Herpesvirus surveillance and discovery in zoo-housed ruminants

Gammaherpesvirus infections are ubiquitous in captive and free-ranging ruminants and are associated with a variety of clinical diseases ranging from subclinical or mild inflammatory syndromes to fatal diseases such as malignant catarrhal fever. Gammaherpesvirus infections have been fully characterized in only a few ruminant species, and the overall diversity, host range, and biologic effects of most are not known. This study investigated the presence and host distribution of gammaherpesviruses in ruminant species at two facilities, the San Diego Zoo and San Diego Zoo Safari Park. We tested antemortem (blood, nasal or oropharyngeal swabs) or postmortem (internal organs) samples from 715 healthy or diseased ruminants representing 96 species and subspecies, using a consensus-based herpesvirus PCR for a segment of the DNA polymerase (DPOL) gene. Among the 715 animals tested, 161 (22.5%) were PCR and sequencing positive for herpesvirus, while only 11 (6.83%) of the PCR positive animals showed clinical signs of malignant catarrhal fever. Forty-four DPOL genotypes were identified of which only 10 have been reported in GenBank. The data describe viral diversity within species and individuals, identify host ranges of potential new viruses, and address the proclivity and consequences of interspecies transmission during management practices in zoological parks. The discovery of new viruses with wide host ranges and presence of co-infection within individual animals also suggest that the evolutionary processes influencing Gammaherpesvirus diversity are more complex than previously recognized.

Certain GHV infections are problematic for captive, zoo, and free-ranging ruminants and have been the focus of numerous investigations aimed at elucidating determinants of their pathogenicity [4,16,[24][25][26][27][28][29][30][31][32]. Screening surveys to assess prevalence of members in the Macavirus genus have been valuable for improving animal management and established the initial foundation of GHV ecology [33,34]. Unfortunately, no vaccines are currently available for any macaviruses; therefore, disease prevention relies solely on the segregation of reservoir and susceptible species. Studies describing new viruses have also contributed to characterization of host-pathogen relationships [9,22]. Yet, much remains unknown about prevalence, diversity, and pathogenic potential of most members.
Although GHVs diverged from other herpesviruses approximately 200 million years ago and continue to drift under varied selection pressures [35,36], members maintain essential core genes with conserved regions that are the basis of several broad range detection assays [12,18,[37][38][39][40]. Nested PCR methods targeting the DNA polymerase (DPOL) and Glycoprotein B genes are the most widely used discovery tools and have helped identify many of the seven unique ruminant GHVs currently recognized by the ICTV. These assays are often used by diagnostic laboratories and have established the DPOL gene as the initial locus for identifying new viruses and taxonomic organization [2,11,22,41]. The current ICTV demarcation criteria for new HV species requires that "(a) their nucleotide sequences differ in a readily assayable and distinctive manner across the entire genome and (b) they occupy different ecological niches by virtue of their distinct epidemiology and pathogenesis or their distinct natural hosts" [42].
To better characterize the genetic diversity of GHVs and their host range, we tested antemortem (blood, nasal or oropharyngeal swabs) or postmortem (internal organs) samples that were collected opportunistically from a large number of healthy or diseased ruminants at the San Diego Zoo and San Diego Zoo Safari Park with a consensus-based herpesvirus PCR for a segment of the DPOL gene. The data describe viral diversity within species and individuals, identify potential host ranges of new viruses, and address the proclivity and consequences of viral infection during management practices in zoo animals. The investigation of viral diversity in such a broad survey greatly expands our knowledge of the host-range of new and previously described viruses, and aids in management of zoo-housed ruminants.

Ethics statement
This research was approved by and carried out in strict accordance with San Diego Zoo Global's Institutional Animal Care and Use Committee's (IACUC) recommendations. All sampling was performed under IACUC protocol number 231.

DNA extractions
All DNA was extracted using the DNeasy blood and tissue kit (Qiagen, Valencia, California, USA) according to the manufacturer's blood or tissue-sample protocol, except that the recommended amounts of sample were first placed with the lysis buffer in 1.5-ml screw-cap FastPrep . Maximum likelihood analysis was also performed using the amino acid alignment in the program PhyML v3.0 [48] with 1000 bootstrap replicates. The nucleotide sequences were aligned using CLUSTALW (total alignment length: 226bp) and phylogenetic analysis was repeated in MrBayes and PhyML using the same parameters used in the amino acid analysis. The General Time-Reversal model with a proportion of invariant sites and gamma distributed rate heterogeneity (GTR +I+G) model was selected as the optimal substitution model for both nucleotide analyses based on Akaike's Information Criterion scores calculated in jModelTest v2.1.10 [49,50]. All trees were visualized with TreeGraphv2.0 [51].

Sanger sequencing
Among 715 animals tested, 210 (29.3%) were PCR positive but only 161 (22.5%) were PCR positive and confirmed with DNA sequencing ( Table 1). The remaining 49 PCR positive cases did not produce an interpretable sequence after repeat attempts to sequence the PCR products. A complete list of the 161 PCR positive animals with confirmed HV sequencing and the samples tested is available in S1 Table. Eleven of the PCR positive animals were tested for HVs twice within a single year ( Table 2). The HV sequences were assigned a numerical identification number and are referred to as DPOL genotypes. All DPOL genotypes and their translated amino acids were subjected to a nucleotide (Table 3) and protein (S2 Table) NCBI BLAST analysis to confirm viral identities. A complete list of host species for each DPOL genotype is included in S2 Table. In total, 44 DPOL genotypes were identified, including 10 identical to nucleotide sequences previously reported in GenBank. Of the 34 newly identified genotypes, 14 had greater than 95% nucleotide identity (range = 96.0-99.4%) to previously reported sequences, and 20 had less than 95% identity (range = 75.5-93.4%) to previously reported sequences ( The 34 DPOL genotypes with less than 100% similarity to previously reported viruses in GenBank were assigned provisional names after their host species and tentatively assigned to Gammaherpesvirinae based on our phylogenetic and BLAST analysis; however, the primary host of these putative viruses remains to be confirmed. A single genotype identified in multiple hosts from the same sub-family or family was named after the sub-family or family; alternatively, a genotype identified in multiple hosts from different families within the order Ruminantia was assigned the name "Ruminantia". All DPOL genotypes are listed in Table 2 with their corresponding GenBank accession numbers.

Phylogenetic analyses
The phylogenetic trees created by MrBayes and PhyML were similar for both amino acid ( Fig  1) and nucleotide (S1 Fig) analyses and support the placement of all DPOL genotypes described here within Gammaherpesvirinae. Our phylogenetic analyses also support the presence of multiple lineages within Gammaherpesvirinae [52], including the genera Macavirus, Rhadinovirus, Lymphocryptovirus, and Percavirus. Many of the nodes within Gammaherpesvirinae strongly supported by Bayesian analysis lacked high support from maximum likelihood analysis; therefore, the more conservative maximum likelihood tree is shown for both amino acid (Fig 1) and nucleotide (S1 Fig) analyses with bootstrap and posterior probability values for each node.

MCF cases
A total of nine GHVs were detected in the 11 clinical MCF cases included in our sample population ( Table 4). Six of the GHVs detected in the clinical MCF cases were also detected in healthy individuals with no clinical signs of disease. Phylogenetic analyses revealed that only four of the nine GHVs isolated from clinical MCF cases grouped within the Macavirus genus (Fig 1). The remaining five GHVs isolated from clinical MCF cases could not be categorized into any single GHV genus, respectively.

Discussion
Our study presents a broad survey of the genetic variation of GHVs within ruminant species in the zoological collections of two facilities. Nearly 30% of 715 opportunistically tested individuals of 96 species at the San Diego Zoo and the San Diego Zoo Safari Park tested positive for GHVs of which only 11 (6.83%) showed clinical signs of MCF disease. Based on Sanger sequencing results of a subset (76.7%) of positive cases, our data confirm the presence of 10 previously discovered GHVs (including BoHV-6 (KJ705001), CaHV-2 (HM216474), Elk GHV

PLOS ONE
(KY612412), Elk GHV (KY612411), Impala HV-1 (AB194012), Oryx rhadinovirus 1 (AY212113), OvHV-2 (HQ450395), Springbok HV-1 (AB194010), Vaal Rhebok HV (AY664876), West Caucasian tur macavirus (HM216457)), and identify 34 novel GHV genotypes in ruminants. This supports many previous observations that GHVs are widespread in asymptomatic hosts and suggests that the potential diversity and host ranges of GHVs are greater than previously known [53]. Our phylogenetic analyses of both nucleotide and translated amino acid data indicate that the novel viral genotypes were members of the family Gammaherpesvirinae; however, the trees also revealed many unresolved polytomies. We attribute the unresolved branch orders within our phylogenetic analysis to the relatively short alignment length of our dataset and suggest that longer DNA sequences be used to resolve the phylogenic relationships in future analyses. Previous studies of GHV phylogeny using longer DNA segments (>3kb) and a multi-gene approach similarly observed polytomies within Gammaherpevirinae clades and also suggest that these polytomies could likely be resolved using even more extensive sequence data [52]. Despite the use of a relatively small DNA sequence fragment there are some strongly supported (i.e. bootstrap and posterior probability >70) clusters within the tree. The high nucleotide identities and phylogenetic confidence among the members of these clusters suggests that many of the GHVs in our sample population may be unique genotypes of the same viral species. However, more data is needed to characterize these putative viral sequences as unique species and confirm their assignment to known genera.
Co-infection was identified in five out of 161 individuals. In three of those animals (Animal ID: 18, 118, and 119), two GHVs were identified in a single tissue, blood, or swab sample. Co -Fig 1. Phylogenetic relationships of translated amino acid sequences from DPOL genotypes detected in ruminant species from the San Diego Zoo or it's Safari Park using PCR. The unrooted phylogenetic tree was constructed using translated amino acid sequences (71 aa) from DPOL genotypes in Table 3 and herpesviruses from GenBank. Accession numbers for each reference sequence are included in the figure. Maximum-likelihood bootstrap values are to the left of the slash ("/") and Bayesian Posterior Probabilities, expressed as a percentage, are to the right at each node. Nodes with Maximum-likelihood bootstrap values less than 50 were collapsed. The numerical DPOL genotype is followed by the provisional common name for new viral species or the previously described virus name from Table 3. DPOL genotypes obtained from clinical MCF cases are indicated with an asterisk ( � ), and references from GHV sub-families recognized by the International Committee on the Taxonomy of Viruses are indicated in bold. Known genera are indicated by color: red (Macavirus), purple (Rhadinovirus), blue (Lymphocryptovirus), and green (Percavirus). Known genus and sub-families are also indicated with brackets.
https://doi.org/10.1371/journal.pone.0246162.g001 Table 4. Summary of PCR and sequencing results from individual animals with clinical signs of MCF. infection of an individual host with multiple HVs has been documented previously [9,[54][55][56]; therefore, it was not surprising to identify multiple HVs within a single host species or individual animal from our sample population, especially given that many of the species had not been previously screened for HVs. Seventeen of the 44 GHVs were identified in more than one species (S2 Table), with four GHVs observed in host species across both families Cervidae and Bovidae. The three species with the highest number of PCR positive individuals sampled [i.e. Ellipsen waterbuck (Kobus ellipsiprymnus ellipsiprymnus), Javan rusa (Rusa timorensis russa), and Indian sambar (Rusa unicolor unicolor)] also had the highest number of unique GHVs. The three genotypes identified in Ellipsen waterbuck (i.e. DPOL genotypes 10, 34, 35) shared 86.8-92.0% nucleotide identity and all clustered together with high support in the phylogenetic analysis (Fig 1). Two of three Ellipsen waterbuck genotypes were only observed in Ellipsen waterbuck (i.e. DPOL genotype 34 and 35), while the third (DPOL genotype 10) was observed in multiple species, including Javan rusa and Indian sambar. The remaining four genotypes observed in Javan rusa (i.e. DPOL genotypes 4, 17, 25, 37) were highly similar (98.3-96.3% nucleotide identity) and clustered together in the phylogenetic analysis with high support. However, Ruminant GHV-3 (DPOL genotype 10) shared only 54.8-57.6% nucleotide identity with the other four Javan rusa DPOL genotypes, and phylogenetic analysis clustered it with other genotypes mostly observed in members of the family Bovidae. The surveyed Indian sambar followed a similar trend with the majority of individuals (7 out of 8 total) infected with two highly similar (97.1% nucleotide identity) GHVs (DPOL genotype 14 and 21), and a single individual infected with DPOL genotype 10. The presence of multiple highly related viruses in a single host species was not surprising given that (1) many of these species have not been surveyed for GHVs previously, and (2) the surveyed population includes animals bred in zoos rather than wild populations. Initial investigations of GHV evolutionary relationships proposed host-adaptation through the co-speciation of host and virus with limited inter-species transfer of viruses [2]; however, later investigations found that many of the GHV lineages were not compatible with a single co-speciational scheme [52]. Co-speciation and duplication events may explain broad evolutionary patterns within the Herpesviridae family as previously suggested [2,57] but the unpredicted complexities of the phylogenetic signal observed in our data could be the result of co-housing animals from geographically separate populations. Thus, the genetic diversity observed within our dataset may be the product of viral host-switching or spillover events from reservoir species to novel hosts given the close proximity of diverse host species in a zoological setting. Animals in this study were not all housed together at the same time, and closer scrutiny of complex individual movement patterns through shared environments may further reveal natural carriers and aberrant hosts. Additionally, future efforts should be aimed at whole genome sequencing in order to tease apart the complex web of evolutionary process shaping GHV diversity and disease processes.
The data further expands the host range of many previously described viruses, including the West Caucasian tur macavirus. This virus was previously identified in West Caucasian tur and its host range may now include four additional species. Our data also suggests that this virus may be associated with MCF in Eastern bongos which are listed as a critically endangered species by the International Union for the Conservation of Nature [58]. Three out of four Eastern bongos sampled had clinical signs of MCF and were infected with West Caucasian tur macavirus. The remaining Eastern bongo was sampled two years after the diseased bongos, and HVs were not detected in the sample at that time. Surprisingly, the Macavirus OvHV-2 was detected in a single domestic goat with clinical signs of MCF. A previous study indicated goats as subclinical carriers of OvHV-2 [59]; however, a recent study also identified MCF in a domestic goat infected with OvHV-2 [17]. Overall, the presence of MCF-related viruses in both healthy and diseased animals of the same species suggests a complex relationship between the virus and host.
Multiple factors that may have influenced viral detection in our study, including but not limited to the intermittence of herpesviral shedding and/or viremia in our study population, and the viral detection limit of the PCR assay. Previous investigations have suggested age, season, and genetic background of the infected individual as potential variables affecting viral shedding and viremia of GHVs [60,61]. Eleven of the 168 individuals with confirmed GHVs by Sanger sequencing were sampled at multiple time points within a single year, including three individuals that were positive at both time points, six that were only positive during the first time point, and two that were only positive during the second time point (Table 2). In one of the animals that was positive during both sampling events, Indian sambar (Rusa unicolor unicolor) #117, the consensus based PCR and Sanger sequencing identified two unique genotypes. In 2006 the nasal swab was positive for the Elk gammaherpesvirus (Genbank# KY612412), and in 2007 the oral/pharyngeal swab was positive for Ruminantia gammaherpesvirus 3 (Genbank# MN599424). Blood samples (Buffy coat) were also tested at both time points in nine of the 11 samples. The blood samples of four individuals were PCR positive for GHVs, but none of them were positive at both time points. These results may be explained by a seasonal influence or other factors that may affect viral shedding and viremia of the viruses detected in these samples. Although, it remains possible that the virus was either not sampled due to sampling error, recently contracted, or present at undetectable levels during the negative sampling events. More research is needed to determine the pathogenesis of the newly discovered viruses as well as the viral detection limit of the PCR primers used in this study. Therefore, the viruses identified in this study are presented as a conservative number of viral species present within a single sample at a single time point.
In summary, the GHV subfamily is an extremely diverse collection with members that have the ability to infect multiple species, and our data provides a snapshot in time of the complex genetic diversity within our sample population. The reported diversity of GHVs increases with sampling of more ruminants, and this diversity may reflect the evolutionary capacity of HVs to adapt to new and varied host conditions. While this study represents the broadest survey of GHV diversity within ruminant species to date, more work is required to define the host-virus relationship and disease complex of the novel viruses presented. A particularly exciting potential for future investigations is correlative studies looking for the existence of one or several common virulence markers that could be targeted in new therapies or vaccines. Future studies may also be directed at sampling wild populations of the species investigated in our study to determine if the viral sequences obtained from animals housed in a zoo reflect the diversity of viruses present in free-ranging populations.
Supporting information S1