Ultrastructural analysis of sequential Cyprinid herpesvirus 3 morphogenesis in vitro

Cyprinid herpesvirus 3 (CyHV-3) is an aAlloherpesvirus, and the aetiological agent of koi herpesvirus disease. Although the complex morphogenic stages of the replication cycle of CyHV-3 were shown to resemble that of other members of the Herpesvirales, detailed analysis of the sequence and timing of these events was not definitively determined. This study describes these features through a time course using cyprinid cell cultures (KF-1 and CCB) infected with CyHV-3 (KHV isolate, H361) and analysed by transmission electron microscopy. Rapid viral entry was noted, with high levels of intracellular virus within 1-4 hours post-infection (hpi). Intra-nuclear capsid assembly, paracrystalline array formation and primary envelopment of capsids occurred within 4 hpi. Between 1-3 days post infection (dpi), intra-cytoplasmic secondary envelopment occurred, as well as budding of infectious virions at the plasma membrane. At 5-7 dpi, the cytoplasm contained cytopathic vacuoles, enveloped virions within vesicles, and abundant non-enveloped capsids; also there was frequent nuclear deformation. Several morphological features are suggestive of inefficient viral assembly, with production of non-infectious particles, particularly in KF-1 cells. The timing of this aAlloherpesvirus morphogenesis is similar to other members of the Herpesvirales but there may be possible implications of using different cell lines for CyHV-3 propagation.