In November 2020, a total of 80 donkeys from a farm located in Chabuchar County of Yili region (Northern Xinjiang, China), reported spontaneous abortions without showing any clinical symptoms. Out of a total population of 700 milk donkeys, 400 were jackass (≥ 4 years) and 200 of 300 were pregnant mares (≥ 4 years) at an estimated seven months of gestation. This farm was established in 2019, and all the milk donkeys were brought in from other farms in the Yili area of Northern Xinjiang without any scheduled vaccination.
The veterinarian of this farm provided lung tissues from three aborted fetuses to identify viral pathogens as potential causes of these abortions. Viral nucleic acids were extracted (Geneaid Biotech Co.) from lung tissues by following the previously described protocol [8]. Based on the previous studies on the abortions of China's Yili mare and donkey [7, 8, 29], PCR was conducted using TIANSeq HiFi Amplification Mix (Tiangen Biotech) to detect the presence of Varicellovirus equidalpha8 (EqAHV-8), EqAHV-1, EqAHV-4, EqAHV-2, and EqAHV-5. The primers used for the PCR analysis are mentioned in Table S1.
The PCR protocol comprised an initial denaturation (94°C for 2 min), proceeded by 35 cycles, each consisting of denaturation (98°C for 10 sec), annealing, and extension (68°C for 30 sec) steps. Lastly, the final extension was also carried out (68°C for 5 min) and the PCR analysis confirmed the existence of EqAHV-1 (ON809533-ON809535) in the lung tissues of three donkey mares' aborted fetuses. However, no evidence of EqAHV-4, EqAHV-2, EqAHV-5, and EqAHV-8 were detected. Thus, the results revealed that the abortion storm in the donkey might have been caused by EqAHV-1.
EqAHV-1 isolation was done on Madin-Darby bovine kidney (MDBK) cells from aborted fetal lung tissue (1:10 PBS, filtered 0.45 mm). The cytopathic impact was visible 36 h after inoculating MDBK cells (nine passages) with the supernatant of an EqAHV-1-positive aborted fetal lung tissue (Fig. 1). The identity of the viral isolate (Chabuchar/2020 strain) was verified via transmission electron microscope (TEM) and whole ORF33 gene sequencing (accession no: ON584564) (Fig. 2).
Horse EqAHV-1 and donkey EqAHV-1 Chabuchar/2020 strains have great sequence similarity, as evidenced by the whole ORF33 sequences of the two strains sharing a nt similarity level of 99.7 to 100% and an amino acid similarity level of 99.5 to 100%. Furthermore, this study also matched the identified sequence with the reference strains of horse EqAHV-1 found in different countries, such as China (ZS-01: MZ561483, ZS-2: MZ561484, ZS-5: MZ561485, ZS-22: MZ561493, ZS-24: MZ561495, ZS-25: MZ561496, etc), UK (Ab1: KU206468, Ab4: AY665713, and Army: KU206477), in Japan (NY03: KF644569, 01c1: KF644578), US (RacL11: MF975656), India (Meerut: MT077857), and Australia (717A-82: KT324733, 438 − 77: KT324734). The results for DNA and aa sequences showed a 100% similarity level. The findings suggest that the ORF33 genes of donkey and horse EqAHV-1 displayed significant genetic conservation. These ORF33 nt sequences were used to construct a phylogenic network, which demonstrated that the identified EqAHV-1 strains were grouped to cluster 1 of EqAHV-1 strains found in horses (Fig. 3).
To determine whether the newly identified donkey EqAHV-1 Chabuchar/2020 carried neuropathogenic or non-neuropathogenic characteristics, a partial PCR amplification was carried out on the ORF30 gene (559 nt) of EqAHV-1 Chabuchar/2020 via respective primers (Table S1). The results demonstrated that the partial ORF30 sequence of the neuropathogenic EqAHV-1 Chabuchar/2020 (ON624152) shared 100% nt and amino acid sequences identity with the referenced strains of EqAHV-1 (OM047215-OM047257), thus suggesting a high degree of genetic conservation. Moreover, similar to the neuropathogenic EqAHV-1 strains ZS-01, ZS-2, ZS-5, ZS-8, ZS-10, ZS-11, ZS-16, and ZS-17 (OM047215-OM047222), the identified donkey EHV1 strain had a G nt at 2254 position (corresponding to D aa at 752 positions of the viral DNA polymerase) (Fig. 4). Therefore, the identified strain was confirmed to be neuropathogenic EqAHV-1.
Furthermore, phylogenetic analysis of the ORF68 genes has categorized all EqAHV-1 strains into VII groups that have a close relationship to the strains' origin [14, 16, 28]. In this study, a phylogenetic tree was developed using ORF68 gene sequences. The analysis showed that the donkey EqAHV-1 in this study and the EqAHV-1 found in abortions of Yili mares in China (ON624153, ON637792-ON637805) formed a distinct branch. They were classed as a new group, designated as group VIII EqAHV-1 (Fig. 5). The results indicated that EqAHV-1, which was detected in abortions between Yili mares and donkeys, may propagate independently in the Yili region of Northern Xinjiang, China.