Ultrastructural analysis of sequential Cyprinid herpesvirus 3 morphogenesis in vitro

Cyprinid herpesvirus 3 (CyHV-3) is an alloherpesvirus, and the aetiological agent of koi 24 herpesvirus disease. Although the complex morphogenic stages of the replication cycle of 25 CyHV-3 were shown to resemble that of other members of the Herpesvirales , detailed 26 analysis of the sequence and timing of these events was not definitively determined. This 27 study describes these features through a time course using cyprinid cell cultures (KF-1 and 28 CCB) infected with CyHV-3 (KHV isolate, H361) and analysed by transmission electron 29 microscopy. Rapid viral entry was noted, with high levels of intracellular virus within 1-4 30 hours post-infection (hpi). Intra-nuclear capsid assembly, paracrystalline array formation and 31 primary envelopment of capsids occurred within 4 hpi. Between 1-3 days post infection (dpi), 32 intra-cytoplasmic secondary envelopment occurred, as well as budding of infectious virions 33 at the plasma membrane. At 5-7 dpi, the cytoplasm contained cytopathic vacuoles, enveloped 34 virions within vesicles, and abundant non-enveloped capsids; also there was frequent nuclear 35 deformation. Several morphological features are suggestive of inefficient viral assembly, with 36 production of non-infectious particles, particularly in KF-1 cells. The timing of this 37 alloherpesvirus morphogenesis is similar to other members of the Herpesvirales but there 38 may be possible implications of using different cell lines for CyHV-3 propagation.


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Cyprinid herpesvirus 3 (CyHV-3) is the official taxonomical classification of koi herpesvirus 43 (KHV) (Waltzek et al. 2005), the highly virulent and economically important aetiological 44 agent of koi herpesvirus disease (KHVD) (Hedrick et al. 2000;2005). The virus has had a 45 devastating impact on to the global koi and carp (Cyprinus carpio Linnaeus, 1758) 46 aquaculture industries since outbreaks were first reported in Israel and the U.S. in 1998 47 (Hedrick et al. 2000;Perelberg et al. 2003). CyHV-3 is a member of the recently formed 48 family Alloherpesviridae (Waltzek et al. 2005) in the order Herpesvirales (Davison et al. 49 2009). This classification has been based on the close phylogenetic relationship of the virus 50 with CyHV-1 (Aoki et al. 2007;Waltzek et al. 2009), the causative agent of carp pox (Sano 51 et al. 1991;1992;Päak et al. 2011 (Hedrick et al. 2000). 59 However, little is known with regards to the timing of the various stages of Alloherpesviridae 60 virion maturation. As with other herpesviruses, CyHV-3 displays differential infection 61 phases, including fatal lytic infection and potential latent infection (Gilad et al. 2003;2004;62 St-Hilaire et al. 2005;Eide et al. 2011;Reed et al. 2014;Sunarto et al. 2014), which can be 63 influenced by temperature (Ronen et al. 2003;St-Hilaire et al. 2005;Sunarto et al. 2014). 64 These different infection states can impact serological and molecular detection sensitivities, 65 Two non-infected flasks per cell line were used as negative controls, which were sampled at 1 138 dpi and 7 dpi. For the seven test flasks, monolayers were inoculated with 3 mL KHV as 139 described above, and sampled at 1, 4 and 8 hpi, and 1, 3, 5, and 7 dpi. Samples were taken by 140 washing the monolayers twice with 10 mL DPBS, and then fixing the cells in-situ with 6 mL 141 2.5% glutaraldehyde (Sigma-Aldrich, UK) in 0.1M sodium cacodylate buffer, pH 7.3. Cells 142 where then scraped into suspension using a rubber policeman and 6 mL (3 mL x 2) of the 143 suspension were centrifuged at 2000 x g for 10 min at 4°C to form a pellet (slow speed 144 centrifugation was used to avoid cell rupture). Pellets were post fixed with fresh 2.5% 145 glutaraldehyde for 2-4 h or overnight at 4°C. The fixative was removed and 2 mL 0.1M 146 sodium cacodylate buffer was added to the pellets, which were detached from the tube wall 147 with a wooden applicator and stored at 4°C until processed.   (Hedrick et al. 2005;Miwa et al. 2007;Miyazai et al. 172 2008). No discernible difference was noted in the sizes of virions obtained from the CCB 173 cells or the KF-1 cells.

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Many cells were devoid of virions and their ultrastructure was normal and similar to the 176 control cells at this stage ( Fig. 1 A). A small number of both CCB and KF-1 cells contained 177 intranuclear paracrystalline capsid arrays at 4 hpi. This was associated with reduction of 178 heterochromatin/euchromatin ratio and chromatin margination ( Fig. 1 B and C). The capsids 179 observed were predominantly devoid of electron dense cores, and were occassionally toroid 180 ( Fig. 1 B-D). The capsids observed within the paracrystalline arrays were predominantly 181 toroid in appearance ( Fig. 1 C). Lamellar bodies, reminiscent of lipofuscin, were occasionally 182 observed regardless of the infection status ( Fig. 1 B).

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Between 4 hpi -1 dpi, virus capsids were observed throughout the nucleus at various stages 184 of maturation in a large number of the infected cells. Capsids were often located below the inner nuclear envelope, occasionally featuring an envelope -primary envelopment ( Fig. 1 E).
cytoplasm of infected cells, some contained an electron dense core, while others were empty 189 ( Fig. 1 F). A this stage, intracytoplasmic secondary enveloped particles within vesicles were 190 already present, with nuclei harbouring large numbers of capsids as described above (Fig 1   191 F). There were no discernable differences in virion formation or cell pathology between  or CCB cells at this stage.   The morphogenesis of CyHV-3 has been described in some detail in cultured cyprinid cells in PrV (Granzow et al. 1997)), a second type with an electron dense core and a third type that 282 is empty. By harvesting infected cell cultures during the first day of inoculation it was 283 possible to determine the earlier capsid formation type in our study which was similar to that 284 described for the third type described by Miwa et al. (2007). These were similar to those 285 previously described for avian and mammalian herpesviruses by Nii (1991) being mostly 286 empty with no electron dense core or toroid in appearance (), and thus were likely to lack 287 DNA at this stage. This is supported by the absence of more mature virions in the cells at this  (Granzow et al. 1997).

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Imported PrV nucleocapsids are found in close proximity to microtubules, and had 300 sometimes already docked at the nuclear pore within 30 min (Granzow et al. 1997;Kaelin et 301 al. 2000). This was not observed in the current study thus may have been missed as cells although no microtubules were observed near these. Interestingly, electron dense and electron 314 lucent virus-like particles were observed in linear arrays, some in close proximity to the 315 nuclear envelope within the first 4 hpi. These resembled capsids and were observed at later 316 stages of infection in cells with disrupted nuclei (7 dpi), but the lack of electron density of 317 some of these structures suggests that no DNA was present to be released at the nuclear pore, 318 or perhaps had already been released.

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As mentioned above, the capsids observed within the nucleus during the first day of infection 320 exhibited all 3 stages of maturation, similar to findings for other herpesviruses (Nii et al. 321 1968; Wolf & Darlington 1971;Nii 1991;Granzow et al. 1997). In addition to this, primary

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The formation of paracrystalline-like arrays of intra-nuclear capsids had previously been 342 reported within the nucleus of infected carp gill epithelial cells (Hedrick et al. 2000), and in  These arrays are typical of herpesvirus infected cells (Nii et al. 1968;Granzow et al. 1997).

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These have been described as pseudocrystals in PrV infected cells, which are hypothesised to 347 dissolve during replication and release individual capsids as they are not found in necrotic 348 cells following replication (Granzow et al. 1997). The current study supports this as these 349 capsid formations were no longer observed after 3 dpi, despite being found in a relatively 350 large number of cells prior to this.

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In contrast to the rapid production of progeny virus of the alloherpesvirus, CCV within 10-12 352 hpi (Wolf & Darlington 1971), release of extracellular infectious virions appears much 353 slower for CyHV-3 and other herpesviruses (i.e. 3-5 dpi) as shown from their growth curves 354 (Ahlqvist et al. 2005;Dishon et al. 2007;Costes et al. 2008;Dong et al. 2011).  (Wolf & Darlington 1971;Hedrick et al. 2000;2005;Miwa et al. 2007;Miyazaki et al. 363 2008). In a recent study, there was elevated expression and abundance of capsid-associated  However, a number of nuclear deformations were observed after 5-7 dpi, that were not 389 observed in non-infected control cells, thus were likely to be associated with elevated virus 390 production and infection and not the senescence of old cells. successful egress and further maturation (Granzow et al. 2004). This can result in thickening 404 (Ghadially 1997) leading to nuclear envelope proliferations, fusions and subsequent abnormal 405 concentric lamellar structures (Nii et al. 1968). These are characteristic cytopathologies 406 observed in CyHV-3 and other herpesviruses (Nii et al. 1968;Wolf & Darlington 1971;Nii 407 1991;Ghadially 1997;Miwa et al. 2007). Disrupted nuclei in the current study contained not 408 only nuclear envelope proliferations in both cell lines, but also occasionally CCB cells 409 contained intra-nuclear vesicles, which were more pronounced at later stages of infection and 410 sometimes resembled those reported in CyHV-3 infected carp cells by Miyazaki et al. (2008).

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In contrast to the results reported by Miwa et al. (2007), primary envelopment within the 412 nuclear envelope was observed more often at the later stages of infection. Furthermore, the 413 production of capsidless particles in the perinuclear envelope, possibly intracisternal L-414 particles, as previously reported for alphaherpesviruses by Granzow et al. (2001), may lead to 415 inefficient viral assembly, and also contribute to the production of non-infectious particles 416 following increased viral infection pressure. Formation of syncytia on the other hand, is 417 thought to result from mutations in glycoprotein genes (Pereira 1994), with an extensive 418 production of intracellular mature and immature virus particles, which with CyHV-3 occurred 419 more often in CCB cells than KF-1 cells, probably due to the latter being more prone to lysis.