Sequence variation in ostreid herpesvirus 1 microvar isolates detected in dying and asymptomatic Crassostrea angulata adults in the Iberian Peninsula: Insights into viral origin and spread

The Portuguese oyster Crassostrea angulata was the main oyster species produced in Europe until the 1970s when mass mortalities almost led to its disappearance. At that time, mortality events were associated with detection of irido-like viruses and no record of herpes-like virus exists. Nowadays, C. angulata is only present in relatively few locations in the Iberian Peninsula, unlike in Asia where production is widespread. In 2011 and 2013, we observed mortality outbreaks of C. angulata adults collected in the Ria Formosa (Portugal) with a cumulative mortality of 47 and 59%, respectively, a few days after the oysters were transferred to indoor conditions. Ostreid herpesvirus 1 (OsHV-1) DNA was detected by PCR in all dying oysters analyzed and a high viral load was observed by real time PCR. Sequence analysis of ORFs 4, 42, 43 and 88 as well as a non-coding region near ORF4 revealed that the OsHV-1 genotype was the microvar (μvar) variant. No nucleotide sequence variation was observed in the regions analyzed in any of the 18 isolates. However, polymorphism was observed in a non-coding region (NC1/NC2 region) located between ORFs 49 and 50. Two genotypes in the NC1/NC2 region differing by several nucleotides were identified during the 2 mortality outbreaks and each genotype was unique to each mortality event. In addition, a third genotype in the NC1/NC2 region of OsHV-1 μvar was observed in asymptomatic adults of C. angulata collected in Carrera River (Spain). OsHV-1 DNA was not detected by PCR in any of the oysters collected in Sado and Mira estuaries. The results obtained in the present study suggest that the NC1/NC2 region may be a highly polymorphic region and hence of particular interest for molecular epidemiological studies. In light of the results of the present and previous studies on the genetic diversity and detection of OsHV-1 in C. angulata, we hypothesize that the virus was introduced recently in Europe from Asia, possibly with the introduction of C. gigas in the 1970s or soon after.