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In 2012, an FHV-1-like virus was isolated from a tiger that presented with clinical signs of sialorrhea, sneezing and purulent rhinorrhea.
The phylogenetic tree based on the TK gene sequences showed that the isolate investigated in this study, was closely related to the ten isolates of FHV-1 (Figure 2), a result consistent with the alignment analysis.All isolates of FeHV-1 appear to be relatively similar, as they antigenically belong to one serotype [9]. TEM of negatively stained samples revealed medium-sized particles with a diameter of approximately 200 nm, an envelope and an electro-dense core (Figure 4), which are characteristics typical of FHV-1. The incubation period of FHV-1 infection as indicated by this challenge test is approximately 6 days, which is consistent with that reported previously [11].
Oligonucleotide primers, reaction systems and conditions used for the amplification of the target genes of the three suspected viruses. La primo-infection a lieu principalement chez les jeunes enfants et les nourrissons puis le virus devient latent dans le ganglion genicule. Isolation was performed with the FK81 cell line, and the virus was identified by PCR, transmission electron microscopy (TEM), and the phylogenetic analysis of the partial thymidine kinase (TK) and glycoprotein B (gB) genes. IntroductionFeline herpesvirus type 1 (FHV-1; felid herpesvirus 1 (FeHV-1), family Herpesviridae, subfamily Alphaherpesvirinae, genus Varicellovirus) is an important pathogenic agent that causes feline viral rhinotracheitis, which is a highly infectious upper respiratory tract infection of felids [1]. The TK gene is a conserved gene, and the target fragment is located in its highly conserved region. Virus Isolation and TEM Examination of the Cell CulturesAt 72 h post-inoculation with trachea homogenates from the tiger positive for FHV-1, FK81 cell cultures showed a distinct cytopathic effect (CPE), characterized by cell rounding, pyknosis, ‘Fleece-Pulling’ to the thyrsoid and degeneration of the cell monolayer (Figure 3). The electron micrograph of cell cultures (Magnification ?40,000) revealed circular or oval herpesvirus-like particles that appeared as spheres and ellipsoids with diameters ranging from 150 to 200 nm (as arrow indicated).
For the IG group cats, pyrexia began at day 4 post-inoculation and continued for 14 days, with a peak of 40.2 °C. Virus Shedding in the Infected CatsFor the IG group cats, positive results appeared on the 6th day post-inoculation.
A minus sign was used to represent negative PCR results, and a plus sign was used for positive results. Additionally, clinical signs, including pyrexia, inappetence, and serous ocular and nasal discharge, were observed in the cats [12], and a long period of virus shedding was observed [13]. Gene Cloning and Sequence AnalysisThe target fragments observed by agarose gel electrophoresis (AGE) were extracted by a DNA Gel Extraction Kit (Axygen, Hangzhou, China), and were cloned into a pGEM-T Easy vector with routine methods [24]. This infection is often fatal to kittens, but adult cats usually survive and exhibit lifelong latency [2,3].
For the EG group and control group cats, fever was detected on the 10th day and the 13th day, respectively (Figure 6).

PCR was used to detect FHV-1 DNA extracted from the specimens, which included nose washing fluid, eye swabs and swallow swabs. The sequencing of the cloned plasmid was performed by BGI Sequencing, and the results were submitted to GenBank for alignment analysis.
Phylogenetic analysis showed that the isolate belonged to the same cluster with other FHV-1 strains obtained from GenBank. Since the first strain of FHV-1 was isolated in America, infected felids have been reported in many countries, including Canada, Switzerland, the United Kingdom, Holland, Hungary and Japan [4].
Subsequently, their nostrils were completely blocked with purulent secretions resulting from thin nasal discharge (Figure 5C). Positive results were found on the 10th day and 16th day for the EG group cats and the control cat, respectively (Table 1). Herpes-like viruses with an envelope and diameters of approximately 200 nm were observed in the culture supernatants of FK81 cells inoculated with samples from the tiger. There have been no documented reports on FHV-1 in the past few years, although the distribution in China was confirmed by serological survey and virus isolation in domestic cats [5].The South China tiger (Panthera tigris amoyensis) is a tiger unique to China and is the most endangered tiger subspecies, as free ranging individuals have not been found in its historic distribution areas for many years [6]. The positive PCR products of TK gene of FHV-1 from trachea homogenates and cell cultures were identified by sequence analysis. These results revealed a baseline of virus shedding in the infected cats after they were inoculated or were exposed to FHV-1.
To aid in the recovery of wild populations, it is common to re-introduce captive individuals into their native range; however, there are fewer than 120 captive South China tigers in China. The 253 bp sequence of TK gene from FHV-1 from trachea homogenates shared 100% homology with FHV-1 isolates. Similar signs and similar clinical courses were observed in the cats in the EG (exposed group) and in the cat of the control group, but the initial clinical signs manifested on the 11th day and the 16th day post-inoculation, respectively. Our finding extends the host range of FHV-1 and has implications for FHV-1 infection and South China tiger conservation.
Furthermore, only a few captive tigers are suitable for reintroduction, and infectious diseases threaten captive tigers. Any sign of disturbance or trouble with the captive populations, in particular any risk of infectious disease, will greatly concern the stakeholders.In June 2012, a South China tiger in Shenzhen Wildlife Zoo presented with sneezing, purulent rhinorrhea, which ended with its death, although treatment including antibiotics had been tried. In the present study, we used molecular methods, virus isolation, TEM examination and an animal challenge experiment to diagnose the cause of death of the South China tiger, and for the first time, we confirmed the infection with FHV-1 in the captive tiger population in China. Electron Microscopy ExaminationThe cultured cells were harvested and fixed in 2.5% glutaraldehyde for transmission electron microscopy (TEM) examination (JEM-1200, Tokyo, Japan).

Phylogenetic Analysis Based on Two Cloned Gene Fragments of FHV-1The glycoprotein B (gB) gene and thymidine kinase (TK) gene have been selected for the study of molecular phylogeny [7,8]. A 253 bp sequence was obtained, and alignment analysis determined that the TK gene cloned in this study shared a high identity (from 99% to 100%) with that of other FHV-1 isolates (Figure 1).
A 566 bp fragment of the gB (glycoprotein B) gene was also cloned, and was found to share 100% identity with that of other FHV-1 isolates. The two sequences have been deposited in Genbank whose accession numbers are ** and **, for TK gene and gB gene fragment separately. Case Description and Sample CollectionBecause of the clinical signs, the tiger was initially diagnosed with rhinotracheitis, and only trachea samples were obtained for laboratory examination after it died.
Polymerase chain reaction (PCR) and reverse transcription polymerase chain reaction (RT-PCR) were used to detect these viruses. ConclusionsIn this study, the authors described the first occurrence of feline herpesvirus type 1 (FHV-1) in a South China tiger in China. Challenge experiments found that the isolate caused typical clinical signs, a long period of virus shedding and efficient transmission in cats.
The study expands the species range for FHV-1 infection and provides new epidemiologic data. Association of Bartonella species, feline calicivirus, and feline herpesvirus 1 infection with gingivostomatitis in cats.
Dramatic decline of wild South China tigers Panthera tigris amoyensis: Field survey of priority tiger reserves.
Characterization of the feline herpesvirus genome and molecular epidemiology of isolates from natural outbreaks and latent infections. The dose response of cats to experimental infection with feline viral rhinotracheitis virus.
Experimental induction of feline viral rhinotracheitis (FVR) virus re-excretion in FVR-recovered cats.

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