Downloaded from www.microbiologyresearch.org by IP: 54.70.40.11 On: Sun, 04 Aug 2019 00:09:01 N-Linked glycosylation is essential for the yellow head virus replication cycle Chumporn Soowannayan, 1,2 Nhuengtida Chanarpakorn, 2,3 Mongkhol Phanthura, 2,3 Nattawoot Deekhlai, 2 Chanon Kunasol 2 and Siriporn Sriurairatana 2 Correspondence Chumporn Soowannayan chumporn@biotec.or.th or csoowannayan@gmail.com Received 18 April 2013 Accepted 13 August 2013 1 National Center for Genetic Engineering and Biotechnology, National Science and Technology Development Agency, Thailand Science Park, Klong Luang, Patumthani 12120, Thailand 2 CENTEX SHRIMP, Faculty of Science, Mahidol University, Rama VI Road, Bangkok, 10400, Thailand 3 Department of Biotechnology, Faculty of Science, Mahidol University, Rama VI Road, Bangkok, 10400, Thailand Yellow head virus (YHV) particles contain a nucleocapsid protein (p20) and two envelope glycoproteins (gp116 and gp64). The glycans attached to the two glycoproteins are N-linked and are complex and high mannose types, respectively. Here, we show that treatment with the N-linked glycosylation inhibitor tunicamycin in YHV-infected black tiger shrimp (Penaeus monodon) resulted in less severe yellow head disease and reduced mortality when compared with untreated control shrimp. Quantitative real-time reverse transcription PCR analysis also revealed lower YHV copy numbers in the haemolymph of treated than control shrimp. This was concurrent with less intense immuno-reactions in tissues of treated versus untreated shrimp using mAbs against all three YHV structural proteins. In addition, transmission electron microscopy of lymphoid organ tissue of the treated and untreated shrimp [eight collected at 36 h and eight at 48 h post-infection (p.i.)] revealed only unenveloped nucleocapsids in all but one of the treated shrimp (collected at 48 h p.i.). By contrast, all the untreated shrimp showed a mixture of many unenveloped and enveloped virions. These results were supported by purification of YHV from the cell-free haemolymph of treated and untreated shrimp followed by YHV structural protein analysis by SDS-PAGE. It revealed three expected structural protein bands (116, 64 and 20 kDa) from the untreated shrimp but no structural protein bands from the tunicamycin-treated shrimp (confirmed by Western blot analysis). Overall, the results indicated that blocking glycosylation with tunicamycin inhibited the formation of mature YHV virions and their subsequent release into shrimp haemolymph, reducing the severity of disease. INTRODUCTION Yellow head virus (YHV) is one of the most important penaeid shrimp viruses. It was first reported as the cause of mass mortality of cultivated native Penaeus monodon in Thailand in 1991 (Boonyaratpalin et al., 1993; Chantanachookin et al., 1993; Flegel et al., 1995; Limsuwan, 1991) and later also in cultivated exotic Penaeus (Litopenaeus) vannamei (Senapin et al., 2010). Other variants that cause less severe disease or no disease were later reported from Australia and from other countries in South-East Asia (Spann et al., 1995, 1997; Wijegoonawardane et al., 2008). In Australia, in the north- eastern states of Queensland and New South Wales, the endemic YHV genotype is called gill-associated virus and is associated with gradual death of P. monodon from 3–3.5 months after stocking (12–15 g in weight) until harvest (Cowley et al., 1999; Spann et al., 1995, 1997), and it is considered to be the main cause of mid-crop mortality syndrome (Oanh et al., 2011). Currently, six genotypic variants of YHV have been identified (Wijegoonawardane et al., 2008), but only type 1 from Thailand and type 2 (gill-associated virus) have been reported to cause disease. YHV is a positive-sense ssRNA virus that has been classified in the order Nidovirales and in the genus Okavirus (Cowley & Walker, 2002; Cowley et al., 1999; Walker et al., 2005; Wongteerasupaya et al., 1995; Ziebuhr et al., 2003). YHV particles contain three structural proteins: a nucleocapsid protein or p20 protein and two envelope glycoproteins (gp116 and gp64) (Cowley et al., 2004; Jitrapakdee et al., 2003). The nucleocapsid protein (smallest of the three) is encoded by ORF2, which is located in the YHV genome at the N terminus of the two viral envelope protein genes. The nucleocapsid protein of Journal of General Virology (2013), 94, 2458–2468 DOI 10.1099/vir.0.054379-0 2458 054379 G 2013 SGM Printed in Great Britain