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The Role of a Secondary Host the Mussel Mytilus spp. in the Transmission of OsHV-1 µVar

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The role of a secondary host the mussel Mytilus spp. in the transmission of OsHV-1 µVar
The Pacific oyster Crassostrea gigas contributes significantly to global aquaculture, however, C. gigas culture has been affected by ostreid herpesvirus-1 (OsHV-1) and variants. The dynamics of how the virus maintains itself at culture sites is unclear and the role of carriers, reservoirs or hosts living in close proximity to C. gigas is unknown. Both wild and cultured mussels Mytilus spp. (Mytilus edulis, Mytilus galloprovincialis and hybrids) are commonly found at C. gigas culture sites. The objective of this study was to investigate if Mytilus spp. can retain the virus and if viral transmission can occur between mussels and oysters. Mytilus spp. living at oyster trestles, 400 – 500m from the trestles and up to 26 km at non culture sites were screened for OsHV-1 μVar by all recommended diagnostic methods. OsHV-1 μVar was detected in wild Mytilus spp. at C. gigas culture sites and more significantly the virus was detected in mussels at non-culture sites. Cohabitation of exposed wild mussels and naïve C. gigas resulted in viral transmission after 14 days, under an elevated temperature regime. These results indicate that mussels act as a carrier of OsHV-1 μVar, however the impact of OsHV-1 μVar on Mytilus spp. requires further investigation.
Key words: Interspecies transmission Cohabitation Temperature Herpesvirus
Mytilus spp.  C. gigas

  • OsHV-1 μVar detected in wild Mytilus spp. at  Pacific oyster culture sites and in mussels at non-culture sites.
  • Interspecies transmission of the virus occurred between mussels naturally exposed to the virus at culture sites and naïve oysters held under an elevated temperature regime in the laboratory.
  • The impact/s of OsHV-1 μVar on Mytilus spp. requires further study.

1.  Introduction
Both wild and cultured mussels Mytilus edulis, Mytilus galloprovincialis and hybrids of both species are abundantly found along most of the Irish coastline (Lynch et al. 2014). One of the main commercial shellfish species cultivated in Ireland is Mytilus edulis L. (Fernández et al. 2015); this species along with the Pacific oyster Crassostrea gigas, has increased in economic importance in recent years. Both wild and cultured mussel stocks are often located in close proximity to cultured C. gigas stocks.  Ostreid herpes virus-1 (OsHV-1) and variants such as OsHV-1 var and OsHV-1 μVar have been associated with C. gigas mortalities in several countries worldwide, including France (Segarra et al. 2010), Australia (Jenkins et al. 2013) Italy (Dundon et al. 2011) and Ireland (Peeler et al. 2012), with an additional Irish genotype also being identified (Lynch et al. 2012). Included in the order Herpesvirales, ostreid herpesvirus-1 (OsHV-1) belongs to the family Malacoherpesviridae, the genus Ostrea virus containing the species ostreid herpesvirus-1 (Davison et al. 2009). The herpesvirus combined with suboptimal environmental factors forms the basis of high economic risk for Pacific oyster culture (Paredes et al. 2013).
Herpes like virus infection has been observed in bivalve species such as the flat oyster Tiostrea chilensis in New Zealand (Hine et al. 1998); Ostrea angasi in Australia (Hine & Thorne 1997); European flat oyster, Ostrea edulis in France (Comps and Cochennec, 1993; Renault et al. 2000a); Manila clam, Ruditapes philippinarum (Renault, 1998, Renault et al. 2001 and Renault and Arzul, 2001); Scallop (Arzul et al. 2001) and Abalone Haliotis diversicolor supertexta (Chang et al. 2005; Tan et al. 2008). Recently OsHV-1 μVar DNA was detected in wild Mediterranean mussels Mytilus galloprovinciallis in California and in cultured M. galloprovinciallis in Italy (Burge et al. 2011; Domeneghetti et al. 2014). More recently OsHV-1 μVar DNA was detected in the Sydney rock oyster Saccostrea glomerata, Sydney cockle Anadara trapezia, blue mussels Mytilus spp., hairy mussel Trichomya hirsuta, whelks Batillaria australis and barnacles Balanus spp. in Australia (Evans et al. 2017).  Filter feeding bivalves such as mussels, oysters and clams may typically harbour various pathogenic organisms such as Herpes and enteric viruses (Venier et al. 2014) as bivalves process large volumes of water during feeding thus maximizing their exposure (Witte et al. 2014). A host that becomes infected, but is not required for the maintenance of the population of a pathogen, can be termed an incidental host (Ashford, 2003). Haydon et al. (2002) defined a reservoir host as ‘one or more epidemiologically connected populations or environments in which the pathogen can be permanently maintained and from which infection is transmitted to the defined target population’. A viral reservoir has been defined ‘as a cell type in which a replication-competent form of the virus accumulates and persists with more stable properties than the main pool of actively replicating virus’ (Blankson et al. 2002).  Reservoirs of infection can be comprised of one or more interacting species (Haydon et al. 2002 cited in Viana et al. 2014).
Recent studies suggest that opportunistic pathogens are associated with Mytilus spp. (Eggermont et al. 2014; Lynch et al. 2014), while climate change and associated stressors may further impact on host: parasite dynamics, and on Mytilus spp. susceptibility to pathogens (Lynch et al. 2014).  Burge et al. (2011) quantified OsHV-1 viral loads in C. gigas and wild M. galloprovincialis in California and viral copy numbers of OsHV-qPCR in positive individuals observed were quite low (mean ± SE; range) in C. gigas (20.31 ± 12.40; 0.005 to 114.4) and in M. galloprovincialis (0.0073 ± 0.006; 0.0005 to 0.025) (Burge et al. 2011).  Domeneghetti et al. (2014) reported that OsHV-1 μVar in C. gigas and M. galloprovinciallis, co-cultured in an Italian lagoon with seawater temperatures of 17.6C, had lower levels of viral DNA in the asymptomatic mussels (Domeneghetti et al. 2014).  Although OsHV-1 μVar DNA was detected in M. galloprovinicialis in both of those studies, transmission of the virus from mussels to oysters and vice versa was not investigated.
Elucidating the role of mussels in viral maintenance and transmission i.e. their ability to act as carriers, reservoirs or incidental hosts, is an important component in understanding viral dynamics. Pathogens maybe transported by water currents naturally or by vector species from areas of infection in adjacent territories. The direct relationship between viral proliferation, transmission and environmental stressors, has been demonstrated particularly temperature influence and increasing temperatures associated with oyster mortality, in field studies (Garcia et al. 2011) and under experimental conditions (Sauvage et al. 2009; Petton et al. 2013; Pernet et al. 2012, 2015; Martenot et al. 2015). C. gigas has a broad temperature tolerance, with a range of –1.8 to 35°C (FAO, 2015). One of the major triggering factors of disease epizootics is temperature, especially for aquatic species (Petton et al. 2013). Oyster mortality events have followed increases in seawater temperature to 19°C (Pernet et al. 2010) with OsHV-1 μVar being persistent in oysters held at a low temperature of 13°C and being reactivated during thermal elevation to 21°C.  In the Pernet et al. (2010) study, low temperature treatments did not improve overall survival of oyster seed infected with OsHV-1 μVar, suggesting that introducing infected oysters into an area of reduced temperature may delay mortality and increase the risk of infection in neighboring stocks when rising temperatures facilitate viral replication (Pernet et al. 2015).
The study of viruses and the role of reservoirs is fundamental for a better understanding of the intrinsic relationship between the virus and host (Appolinário et al. 2015). The objectives of this study were (a) to determine if OsHV-1 μVar was present in Irish wild Mytilus spp. living in close proximity to oyster trestles, approximately 400 m to 500 m from the oyster culture site and up to 26 km from the oyster farms; (b) investigate virus development and mussel and oyster performance under an elevated temperature regime in the laboratory and (c) establish if the virus could be transmitted between naturally exposed mussels and naïve C. gigas under different temperature regimes.  Findings from this study will contribute to a more comprehensive understanding of the underlying relationship between viral proliferation among cultivated oysters and wild mussel species and provide an insight into the interspecific species interactions and transmission that occurs in a marine environment.
2.  Materials and Methods:
2.1 Study sites and field sampling
As the sites used in the study contain a mixture of Mytilus edulis, Mytilus galloprovincialis and hybrids mussels, (with the exception of Carlingford Lough where M. edulis is exclusively found), all the following descriptions refer to Mytilus species (spp.). Wild mussels from both culture and non-culture (control) sites were screened.
2.1.1 Field survey 1
The first field survey, to determine if OsHV-1 μVar could be detected in mussels from hybrid zones (Mytilus edulis, Mytilus galloprovincialis and hybrids of both parent species) in Ireland took place in 2014 with (a) a single mussel sample (n=30) being collected in late June from Goat Island, approximately 5 km west of Ardmore, County Waterford (51° 57’04” N, 7° 43’23” W) and approximately 26 km from a OsHV-1 μVar endemic C. gigas culture site, (b) a single mussel sample (n=250) being collected in July from Garrettstown, Kinsale, Co. Cork (51° 38’16.93” N, 8° 34’21.41” W), which is approximately 22 km from a OsHV-1 μVar endemic C. gigas culture site from which (n=30) mussels were initially screened. An additional Garrettstown mussel sample (n=30) was screened after the mussels collected in July were held in the laboratory for three weeks and held at 13°C and (c) several mussel samples were collected in July on the south east coast of Ireland at Ballymacoda Bay, Youghal Co. Cork (51° 53′ 24″ N, 7° 55′ 57″ W).  At that site, wild mussels were sampled at a rocky outcrop on the mid to lower intertidal approximately 400 m from a C. gigas OsHV-1 μVar designated culture site, a site where significant oyster mortalities occurred in 2010 (  An initial sample (n=42) of adult sized mussels Mytilus spp. was collected in mid-July and a second random sample (n=167) of Mytilus spp. was collected in late-July.
2.1.2 Field survey 2
Further sampling and screening of mussels took place as part of a second larger field study between 2013-2015 at two C. gigas culture sites: (d) Carlingford Lough (54.0733° N, 6.1994° W), on the east coast of Ireland in the Irish Sea and (e) Dungarvan Harbour (52°03’ N, 007°35’ W), on the south east coast in the Celtic Sea. OsHV-1 μVar  is endemic at both sites since 2009 (  Dungarvan is a known hybrid zone, while only M. edulis is found at Carlingford Lough.  At both culture sites, mussels were collected from the C. gigas PVC culture bags at the oyster trestles on the mid to lower intertidal and approximately 500 m from the trestles at the high shore. At Carlingford Lough, sampling took place on nine occasions, every two weeks from July to October in 2013 and on eight occasions, every two weeks from April to August in 2015. Similarly, at Dungarvan Harbour, sampling took place on nine occasions, every 2 weeks from April to August in 2015. All mussel samples were returned to the laboratory and processed immediately for histology and PCR analysis to screen for OsHV-1 µVar.
2.2. Laboratory trials
Naïve Pacific oyster spat (<3 months n=600; less than 1-year-old) were obtained from a hatchery at New Quay, Galway Bay (53°09´16.27´´N, 9°04´58.19´´W). Oysters from this hatchery have been used as control oysters in previous studies as the location is deemed uninfected with the herpes virus OsHV-1µvar and variants ( C. gigas spat were kept in a holding tank (50L) in a constant temperature room (~14°C) at a salinity of 35 for 2-3 days until the laboratory cohabitation trials began.  The mussel stocks used in the laboratory transmission trials comprised of Mytilus spp. from Ballymacoda Bay (Trial 1) and M. edulis from Carlingford Lough (Trial 2).
2.2.1 Laboratory cohabitation transmission Trial 1: naïve C. gigas and Mytilus spp. from a OsHV-1 μVar endemic culture site held together at 21ºC to determine if the virus can be transmitted between the two species
The combined effects of an elevated seawater temperature (21ºC) on oyster and mussel performance and OsHV-1 μVar pathogenicity were investigated in a laboratory transmission trial. A total of 3 x 10L stand-alone control tanks, each containing 40 naïve Galway Bay C. gigas, and 3 experimental (10L) tanks, each containing 40 naïve Galway Bay C. gigas and 40 randomly sampled wild Mytilus spp. obtained from the Ballymacoda site, which would have experienced a mean seawater temperature in the field of approximately 16ºC were used. The control and experimental tanks were placed in a constant temperature (CT) room at 21ºC and a salinity of 35. The trial ran for 7 days and the tanks were checked several times daily for moribund mussels and oysters, which were removed immediately, processed and screened for OsHV-1 μVar.
2.2.2 Laboratory cohabitation transmission Trial 2: naïve C. gigas and exposed Mytilus spp. from a OsHV-1 μVar endemic culture site held under an increasing temperature regimes to determine if transmission could occur between the two species.
The combined effects of increasing seawater temperature (14ºC, 21ºC and 28ºC), at a salinity of 35 on oyster and mussel performance and OsHV-1 μVar pathogenicity was investigated in a laboratory trial with wild M. edulis collected from a OsHV-1 μVar endemic oyster culture site and hatchery reared naïve C. gigas spat. The M. edulis were randomly collected from in, and around the oyster trestles during the seasonal field sampling conducted at Carlingford Lough in the summer of 2015 (Section 2.1). Two control tanks, each containing 30 naïve Irish hatchery C. gigas, and 3 experimental tanks, each containing 30 naïve hatchery C. gigas and 30 exposed M. edulis were used. The control tanks and experimental tanks were placed in two separate constant temperature (CT) rooms to avoid aerosol contamination. The temperature was initially set in both CT rooms at 14ºC (Day1-14); increased to 21ºC (Day 15-20) and subsequently increased to 28ºC (Day 21-29) in both CT rooms.
The trial ran for 29 days and the tanks were checked several times daily for moribund or dead oysters and mussels, which were removed and subsequently screened for OsHV-1 μVar.  All moribund or dead individuals were screened over the trial and the remaining living oysters and mussels were also screened in both the experimental and control tanks at the end of the trial. A sampling regime of two oysters per tank per day was carried out in the experimental tanks from Days 2-7. Screening recommenced in both mussels and oysters at Day 13 to the end of the trial on Day 29 with more randomised screening of approximately 6 mussels or 6 oysters being sampled every other day providing an additional 72 living oysters and 71 living mussels from the experimental tanks and 44 living oysters from the control tank being screened. Processing of animals from each CT room was carried out in separate laboratories to avoid cross contamination.
2.6 Molecular diagnostic screening of Mytilus spp. and C. gigas
2.6.1 DNA extraction and polymerase chain reaction (PCR)
Mytilus spp. sampled during the first field study (2014) at (a) Ardmore (n=30) and (b) Garrettstown (n=60) were screened by PCR for the herpes virus using DNA extracted from gill tissue. From the initial sample (n=42) collected at (c) Ballymacoda, a subsample of 12 mussels were randomly screened by PCR using gill tissue, haemolymph and shell cavity fluid from each individual.   DNA was extracted using the Chelex-100 method (Walsh et al. 1991). All Mytilus spp. sampled during the second field study (2013-2015) at (d) Carlingford Lough and (e) Dungarvan Harbour were screened by PCR for the herpes virus (n=646) using gill tissue. This screening comprised of 45 Mytilus spp. individuals in 2013 and 601 Mytilus spp. in 2015 with 240 in total from Carlingford lough and 361 from Dungarvan Harbour. Gill tissue (5mm2) was excised and DNA extraction (Qiagen Blood and Tissue kit) was carried out. Mytilus spp. were also screened by PCR for the herpes virus during the laboratory cohabitation transmission trials Trial 1 (n=42) and Trial 2 (n=90).
PCRs (Renault et al. 2000b) using different primer pairs (OHVA/OHVB, OHVC/OHVD) (Lynch et al. 2013) were used on all of the mussels sampled from the field sites.  Expected amplified PCR products were 385-bp and 296-bp respectively.  Negative controls consisted of deionized distilled water and positive controls were OsHV-1 μVar DNA.  DNA was visualized on a 2% agarose gel and was quantified and the quality checked, using a spectrophotometer (Thermo- Scientific NanoDrop 1000 spectrophotometer).
2.6.2 Quantitative polymerase chain reaction (qPCR)
A subsample of PCR positive results observed in the mussels from both the second field trial (n=7) and experimental laboratory cohabitation transmission trials (n=7) were rescreened by quantative real-time (qPCR) to detect the viral load and mean quantification (range min-max) using HVDP-F and HVDP-R primers (Pepin et al. 2008), which amplify a 197-bp fragment. All samples were tested in duplicate. Negative controls consisting of deionized distilled water were included in the assay.
2.6.3 Sequencing
Direct Sanger sequencing on PCR products (385-bp) amplified from the samples using the OHVA/OHVB primers was carried out to ensure the amplified sequences were being recovered from the OsHV-1 genome. DNA isolated from PCR products amplified from separate mussel individuals (n=5) from each site were pooled into replicates (4-5) to increase the DNA concentration. Both the forward and reverse strands of DNA samples were sequenced commercially (Eurofins MWG Operon). Each sequence was matched against a nucleotide database ( to confirm the individual sequences were from the OsHV-1 and variants genome.
2.6.5 Histological screening
A subsample of mussels (n=20) that were collected at Carlingford Lough in 2013 were prepared for histological analysis and in situ hybridisation (ISH). For each animal, a 5mm cross section section of digestive gland, gonad, gills and mantle was removed and a 3-mm cross section of the visceral mass was excised in front of the pericardial region for histological analysis and immediately fixed in Davidson’s solution at 4ºC for 48h after which they were placed in 70% ethanol. Samples were processed (Shandon Citadel 1000) and microtomed, 5 µm tissue sections for histology and 7µm for in situ hybridisation (ISH). Tissue sections were stained with haematoxylin and eosin while a DIG labeled probe was used for ISH. Sections were viewed with a Nikon Eclipse 80i and images were captured using NIS elements software (at 100X, 200X, 400X and 1,000X. The presence of any abnormal and/or viral cells (Renault et al. 1994a; Renault and Novoa, 2004) was noted.
2.6.6 In situ hybridisation (Digoxygenin (DIG) labelled probe)
In situ hybridisation (ISH) analysis was conducted in accordance with the Ifremer protocol (Renault and Lipart, 1998; Lipart and Renault, 2002; Segarra et al. 2016), however, the OHVA/B and OHVC/D primer pairs were used to create the probe (Lynch et al. 2013). In situ hybridisation was conducted on the 20 Carlingford samples on silane-prep™ slides. A digoxygenin (DIG) labelled probe was produced using OsHV-1 primers and OsHV-1 DNA as a template which was used to hybridise to the target viral DNA within the tissue samples. The slides were mounted using Eukiit mounting medium and cover slips. As with histology, slides were viewed using a microscope (100X, 200X, 400X and 1,000X). (Nikon Eclipse 80i) and images were captured using NIS elements software.
2.6.7 Data analysis
Statistical analyses were carried out using the IBM SPSS Statistics version 23. Statistical significance was determined using P<0.05. Yates’s continuity correction chi-square test was performed to compare percentage (%) mortality between experimental and control groups. Fisher’s exact test was used to compare percentage (%) infected between experimental and control groups and to compare percentage (%) mortality in infected and non-infected animals. Fisher’s exact test was used when one or more cells had an expected count of less than 5
3.  Results
3.1 Seasonal Field Sampling
3.1.1 Field Survey 1
In the first field trial conducted in 2014, 3.3% (1/30) prevalence of OsHV-1 μVar was observed in the Mytilus spp. by PCR from (a) Ardmore. In the (b) Garrettstown mussels, results from the initial sample taken indicated the presence of one infected individual 3.3% (1/30) while the second mussel sample, taken after mussels were placed into holding tanks for three weeks in the laboratory at 13°C, showed a significant increase in virus detection with 46.7% (14/30) prevalence. At (c) Ballymacoda, all mussels (n=12) screened were positive for OsHV-1 μVar in each sample type, gill tissue, haemolymph and shell cavity fluid, taken from each mussel.
3.1.2 Field Survey 2
OsHV-1 μVar DNA was detected in Mytilus spp. at (c) Carlingford Lough in 24% of the mussels screened at the oyster trestles (11/45) in 2013 and 2% (6/240) in 2015. The six Carlingford Lough mussels had a viral load range of 222.70 to 1,565.37 viral copies (n=6) with a mean value of 679.623 viral copies. qPCR was not conducted on the 2013 samples. In the 2015 field study, prevalence of infection in C. gigas seed were minimal at Carlingford Lough at 10% (25/240).  During the second field trial (2013-2015), a single mussel (0.2%, n=361) was positive for OsHV-1 μVar and had a viral load of 123.20 viral copies at the trestles at (e) Dungarvan Harbour.
3.2 Laboratory cohabitation transmission Trial 1: naïve C. gigas held with Mytilus spp. collected 400m from a OsHV-1 µVar endemic culture site
In laboratory Trial 1, the initial sample (n=30) of Galway C. gigas was negative for OsHV-1 µVar, however, the initial sample (n=12) of Mytilus spp. from Ballymacoda was positive (100% prevalence) using representative DNA’s from (i) gill tissue (n=12), (ii) haemolymph (n=12) and (iii) the shell cavity fluid (n=12). No oyster or mussel mortalities were observed in the 7-day trial. During the trial OsHV-1 μVar was not detected in the control oysters (0/15), however, 10% (3/30) of the experimental mussels were positive and 100% (30/30) of the experimental oysters (i.e. naïve oysters exposed to infected mussels) were positive for OsHV-1 μVar.
3.3 Laboratory cohabitation transmission Trial 2: naïve C. gigas and exposed Mytilus spp. collected at oyster trestles held at various increasing temperatures over a 4 week period in 2015
In laboratory Trial 2, mortality rates were 26.7% (16/60) in the control group (oysters) and 10% (18/180) in the experimental group (9% (8/90) in the experimental oysters and 11% (10/90) in the experimental mussels. 14.1% (34/240) of the total animals tested, died over the duration of the study (Figure 1). Yate’s continuity corrected chi-square test indicated that the percentage (%) mortality differed significantly between the control and experimental groups (χ 2 (1) =8.96, P<0. 01). Of the 16 (26.7%) oysters which died in the control groups, all were being held at 28C. Of the 18 (20%) animals (mussels and oysters), that died in the experimental groups, 3 mortalities occurred in the oysters at 14C and 5 oyster and 10 mussel mortalities occurred at 28C (Figure 2 & 3).
The initial samples (n=30) of Galway C. gigas and Carlingford Lough M. edulis were both negative for OsHV-1 μVar. In the experimental groups 3.9% (7/180) of animals (mussels (n=2) and oysters (n=5)) were positive for OsHV-1 μVar compared to 3.3% (2/60) in the control oyster group (Figure 4 & 5). p. Fisher’s exact test indicated that there was no significant difference in prevalence (%) of the virus between treatments (experimental and control groups) (P>0.05). Of the 7 infected animals in the treatment groups, a single oyster was infected at 14C (alive at the end of the study), and the remaining 6 (4 oysters and 2 mussels) were infected at 28C (5 were alive and 1 mussel was dead at the end of the study). The 2 infected control animals occurred at 28C, were found moribund at the end of the study. Of the positive oysters (n=5) and mussels (n=2), higher viral loads were observed in the oysters (n=5, 384.2-3,676.67 with an average of 1,081.61) than the mussels (n=2, 416.74-660.32 with an average of 538.53). Fisher’s exact test revealed that there was no statistically significant difference in percentage (%) mortality between individuals with the virus and those without (oysters and mussels) (P>0. 05).
3.5 Histological examination and In situ hybridisation
Abnormal cells consistent with the presence of OsHV-1 μVar were observed in the Mytilus spp. histology in individuals that were positive for OsHV-1 μVar by PCR. In Mytilus spp., collected from the field (Ballymacoda), abnormal cells, which were enlarged, rounded with the nuclei disrupted or pushed to the edge of the cell were detected in half (10/20) of the histology samples screened and were mostly observed throughout the connective tissue in the mantle and to a lesser extent in the gills and digestive tubules. ISH detected a positive signal for OsHV-1 μVar in 20% (4/20) of the mussels screened using this method (Figure 6 &7).
In this study, OsHV-1 μVar was detected in wild Mytilus spp. and Mytilus edulis in internal tissues and hemolymph but also within the shell cavity fluid. Similar pathologies to those previously reported in oysters and associated with herpes virus infection (Renault et al. 1994a; Renault and Novoa, 2004) were observed in the mussel tissues and positive signals in the ISH were noted.  Although these histological changes and abnormalities associated with ostreid herpesvirus have not been previously described for Mytilus spp., similar changes have formerly been observed in a non-ostreid bivalve species, the Manila clam Ruditapes philippinarum (Renault et al. 2001a).  Of significance, this study reports the first transmission of this virus from both Mytilus edulis and Mytilus spp. to naïve C. gigas, which occurred in the laboratory under an increased and increasing temperature regime, however, it must also be highlighted that replication of the virus (14x) also occurred in mussels, which were held in the laboratory at 13°C during a three week period. The virus was detected in mussels cohabiting with oysters within the oyster bags at the trestles but it was also detected in wild mussels located 400m and 500m from the oyster trestles and up to 26 km from the oyster farms at non-culture sites, thus highlighting the range extension of the virus from endemic sites.  The impact of the virus on mussel health is unclear, as OsHV-1 μVar was detected at an overall low prevalence in mussels and at a low viral load for those screened by qPCR and without associated mortality in the laboratory trials, however cytopathic effects were observed in the mussels similar to those observed in C. gigas, which have been associated with oyster mortalities. A low prevalence of OsHV-1 μVar was observed in oyster populations in the field at the same time, possibly due to low seawater and air temperatures experienced at those sites for the duration of the trials.
According to the World Organisation for Animal Health (OIE), a ‘suspected’ case of infection with microvariants, can exist without evidence of mortality, whereas a ‘confirmed’ case occurs when detection by histology, transmission electron microscopy, or PCR is followed by sequencing confirmation.  In this study, detection of OsHV-1 μVar DNA was established by PCR in mussel samples from all five study sites, qPCR in mussel samples from two of the study sites, and visualised by histology and ISH in mussels from a single site. The DNA was confirmed by direct sequencing to be that of OsHV-1 μVar in samples from each study site. From the qPCR analysis we can conclude that both field sampled and laboratory experimental living and dead Mytilus spp. displayed detectable quantities of OsHV-1 μVar. The mussels used in the laboratory cohabitation transmission Trial 2 were exclusively from Carlingford Lough, which represents the first record of OsHV-1 μVar in M. edulis. In contrast to our study, OsHV-1 DNA was previously detected in samples of Mytilus spp. in Australia with a 20% (n=1/5) prevalence and viral concentrations below the quantification limit of the qPCR assay (<12 DNA copies per PCR reaction) (Evans et al. 2017), however, this was not the case in this study where up to 1,565.37 viral copies were detected in Irish mussels.  In addition, abnormal cells consistent with the pathology associated with OsHV-1 μVar infection were visualised in the mussel histology. The cytopathic effects (abnormal cell morphology) observed in infected mussels were similar to those visualised in infected oysters in previous studies (Renault et al. 1994a; Renault and Novoa, 2004; Renault et al. 2000a).
This study demonstrated that elevated temperatures exacerbated OsHV-1 μVar prevalence and transmission between mussels and oysters but mortality was relatively low in both species. During the summer of 2015 all monthly mean air temperatures were below their long term average (LTA) ( OsHV-1 μVar is generally detected in dying oysters when seawater temperatures are >16°C (Pernet et al. 2012). Higher temperatures appear to act as a stressor for C. gigas and exacerbate virus proliferation and transmission; and detection and mortality are associated with a marked increase in mean daily seawater temperature (Garcia et al. 2011). Previous studies of Mytilus spp. have also observed that temperature is also a key driver of biological response (Hu et al. 2015) with elevated temperatures negatively impacting on the immune response in M. edulis by significantly increasing the antibacterial activity of cell-free haemolymph (Ellis et al. 2015). C. gigas has a broad temperature tolerance, with a range of –1.8 to 35°C (FAO, 2015), however higher temperatures exacerbate virus proliferation, transmission and mortality as was observed in this present study. OsHV-1 μVar is known to be persistent in oysters held at temperatures as low as 13°C and reactivated during thermal elevation to 21°C (Pernet et al. 2015), this was confirmed by the findings of our study following elevation of temperature during the laboratory trial. However, our results would further indicate that the virus is replicating also at 13°C in the mussels. OsHV-1 μVar was detected in a small percentage of the control living oysters, however, this may have been attributed to background aerosol contamination as the initial sample was negative and this population was disease free. Elevated temperature alone may not result in increased mortalities for example C. gigas that experienced prolonged high temperature (21 °C for 14 days) in the Inland Sea, Wales, did not experience mass mortalities (Malham et al. 2009).  However, in the context of a changing climate, with future increases in seawater temperatures predicted, the ecological sustainability of marine fisheries and aquaculture may be threatened (Dang et al. 2012), it is acknowledged that disease transmission may be facilitated by continued ocean warming (Vezzuilli et al. 2013).
Consideration of these results should be given in areas where both mussel and oyster culture occur and for the movement of mussels from OsHV-1 μVar infected sites to sites where Pacific oysters may be cultured and are free of infection. Even with these viral loads and low number of animals testing positive, transmission of the virus was still effected to naive oysters, which highlights the risks involved with the movement of shellfish for aquaculture and the unintentional introduction of pathogens in nontypical hosts. Wild mussels approximately 400m from a C. gigas culture site successfully transmitted OsHV-1 μVar to naïve C. gigas in the laboratory, thus indicating that the virus is viable and is being maintained outside the known host. In addition to oysters, Herpesviruses infect fish, amphibians and other invertebrates and it has been identified in many of the other major aquaculture species, such as Common carp C. carpio, Atlantic salmon S. salar, Channel catfish I. punctatus, Europeaneel  A. anguilla and White sturgeon A. ransmontatus with certain herpes viruses being capable of causing serious disease in non-reservoir host species (van Beurden and Engelsma, 2012). Indeed, viral pathogens are often highly infectious and easily transmissible (Renault, 2008) among different susceptible species.
The full process of how the virus might maintain itself at Pacific oyster culture sites is unclear and the effects of OsHV-1 μVar infection on the mussel population in Ireland, in particular those at infected oyster culture sites, is yet to be determined. Mytilus spp. appears to act as a reservoir or carrier for OsHV-1 μVar but the nature of this interaction and impacts on mussel health need to be investigated further. What is certain from the findings of this study is that OsHV-1 μVar has the ability to be associated with other cohabiting bivalve species even at a distance from a disease “hot spot” such as an OsHV-1 μVar endemic C. gigas culture site, which highlights a highly efficient transmission and dispersal strategy for this pathogen.
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