus 1 (OsHV-1) is one of the most economically important pathogens of Pacific oysters. Understanding the pathogenesis of this virus is critical to developing tools to control outbreaks on shellfish farms. OsHV-1 is genetically related to vertebrate herpesviruses, which have a lytic and a latent stage, with the latent stage capable of being reactivated to the lytic stage. Here, OsHV-1 latency in Pacific oysters was investigated in experimentally and naturally infected oysters. Lytic infection in one-year-old oysters injected with the Tomales Bay strain of OsHV-1 was detectable between 1 and 4 days post-injection (dpi) but was not detectable after 5 dpi. The injected oysters shed 1 × 102 to 1 × 104 DNA copies/ml into the water during the 4-day acute phase. Lytic shedding was not detectable in two-year-old oysters injected similarly with the same strain of OsHV-1; however, the OsHV-1 genome was detectable by qPCR in the adductor muscle, gill, mantle, and hemocytes within the first 3 dpi, after which it became undetectable. No OsHV-1 was detectable in the adductor muscle, gill, or mantle from experimentally infected oysters on days 15 and 21 post-injection or from oysters sampled 9 months after surviving an OsHV-1 mortality event; however, OsHV-1 DNA could be detected in hemocytes of both experimentally infected oysters at 21 dpi and naturally infected oysters using nested PCR. In addition, lytic viral gene transcription was detectable in hemocytes of experimentally infected oysters between 1 and 21 dpi and in hemocytes of naturally infected oysters. Furthermore, OsHV-1 reactivation from latency was induced in experimentally infected oysters at 21 dpi and in naturally infected oysters 12 months after an OsHV-1 outbreak.

Genome-wide allele frequency studies in Pacific oyster families identify candidate genes for tolerance to ostreid herpesvirus 1 (OsHV-1).

BACKGROUND: Host genetics influences the development of infectious diseases in many agricultural animal species. Identifying genes associated with disease development has the potential to make selective breeding for disease tolerance more likely to succeed through the selection of different genes in diverse signaling pathways. In this study, four families of Pacific oysters (Crassostrea gigas) were identified to be segregating for a quantitative trait locus (QTL) on chromosome 8. This QTL was previously found to be associated with basal antiviral gene expression and survival to ostreid herpesvirus 1 (OsHV-1) mortality events in Tomales Bay, California. Individuals from these four families were phenotyped and genotyped in an attempt to find candidate genes associated with the QTL on chromosome 8. RESULTS: Genome-wide allele frequencies of oysters from each family prior to being planting in Tomales Bay were compared with the allele frequencies of oysters from respective families that survived an OsHV-1 mortality event. Six significant unique QTL were identified in two families in these genome-wide allele frequency studies, all of which were located on chromosome 8. Three QTL were assigned to candidate genes (ABCA1, PIK3R1, and WBP2) that have been previously associated with antiviral innate immunity in vertebrates. CONCLUSION: The identification of vertebrate antiviral innate immunity genes as candidate genes involved in molluscan antiviral innate immunity reinforces the similarities between the innate immune systems of these two groups. Causal variant identification in these candidate genes will enable future functional studies of these genes in an effort to better understand their antiviral modes of action.

Evaluation of Histone Demethylase Inhibitor ML324 and Acyclovir against Cyprinid herpesvirus 3 Infection.

Cyprinid herpesvirus 3 (CyHV-3) can cause severe disease in koi and common carp (Cyprinus carpio). Currently, no effective treatment is available against CyHV-3 infection in koi. Both LSD1 and JMJD2 are histone demethylases (HD) and are critical for immediate-early (IE) gene activation essential for lytic herpesvirus replication. OG-L002 and ML324 are newly discovered specific inhibitors of LSD1 and JMJD2, respectively. Here, HD inhibitors were compared with acyclovir (ACV) against CyHV-3 infection in vitro and in vivo. ML324, at 20-50 µM, can completely block ~1 × 103 PFU CyHV-3 replication in vitro, while OG-L002 at 20 µM and 50 µM can produce 96% and 98% inhibition, respectively. Only about 94% inhibition of ~1 × 103 PFU CyHV-3 replication was observed in cells treated with ACV at 50 µM. As expected, CyHV-3 IE gene transcription of ORF139 and ORF155 was blocked within 72 h post-infection (hpi) in the presence of 20 µM ML324. No detectable cytotoxicity was observed in KF-1 or CCB cells treated for 24 h with 1 to 50 µM ML324. A significant reduction of CyHV-3 replication was observed in ~6-month-old infected koi treated with 20 µM ML324 in an immersion bath for 3-4 h at 1-, 3-, and 5-days post-infection compared to the control and ACV treatments. Under heat stress, 50-70% of 3-4-month-old koi survived CyHV-3 infection when they were treated daily with 20 µM ML324 in an immersion bath for 3-4 h within the first 5 d post-infection (dpi), compared to 11-19% and 22-27% of koi in the control and ACV treatments, respectively. Our study demonstrates that ML324 has the potential to be used against CyHV-3 infection in koi.

Differential Mortality and High Viral Load in Naive Pacific Oyster Families Exposed to OsHV-1 Suggests Tolerance Rather than Resistance to Infection.

Pacific oysters, Crassostrea gigas, are one of the most productive aquaculture species in the world. However, they are threatened by the spread of Ostreid herpesvirus-1 (OsHV-1) and its microvariants (collectively "µvars"), which cause mass mortalities in all life stages of Pacific oysters globally. Breeding programs have been successful in reducing mortality due to OsHV-1 variants following viral outbreaks; however, an OsHV-1-resistant oyster line does not yet exist in the United States (US), and it is unknown how OsHV-1 µvars will affect US oyster populations compared to the current variant, which is similar to the OsHV-1 reference, found in Tomales Bay, CA. The goals of this study were to investigate the resistance of C. gigas juveniles produced by the Molluscan Broodstock Program (MBP) to three variants of OsHV-1: a California reference OsHV-1, an Australian µvar, and a French µvar. This is the first study to directly compare OsHV-1 µvars to a non-µvar. The survival probability of oysters exposed to the French (FRA) or Australian (AUS) µvar was significantly lower (43% and 71%, respectively) than to the reference variant and controls (96%). No oyster family demonstrated resistance to all three OsHV-1 variants, and many surviving oysters contained high copy numbers of viral DNA (mean ~3.53 × 108). These results indicate that the introduction of OsHV-1 µvars could have substantial effects on US Pacific oyster aquaculture if truly resistant lines are not achieved, and highlight the need to consider resistance to infection in addition to survival as traits in breeding programs to reduce the risk of the spread of OsHV-1 variants.

First evaluation of resistance to both a California OsHV-1 variant and a French OsHV-1 microvariant in Pacific oysters.

BACKGROUND: Variants of the Ostreid herpesvirus 1 (OsHV-1) cause high losses of Pacific oysters globally, including in Tomales Bay, California, USA. A suite of new variants, the OsHV-1 microvariants (µvars), cause very high mortalities of Pacific oysters in major oyster-growing regions outside of the United States. There are currently no known Pacific oysters in the United States that are resistant to OsHV-1 as resistance has yet to be evaluated in these oysters. As part of an effort to begin genetic selection for resistance to OsHV-1, 71 families from the Molluscan Broodstock Program, a US West Coast Pacific oyster breeding program, were screened for survival after exposure to OsHV-1 in Tomales Bay. They were also tested in a quarantine laboratory in France where they were exposed to a French OsHV-1 microvariant using a plate assay, with survival recorded from three to seven days post-infection. RESULTS: Significant heritability for survival were found for all time points in the plate assay and in the survival phenotype from a single mortality count in Tomales Bay. Genetic correlations between survival against the French OsHV-1 µvar in the plate assay and the Tomales Bay variant in the field trait were weak or non-significant. CONCLUSIONS: Future breeding efforts will seek to validate the potential of genetic improvement for survival to OsHV-1 through selection using the Molluscan Broodstock Program oysters. The lack of a strong correlation in survival between OsHV-1 variants under this study's exposure conditions may require independent selection pressure for survival to each variant in order to make simultaneous genetic gains in resistance.

Single and multiple phenotype QTL analyses of downy mildew resistance in interspecific grapevines.

KEY MESSAGE: Downy mildew resistance across days post-inoculation, experiments, and years in two interspecific grapevine F1 families was investigated using linear mixed models and Bayesian networks, and five new QTL were identified. Breeding grapevines for downy mildew disease resistance has traditionally relied on qualitative gene resistance, which can be overcome by pathogen evolution. Analyzing two interspecific F1 families, both having ancestry derived from Vitis vinifera and wild North American Vitis species, across 2 years and multiple experiments, we found multiple loci associated with downy mildew sporulation and hypersensitive response in both families using a single phenotype model. The loci explained between 7 and 17% of the variance for either phenotype, suggesting a complex genetic architecture for these traits in the two families studied. For two loci, we used RNA-Seq to detect differentially transcribed genes and found that the candidate genes at these loci were likely not NBS-LRR genes. Additionally, using a multiple phenotype Bayesian network analysis, we found effects between the leaf trichome density, hypersensitive response, and sporulation phenotypes. Moderate-high heritabilities were found for all three phenotypes, suggesting that selection for downy mildew resistance is an achievable goal by breeding for either physical- or non-physical-based resistance mechanisms, with the combination of the two possibly providing durable resistance.

Computer Vision for High-Throughput Quantitative Phenotyping: A Case Study of Grapevine Downy Mildew Sporulation and Leaf Trichomes.

Quantitative phenotyping of downy mildew sporulation is frequently used in plant breeding and genetic studies, as well as in studies focused on pathogen biology such as chemical efficacy trials. In these scenarios, phenotyping a large number of genotypes or treatments can be advantageous but is often limited by time and cost. We present a novel computational pipeline dedicated to estimating the percent area of downy mildew sporulation from images of inoculated grapevine leaf discs in a manner that is time and cost efficient. The pipeline was tested on images from leaf disc assay experiments involving two F1 grapevine families, one that had glabrous leaves (Vitis rupestris B38 × 'Horizon' [RH]) and another that had leaf trichomes (Horizon × V. cinerea B9 [HC]). Correlations between computer vision and manual visual ratings reached 0.89 in the RH family and 0.43 in the HC family. Additionally, we were able to use the computer vision system prior to sporulation to measure the percent leaf trichome area. We estimate that an experienced rater scoring sporulation would spend at least 90% less time using the computer vision system compared with the manual visual method. This will allow more treatments to be phenotyped in order to better understand the genetic architecture of downy mildew resistance and of leaf trichome density. We anticipate that this computer vision system will find applications in other pathosystems or traits where responses can be imaged with sufficient contrast from the background.