Virus isolation and identification
One FHV-1 strain, designated FHV-1 CH-B, was successfully isolated from a British shorthair cat. After purification, the stable CPE was observed when five additional continuous cultures, characterized by cell rounding, pyknosis and focal-like forms, eventually necrosis and shed completely (Fig. 2a). The supernatants of all five purified cell cultures were tested to be positive for FHV-1 using specific primers HerpF and HerpR targeting FHV-1 TK gene, and the nucleotide sequences of obtained 292-bp fragments showed 100% identity with that of FHV-1 reference strains. FHV-1 CH-B strain was further identified via IFA using anti-FHV-1 antibody, bright green fluorescence was found in infected cells subject to fluorescence microscope, in contrast, no fluorescence was found in control cells (Fig. 2a). TEM of negatively stained isolate revealed spherical virus particles with a diameter of 140 nm, numerous spikes on the surface of envelope, which accord with the structural characteristics of herpesvirus (Fig. 2b).
The nearly complete genome sequences were successfully sequenced and deposited in GenBank under accession number MT813047. The genome of isolate CH-B was 134,779 bp in length and contained 76 open reading frames (ORFs). We predicted and verified that the genomic organization of isolate CH-B was identical to other FHV-1 reference stains previously described (data not shown). Comparative genomic analysis showed that the higher nucleotide identities were found between the isolate CH-B and FHV-1 reference sequences retired from GenBank, with a mean value of 98.14% (97.7% -98.8%). A neighbor-joining (NJ) tree including canine herpesvirus type 1 (CHV-1) as an outgroup was generated based on the complete genome nucleotide sequences (Fig. 3), and showed that the isolate CH-B was more closely related to FHV-1 reference strains and form a group distinct from CHV-1. Moreover, the NJ tree suggested that FHV-1 clusters primarily correlated with geographic location.
Optimal infectious dose
To determine the optimal infectious dose of FHV-1 CH-B strain, domestic cats were inoculated intranasally with different doses of viruses, and were evaluated via various indicators. Cats in group A (107 TCID50), group B (106 TCID50) and group C (105 TCID50) showed initial clinical signs at days 2, 4 and 5 post inoculation (dpi), respectively, accompanied by an increase of rectal temperature (>103℉) and significant weight loss (Fig. 4ab). These cats presented with nasal serous secretion, sneezing, dyspnoea and anorexia. Subsequently, the tested cats showed more severe clinical signs gradually, which manifested on increasing palpebral and nasal purulent secretion, symblepharon and skin ulcers of the nose. As the disease progressed, the clinical scores were more than 5, and cats in group A, B and C began to die at 6, 10 and 14 dpi, respectively (Fig. 4c). While no clinical sign was observed within cats in group D (104 TCID50) except for mild anorexia at 7 dpi. At 15 dpi, the survival rates in group A, B, C, D and E were 0/4, 0/4, 3/4, 4/4 and 3/3 (Fig. 4d).
Nasal and conjunctival swabs were collected daily, and were used to test for FHV-1 shedding by PCR. The monitoring results (Fig. 4e) showed that FHV-1 shedding originally occurred in nasal cavity of infected cats, and then in eyes. Cats inoculated with different doses of FHV-1 showed different characteristics of viral shedding. Cats in group A, B and C began to release viruses via nasal or ocular discharge at 3 dpi, while viral shedding was detected at 7 dpi for cats in group D. Control cats in group E remained healthy throughout the trial.
From the above comparison and contrast, cats inoculated with excessive doses (107 TCID50 and 106 TCID50) of FHV-1 CH-B strain showed acute infection and high mortality rate, while low-dose group (104 TCID50) did not show distinct clinical signs, only released viruses. In contrast, cats inoculated with 105 TCID50 of CH-B strain not only showed typical upper respiratory track and ocular symptoms, but also could replicate the progress of disease development. So, we choose 105 TCID50 as the optimal infectious dose to develop FHV-1 experimental infection model.
FHV-1 experimental infection model
Domestic cats inoculated intranasally with the optimal infectious dose (105 TCID50) of CH-B strain (Artificial inoculation group) replicated the disease progress of FVR, from acute infection phase to lifelong latency. Artificial inoculated (AI) cats begin to sneeze and cough at 5 dpi, accompanied by depression and anorexia. With the progression of disease, AI cats showed typical clinical symptoms of FVR (at 10 dpi), including fever, dyspnea, serous or purulent discharge in nasal cavity and eyes, and nasal skin ulcer, followed by deterioration (at 13 dpi) and death (Fig. 5a). Oculonasal purulent discharge and other clinical signs of most of survivors decreases and disappear at 20 dpi, and almost fully recovering at the end of study. Notably, clinical signs for most of cohabiting cats initially appeared at 10 dpi, and peaked at 16 dpi. The changes of clinical score, rectal temperature and body weight were showed in Fig 5bcd. At the peak of disease, the clinical score was >5, for AI cats, the rectal temperature was more than 103℉, and the weight loss rate was >4%. The survival rates in three groups were 9/15, 7/9 and 9/9 at the end of study (Fig. 5e).
Antibody responses to FHV-1 infection
Before starting the experiment (5 days prior to inoculation), all cats were negative for anti-FHV-1 antibodies based on the negative ELISA results. All serum samples from CH-B challenged cats (Artificial inoculation group) showed a positive reaction with very low antibody titers in the ELISA at 5 dpi, and peaked at 15 dpi (Fig. 6). For cohabitation infected cats, the anti-FHV-1 antibodies were not detected by ELISA or very low until 10 dpi, and then increased rapidly by 20 dpi. All the surviving cats in these two groups remained seropositive until the end of the experiment. Furthermore, all mock-inoculated cats (negative control) were serologically negative for FHV-1 throughout the study.
Inflammatory responses to FHV-1 infection
The white blood cells (WBC) in AI cats significantly rose compared with control cats at 5, 10 and 15 dpi, although the WBC in most of AI cats did not exceed the upper limits of normal (ULN) (Fig. 7b). There was a significant increase in blood lymphocyte (LYM) in AI cats at 15, 20 and 25 dpi, compared with controls (Fig. 7c). Furthermore, from 5 to 20 dpi, serum SAA contents in AI cats exceed the ULN, and were significantly higher than that in controls (Fig. 7a). For cohabiting cats, the significant increases of WBC and SAA were observed from 10 to 25 dpi, and no significant change in LYM appeared.
We examined virus shedding and viral titres in all cats by collecting nasal and ocular swabs at each time point. As shown in Fig. 8, virus shedding in nasal cavity were detectable in 8 of 15 AI cats at 1 dpi by quantitative PCR, and in all AI cats at 3-21 dpi. Viral DNA copies in nasal swabs maintained in a higher level (ranging from 105.68 copies/ml to 106.51 copies/ml) at 3-15 dpi, while decreased rapidly after 17 dpi. Viral DNA was detected in unilateral ocular swabs collected from a few AI cats at 3 dpi, and in ambilateral eyes of all AI cats from 7 to 21 dpi, with the viral loads (ranging from 105.91 copies/ml to 106.66 copies/ml) peaking between 9-15 dpi. Furthermore, AI cats released more virion through eyes at acute infection period (9-15 dpi). No detectable viral DNA was found in nasal cavity and eyes of all AI cats from 23 to 25 dpi, except one cat with low viral loads of 102.17 copies/ml in left eye at 23 dpi.
We also examined viraemia and viral replication in the primary tissues (Fig. 9), including turbinate, conjunctiva, cornea, trigeminal ganglia (TG), ciliary ganglia (CCG), optic nerve, olfactory bulb, trachea, tonsils, lung, liver, kidney, spleen and intestine. Viral loads were detectable at 5, 10, 13 (dead of infection) and 15 dpi in turbinate, conjunctiva, cornea, TG, CCG, optic nerve and olfactory bulb of all AI cats, and viral DNA copies reached peaks at 13 dpi. Furthermore, we only detected viral loads in trachea (103.28 copies/ml) and tonsils (102.35 copies/ml) collected from three cats died on 13 dpi. Only one cat died on 13 dpi showed detectable viral DNA in lung. Other tissues were tested to be negative for FHV-1 at each time point. Viral DNA were still detected in TG of all AI cats at 25 dpi with a mean viral load of 102.27 copies/ml, while no viral DNA was detected in other tissues at 25 dpi with very few exceptions.
For in-contacted cats, viral DNA were detectable in nasal cavity and eyes from 7 to 19 dpi, the change of viral loads coincided with AI cats (Fig. S1). Same as AI cats, viral loads were detectable in turbinate, conjunctiva, cornea, TG, CCG, optic nerve, olfactory bulb, trachea and tonsils, but not in lung and other tissues (Table S2). No viral DNA were detected in all swabs and tissues from all uninfected control cats.
Pathology and histopathology
Cats died of infection and euthanized cats were dissected according to the standard operational procedures, subjected to pathological and histopathological examinations. No tissue lesion was observed in any of cats at 5 dpi. Compared with control cats, AI cats at 10 dpi displayed mild lesions, including lung congestion and tracheal mucous secretions. Severe tissue lesions, including multifocal necrosis throughout the lung, dark-reddish purulent secretions in trachea, and tonsils severe congestion and necrosis, were observed in AI cats at 13 or 15 dpi (Fig. 10). The obvious lesions were not observed in other tissues. Histopathological examination at 13 dpi showed that strain CH-B caused coalescing severe interstitial pneumonia with diffuse lesions, characterized by destroyed alveoli, thickened alveolar septa accompanied with infiltration of inflammatory cells (including macrophages and lymphocytes), and aggregation of inflammatory cells in partial alveolar cavities (Fig. 10c). More seriously, the destruction of alveoli was observed microscopically in 30%-50% of the lung tissue. Alveolar wall dissolves, and adjacent alveoli fuse to form a large cavity accompanied with infiltration of inflammatory cells and fibrin exudation. Microscopically, the thickened mucous layer accompanied with infiltration of macrophages and lymphocytes, and hyperaemia and haemorrhage were observed in diseased trachea of AI cats (Fig. 10b). Furthermore, in necrotic tonsil of AI cats, there were large amounts of inflammatory cells (Fig. 10a). The lung and trachea from cohabiting cats at 15 and 18 dpi displayed moderate interstitial pneumonia and tracheitis (data not shown). Immunohistochemistry (IHC) examination showed that viral antigens for FHV-1 were found in alveolar epithelia and tracheal epithelial cells in areas of lung and trachea lesions from AI cats and cohabiting cats (Fig. 10d). The mock-infected cats showed no histopathological change and viral antigen.