Further evidence of Chelonid herpesvirus 5 (ChHV5) latency: high levels of ChHV5 DNA detected in clinically healthy marine turtles

Introduction

Historically, viral infections associated with abnormal cell growth have been reported and studied directly from tumours (Work et al., 2001). Among others, several herpesviruses can lead to tumour formation in humans (Hausen zur, 1991) and other vertebrates such as reptiles (Ariel, 2011). The marine turtle disease fibropapillomatosis (FP), has been linked to infection by the Chelonid herpesvirus 5 (ChHV5) also commonly known as the Chelonid fibropapilloma-associated herpesvirus (CFPHV) (Herbst, 1994). Despite, all attempts at culturing ChHV5 in vitro have been unsuccessful (Lu et al., 1999; Work et al., 2009). Thus, confirmation of ChHV5 as the primary causal agent of FP tumours remains inconclusive as defined by the four Koch’s postulates that establish relationships to identify the causative agent of a disease.

Several studies have used quantitative real time PCR (qPCR) methods to assay the levels of ChHV5 DNA in marine turtle tissues, in an attempt to shed further light into its potential relevance for FP. While most of such qPCR assays have exclusively targeted the skin tumours (Work et al., 2009), conjunctival growths, and/or tumours from visceral organs (Curry et al., 2000) sampled from FP affected turtles, non-tumour skin tissue samples of infected animals have been used to demonstrate that ChHV5 is always found at higher level in FP tumours than in healthy tissue samples from affected animals (Quackenbush et al., 2001). Although this has been interpreted as further evidence for ChHV5s role in FP, little attention has been given to the levels of ChHV5 in clinically healthy turtles, whether from feeding grounds shared with FP affected individuals or from species’ populations in which FP has never been observed. Only a handful of research studies, exclusively focused on populations historically documented with individuals infected by the FP disease, have described the detection of herpes viral DNA from clinically healthy marine turtles (Herbst et al., 2008).

In a recent series of studies we documented the presence of ChHV5 DNA sequences in a global sample set of five species of clinically healthy marine turtles, using sequenced PCR amplicons (Alfaro-Núñez & Gilbert, 2014; Alfaro-Núñez et al., 2014). In the same studies, we also investigated the possibility that these viral amplicons have been integrated in the turtle genome (Wang et al., 2013), and finding no evidence for this hypothesis, we confirmed the presence of ChHV5 DNA in healthy individuals. We did emphasise the presence of Glycoprotein B (gB) amplicons, a well-characterized protein involved in the viral cell entry, in clinically healthy turtles. This immediately begs questions as: (i) if ChHV5 is the etiological agent for FP, how can it be found in asymptomatic animals; and (ii) how do levels of ChHV5 in healthy animals compare to those originating from FP samples? To investigate this, we aimed to determine the relative concentration of ChHV5 Glycoprotein B (gB) to host nuclear DNA ratio in a sub-sample set from a recently published work (Alfaro-Núñez & Gilbert, 2014) including FP positive and negative sea turtle tissue samples using a real-time polymerase chain reaction (qPCR) assay in order to provide further evidence of the ChHV5 latency distinctive feature.

Materials and Methods

Results

Consistent with previous observations (Quackenbush et al., 2001; Work et al., 2009), we observed that the highest viral loads (average ratio of 15.61, s ± 12.5) were in FP tumour samples, while the ratio of viral to nuclear DNA in FP non-tumour samples from FP infected animals were low to undetectable (average ratio of 0.02, s ± 0.00). However, tissue samples from clinically healthy animals were found to contain significantly higher viral loads of gB (average ratio of 6.03, W-t, p = 0.008, t-t, p = 0.002) than non-tumour samples from FP exhibiting turtles (Fig. 1).

Although the starting tissue mass was only estimated to be similar between all samples, the estimated nuDNA levels were overall consistent (Fig. 2A). Based on standard curve, we estimate that the median of gB is approximately 4.9^E+04 copies/µL DNA extract in FP tumours, 1.8^E+01 copies/µL in non-tumour tissue from FP animals and 5.8^E+04 copies/µL in clinically healthy turtles (Fig. 2B).

Discussion

ChHV5 has been associated as primary etiological agent of FP by three lines of evidence. First, every tumour analysed by polymerase chain reaction (PCR) has yielded ChHV5 sequences (Herbst et al., 2004; Alfaro-Núñez & Gilbert, 2014), second, tumours occasionally show epidermal viral inclusions with ultrastructural similar to herpesvirus-like particles (Jacobson et al., 1991); and third, experimental transmission of the disease using cell-free tumour extracts inoculated into healthy turtles resulted in the development of FP disease (Herbst et al., 1995). Nevertheless, the role of ChHV5 as causal agent for FP remains inconclusive as; (i) in vitro culturing has been repetitively unsuccessful and (ii) ChHV5 DNA can be also found in asymptomatic turtles (Alfaro-Núñez & Gilbert, 2014).

The observation of higher levels of ChHV5 DNA in tumour versus non-tumour samples from FP exhibiting turtles was expected, as ChHV5 DNA burdens are increased within tumour tissues (see Table 2 and Data S1 —copy number loads of gB and nuDNA per turtle sample grouped by health status tissue). What is remarkable though, is that on average, clinically healthy turtle samples exhibited viral to host DNA ratios that were significantly higher than those within non-tumour affected tissues of FP exhibiting turtles (Fig. 2B). A key fact in this regard is that no visually observable FPs were reported for any of the ‘healthy’ turtles sampled during the tissue collection. Although ChHV5 DNA has previously been detected in skin samples from clinically healthy green, loggerhead and olive ridley turtles (Herbst et al., 2008), these animals were from populations historically well documented containing FP infected turtles.

We reiterate that the small number of samples analysed in this study represent a sub-sample set from a larger number of turtle populations recently published work (Alfaro-Núñez & Gilbert, 2014). Thus, all samples analysed here have been already detected viral DNA positive by PCR providing further evidence of actual presence of the herpesvirus. Our results however provide first evidence of viral loads in multiple sub-samples of clinically affected and healthy turtles from distant ocean basins across four species. In fact, the ratios of gB to turtle nuDNA were actually very high (>10) in six of the clinically healthy turtles (Fig. 1). These samples correspond to four hawksbill turtles from Principe Island, Western Africa, which are known to share feeding grounds with green turtle FP exhibiting populations (Duarte et al., 2012); one leatherback from Ghana and one loggerhead in the Mediterranean (Table 2). FP has rarely been documented in hawksbill (Herbst, 1994; D’Amato & Moraes-Neto, 2000) and leatherback (Huerta et al., 2000) species, so the detection of ChHV5 gB DNA in these two species represent a quandary. Nuclear DNA levels were consistent among samples and routine use of negative and positive controls performed as expected, therefore, it is not suspected that these results are artefact of procedure. Furthermore, as with most alphaherpesviruses ChHV5 primarily appears to target skin tissue (Kang et al., 2008). Thus, the presence of ChHV5 gB DNA in skin from clinically healthy animals may represent an early stage of infection which remains in latency, and detection of viral concentrations in tumour-free animals may be a means of identifying animals that will eventually develop FP tumours (Quackenbush et al., 2001) subjected to the appropriate co-factors triggering the infection. The mechanism underlying this phenomenon remains unclear and was not addressed in this study. There is no evidence that immunosuppression is needed for FP development, but there is evidence that FP does lead to immunosuppression in Hawaiian green turtles (Work et al., 2001).

The gB gene selected for this study is an envelope protein that is only found in large amounts in intact virions undergoing lytic replication (Zhu et al., 2004). As in FP in turtles, skin tumours are the only tissues where lytic replication with intact virions has been consistently documented (Work et al., 2009), one would expect gB levels to be relatively high in these tissues. Thus, we were anticipating and expecting high levels of ChHV5 gB DNA in FP tumours, followed by relatively low in FP non-tumours and extremely low to absent in healthy tissue samples. However, high levels of herpesviral DNA in skin from clinically healthy animals suggest another not described process in regards to development of ChHV5 latency.

Many herpesviruses are maintained in sensory neurons in a latent state in which no clinical disease is produced and characterised by the repression of most viral lytic cycle genes and the abundant expression of certain types of viral RNA (Kramer et al., 1998). Such repression of viral protein synthesis during latency has led to the general view of latency as being a quiescent and antigenically silent infection that is ignored by host immunity (Ramachandran et al., 2010). However, very low levels of gene transcripts and proteins from all kinetic classes of herpes simplex virus have been detected in latently laboratory infected rodents (Feldman et al., 2002). Previous studies have detected nucleosomes on the herpes simplex virus (HSV) genome during a lytic infection (Deshmane & Fraser, 1989) that are not arranged in an equally spaced array like in cellular DNA. However, as in cellular DNA, the promoter regions of several viral genes have been shown to be associated with nucleosomes containing modified histone proteins that are generally found associated with actively transcribed genes (Oh & Fraser, 2008). Because no histones were detected inside HSV type 1 capsids, the viral genome probably starts to associate with histones after being transported from infecting virions into the host nucleus. This represents evidence that a herpesvirus genome can remain intact in the host nucleus disassociated from the viral envelope protection. Thus, we propose that a similar situation exists in turtles latently infected with ChHV5, where our data provides evidence of gB DNA being consistently present in clinically healthy marine turtles across four different species. We caution here that our methods detect only a portion of the gB DNA and does not imply production of lytic virus in normal skin from FP free animals.

While Koch’s postulates are still regarded as the most compelling proof of causation, there are documented instances where disease agents cannot be cultured, yet are now known to be causative (Lipkin, 2009), such as the herpes simplex and polio viruses. Although a 21st century adaptation of Koch’s postulates (Fredericks & Relman, 1995) have been proposed to reflect the introduction of culture-independent molecular methods, these updated criteria are also not yet fulfilled by ChHV5. Thus, ChHV5’s association with FP remains tantalising, yet unsolved.

In summary, our findings offer evidence that clinically healthy marine turtles from distant sites worldwide where FP has not been reported yet across species carry ChHV5 DNA through persistent latent infections, often at very high levels. This in turn provides additional evidence for hypotheses (Herbst & Klein, 1995; Van Houtan, Hargrove & Balazs, 2010) that ChHV5 is a near ubiquitous virus and alone does not cause infection, but requires one or more possibly environmental or immune related co-factors or triggers to induce FP.

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