Quantifying Ostreid herpesvirus (OsHV-1) genome copies and expression during transmission

Understanding the pathogenic potential of a new pathogen strain or a known pathogen in a new locale is crucial for management of disease in both wild and farmed animals. The Ostreid herpesvirus-1 (OsHV-1), a known pathogen of early-life-stage Pacific oysters, Crassostrea gigas, has been associated with mortalities of juvenile oysters in many locations around the world including Tomales Bay, California. In two trials, the California OsHV-1 strain was transmitted from infected juvenile C. gigas to naïve C. gigas larvae. Survival of control larvae was high throughout both trials (97-100%) and low among those exposed to OsHV-1. No OsHV-1-exposed larvae survived to day 9 in trial 1, while trial 2 was terminated at day 7 when survival was 36.90 ± 8.66%. To assess the amount of OsHV-1 DNA present, we employed quantitative polymerase chain reaction (qPCR) assays based on the A fragment and OsHV-1 catalytic subunit of a DNA polymerase δ (DNA pol) gene. Viral genome copy numbers based on qPCR assays peaked between 3 and 5 days. To measure the presence of viable and actively transcribing virus, the DNA pol gene qPCR assay was optimized for RNA analysis after being reverse transcribed (RT-qPCR). A decline in virus gene expression was measured using RT-qPCR: relative to earlier experimental time points copy numbers were significantly lower on day 9, trial 1 (p < 0.05) and day 7, trial 2 (p < 0.05). Peaks in copies of active virus per genome occurred during two periods in trial 1 (days 1 and 5/7, p < 0.05) and one period in trial 2 (day 1, p < 0.05). Transmission electron microscopy confirmed OsHV-1 infection; herpesvirus-like nucleocapsids, capsids, and extracellular particles were visualized. We demonstrated the ability to transmit OsHV-1 from infected juvenile oysters to naïve larvae, which indicates the spread of OsHV-1 between infected hosts in the field and between commercial farms is possible. We also developed an important tool (OsHV-1-specific RT-qPCR for an active virus gene) for use in monitoring for active virus in the field and in laboratory based transmission experiments.