Investigating the interactions of Cyprinid herpesvirus-3 with host proteins in goldfishCarassius auratus

Abstract

Keywords: antibody, electrospray ionization mass spec-trometry, koi herpesvirus disease, protein purification.Cyprinid herpesvirus-3 (CyHV-3) is the aetiologi-cal agent of a serious and deadly disease, termed‘koi herpesvirus disease’ (KHVD), that causes highmortality in koi and common carp Cyprinus carpioL. (Pikarsky et al. 2004; Ilouze et al. 2008). Sincethe initial identification of CyHV-3 in Europe(Bretzinger et al. 1999), the USA and Israel (Hed-rick et al. 2000), and Japan (Sano et al. 2004),there is a great effort to limit the spread ofCyHV-3. Recently, the focus of prevention hasshifted to understanding how the virus propagatesand what carriers exist for the virus. Environmen-tal factors such as mating (Uchii et al. 2011),feeding activities (Kielpinski et al. 2010; Minam-oto et al. 2011; Fournier et al. 2012) and proxim-ity in aquaculture (Dishon et al. 2005) arecontributing factors in the spread of CyHV-3.Earlier studies suggested that immunization withattenuated virus may prevent the spread ofCyHV-3 (Ronen et al. 2003; Perelberg et al.2005), but current studies suggest that CyHV-3spread is due to subclinical carriers (Bergmann K Eide et al. 2011), which can infectna€ive fish (St-Hilaire et al. 2005). Recent studieshave shown that CyHV-3 DNA exists indifferent species of healthy looking Cyprinid fishthat include grass carp Ctenopharyngodon idella,goldfish Carassius auratus and blue back ideLeuciscus idus, as well as in non-Cyprinid fish thatinclude Russian sturgeons Acipenser gueldenstaedtii,Atlantic sturgeons A. oxyrinchus and Ancistrus sp.,suggesting that these fish may be potential carriersfor the CyHV-3 (Bergmann et al. 2009; Kempteret al. 2009). In addition, cohabitation studies haveshown that CyHV-3 can be transferred to goldfishfrom infected koi fish (El-Matbouli, Saleh & Soli-man 2007), but goldfish do not show clinicalsigns of the disease (Bergmann et al. 2010a).The genome of CyHV-3 consists of 295 kB(Aoki et al. 2007) that codes for 156 unique openreading frames (ORFs) that are all transcribed intomRNA transcripts (Ilouze, Dishon & Kotler2012). Mass spectrometry (MS) identified 40CyHV-3 proteins incorporated into mature virions(Michel et al. 2010). Some of the CyHV-3 pro-teins have been further characterized, such asORF81, a component of the viral envelope(Rosenkranz et al. 2008) and genes involved inDNA synthesis (Ilouze et al. 2006; Fuchs et al.2011). Recently, Sunarto et al. (2012) showedthat CyHV-3 transcribes a potential immune sup-pression gene, ORF134, a homolog of interleu-kin-10 (IL-10). CyHV-3 has also been shown tomodulate the innate immune response pathway invitro (Adamek et al. 2012) and in vivo (Adameket al. 2013). Gene expression analysis usingmicroarray chips of two common carp lines thatdiffer in mortality rates during CyHV-3 infectionidentified 581 genes in line ‘R3’ and 107 genes inline ‘K’ that are differentially transcribed duringCyHV-3 infection (Rakus et al. 2012). The eluci-dation of interaction partners is often used to