Background: A major outbreak of human enterovirus 71-associated hand, foot and mouth disease in Sarawak in 1997 marked the beginning of a series of outbreaks in the Asia Pacific region. Some of these outbreaks had unusually high numbers of fatalities and this generated much fear and anxiety in the region.

During the 2010−11 summer outbreak of ostreid herpesvirus 1 (OsHV-1) in New Zealand, an opportunistic longitudinal field study was conducted. OsHV-1 PCR-negative oyster spat (Crassostrea gigas) were relocated to an OsHV-1 PCR-positive area of the North Island of New Zealand that was experiencing juvenile oyster mortalities. Over a period of 13 d, spat were monitored for mortality, sampled for histopathology, and tested for the presence of OsHV-1 using real time PCR and Vibrio culture. Histopathology showed some evidence of tissue pathology; however, no consistent progressive pathology was apparent. Field mortalities were evident from Day 6 on. After 5 and 7 d of exposure, 83 and 100% of spat, respectively, tested positive for the virus by real time PCR. Vibrio species recovered during the longitudinal study included V. splendidus and V. aestuarianus. This study offers insight into the rapidity of onset and virulence of the virus in naïve oyster spat in New Zealand waters. KEY WORDS: Ostreid herpesvirus 1 · Vibrio · Crassostrea gigas Resale or republication not permitted without written consent of the publisherDis Aquat Org 109: 231-239, 2014 al. 1972). In 1999, Le Deuff and Renault undertook initial molecular characterisation of the virus infecting Pacific oysters (Le Deuff & Renault 1999). This was followed up by the identification of the first variant (OsHV-1 Var) (Arzul et al. 2001a,b) and subsequent whole-genome sequencing of the ostreid herpesvirus 1 (OsHV-1; GenBank AY509253) (Davison et al. 2005). Segarra et al. (2010) published sequencing data on the emergence of the now problematic microvariant (OsHV-1 µVar).Several studies have identified other pathogens associated with major OsHV-1 mortality events in Europe. Vibrio species, including V. splendidus, V. aestuarianus and V. harveyi, have been isolated in association with OsHV-1 mortalities , Dégremont 2011, Schikorski et al. 2011a. Whilst Vibrio species are likely to be opportunistic pathogens, the significance of regular detection in association with OsHV-1 should not be overlooked. The major oyster mortality events in Europe are considered to be multi-factorial, with OsHV-1, Vibrio species and environmental conditions (e.g. increased water temperatures) all believed to contribute (Sauvage et al. 2009, Segarra et al. 2010, De Decker & Saulnier 2011, De Decker et al. 2011.During the summer of 2010−11 in New Zealand, OsHV-1, Vibrio species and warm water temperatures appeared to contribute to the deaths of juvenile C. gigas on the North Island of New Zealand. During the mortality event, the oyster industry provided access to pre-planned movements of apparently healthy hatchery-reared spat to an oyster growing site on the North Island where oyster mortalities attributed to OsHV-1 were occurring. This provided a unique opportunity to conduct a longitudinal study. This paper describes the molecular characterisation of the New Zealand OsHV-1 virus and results of the longitudinal study. MATERIALS AND METHODS Longitudinal studyApproximately 17 0...

Ultracentrifugation in sucrose density gradient remains the most commonly used technique for hRSV purification. However, the high viscosity and hyper-osmotic property of sucrose can cause damage to the extremely labile virus leading to loss of infectivity. To overcome these limitations, an alternative purification technique was developed using iodixanol as gradient medium, incorporating MgSO4 as a stabilizing agent and EDTA to disaggregate the virus prior to infectivity assay. Virus particles were banded at the 20–36% interface after purification of polyethylene glycol-concentrated viruses by rate zonal ultracentrifugation on a 20–52% discontinuous iodixanol gradient. The presence of the virus was confirmed by viral fusion glycoprotein content using ELISA. After further purification by buoyant density ultracentrifugation on a 20–52% continuous gradient, the virus was recovered in the region of density 1.15–1.19 g/ml and this was confirmed by the coincidence of the infectivity titre, viral genome and fusion glycoprotein peaks. Analysis of recovery rates showed that the use of iodixanol increased the virus yield up to 69%. Iodixanol was also found to be non-toxic to HeLa cells used in infectivity assay, eliminating the need of its downstream removal by dialysis.

Megalocytiviruses have been associated globally with severe systemic disease and economic loss in farmed food fish and ornamental fish. The viruses have been spread internationally by translocation of live fish. In New Zealand, megalocytiviruses are regarded as exotic. A potential pathway for introduction has been identified, namely imported ornamental fish. In the present study, real-time PCR assays were developed for detection of megalocytiviruses using a conserved major capsid protein gene. A SYBR green assay was developed to target all known megalocytiviruses. A second real-time PCR assay using a molecular beacon was developed to specifically target gourami, Trichogaster trichopterus, iridovirus, a species of iridovirus previously linked to ornamental fish imports in Australia. The analytical sensitivity for the SYBR green and molecular beacon assays were 10 and 100 fg, respectively. The analytical specificity of the real-time PCR assays determined using genomic DNA templates from three target viruses, 12 non-target viruses and 25 aquatic bacterial species were 100%. The intra-run and inter-run coefficients of variation of both assays were <5%. The real-time PCR assays developed in this study provide rapid, sensitive, and specific detection of megalocytiviruses and gourami iridovirus.

Subgroup A respiratory syncytial viruses present in respiratory secretions and low passage level cell culture isolates were found to be markedly less susceptible to neutralization with monoclonal antibodies (MAbs) to the F glycoprotein than the cell culture adapted A2 virus strain. Low passage virus isolates collected over a 20 year period and belonging to several sub-group A lineages were refractory to neutralization with antibodies recognizing two major neutralizing antigenic sites located sub-terminally at opposite ends of the F(1) glycoprotein sub-unit. On further passage in cell culture, virus isolates exhibited both increased infectivity titers and increased susceptibility to neutralization by antibodies to both antigenic sites. The consensus nucleotide sequence of the membrane associated proteins M and of the SH, G and F glycoprotein genes, and their intergenic regions were compared for neutralization resistant and susceptible stocks of one virus strain, R17532. No changes were observed in the known monoclonal antibody epitopes on the F glycoprotein. In line with this, the increase in susceptibility was not found to be associated with any increased binding of monoclonal antibody to isolated F glycoprotein in a BIAcore assay, thus excluding the possibility that passage in cell culture selected for viruses with mutations in the antibody binding sites. M and SH genes were conserved but a number of sites in the G and F glycoprotein genes were found to vary on adaptation to cell culture suggesting that change in susceptibility to neutralization was associated with a change in the prevalent quasispecies present in the virus population. The genetic basis of phenotypic change in susceptibility remains to be determined.