Elephant Endotheliotropic Herpesvirus 1, 4 and 5 in China: Occurrence in Multiple Sample Types and Implications for Wild and Captive Population Surveillance

Elephant endotheliotropic herpesviruses (EEHVs) are important causes of death in both captive and wild Asian elephants (Elephas maximus). Nothing is known about the prevalence of EEHVs in wild or domestic elephants in China. To determine if EEHVs are present in elephants in China, 126 wild elephants from three populations and 202 captive individuals from zoos (n = 155) and the Wild Elephant Valley (n = 47) were screened using semi-nested polymerase chain reaction assays with EEHV-redundant and EEHV1/4/5-specific primers. EEHV1B and EEHV4 were detected in samples from both wild (EEHV1B:8/126; EEHV4:2/126) and captive (EEHV1B:5/155; EEHV4:9/155) elephants, while EEHV1A (six cases) and EEHV5 (one case) were only present in the captive elephants from the Wild Elephant Valley. EEHV1 was detected in blood and trunk and oral swabs; EEHV4 was detected in trunk and oral swabs as well as feces; EEHV5 was found in trunk and oral swabs. No significant age or sex association with EEHV1A, EEHV1B, or EEHV5 positivity was observed. An age association with EEHV4 positivity was found, with all unweaned elephants being EEHV4 positive, but an association with the sex of the elephant was not observed. These findings represent the first documentation of EEHV presence in captive and wild elephants in China. These findings also document EEHV1B and EEHV4 shedding in feces and demonstrate the utility of fecal screening as a tool for investigating EEHV4 infection in wild populations of elephants. It is recommended that EEHV testing be included in surveillance programs for captive and wild elephants in China.

Infections with EEHVs, in most cases, result in a subclinical but likely lifelong infection with Samples were collected from 202 captive Asian elephants from 2018 to 2020, representing approximately 63% of the entire population of captive elephants in China. Samples were collected from two sources: elephants from the Wild Elephant Valley (n = 47) and elephants in traditional zoos (n = 155) ( Table 1).
The Wild Elephant Valley (100 • 51'33.2172", 22 • 10'39.3636") facility is located within the Pu'er-Mengyang Asian elephant population area ( Figure 1). It is a tourist attraction and contains an Asian elephant rescue center. It has a mixture of imported elephants that are occasionally moved between institutions as well as captive-bred and rescued elephants. The elephants in this facility are allowed into their natural environment to forage and roam during the day accompanied by their mahouts; thus, they cannot have active direct contact with wild elephants during this time. However, during the night, wild elephants may visit their enclosure, and Wild Elephant Valley elephants can have physical contact with wild elephants through the bars of their enclosures; therefore, the two populations are not fully isolated. The origin, age, and sex of Wild Elephant Valley elephants and zoo elephants were recorded based on clinical records, and the elephants were categorized into groups according to their age. The age groups were based on Arivazhagan and Sukumar [34] with minor modification: adult (>15 y); sub-adult (5 < X ≤ 15 y); juvenile (3 < X ≤ 5 y); unweaned elephants (≤3 y).
The majority of Asian elephants in zoos were imported from other Asian elephant range countries with a small number of captive-bred elephants. These animals are routinely moved among zoos. Only fecal samples were taken from the zoo elephants (n = 155).
Elephants were classified as herpesvirus virus positive if herpesvirus DNA was detected in one or more samples.

Wild Populations
In 2017, fecal samples from three wild Asian elephant populations (i.e., Pu'er-Mengyang (n = 81), Mengla (n = 26), and Shangyong (n = 19)) were collected ( Figure 1). A hundred and twenty-six individuals were identified by microsatellite markers [35], and their fecal samples were used in this study. The tested individuals were aged by bolus diameter according to the rearranged Von Bertalanffy growth equation: t = (t − ) × ln 1 − ; t is the mean age, t0 is the theoretical age at which an elephant's bolus would have a length measurement of 0 if the same growth pattern was applied from birth to later life, K is the Brody growth coefficient, and L∞ is the theoretical maximum size of the bolus diameter [36,37]. Age groups were classified in the same way as captive elephants. At the time of sampling, there was approximately 280 wild individuals in China. The approximate number of each sampled population is shown in Table 1.

DNA Extraction
Fecal DNA was extracted using the QIAamp DNA Stool Mini Kit (Qiagen, Hilden, Germany), and DNA from blood and trunk and oral swabs was extracted using the DNeasy Blood & Tissue Kit (Qiagen, Germany) with a minor modification to the manufacturer's protocols. The centrifugation time of the fecal samples after digestion was extended to 25 min to assure that all undigested material was pelleted. All DNA samples were stored at −20 °C until analysis. The origin, age, and sex of Wild Elephant Valley elephants and zoo elephants were recorded based on clinical records, and the elephants were categorized into groups according to their age. The age groups were based on Arivazhagan and Sukumar [34] with minor modification: adult (>15 y); sub-adult (5 < X ≤ 15 y); juvenile (3 < X ≤ 5 y); unweaned elephants (≤3 y).
The majority of Asian elephants in zoos were imported from other Asian elephant range countries with a small number of captive-bred elephants. These animals are routinely moved among zoos. Only fecal samples were taken from the zoo elephants (n = 155).
Elephants were classified as herpesvirus virus positive if herpesvirus DNA was detected in one or more samples.

• Wild Populations
In 2017, fecal samples from three wild Asian elephant populations (i.e., Pu'er-Mengyang (n = 81), Mengla (n = 26), and Shangyong (n = 19)) were collected ( Figure 1). A hundred and twenty-six individuals were identified by microsatellite markers [35], and their fecal samples were used in this study. The tested individuals were aged by bolus diameter according to the rearranged Von Bertalanffy growth equation: t = t 0 − 1 k × ln 1 − L L ∞ ; t is the mean age, t 0 is the theoretical age at which an elephant's bolus would have a length measurement of 0 if the same growth pattern was applied from birth to later life, K is the Brody growth coefficient, and L ∞ is the theoretical maximum size of the bolus diameter [36,37]. Age groups were classified in the same way as captive elephants.
At the time of sampling, there was approximately 280 wild individuals in China. The approximate number of each sampled population is shown in Table 1.

DNA Extraction
Fecal DNA was extracted using the QIAamp DNA Stool Mini Kit (Qiagen, Hilden, Germany), and DNA from blood and trunk and oral swabs was extracted using the DNeasy Blood & Tissue Kit (Qiagen, Germany) with a minor modification to the manufacturer's protocols. The centrifugation time of the fecal samples after digestion was extended to 25 min to assure that all undigested material was pelleted. All DNA samples were stored at −20 • C until analysis. protein-coupled receptor (EEHV1-vGPCR) primer sets [29,38] (Appendix A) and Qiagen HotStarTaq Master Mix Kit (Qiagen, Germany). Samples were first tested by redundant EEHV POL and EEHV1, 4, and 5 POL primer sets for EEHV subtype identification. Then, the EEHV1 positives in the first screen were amplified by EEHV1-vGPCR primer sets for further phylogenetic analysis.
The DNA amplifications were carried out on a Veriti 96-Well Thermal Cycler (Applied Biosystems, Singapore) using the following conditions: One cycle of 95 • C for 15 min, followed by 35 cycles of 94 • C for 30 s, 55 • C for 90 s, and 72 • C for 60 s; then, the samples were held at 72 • C for 5 min and immediately chilled at 4 • C. Two microliters of the solution containing the purified DNA was used in a 20 µL reaction. For the second and third round PCR reactions, 2 µL of the previous 20 µL PCR reaction was used. The amplification products were separated by electrophoresis on 1.5% agarose gels containing SYBR gel stain (Invitrogen, Waltham, MA, USA) and visualized under ultraviolet light. Positive samples were sent for sequencing (BGI, Beijing, China).

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Analytical sensitivity of the PCR assay To determine the analytical sensitivity of the PCR assay, plasmids containing U38(POL) fragments of EEHV1, 3, and 4 were synthesized. The plasmid DNA was added into the EEHV-negative DNA in this study. The 10-fold serial dilutions of the synthesized plasmid DNA were created, making the final concentration of the plasmid DNA in the EEHVnegative DNA starting from 10 5 to 10 copies/µL. The products were then amplified by the PCR assay employed in the paper.

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Inhibitor test for the fecal DNA samples Given that a zoo elephant diet is different from that of wild elephants as well as the Wild Elephant Valley elephants and that a very low prevalence of EEHV DNA was detected in feces from zoo elephants, we sought to determine if it might contain inhibitors. The plasmid DNA used in the PCR sensitivity test was added to 105 EEHV-negative fecal DNA samples and amplified as described above. Of these, forty samples were from the Wild Elephant Valley (n = 44), forty-two DNA samples were from randomly selected zoo elephants (n = 155), and twenty-three DNA samples were from randomly selected wild elephants (n = 126). The final plasmid concentration in the PCR system was 500 copies/µL.

Age and Sex Differences
Information on the age composition and sex of elephants in the Wild Elephant Valley is well documented. Thus, the association between age or sex on EEHV infection of elephants in the Wild Elephant Valley was explored. Sex and age data (n = 47) for the elephants in the Wild Elephant Valley in 2020 were used in the analysis (Appendix B). Significant tests for associations between EEHV-positive elephants and the age and sex of the tested elephants were based on the Chi-squared test and Fisher's exact test (p ≤ 0.05) [39,40].

Phylogenetic Analysis
Sequences were analyzed in MEGAX and were compared with the EEHV sequences in GenBank using NCBI BLAST. For the EEHV1 analysis, a vGPCR (U51) locus was used. All of the U51 fragments of EEHV1 in the NCBI database [41] were downloaded and aligned in MEGAX [42] with the sequences found in this study. Aligned sequences were then trimmed and translated into protein sequences. Two EEHV6 and one EEHV2 vGPCR sequences were added to the analysis. Identical sequences were then removed using the DISTANCE tool, and one representative sequence was retained for each set of identical sequences. The result was used to construct a neighbor-net by ProteinMLdist using SplitsTree4 [43], and the bootstrap value was obtained using 1000 replicate bootstrapping. The information on the country of discovery for all the sequences involved in the analysis was summarized in a bar chart (Numbers, 2021, Apple). According to Long, Latimer, and Hayward [8] and Sripiboon et al. [44], the delineation of clusters is based on amino-acid polymorphisms for the U51 (vGPCR) protein. We analyzed the cluster distribution, and then used Keynote For the EEHV4 and 5 phylogenetic analyses, the DNA POL(U38) locus was used. All the U38 sequences of EEHV2/3/4/5/6/7 in NCBI were downloaded and aligned with ClustalW in MEGAX with the sequences found in this study [41,42], trimmed, and translated into protein sequences. Two EEHV1 U38 sequences were added to the analysis. The identical sequences were then removed using the DISTANCE tool, and one representative sequence was retained for each set of identical sequences. The best model for the maximum likelihood tree was selected according to the MODELS in the MEGAX recommendation. Jones Taylor Thornton Model plus Gamma Distributed (JTT + G) was utilized for EEHV4 and EEHV5 U38 locus maximum likelihood tree construction. The bootstrap value was obtained using 1000 replicate bootstrapping.

Detection of EEHV in Different Sample Types of Wild Elephant Valley Elephants
A comparison of the PCR results for the feces, blood, and trunk and oral swabs from 47 valley elephants is shown in Table 2. Overall, fourteen elephants (29.8%; 14/47) were positive for EEHVs. Positive cases were detected in imported, rescued elephants, and in captive breeding elephants. Nine of the elephants were infected with a single EEHV including two EEHV1A, two EEHV1B, four EEHV4, and one EEHV5. Co-infections were found in five elephants (EEHV1A/EEHV4, n = 4; EEHV1B/EEHV4, n = 1).  Only EEHV4 was detected in feces. EEHV1A and EEHV1B were detected in blood, and EEHV1A, EEHV1B, EEHV4, and EEHV5 were detected in both trunk and oral swabs. Four of the five co-infected elephants showed PCR positivity for two samples, each detected a separate EEHV type/subtype, while one elephant had three PCR-positive samples: two samples detected EEHV1 and another sample showed EEHV4 positivity. Excluding co-infected elephants, five elephants with two sample types showed PCR positivity simultaneously, and one elephant with three sample types showed PCR positivity at the same time.
A total of nine cases of EEHV4 positivity (19.1%; 9/47) were detected in feces and trunk and oral swabs, and no EEHV4 positivity was found in the blood. The fecal samples from two EEHV4-positive elephants were not collected, and four EEHV4-positive elephant fecal samples collected were detected positive (57.1%; 4/7). Three cases (33.3%; 3/9) were detected in trunk swabs, and six (66.7%; 6/9) were detected in oral swabs. Among them, one (V36) was positive for EEHV4 in feces and trunk and oral swabs; one (V38) was positive in feces and oral swabs; one (V39) was positive in feces and trunk swabs. The remaining six cases were all positive for a single sample type.

Detection of EEHV in Fecal Samples from Captive and Wild Elephants
The screening results for EEHVs in the fecal samples from captive and wild elephants are shown (Table 3). This sub-study includes fecal sample testing results in the Wild Elephant Valley from the previous sub-study, thus comparing the presence of EEHV in feces in different Asian elephant populations in China. A total of 16 EEHV infections from 16 elephants were detected in the 325 wild and captive elephants screened with a total prevalence of 4.92%. Two EEHVs were detected: EEHV1B, and EEHV4.

• Wild Populations
A total of ten wild elephants were found to be infected with an EEHV (7.94%; 10/126). There were eight EEHV1B (6.35%: 8/126) and two EEHV4 (1.59%; 2/126) positive elephants, and EEHV1A was not detected. According to the fecal bolus diameters, seven EEHV1B-positive elephants and all EEHV4-positive elephants were sub-adults between 5 and 15 years old. A single EEHV1B-positive elephant was an adult. EEHV1B was detected in the population inside China (i.e., Pu'er-Mengyang) and the populations traveling between China and Laos (i.e., Shangyong and Mengla). The two EEHV4 positives were detected from two elephants in the Mengla population.

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Analytical sensitivity of the PCR assay and prevalence of inhibitor samples from the Wild Elephant Valley, zoo, and wild elephants All PCR assays were able to detect a hundred copies and occasionally (2/16 times) detected ten copies. Two of the forty Wild Elephant Valley samples (5.00%; 2/40) contained inhibitors, five of the forty-two zoo samples (11.90%; 5/42) contained inhibitors, and none of the twenty-three wild samples (0.00%; 0/23) tested contained inhibitors.

Age and Sex Differences
The positivity rate for the age groups of each virus in the Wild Elephant Valley is shown in Figure 2. There were no significant association between EEHV1A, 1B, or 5 positivity and age (p = 0.234 > 0.05; p = 0.183 > 0.05; p = 0.921 > 0.05); however, a significant association between EEHV4 positivity and age was observed (p = 0.000 < 0.05). Further pairwise comparison of the EEHV4-positive rate between age groups showed that there were significant differences in infection rates between the non-weaned group and the subadults, as well as between the non-weaned group and the adult group (p = 0.005 < 0.05; p = 0.000 < 0.05). An association between EEHV1A, EEHV1B, and EEHV4 infection rate and sex was not detected (p = 0.947 > 0.05; p = 0.726 > 0.05; p = 0.106 > 0.05; p = 0.844 > 0.05).

Wild Populations
A total of ten wild elephants were found to be infected with an EEHV (7.94%; 10/126). There were eight EEHV1B (6.35%: 8/126) and two EEHV4 (1.59%; 2/126) positive elephants, and EEHV1A was not detected. According to the fecal bolus diameters, seven EEHV1B-positive elephants and all EEHV4-positive elephants were sub-adults between 5 and 15 years old. A single EEHV1B-positive elephant was an adult. EEHV1B was detected in the population inside China (i.e., Pu'er-Mengyang) and the populations traveling between China and Laos (i.e., Shangyong and Mengla). The two EEHV4 positives were detected from two elephants in the Mengla population.

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Analytical sensitivity of the PCR assay and prevalence of inhibitor samples from the Wild Elephant Valley, zoo, and wild elephants All PCR assays were able to detect a hundred copies and occasionally (2/16 times) detected ten copies. Two of the forty Wild Elephant Valley samples (5.00%; 2/40) contained inhibitors, five of the forty-two zoo samples (11.90%; 5/42) contained inhibitors, and none of the twenty-three wild samples (0.00%; 0/23) tested contained inhibitors.

Age and Sex Differences
The positivity rate for the age groups of each virus in the Wild Elephant Valley is shown in Figure 2. There were no significant association between EEHV1A, 1B, or 5 positivity and age (p = 0.234 > 0.05; p = 0.183 > 0.05; p = 0.921 > 0.05); however, a significant association between EEHV4 positivity and age was observed (p = 0.000 < 0.05). Further pairwise comparison of the EEHV4-positive rate between age groups showed that there were significant differences in infection rates between the non-weaned group and the subadults, as well as between the non-weaned group and the adult group (p = 0.005 < 0.05; p = 0.000 < 0.05). An association between EEHV1A, EEHV1B, and EEHV4 infection rate and sex was not detected (p = 0.947 > 0.05; p = 0.726 > 0.05; p = 0.106 > 0.05; p = 0.844 > 0.05).

Phylogenetic Analysis
We analyzed the amplification results of vGPCR locus of all EEHV1-positive samples and the POL locus of EEHV4 and EEHV5 positive samples obtained in this study. Only

Phylogenetic Analysis
We analyzed the amplification results of vGPCR locus of all EEHV1-positive samples and the POL locus of EEHV4 and EEHV5 positive samples obtained in this study. Only the POL fragment of samples from one elephant (V15) of the Wild Elephant Valley was amplified, and the amplification of vGPCR fragments failed. Therefore, this elephant (V15) was not included in the analysis.
As shown in Table 4, a total of seven strains (CN01-CN07) were identified, of which EEHV1A contained three strains (CN01-CN03), EEHV1B contained two strains (CN04-CN05), and EEHV4 and EEHV5 each had one strain (i.e., CN06 and CN07). CN01 and CN05 were novel strains, different from those found in other countries. The other five strains were identical to the strains found in Asian elephants in Thailand and North America (Table 4). The distribution of each EEHV strain found in this study for each population is shown in Figure 3. Seven strains were present in the Wild Elephant Valley area, and four were present only in the Wild Elephant Valley population (CN01-CN03 and CN07). The other three strains were also detected in other populations, and only 1-2 strains were present in each population. Strain CN04 was distributed in all populations tested. In addition to strain CN04, CN06 was present in the wild elephant population in Mengla and CN05 in the wild elephant population in Shangyong, while only CN04 was present in the zoo elephant population and wild elephants from Pu'er-Mengyang. In addition, CN04 and CN05 were found in both wild and captive populations. The distribution of each EEHV strain found in this study for each population is shown in Figure 3. Seven strains were present in the Wild Elephant Valley area, and four were present only in the Wild Elephant Valley population (CN01-CN03 and CN07). The other three strains were also detected in other populations, and only 1-2 strains were present in each population. Strain CN04 was distributed in all populations tested. In addition to strain CN04, CN06 was present in the wild elephant population in Mengla and CN05 in the wild elephant population in Shangyong, while only CN04 was present in the zoo elephant population and wild elephants from Pu'er-Mengyang. In addition, CN04 and CN05 were found in both wild and captive populations. Genetic analysis was conducted to identify the variation and discover the relatedness between the isolates found in our study and other studies. For the EEHV1-positive samples, based on 159 amino acid sequences, the neighbor-net showed that the five strains in this study plus the 93 EEHV1 vGPCR (U51) sequences belonging to different individuals found in the NCBI database belonged to a total of 17 groups (Figure 4). The strains found in China were concentrated in Cluster B, C, and D with two strains each in Cluster B and D and one strain in Cluster C. CN01 and CN02 were in Group 5 and Group 4 in Cluster D, and Group 4 also contained strains from Thailand, North America, India, and Indonesia. CN03 was in Group 2 in Cluster C, and Group 2 also contained strains from Thailand and North America. CN04 and CN05 were in Group 3 and Group 1 in Cluster B, and Group 3 also contained strains from Thailand, North America, and Europe.
The maximum likelihood phylogenetic tree constructed based on the POL(U38) gene locus of the EEHV4 and EEHV5 strains found in this study and EEHV1-5 and EEHV7 found in other studies is shown in Figure 5. Based on the 63 amino acid sequences, the EEHV4 and EEHV5 genomes were well conserved, and the CN06 and the reference EEHV4 POL genome (KX139432) found in Thailand only had 1.5% differences, and CN07 was identical to the reference EEHV5 genomes. Genetic analysis was conducted to identify the variation and discover the relatedness between the isolates found in our study and other studies. For the EEHV1-positive samples, based on 159 amino acid sequences, the neighbor-net showed that the five strains in this study plus the 93 EEHV1 vGPCR (U51) sequences belonging to different individuals found in the NCBI database belonged to a total of 17 groups (Figure 4). The strains found in China were concentrated in Cluster B, C, and D with two strains each in Cluster B and D and one strain in Cluster C. CN01 and CN02 were in Group 5 and Group 4 in Cluster D, and Group 4 also contained strains from Thailand, North America, India, and Indonesia. CN03 was in Group 2 in Cluster C, and Group 2 also contained strains from Thailand and North America. CN04 and CN05 were in Group 3 and Group 1 in Cluster B, and Group 3 also contained strains from Thailand, North America, and Europe.
The maximum likelihood phylogenetic tree constructed based on the POL(U38) gene locus of the EEHV4 and EEHV5 strains found in this study and EEHV1-5 and EEHV7 found in other studies is shown in Figure 5. Based on the 63 amino acid sequences, the EEHV4 and EEHV5 genomes were well conserved, and the CN06 and the reference EEHV4 POL genome (KX139432) found in Thailand only had 1.5% differences, and CN07 was identical to the reference EEHV5 genomes. Viruses 2022, 14, x FOR PEER REVIEW 11 of 17

Discussion
The risks posed by the EEHVs to captive and wild Asian elephants are widely recognized. Surveys of captive, semi-captive, and, to a lesser extent, wild elephants have been conducted in several countries, and surveillance for captive and semi-captive elephants have been developed to suit local conditions [2,14,15]. China is an Asian elephant range country and has a captive Asian elephant population; however, the EEHV status of China's wild and captive elephants is not known.

Types, Subtypes, and Strains Identified and the Potential Implications for the Occurrence of EEHV-HD
This large-scale study reports the findings of the first survey for the presence of EEHV in captive and wild Asian elephants in China. In this survey, all EEHV types and subtypes (EEHV1A, 1B, 4, and 5) found in Asian elephants elsewhere were found in captive Asian elephants and fewer types (EEHV1B and 4) were found in the wild populations. Most of the EEHV strains identified had been previously documented in elephant populations outside of China; however, novel EEHV1A and EEHV1B strains are described here for the first time. Sequences from the EEHV1A and EEHV1B genetic Groups 6-17, which contained the majority of sequences from Asian elephants sampled in India, were not found in the Chinese populations. Additional testing of elephants in China will be required to determine if these findings represent geographical trends in EEHV1A and 1B distributions or if they are the result of overall low sample sizes of sequenced EEHV viruses.
EEHV-HD is caused predominately by strains of EEHV1 but occasionally also by EEHV4 and EEHV5 [5,7,8,38]. Therefore, finding these strains in the captive population of elephants held at the Wild Elephant Valley facility suggests that calves born at these facilities are at risk of developing EEHV-HD. In addition, given that elephants are routinely moved among institutions in China, including the Wild Elephant Valley facility, it is likely that these strains of EEHV are present in other captive collections of elephants as well and pose the same threat to calves in their collection. Contact between wild elephants and captive elephants at the Wild Elephant Valley facility, although minimal and restricted, may allow EEHV strains present in the captive population to spread to wild elephants in this part of China.

Using Multiple Sampling Sites Improved the Detection Rate of EEHV Infections and Types
This is only one of a few studies where it was possible to compare the findings of PCR screening of blood, oral swabs, and trunk swabs for EEHV DNA in the same elephant, and this is the first study where the results of the PCR screening of these samples were compared to the PCR screening of DNA from elephant feces. Our results show, as has been noted previously, that if PCR screening is to be used as a method to detect an active EEHV infection, that screening samples from trunk swabs, oral swabs, and blood will detect more active infections then either one alone and will be more likely to detect infections with multiple EEHV types. Combining the results of testing fecal DNA with testing of one or all three other samples also improved the chance of detecting active virus infections in the elephants and detecting infections with multiple genotypes in this study. Therefore, we recommend that future investigations and routine screening protocols for active EEHV infections in elephants include testing samples from blood, oral swabs, trunk swabs, and fecal DNA whenever possible.
We chose to screen elephants for active EEHV infection by screening fecal DNA for EEHV DNA, because if EEHV DNA could be detected in this sample, it would mean that the presence of EEHV types and possibly even the prevalence of active EEHV infections in wild elephants could be determined, and collecting a fecal sample would be easier and safer than collecting blood and oral and trunk swabs from captive elephants [30,33]. Our results show that active EEHV infections can be detected in elephants by screening fecal DNA. However, a single fecal sample, as with samples from other sites, did not detect all active EEHV infections, and not all EEHV types were detected in fecal DNA [31,33]. While our data set was relatively small, it provides proof that screening of a single fecal DNA sample will be able to detect some, by far not all, elephants actively infected with EEHV1B and EEHV4, and given that EEHV1A DNA was never detected in an elephant fecal sample, it may not be able to detect elephants with active EEHV1A infections. Therefore, as discussed above, when testing elephants for EEHV1A, 1B, and 4 active infections, the chances of detecting them are improved by testing a combination of oral and trunk swabs and blood in conjunction with feces. However, testing feces can provide valuable information about the presence or absence of EEHV1B and EEHV4 types in populations where blood and oral and trunk swabs cannot be obtained. The value of screening fecal DNA to detect active EEHV5 infections in Asian elephants could not be determined by this study as only one elephant with an active EEHV5 infection was identified and EEHV5 DNA was not detected in its fecal DNA.

Impact of Age on Prevalence of Active EEHV Infections
Our findings are similar to a previous study that showed that viremia and virus shedding of elephants infected with EEHV1A or 1B is most common in juvenile elephants between 1 and 5 years of age [19]. Comparable studies of the effect of age on virus shedding in elephants infected with EEHV4 and 5 have not been conducted. However, our study suggests that younger elephants are also more likely to have active EEHV4 infections than older animals, as of the nine positive cases of EEHV4 in the Wild Elephant Valley, six were unweaned elephants. However, this may be a result of the sampling constraints. Further validation by future studies is needed.

Possible Explanations for Variations in Fecal EEHV Prevalence in the Three Elephant Populations Studied
The prevalence of EEHV PCR positive elephants, based on testing of fecal DNA screening, in Chinese zoological collections was low (1.29%) compared to the Wild Elephant Valley (9.09%) and wild populations (7.94%). The reason for this is not known but could reflect a higher percentage of samples containing inhibitors in the fecal samples from the zoo animals compared to those from the Wild Elephant Valley and from the wild elephants. It might also reflect the quality of the DNA in these samples, and though not tested, DNA degradation may have occurred more commonly during the sample collection and storage of the feces from the zoo elephants compared to the samples from the Wild Elephant Valley elephants and wild elephants. Including an internal control for DNA quality, such as testing for the elephant TNF gene, would address this question in future studies.

Molecular versus Serological Screening of Asian Elephants for EEHV
In this study, we used PCR assays to detect active infections in the elephants that we studied instead of screening for infection using serology, as the only population of elephants that we could obtain blood samples from was the captive population housed at the Wild Elephant Valley. Using molecular screening also allowed us to compare the sequences of the detected types and subtypes to those reported in other countries [15]. Screening elephants with PCR assays, however, has its limitations, as it only detects elephants with active infections and not latently infected elephants that are not viremic or shedding EEHV.
In recent years, there have been great advances in the development of sensitive and specific serological assays that are able to identify elephants that are infected with EEHVs. These findings show that the prevalence of virus infection in the populations that have been tested using serology far exceeds that detected using molecular methods screening for viral DNA in blood or other samples [21,26,27]. In fact, it has been suggested that EEHV infections may be ubiquitous in Asian elephants greater than five years old [21,27]. It is therefore extremely likely that the overall prevalence of EEHV-positive elephants in the captive and wild populations that we studied is considerably higher than what we were able to detect. It is hoped that as soon as these serological assays are further refined and become more widely available, they may be able to be used for future studies and screening protocols in China, especially as they appear to be particularly useful for identifying naive elephant calves that would be at the highest risk for developing disease [21,27].

Implications of These Findings for Asian Elephants in China
Overall, there are relatively few captive Asian elephants in China, and it is difficult and costly to acquire new stock. Therefore, it is important to do everything possible to protect the health of the Asian elephants that are present in captive populations in China and ensure that as many elephant calves that are born to this population survive to reproduce. To protect calves born to captive elephants from EEHV disease, we propose that the screening recommendations of Luz and Howard [20] be followed at least in juvenile elephants. Based on these recommendations, we suggest elephants less than 8 years old should have blood samples and preferably trunk swab, oral swab, or and feces tested by PCR for EEHVs at weekly intervals and 2-4 times per year after that. Elephant calves that are found to be infected with an EEHV and found to have a rising virus titer or are experiencing signs consistent with EEHV disease should be treated with aggressive supportive care and an antiviral drug [45,46]. In addition, all sick and dead calves should be routinely tested for EEHV infection. In order to address the need for such testing and fill the vacancy of bodies in China that can perform official EEHV testing rather than research, it is recommended that a laboratory that is able to perform these assays be established and that they be a repository for data generated through this testing. Moreover, as antiviral drugs are costly and may not be easily sourced in an emergency situation where an elephant calf is stricken with disease caused by an EEHV, we recommend that enough antiviral medication to treat one or more calves be stockpiled in China and made available to captive elephant collections as needed.
The impact of EEHVs on the wild Asian elephant populations in China is unknown, but at present it seems to be minimal. The wild Asian elephant populations have maintained a strong annual growth rate of 3-5% in recent years as a result of aggressive conservation efforts [47]. However, as Asian elephant populations increase in size within the confines of the current sanctuaries, new stressors may arise impacting EEHV shedding and transmission [48], and dead wild elephant calves have been found. We therefore recommend testing tissue or blood samples for all dead wild elephants found during routine patrols or samples from live wild elephants obtained by chance. In addition, to obtain information on EEHV-related dynamics in wild elephant populations, we recommend conducting regular surveys for EEHV fecal shedding in these populations.  Institutional Review Board Statement: The animal study protocol was approved by the Ethics Committee of College of Life Science, Beijing Normal University (protocol code CLS-EAW-2020-015 and date of approval: 4 April 2020).

Informed Consent Statement: Not applicable.
Data Availability Statement: Genomic data reported in this study are available at GenBank, Accession numbers: OL799261-OL799267, OL845859.

Conflicts of Interest:
The authors declare no conflict of interest.