Effects of Mustelid gammaherpesvirus 1 (MusGHV-1) Reactivation in European Badger (Meles meles) Genital Tracts on Reproductive Fitness

Reactivation of latent Gammaherpesvirus in the genital tract can lead to reproductive failure in domestic animals. Nevertheless, this pathophysiology has not received formal study in wild mammals. High prevalence of Mustelid gammaherpesvirus 1 (MusGHV-1) DNA detected in the genital tracts of European badgers (Meles meles) implies that this common pathogen may be a sexual transmitted infection. Here we used PCR to test MusGHV-1 DNA prevalence in genital swabs collected from 144 wild badgers in Ireland (71 males, 73 females) to investigate impacts on male fertility indicators (sperm abundance and testes weight) and female fecundity (current reproductive output). MusGHV-1 reactivation had a negative effect on female reproduction, but not on male fertility; however males had a higher risk of MusGHV-1 reactivation than females, especially during the late-winter mating season, and genital MusGHV-1 reactivation differed between age classes, where 3–5 year old adults had significantly lower reactivation rates than younger or older ones. Negative results in foetal tissues from MusGHV-1 positive mothers indicated that cross-placental transmission was unlikely. This study has broader implications for how wide-spread gammaherpesvirus infections could affect reproductive performance in wild Carnivora species.


Introduction
The herpesviridae comprise four subfamilies, the alphaherpesvirinae, betaherpesvirinae, gammaherpesvirinae [1], and the newly assigned deltaherpesvirinae [2], which can all maintain lifelong infection, and exhibit prolonged latency and repeated reactivation. Infection typically causes no, or only mild subpatent, disease [3], although serious symptoms may be observed in immuno-compromised trade-offs [44], infanticide in Southern England [42] (but not in Ireland [39]), and high neonatal cub mortality due to coccidiosis [45]; but no single suggested cause can fully explain the observed phenomenon [42]. Surprisingly, the potential for this reduced fecundity, infertility, or abortigenic disease to be linked to STIs [46], has been largely ignored in badgers (but see [47]).
The Mustelid gammaherpesvirus 1 (MusGHV-1), a member of the genus Percavirus, was first isolated from lung tissue of a wild badger in 1996 without obvious association to disease [48]. It is highly prevalent amongst wild badgers in the UK [49] and Ireland [50], where MusGHV-1 DNA isolation from blood shows near 100% infection rate at some localities. Nevertheless, unlike alphaherpesviruses that reside in neuron cells during latency, gammaherpesviruses reside in B-lymphocytes (EBV, MHV-4, EHV-5) [4,51,52] or monocytes (BHV-4) [53], and therefore it is not possible to differentiate latency from reactivation when isolating viral DNA from whole blood samples. Confirming sexual transmission in a free-ranging population without using manipulative experiments is extremely challenging, but indirect evidence permits inference of horizontal and/or vertical transmission, for example, by isolating the pathogen DNA using PCR from swabs of genital tracts, placenta tissues, or foetal tissues. A population-wide screening survey revealed a reactivation rate of 55% in genital tract mucosae of badgers in a high density population in the UK [47], indicating a high likelihood of sexual transmission. This same study also linked reactivation of genital MusGHV-1 to reduced future reproductive success in female badgers [47], but did not investigate the factors contributing to herpesvirus reactivation. Here, we use badger samples collected post-mortem from across the Republic of Ireland under a national culling programme for the management of bovine tuberculosis to investigate: 1.
Patterns of MusGHV-1 reactivation in badger genital tracts by comparing prevalence among females culled at the start of the primary mating season with those culled at the end (where many females are pregnant at the beginning, but all will have given birth by the end); 2.
Differences in MusGHV-1 reactivation between males and females, and between different age groups; 3.
Effects of MusGHV-1 reactivation on female reproductive success, male testes weight, and sperm abundance; 4.
The potential for vertical transmission of MusGHV-1 to the foetus via the placenta during gestation, or via the maternal vaginal tract during parturition; and 5.
The potential for horizontal transmission through semen.
We discuss our results in the framework of potential impacts of MusGHV-1 infection on badger population dynamics and reproductive health and in the broader context of carnivore conservation.

Interactions of Genital MusGHV-1 Reactivation with Body Weight
There was no overall body weight difference between MusGHV-1 positive and negative individuals, with the exception of females in winter. As pregnancy is energetically costly, we further separated females into pregnant and nonpregnant, and evidenced that MusGHV-1 was associated with lighter bodyweight only in pregnant females, but not for those that had already given birth or had not been pregnant; nevertheless, this effect was not statistically significant, perhaps due to the small sample sizes (t = 1.8691, df = 4.8914, p-value = 0.122; t-test) (Figure 1). Pathogens 2020, 9, x FOR PEER REVIEW 5 of 17

Interactions of Genital MusGHV-1 Reactivation with Body Weight
There was no overall body weight difference between MusGHV-1 positive and negative individuals, with the exception of females in winter. As pregnancy is energetically costly, we further separated females into pregnant and nonpregnant, and evidenced that MusGHV-1 was associated with lighter bodyweight only in pregnant females, but not for those that had already given birth or had not been pregnant; nevertheless, this effect was not statistically significant, perhaps due to the small sample sizes (t = 1.8691, df = 4.8914, p-value = 0.122; t-test) (Figure 1).

Effects of Genital MusGHV-1 Reactivation on Reproductive Fitness
Relative sperm abundance ranged from 0 to 160.38 × 10 5 / mL with an average of 43.89 × 10 5 spermatozoa/mL (±40.18 × 10 5 ) of equally diluted semen collected from 39 males. However, there was no significant effect of genital MusGHV-1 reactivation on relative testicular weight (p = 0.82), but a trend-albeit not significant (p = 0.085)-for positive males to have higher sperm abundance ( Figure 2).

Interactions of Genital MusGHV-1 Reactivation with Body Weight
There was no overall body weight difference between MusGHV-1 positive and negative individuals, with the exception of females in winter. As pregnancy is energetically costly, we further separated females into pregnant and nonpregnant, and evidenced that MusGHV-1 was associated with lighter bodyweight only in pregnant females, but not for those that had already given birth or had not been pregnant; nevertheless, this effect was not statistically significant, perhaps due to the small sample sizes (t = 1.8691, df = 4.8914, p-value = 0.122; t-test) ( Figure 1).

Potential for Horizontal and Vertical Transmission
One (20%) seminal plasma sample from genital MusGHV-1 negative males tested positive for MusGHV-1, but interestingly none of the five samples from genital MusGHV-1 positive males contained cell-free MusGHV-1 DNA. From 18 well-preserved pregnant females, we dissected a total of 43 foetuses (partial dissection of 33 and full dissection of 10 foetuses), which all appeared to be healthy and normally developed, irrespective of the mother's herpes infection status. Three out of 10 placentas tested were positive for MusGHV-1 DNA, (one from a genital MusGHV-1 negative mother; two from mothers with partial foetal loss-one tested positive for genital MusGHV-1, one negative); however, no MusGHV-1 DNA was detected in any foetal tissues.

Abnormalities in Female Reproductive Tracts Associated with Genital MusGHV
In 25% (7/28) of females (three young and four old adults) that were pregnant or showed signs of recent pregnancy, we recorded possible abortions (enlarged uterus but no foetuses/placental scar(s) present, but with signs of reabsorption, such as dark, amorphous mass of dead cells in the enlarged uterus [41]) of entire 14.3% (4/28) or partial (10.7%; 3/28) litters. All four females (three were >5 years old, one was <3 years old) with complete litter loss showed no sign of lactation and tested positive for genital MusGHV-1 reactivation Furthermore, one of the four pregnant MusGHV-1 positive females had reddened cyst-like gross lesions surrounding the cervix; one had submucosal haemorrhage around the cervix, and one had the remains of an absorbed foetus (in addition to two healthy ones) in her uterus; only one showed no abnormalities in her reproductive tract.
We also observed that 48.38% females (30/62, 40 from winter and 22 from spring) and 52.5% males (21/40, all from winter) displayed macroscopic lesions that resembled lymphoid hyperplasia on the epithelium of the lower genital tracts or around the cervix in females, and the mucosal epithelium of the outer urethra in males. These gross lesions took the form of vesicle-like ( Figure 3A), congestive and oedemic ( Figure 3B), or pigmented dotted scars ( Figure 3C), but were not always associated with genital MusGHV-1 reactivation (Supplementary Material S2). We only tested one lesion tissue ( Figure 3B) for MusGHV-1 and the result was positive, but the genital swab of this specific animal tested negative.
Pathogens 2020, 9, x FOR PEER REVIEW 7 of 17 haemorrhage around the cervix, and one had the remains of an absorbed foetus (in addition to two healthy ones) in her uterus; only one showed no abnormalities in her reproductive tract. We also observed that 48.38% females (30/62, 40 from winter and 22 from spring) and 52.5% males (21/40, all from winter) displayed macroscopic lesions that resembled lymphoid hyperplasia on the epithelium of the lower genital tracts or around the cervix in females, and the mucosal epithelium of the outer urethra in males. These gross lesions took the form of vesicle-like ( Figure 3A), congestive and oedemic ( Figure 3B), or pigmented dotted scars ( Figure 3C), but were not always associated with genital MusGHV-1 reactivation (Supplementary Material S2). We only tested one lesion tissue ( Figure 3B) for MusGHV-1 and the result was positive, but the genital swab of this specific animal tested negative. Histopathological examination confirmed that all seven samples from animals with gross lesions investigated (females: one cervix and four inner genital tract tissue samples from females; males: two inner genital tract tissue samples) showed inflammation, with five out of the six genital tract tissues exhibiting epithelial hyperplasia (Supplementary Material S3). Among these, only one male mucosal tissue collected from the base of the penis had intra-nuclear inclusion bodies in the epithelium, likely indicating viral infection (Figure 4). Histopathological examination confirmed that all seven samples from animals with gross lesions investigated (females: one cervix and four inner genital tract tissue samples from females; males: two inner genital tract tissue samples) showed inflammation, with five out of the six genital tract tissues exhibiting epithelial hyperplasia (Supplementary Material S3). Among these, only one male mucosal tissue collected from the base of the penis had intra-nuclear inclusion bodies in the epithelium, likely indicating viral infection (Figure 4).
Genital discharge was present in 74.2% (23/31) of females during spring, and 7.5% (3/40) of females and none of the males (0/40) in winter, but was not associated with MusGHV-1 reactivation. A cervical mucus plug (CMP) was observed in 44% (8/18) of the pregnant females, none of which tested positive for genital MusGHV-1 reactivation (compared to 40% genital MusGHV-1 prevalence in the remaining 10 pregnant females without a CMP).
Histopathological examination confirmed that all seven samples from animals with gross lesions investigated (females: one cervix and four inner genital tract tissue samples from females; males: two inner genital tract tissue samples) showed inflammation, with five out of the six genital tract tissues exhibiting epithelial hyperplasia (Supplementary Material S3). Among these, only one male mucosal tissue collected from the base of the penis had intra-nuclear inclusion bodies in the epithelium, likely indicating viral infection (Figure 4). Genital discharge was present in 74.2% (23/31) of females during spring, and 7.5% (3/40) of females and none of the males (0/40) in winter, but was not associated with MusGHV-1 reactivation. A cervical mucus plug (CMP) was observed in 44% (8/18) of the pregnant females, none of which tested positive for genital MusGHV-1 reactivation (compared to 40% genital MusGHV-1 prevalence in the remaining 10 pregnant females without a CMP).
Nevertheless, MusGHV-1 reactivation rates fluctuated over a badger's lifetime: middle aged (3-5 years old) individuals had lower reactivation rates than young (2-3 years old) and over 5 year old animals, and all MusGHV-1 positive pregnant female badgers belonged to the old age group. This mirrors results from humans and some domestic animals where primary infection occurs predominantly during infancy [59], with initial reactivation in mucosae near the entry portal [60] and repeated reactivation throughout life [51], which increases with aging due to declining immune-competency [14].
Overall, we found no difference in the body weights of genital MusGHV-1 positive and negative individuals suggesting that MusGHV-1 reactivation does not affect-nor was triggered by-host body condition, mirroring results from in England [47]. We did, however, identify a nonsignificant trend in pregnant females, where genital MusGHV-1 positive individuals had lighter body weights than those that tested negative (Figure 1), indicating that during pregnancy, reactivation may lead to decreased body condition [61], or-conversely-that reduced body condition during pregnancy may promote MusGHV-1 reactivation [62].

Sex-Specific Seasonal Patterns in Genital MusGHV-1 Reactivation
Our study shows that male badgers are significantly more at risk of genital MusGHV-1 reactivation than females, particularly at the beginning of postpartum mating season (end of January and early February). Latent sexually transmittable pathogens likely evolved to be reactivated during their host's mating season to maximize host-to-host transmission [63]; a pattern well-established for cyprinid herpesvirus CyHV-3 in wild common carp [64]. The main mating season in badgers coincides with the postpartum oestrus (in the UK and Ireland typically in the middle of February: [37,65], but males start their "rutting behaviour" several weeks earlier from the middle of January [66], coinciding with a rise Pathogens 2020, 9, 769 9 of 17 in their testosterone levels [67]. Testosterone, however, acts as an immune-suppressant [68] and may thus be partly responsible for the corresponding peak in male MusGHV-1 prevalence rates, mirroring results in domestic cats where males also have a higher risk than females to be infected with the feline gammaherpesvirus FcaGHV-1 [33,69], and entire males are at higher risk of infection with FcaGHV-1 than neutered males [70]. Female badger MusGHV-1 reactivation rates in the genital tract, in contrast, are higher in spring at the end of the main mating season than at its start in January. This could be due to the physiological stress linked to late-term pregnancy, birthing, lactation [71], and postpartum mating [72], but may also arise due to (re-)infection through mating with males shedding MusGHV-1 in their genital tract (e.g., equid alphaherpesvirus 3 reported in seropositive mares re-shed after mating with virus-shedding stallions [73]), where our results show that over 80% MusGHV-1 prevalence in genital tracts of male badgers, and 10% semen contained viral DNA.

Effects of Genital MusGHV-1 Reactivation on Reproductive Fitness
Although reproductive failure [41] and premature cessation of lactation [39] are often reported in badgers, specific studies investigating potential links to reproductive tract diseases are lacking. MusGHV-1 reactivation did not decrease male sperm production; in fact, there was a nonsignificant trend for MusGHV-1 positive males to have a higher sperm count than MusGHV-1 negative ones (Figure 2A), although we found no difference in testicular weight connected to genital herpes ( Figure 2B). This suggests that, in males, genital MusGHV-1 reactivation does not cause sterility, although effects on sperm morphology, motility, and viability remain unknown (see also humans: [74]).
In females, in contrast, genital MusGHV-1 reactivation has a negative effect on reproductive fitness, manifesting in failure to carry pregnancy to term as seen in in other species [15]. The abortion rate (entire litter: 14.3% and partial 10.7%) confirmed in this study is considerably higher than the <1% previously reported in Irish badgers [37,39]. This is likely due to the concentrated sampling immediately before the main birthing period in this study, which appears to be more likely to observe abortions through gross examination, but comparable to rates reported from England (complete litter loss: 20-33% [42], partial loss: 10-14.3% [42]). In winter, genital MusGHV-1 prevalence rates were significantly higher in females that did not reproduce than in those carrying healthy foetuses or having carried their pregnancy to term. All four females with signs of spontaneous abortion of their full litters, as well as one out of the three females with late partial abortion tested MusGHV-1 positive. This further substantiates the negative association of genital MusGHV-1 reactivation with badger female reproductive fitness. In addition, 4/5 of these were old, potentially indicating that aging in in combination with increasing stress when approaching parturition [75] may favour latent MusGHV-1 reactivation, thus increasing the likelihood of abortion [76].
In domestic animals, gammaherpesvirus-associated reproductive failure has frequently been shown to be associated with bacterial coinfection [19][20][21][22]. During pregnancy, cervical mucous plugs (CMPs) likely form a physical and immunological barrier to prevent microbes ascending into the uterus from the lower reproductive tract (e.g., humans: [77]; mares: [78]). While we did not investigate potential differences in the genital microbiome community structure, all pregnant females with a CMP tested MusGHV-1 negative, compared to a 40% prevalence of genital MusGHV-1 reactivation in females without a CMP, suggesting that reproductive success may be affected by complex relationships between MusGHV-1 reactivation, bacterial microbiome dynamics, and host immunity [79].

Genital Lesions
Both sexes commonly had gross lesions in their genital tracts resembling the lymphoid hyperplasia and hyperaemia of the vaginal mucosa characteristic of female dogs infected with CHV-1 [80], and similar to the progress of equine coital exanthema caused to horse genitalia by EHV-3 infection [81,82]. In the case of badgers, however, we did not find a significant correlation between the presence of gross genital lesions and MusGHV-1 reactivation, nor with female reproductive success, although intranuclear inclusion bodies-an indicator of herpesviral lytic infection [83]-were found during histopathological examination in epithelial cells of the genital mucosa of one male with epithelial hyperplasia. This may be due to lytic infection and virion-shedding not necessarily being synchronized with the formation of lesions, as reported from experimental CvHV-2 reactivation in reindeer [20].

Vertical Transmission of MusGHV-1
Our findings suggest that vertical transmission of MusGHV-1 through the placenta is possible but unlikely. This corroborates other studies on gammaherpesvirus transmission (e.g., MHV-4 transmission in mice [13]; EBV in humans [82]), but contradicts a study of BHV-4 transmission in cattle, where intrauterine infection was found in 4/7 stillbirth foetuses [76]. Nevertheless, primary infection in foetuses may likely happen during parturition, or through close postnatal contact with the mother, explaining results from a previous study that found 75% 3-month old prepubescent cubs presenting positive for genital MusGHV-1 [47].

The Significance of Gammaherpesviruses as Drivers of Wildlife Population Dynamics
Gammaherpesviruses are of increasing concern in humans [51,58] and animals (livestock: [15], pets: [84], wild [28,31,48,69], and captive wildlife populations: [26]). EBV is prevalent in 90% of the human population, and is associated with diseases such as infectious mononucleosis, childhood leukaemia from maternal infection and, more rarely, lymphoma [51,85,86]. Bovine herpesvirus 4 is associated with abortion in cattle [15], and the widespread equid herpesvirus 2 and 5 are associated with rare diseases such as fatal equine multinodular pulmonary fibrosis [52], and there is increasing evidence of their connection to other reproductive disorders [25]. Most recently, infections by a novel gammaherpesvirus Leporid gammaherpesvirus 5 (LeHV-5) were identified in wild Iberian hares (Lepus granatensis) with genital and skin lesions, and will likely intensify the recent population decline [87].
Although gammaherpesvirus associated diseases have rarely been reported in wild animals, this observation might be due to the difficulty of diagnosis in the field, and to asymptomatic infection, especially for abortigenic diseases. However, reports of novel herpesviruses in wildlife have strengthened the assumption of ubiquity across the animal kingdom. Broad surveys of occurrence and in-depth research on associated diseases in wildlife are warranted to enhance understanding of gammaherpesvirus effects on epidemiology, pathogenicity, evolution, and disease ecology. As theory predicts [88,89] and empirical studies evidence [90,91], STI-induced sterility can constitute an important driver of population dynamics, and superficially benign infections without clinical manifestation may in fact drive already endangered wildlife populations to extinction.

Source of Post-Mortem Badgers
Samples were collected at the Irish Equine Centre (IEC) between 23rd and 26th April 2019 (lactation/end of primary mating season) and 3rd-7th February 2020 (end of gestation/onset of the primary postpartum mating season) post-mortem from 144 badgers (71 males and 73 females) from across the Republic of Ireland culled under licence by the National Parks and Wildlife Service, with powers conferred by the Wildlife Acts 1976 to 2010 in the context of a national removal scheme to control bovine tuberculosis led by the Department of Agriculture, Food and the Marine (DAFM) [92] (see Table 2 for details). Carcasses were preserved in a cold room at 0-2 • C as soon as possible after death and were examined between 1 and 10 days after death (average = 4.58 ± 1.95 days, median = 4 days). For each badger, culling site, sex, weight, and approximate age group (estimated from tooth wear: [93] and categorized as cubs: <1 year old; yearling: <2 years old; young adult: 2-3 years old; middle-aged adult: 3-5 years old; old adult: >5 years old) were recorded. All badgers were examined for genital discharge and lesions in the reproductive system.

Assessment of Reproductive Fitness
In females, appearance and interior of the uterus were examined and reproductive status was classified as follows: pregnant (carrying developing foetus(es)), recently pregnant (enlarged and thickened uterus with dark pigmented placental scars and mammary development/lactation), recently pregnant but aborted (enlarged and thickened uterus with no or pale placental scars, or no mammary development), not pregnant (determined by opaque round uterine appearance, absence of placental scars and small nonelongated teats; or thin translucent uterus with no evidence of uterine thickening, placental scars, or mammary development) [39]. Uterine flushing with 0.9% saline solution and following examination of the flush exudate under a dissecting microscope at 4× magnification were used to determine recent, successful mating (i.e., presence and number of blastocysts in uterus). Litter size was determined through direct counts of foetuses if the female was pregnant, or placental scar counts if it had already given birth [94]. Lactational status was determined peripartum in winter by gross and histological examination of the mammary gland tissues, and postpartum in spring by mammary development and presence of milk [39].
In males, one testis was collected using sterile scissors, and after removing all attached fat, muscles and blood vessels, was weighed in g using a precision balance [67]. The epididymis was then detached and placed on a sterile petri dish, before being macerated and mixed with 2.5 mL 1× PBS to release the semen. The diluted semen was transferred to a sterile 2 mL microcentrifuge tube and stored at 4 • C before processing. Relative abundance of sperm was determined using a haemocytometer (1:10 diluted by 1× PBS) under a microscope at 40× magnification [95].

Assessing Potential for Vertical and Horizontal Transmission of MusGHV-1
To investigate, if MusGHV-1 can be transmitted vertically prebirth (transplacentally), we collected maternal placentas and foetal brains, lungs, spleens, and livers from eight pregnant females (four females that tested MusGHV-1 positive in genital swabs and four females that tested negative in genital swabs-i.e., still with potential for latent infection or reactivation in other body-sites). To investigate if horizontal transmission through cell-free MusGHV-1 DNA in semen is plausible, we selected diluted semen samples from 10 males (five genital swab MusGHV-1 positive and five genital swab-negative control samples to elucidate if potential cell-free viral DNA originates from sperm or from epithelial cells in the genital tract), and then collected seminal plasma from the supernatant after centrifugation at 1500 rpm for 10 min. All swabs, tissue, and semen samples were frozen immediately and stored at −20 • C before being shipped frozen and packaged in dry ice to the University of Oxford for DNA extraction and PCR.

Histopathological Examination
Mammary gland tissues from all females with recent pregnancy but uncertain lactation status were collected to confirm lactation by histological examination. Representative lower genital tract tissue samples with lesions were collected from five females and two males (according to availability) and fixed in 10% neutral buffered formalin. These formalin-fixed tissues were trimmed, dehydrated through a graded ethanol series, and then infiltrated with paraffin. These embedded tissues were then sliced at 4 µm and stained with haematoxylin and eosin (H&E) for microscopic examination.

MusGHV-1 DNA Detection in Genital Tracts, Semen, and Tissues
To determine MusGHV-1 reactivation in badger genital tracts (i.e., presence of cell-free MusGHV DNA), lower genital tracts were swabbed with sterile cotton tops with wooden shafts (Dynarex, New York, USA) to secure virions without damaging the epithelium. Any swabs contaminated with blood (from culling/prior dissection) were centrifuged at 1500 rpm for 10 min to eliminate circulating white blood cells that could contain latent MusGHV-1. Samples were then placed in empty 2 mL sterile microcentrifuge tubes and stored at −20 • C.
A ca. 25 mg subsample of each tissue was taken, and each swab was reconstituted with 400 µL sterile doubly distilled water, vortexed gently at room temperature for 10 min, before decanting a 200 µL aliquot from the supernatant. DNA was processed using DNeasy Blood and Tissue Kit (Qiagen), following the manufacturer's instructions. Purified DNA was then eluted in 100 µL of the buffer provided.
Purified DNA was screened for MusGHV-1 using a specific primer pair (former primer: 5 -CCA AGC AGT GCA TAG GAG GT-3 ; reverse primer: 5 -TGG ACT TCT CCA ACA TGC GTC GCC CTT C-3 ) modified from King et al. 2004 [50], targeting 771 base pairs of a partial DNA polymerase gene. For each reaction, a total of 20 µL PCR solution was mixed with 10 µL HotStartTaq Master Mix (Qiagen) containing 1 unit of HotStartTaq DNA Polymerase, 12 µM of MgCl 2 , and 1.6 µM of each dNTP), 0.5 µM of front and reverse primer, 2 µL Coralload gel loading dye, and 5 µL DNA template. Amplification conditions were kept at 95 • C for 5 min to activate DNA polymerase, followed by 45 cycles of denaturation at 95 • C for 45 s, primer annealing at 60 • C for 45 s, and chain elongation at 72 • C for 1 min, followed by a final extension at 72 • C for 10 min. Finally, the PCR products were loaded in 2% agarose gel to check amplification results under UV light. Representative PCR products with positive results were genotyped by Sanger sequencing (Zoology Sequencing Facility, University of Oxford), and one representative sequence was submitted to the NCBI Genbank under accession number MT332102. Trimmed sequences were compared to previously published sequences on GenBank by the online NCBI Blast service.

Statistical Analysis
All sample details and variables are shown in Supplementary Material S1. Statistical analyses were performed using R Studio software (version 1.21335). Prevalence of MusGHV-1 was calculated as the percentage of PCR positive cases over the total number of samples examined, and 95% upper and lower confidence intervals were calculated using the Wilson method [96]. Univariate analysis using Chi-square tests with Yates continuity correction and Fisher's exact test (if any column of the contingency table was less than 5) were used to investigate if age, sex, or age groups were risk factors for genital MusGHV-1 reactivation in badgers, and whether genital MusGHV-1 reactivation affects female reproductive fitness. To investigate whether there was an interaction between MusGHV-1 reactivation and badger weight, herpes-positive and negative captures of each sex, in each culling period, were compared separately with t-tests to standardize for sexual and seasonal weight differences [39]. To investigate effects of genital MusGHV-1 on testes weight and relative sperm concentration, herpes-positive and negative males were compared using t-tests. Logistic regression multivariable analysis and odds ratio were used to estimate the size effect of different risk factors on MusGHV-1 reactivation, and the area under receiver operating characteristic curve (AUROC) was calculated for diagnostic ability of the final model [97]. Due to data availability, age group analysis was only applied to samples collected in winter, and yearlings were excluded (due to very low sample sizes). We are currently working on a different (n = 251) dataset from a UK population, where we show that age has strong correlation to MusGHV-1 prevalence (quadratic term, adjusted R 2 = 0.602, p < 0.001). We use adjusted R 2 as effect size, predictor = 1, significance level = 0.05 to calculate the minimum size required to reach 0.8 statistical power [98], and determined that n = 8 has sufficient statistical power to reach a valid statistical result.