Feline Herpesvirus Infection of Snow Leopard

Feline herpesvirus type 1 (FHV-1) is a common causative agent of domestic cats rhinotracheitis and gradually threatens the wild felid worldwide. The endangered snow leopard belongs to the family Felidae and is also the top predator on the Tibetan Plateau. Herein, FHV-1 was identied and isolated from three dead snow leopards with symptom of sneezing and rhinorrhea. To explore the relationship between FHV-1 and their death, histopathology and molecular biology was performed. The organs and nasal swabs were collected for examinations of histopathology, the nucleic acid of the pathogen, viral isolation, and sequence analysis. The results reveal that all three snow leopards were infected with FHV-1. The rst case primarily died of old cerebral infarction and secondary non-suppurative meningoencephalitis probably caused by FHV-1. The second case mainly died of renal failure accompanied by interstitial pneumonia caused by FHV-1. The third case was doubted to be related to the reactivation of latency of FHV-1. The gD and gE gene sequence alignment of the FHV-1 isolate strain revealed that the isolated strain originated from a domestic cat. Therefore, FHV-1 infection can cause different lesions of snow leopards and shows a high risk for the wild felid. We should focus more on protecting felid against threatening of FHV-1 infection originating from domestic cats.

The snow leopard (Panthera uncial, family Felidae) is a kind of rare and endangered animal and is known as the top predator on the Tibetan Plateau and its surrounding mountain ranges 13 . Its ranges these countries including Afghanistan, Bhutan, China, India, Kazakhstan, Kyrgyzstan, Mongolia, Nepal, Pakistan, Russia, Tajikistan, and Uzbekistan. It is estimated that there are only approximately 4,000-7,000 individual snow leopards living in the wild now (Snow Leopard Network, 2014). Chinese Snow leopards are primarily distributed in Qinghai Province, Tibet, and Xinjiang autonomous regions. They are also found in Gansu, Sichuan and Yunnan Provinces, and Inner Mongolia autonomous region 4 . In the past two decades, signi cant progress has been made in the conservation of snow leopards in China and poaching has been effectively curbed. With the development of tourism, pet infectious diseases have become a new threat for snow leopards, such as canine distemper 14 .
In December 2019, three snow leopards in the Qinghai-Tibet Plateau wild zoo had sneezing and rhinorrhea. Nasal swabs were collected and subjected to detection of potential pathogens such as canine distemper virus, FHV-1, feline calicivirus, mycoplasma, and chlamydia using real-time uorescence quanti cation polymerase chain reaction (PCR). The results showed that FHV-1 was positive in three nasal swabs. Furthermore, virus isolation, immunohistochemistry, and other methods were used to analyze the characteristics of the snow leopard-origin FHV-1 strain. We rstly con rmed the infection and death cases with FHV-1 in the captive snow leopards in China.

Results
Pathological changes relative to FHV-1 In this study, the tissues xed with formalin were histopathologically examined including the brain and tonsils of No. 1 and the lung, heart, liver, spleen, and kidney of No. 2.
In the case of No. 1, meningeal congestion and liquefactive necrosis region with a diameter of approximately 2 cm × 0.5 cm × 1 cm in the right cerebral hemisphere were observed (Fig. 1). Moreover, no obvious abnormality was observed in the other organs. The results of histopathology demonstrated meningeal hyperemia and hemorrhage, liquefactive necrosis of the cerebral cortex, a massive collection of foam cells, and hemosiderosis. Capillary hyperemia, bleeding foci, edema, vascular cuff reaction (lymphohistiocytic perivascular in ltrates), neuron necrosis, neurophagia, and demyelination reaction were visible in the brain parenchyma (Fig. 2). Therefore, the pathological diagnosis was old cerebral infarction and secondary non-suppurative meningoencephalitis.
In the case of No. 2, the bladder accumulated and dilated (20 cm × 10 cm in diameter), was full of 1050 mL dark red urine. The congestion presented on the surface of urinary bladder mucosa ( Fig. 3A and 3B).
The renal pelvis of the right kidney had effusion and dilation. The lung presented dull-red, swelling, and liquid leakage from the section (Fig. 3C). Moreover, there was no obvious abnormality in other organs.
Microscopic examination revealed coagulation necrosis of massive glomerulus and tubules in the renal cortex, and bilirubin deposition in tubule epithelial cells (Fig. 4A). The bladder had severe autolysis and the mucosa structure was disordered and homogeneous. Pulmonary alveoli were expanded and lled with pink exudation. Many lymphocytes and exfoliative cells lled the bronchioli ( Fig. 4B and 4C). Therefore, we can conclude that the pathological diagnosis was an acute postrenal renal failure owing to urinary retention and interstitial pneumonia.

Detection of suspicious pathogens and FHV-1 distribution and location in tissues
The detection of suspicious pathogens in nasal swabs is shown in Table 1.  The FHV-1 was found to be distributed in lungs and nasal swabs by PCR test. Liver, spleen, kidney, and tonsils were negative. The details are shown in Table 1.
The immunohistochemistry results showed that lung of No. 2 was positive and FHV-1 was primarily located in the cytoplasm of epithelial cells of a bronchial bronchiole and exfoliative cells (Fig. 4D).

Viral isolation and detection
At 36 h post-inoculation with nasal swab supernatant of No. 2, FK81 cells showed an obvious cytopathic effect (CPE), characterized by round, pyknosis, fusion, aggregating like "Fleece-Pulling" (Fig. 5A). PCR revealed that culture inoculating with nasal swab was positive for FHV-1. Much positive signal (green) of FHV-1 was located in the cytoplasm of FK81 cells by indirect immuno uorescence (Fig. 5B).
Phylogenetic analysis based on gD and gE genes of FHV-1 In this study, the three isolates shared 100% identity on gD (GenBank accession number: OK087390) and gE gene; therefore, the FHV-1 isolates from snow leopard were named SL/QH/2019. According to the alignment, SL/QH/2019 shared 99.9% identity with isolates of FHV-1 from cats and 99.7% identity with isolates of FHV-1 from tiger in China on gD gene. However, SL/QH/2019 shared a low identity (from 30.6-50.8%) with canine herpesvirus type 1 (CHV-1) on the gD gene (Fig. 6A).
The phylogenetic tree based on the gD and gE gene sequences showed that the isolate investigated in this study was closely related to the isolates of FHV-1 from cats ( Fig. 7A and 7B), a result consistent with the alignment analysis.

Discussion
FHV-1 existing in snow leopards has been found by next-generation sequencing using serum and rectal swab samples 5 ; however, there is no clinical case report about snow leopards. Herein, three snow leopards infected with FHV-1 presented different lesions and clinical passages. FHV-1 often causes feline viral rhinotracheitis, ocular disease, ulcerative dermatitis, and pneumonia 3 ; 7 ; 15 . The pathogenesis of FHV-1 is based on two different mechanisms 16 . The rst mechanism is that FHV-1 as a cytolytic virus can damage the epithelial cells of mucosae and cornea leading to ulceration. The second mechanism is immune-mediated reaction driven by antigenic stimulation 16 . In this study, all of the snow leopards with obvious sialorrhea and sneezing symptoms have been rst con rmed to be infected with FHV-1 using real-time qPCR.
In the case of No. 1, foam cells and hemosiderosis showed that cerebral infarction was old 17 ; 18 . The meningoencephalitis was in the acute stage and an obvious demyelination reaction was visible in the white matter according to the clinical neural symptom of No. 1. It has been reported that one whitehanded gibbon died of cerebral infarction and myocardial brosis with herpes simplex and Epstein-Barr virus has been reported; however, the viral infection was not considered the main cause of death 19 . In cats and dogs, non-suppurative meningoencephalitis is frequently found and these pathogens include porcine herpesvirus 1, parvovirus, feline infectious peritonitis virus, feline leukemia virus, West Nile virus, and encephalomyocarditis virus are found in the central nervous system of dogs and cats with nonsuppurative meningoencephalitis by immunohistochemistry. Therefore, the primary or virus-triggered secondary immune-mediated mechanisms cannot be ignored 20 . FHV-1 has also been reported as a causative agent of severe nonsuppurative meningoencephalitis in domestic cats 21 . In this study, though FHV-1 was not detected in organs other than the lung of No. 1 using PCR, the vascular cuff reactions and demyelinating lesions were generally suggestive of a viral etiology in the brain 22 . Additionally, herpesvirus is a common causative agent in humans and animals [23][24][25] . Thus, the FHV-1 infection may be relative to the non-suppurative meningoencephalitis of No. 1.
In the case of No. 2, lung, kidney, and urinary bladder presented the obvious pathological change.
Histopathology examination showed renal failure owing to uroschesis and interstitial pneumonia. Similarly, only the lung was positive for FHV-1 using PCR and immunohistochemistry. Owing to the cataract of the right leg, the activity of No. 2 might be limited which would induce neurothlipsis of the urinary bladder 26 . This may lead to acute postrenal renal failure, according to the symptoms of anuria.
To date, urinary system diseases with FHV-have not been reported. Using a pathological examination, immunohistochemistry, and PCR, pneumonia with FHV-1 infection became clear. Firstly, the lung was characterized by interstitial pneumonia, which was primarily caused by a viral infection in morphology, and immunohistochemistry demonstrated the antigen of FHV-1 located in the epithelial cells of a bronchiole 22 . Secondly, detection of the gD gene was also positive. According to the report, FHV-1 targets both respiratory epithelial cells and pneumocytes and enters the lung. Moreover, the FHV-1 can make infectious cells dead via apoptosis or inducing neutrophil in ltration 3 . In this study, FHV-1 was mainly located in the epithelial cells of the bronchiole with little neutrophil in ltration. However, massive necrotic cast-off cells are visible in the bronchiole. That also proves that the pathway of FHV-1 shedding is primarily the respiratory tract. Therefore, it is considered that No. 2 died of the combined effect of renal failure and FHV-1 pneumonia.  27 . In this case, FHV-1 poses a greater threat to sick snow leopards, similar to that FHV-1 primarily affects kittens and juvenile cats and persists lifelong of the host 1 . Therefore, the animal recovered from FHV-1 infection should be fed separately for further observation.
Furthermore, a nasal swab of No. 2 was used for the isolate of FHV-1 in F81 cells, and the obvious CPE was visible. Immunohistochemistry showed that FHV-1 isolate replicated in cells. The nucleic acid of FHV-1 was also detected in infected cells. The results further con rmed that snow leopards were infected with FHV-1.
Research shows that FHV-1 has only one serotype and is relatively homogenous genetically 1 . To explore the infectious origin, the sequence of gD and gE genes was analyzed and phylogenetic trees were constructed. The gD protein probably had host-selective and stimulated the host to produce high cellular immunity and anti-gD antibody 28 . While the gE protein is mainly related to the virulence of FHV-1 29 . The results showed SL/QH/2019 was highly homologous with the mainly epidemic isolates of cats in China.
Thus, the origin of FHV-1 infecting snow leopards was probably from feral cats in the zoo. Additionally, the identity between these isolates and tiger isolate is lower compared to cat isolates. The genomic variation is not necessary for cross-species transmission of FHV-1 and FHV-1 from a domestic cat that can directly infect other wild felids.
The severity and symptoms of FHV-1 infection are not relative to the viral genome variants, and it is most likely due to other factors such as host response 30 . Therefore, the different pathological changes of No. 1 and No. 2 snow leopards may be the result of different background diseases.

Conclusions
In this study, the rst clinical cases of snow leopards dying of FHV-1 infection are described. The relationship between FHV-1 and the causes of death of three snow leopards has been explored using multiple methods. FHV-1 may induce massive death of wild felid with severe background disease. Therefore, administrators of the zoo and natural reserves should focus more on the threat of FHV-1 from domestic cats.

Methods And Materials
Case Descriptions Three dead snow leopards in the Qinghai-Tibet Plateau Wild Zoo presenting with sneezing and rhinorrhea were numbered 1, 2, and 3. Their clinical information is shown in Table 1.

Sample collection and pathological examination
After their deaths, an autopsy was performed for No. 1 and No. 2 snow leopards. The details of samples are shown in S1.
The frozen samples were sent for PCR examination. The samples xed by 4% neutral formalin were processed to the para n section and were stained using hematoxylin-eosin for pathological examination. Additionally, the xed tissues were immunohistochemically stained with murine monoclonal antibody 4G12 of FHV-1 antigen (ProtTech, China), and observed under an optical microscope and photographed.

PCR Assays
According to the symptoms, we rstly detected the potential pathogens including canine distemper virus (CDV), FHV-1, feline calicivirus, mycoplasma, and chlamydia in nasal swabs using T8 real-time uorescence quantitative PCR instrument and its commercial kit (Manufactured by Beijing Anheal Laboratories Co., Ltd. China).
Furthermore, viral genomic DNA was extracted from nasal swabs and tissue samples using a DNA Viral Genome Extraction Kit (D2400, Solarbio, China), subjected to PCR. The complete genome of the glycoprotein D (gD) gene and glycoprotein E (gE) gene of FHV-1 were ampli ed. The primer sequences and conditions of PCR are shown in S2.

Virus isolation
The nasal swab from No. 2 was made into supernatant using phosphate buffer solution (0.1 mol/L, pH 7.4, PBS) and ltered using 0.22 µm lter membrane for sterilization. The viral culture refers to the method described by Zhang 31 . The F81 cells were selected for replication of FHV-1. F81 cells infected with FHV-1 were detected using PCR and indirect immuno uorescence using murine monoclonal antibody 5H8 of FHV-1 antigen (ProtTech, China).

Sequence analysis and phylogenetic tree construction
The sequences of the gD and gE genes were analyzed and compared with other FHV-1 strains published in NCBI using MegAlign (7.1). The phylogenetic trees were constructed based on the gD and gE genes using MEGA-7 software (7.0).

Ethics statement
The authors con rm that the ethical policies of the journal, as noted on the journal's author guidelines page, have been adhered to. All relevant guidelines for the use of animals in scienti c studies were followed. The study did not include any experimentation on animals or humans, and samples were taken from natural dead animals that was approved by the owner of animal.  Figure 1 Gross observation of brain of No.1. Meningeal vascular is congested, and a liquefactive necrosis region approximately 2 cm × 0.5 cm × 1 cm in diameter is located on the right cerebral hemisphere (*).