Epidemiological Surveillance of Respiratory Diseases in Urban Stray Cats in Shanghai.

Urban stray cats are cats without owners that survive in the wild for extended periods of time. They are one of the most common stray animals in cities, and as such, monitoring the pathogens carried by urban stray cats is an important component of urban epidemiological surveillance. In order to understand the prevalence of respiratory diseases in urban stray cats in Shanghai and provide scientific evidence for the development of targeted prevention and control strategies for respiratory diseases in stray cats, we collected 374 ocular, nasal, and oropharyngeal swabs from urban stray cats in Shanghai from January 2022 to December 2022. After RNA extraction, we used real-time PCR to detect six respiratory pathogens, including influenza A virus, feline calicivirus, feline herpesvirus type 1, Mycoplasma, Chlamydia, and Bordetella bronchiseptica. The results showed that among the 374 samples, 146 tested positive, with a positivity rate of 39.04%. The highest positivity rate was observed for Mycoplasma felis at 18.72% (70/374), followed by Chlamydia felis at 11.76% (44/374), feline calicivirus at 3.74% (14/374), feline herpesvirus 1 at 3.48% (13/374), Bordetella bronchiseptica at 1.34% (5/374), and influenza A virus was not detected. The highest positivity rate for Mycoplasma felis was in Minhang District at 31.94% (23/72), while Chlamydia felis and Bordetella bronchiseptica had the highest positivity rates in Jiading District at 23.53% (8/34) and 5.88% (2/34), respectively. The highest positivity rates for feline calicivirus and feline herpesvirus 1 were both observed in Qingpu District, at 14.46% (12/83) and 9.64% (8/83), respectively. A total of 36 samples showed mixed infections with two or more pathogens, with Mycoplasma felis being involved in 32 of these mixed infections, with the highest number of mixed infections being with Chlamydia felis at 25 samples. Respiratory pathogen positivity was detected throughout the year, with peak detection rates in summer and winter. The positivity rates of cat respiratory pathogens in different seasons showed statistical differences (χ2 = 27.73, p < 0.01). There was no statistical difference in the positivity rates of respiratory pathogens between cats of different genders (χ2 = 0.92, p > 0.05). The positivity rates of respiratory pathogens in cats of different age groups showed statistical differences (χ2 = 44.41, p < 0.01). Mycoplasma felis and Chlamydia felis were the main pathogens causing respiratory infections in stray cats, with Mycoplasma felis showing a much higher positivity rate than other respiratory pathogens and often co-infecting with Chlamydia felis and feline calicivirus. The positivity rate of Mycoplasma felis was high in summer, autumn, and winter, with no statistical difference between seasons. These results indicate a serious overall prevalence of respiratory pathogens in urban stray cats in the Shanghai area, showing seasonal trends and mixed infections with other pathogens. These findings suggest the need for comprehensive prevention and control measures to address respiratory pathogen infections in urban stray cats in the Shanghai area.

Ultrasensitive and visual detection of Feline herpesvirus type-1 and Feline calicivirus using one-tube dRPA-Cas12a/Cas13a assay.

BACKGROUND: Feline herpesvirus type 1 (FHV) and Feline calicivirus (FCV) are the primary co-infecting pathogens that cause upper respiratory tract disease in cats. However, there are currently no visual detection assays available for on-site testing. Here, we develop an ultrasensitive and visual detection method based on dual recombinase polymerase amplification (dRPA) reaction and the hybrid Cas12a/Cas13a trans-cleavage activities in a one-tube reaction system, referred to as one-tube dRPA-Cas12a/Cas13a assay. RESULTS: The recombinant plasmid DNAs, crRNAs, and RPA oligonucleotides targeting the FCV ORF1 gene and FHV-1 TK gene were meticulously prepared. Subsequently, dual RPA reactions were performed followed by screening of essential reaction components for hybrid CRISPR-Cas12a (targeting the FHV-1 TK gene) and CRISPR-Cas13a (targeting the FCV ORF1 gene) trans-cleavage reaction. As a result, we successfully established an ultra-sensitive and visually detectable method for simultaneous detection of FCV and FHV-1 nucleic acids using dRPA and CRISPR/Cas-powered technology in one-tube reaction system. Visual readouts were displayed using either a fluorescence detector (Fluor-based assay) or lateral flow dipsticks (LDF-based assay). As expected, this optimized assay exhibited high specificity towards only FHV-1 and FCV without cross-reactivity with other feline pathogens while achieving accurate detection for both targets with limit of detection at 2.4 × 10- 1 copies/µL for the FHV-1 TK gene and 5.5 copies/µL for the FCV ORF1 gene, respectively. Furthermore, field detection was conducted using the dRPA-Cas12a/Cas13a assay and the reference real-time PCR methods for 56 clinical samples collected from cats with URTD. Comparatively, the results of Fluor-based assay were in exceptional concordance with the reference real-time PCR methods, resulting in high sensitivity (100% for both FHV-1 and FCV), specificity (100% for both FHV-1 and FCV), as well as consistency (Kappa values were 1.00 for FHV-1 and FCV). However, several discordant results for FHV-1 detection were observed by LDF-based assay, which suggests its prudent use and interpretaion for clinical detection. In spite of this, incorporating dRPA-Cas12a/Cas13a assay and visual readouts will facilitate rapid and accurate detection of FHV-1 and FCV in resource-limited settings. CONCLUSIONS: The one-tube dRPA-Cas12a/Cas13a assay enables simultaneously ultrasensitive and visual detection of FHV-1 and FCV with user-friendly modality, providing unparalleled convenience for FHV-1 and FCV co-infection surveillance and decision-making of URTD management.

Immunogenicity evaluation of a bivalent vaccine based on a recombinant rabies virus expressing gB protein of FHV-1 in mice and cats.

Feline viral rhinotracheitis (FVR) is caused by the feline herpesvirus-1 (FHV-1), which commonly results in upper respiratory symptoms, and can result in death in the kittens and weak cats. Rabies is an infectious disease with zoonotic characteristics highly relevant to public health and also poses a serious threat to cats. Vaccines are the most effective method to control the spread of both FHV-1 and RABV and have the advantage that they produce long-term specific immune responses. In this study, we constructed a bivalent vaccine against FHV-1 and rabies virus (RABV) simultaneously. The vaccine was constructed by cloning FHV-1 gB into a RABV based vector, and the recombinant RABV (SRV9-FHV-gB) expressing the FHV-1 gB protein was rescued. The growth characteristics of SRV9-FHV-gB were analyzed on NA and BSR cells. To assess the immunogenicity of the vaccine, mice and cats were immunized with SRV9-FHV-gB supplemented with Gel02 adjuvant. The SRV9-FHV-gB exhibited the same growth characteristics as the parent virus SRV9 in both BSR cells and NA cells. The safety of SRV9-FHV-gB was evaluated using 5-day-old and 14-day-old suckling mice. The results showed that mice infected with the SRV9-FHV-gB survived for longer than those in the SRV9 group. Mice immunized with inactivated SRV9-FHV-gB produced high titers of specific antibodies against FHV-1 and neutralizing antibodies against RABV. Cats that received three immunizations with SRV9-FHV-gB also produced neutralizing antibodies against both FHV-1 and RABV. This study represents the first time that a bivalent vaccine targeting FHV-1 and RABV has been constructed, laying the foundations and providing inspiration for the development of other multivalent vaccines.

A bacterium-like particle vaccine displaying protective feline herpesvirus 1 antigens can induce an immune response in mice and cats.

Feline herpesvirus 1 (FHV-1) is a highly transmissible virus that mainly causes ocular and upper respiratory infections in cats and seriously threatens the health of domestic cats and captive or wild cats (such as tigers, cheetahs, and lions). Vaccination is crucial to reduce the incidence rate and mortality of cats infected with FHV-1. In this study, three bacterium-like particles (BLPs) displaying the gB, gC, and gD proteins of FHV-1 were constructed based on a gram-positive enhancer matrix-protein anchor (GEM-PA) surface display system. Indirect immunofluorescence assay, western blot, and electron microscopy results showed that gB, gC or gD protein of FHV-1 was successfully displayed on the surface of GEM particles. Additionally, we designed one more BLPs, designated gB&gC&gD-GEM, which consisted of a mixture of gB-GEM, gC-GEM, and gD-GEM at a protein content ratio of 1:1:1. Mice were immunized with the four BLPs mixed with Gel02 adjuvant, and the results indicated that neutralizing antibody level in the gB&gC&gD-GEM group was superior than those in the other groups. Moreover, gB&gC&gD-GEM significantly increased the secretion of cytokines, as well as the activation and maturation of B cells. It also boosted the production of central memory T cells among CD4 + and CD8 + T cells. Moreover, gB&gC&gD-GEM mixed with Gel02 adjuvant provoked an antibody response in cats. In conclusion, the BLPs vaccine prepared from gB&gC&gD-GEM induced specific humoral and cellular immune responses to FHV-1 and be used as a potential vaccine candidate for the control of FHV-1 infection in cats.

Development and application of a dual ERA method for the detection of Feline Calicivirus and Feline Herpesvirus Type I.

Feline calicivirus (FCV) and feline herpesvirus type I (FHV-1) are the most common viral pathogens responsible for cat respiratory diseases, and coinfection with these two pathogens is often found. In veterinary clinics, the main diagnostic methods for FCV and FHV-1 are test strips and polymerase chain reaction (PCR). However, the sensitivity of test strips are not sufficient, and PCR is time-consuming. Therefore, developing a rapid and high-performance clinical diagnostic test is imperative for the prevention and treatment of these diseases. Enzymatic recombinase amplification (ERA) is an automated isothermal nucleic acid amplification technique that maintains a constant temperature, and is both rapid and highly accurate. In this study, a dual ERA method was developed using the Exo probe for a differential detection of FCV and FHV-1. This dual ERA method demonstrated high performance with the detection limit of 101 copies for both viruses, and no cross-reactions with feline parvovirus virus and F81 cells. To test the utility of the method for clinical applications, 50 nasopharyngeal swabs from cats with respiratory symptoms were collected and tested. The positive rates of FCV and FHV-1 were 40% (20/50, 95% confidence interval [CI], 26.4 to 54.8%) and 14% (7/50, 95% CI, 5.8 to 26.7%), respectively. The rate of coinfection with FCV and FHV-1 was 10% (5/50, 95% CI, 3.3 to 21.8%). These results were in agreement with those found using quantitative real-time PCR. Therefore, this dual ERA method is a novel and efficient clinical diagnostic tool for FCV and FHV-1 detection.

Development of a triple NanoPCR method for feline calicivirus, feline panleukopenia syndrome virus, and feline herpesvirus type I virus.

BACKGROUND: Feline calicivirus (FCV), Feline panleukopenia virus (FPV), and Feline herpesvirus type I (FHV-1) are the three most common pathogens in cats, and also are the main pathogens leading to the death of kittens. Here, by a combination of gold nanoparticles and conventional PCR, we established a novel triple NanoPCR molecular detection method for clinical detection. RESULTS: The triple NanoPCR molecular detection is able to detect 2.97 × 101copies/µL FCV recombinant copies plasmid per reaction, 2.64 × 104copies/µL FPV recombinant copies plasmid per reaction, and 2.85copies/µL FHV-1 recombinant copies plasmid per reaction at the same time. The sensitivity of each plasmid is 100 times, 10 times, and 100 times higher than conventional PCR, respectively. The clinical results showed that among the 38 samples, the positive rates of FCV, FPV, and FHV-1 in a NanoPCR test were 63.16, 31.58, and 60.53%, while in a conventional PCR were 39.47, 18.42, and 34.21%. CONCLUSIONS: In this report, it is the first time that NanoPCR assays are applied in the detection of FCV, FPV, and FHV-1 as well. This sensitive and specific NanoPCR assay can be widely used in clinical diagnosis and field monitoring of FCV, FPV, and FHV-1 infections.

Cross-species transmission of feline herpesvirus 1 (FHV-1) to chinchillas.

BACKGROUND: Herpesviruses are a class of double-stranded DNA viruses found in both vertebrates and invertebrates. They are usually highly host-specific and do not easily spread across species. Chinchillas have gradually entered the Chinese pet market in recent years, but references to viral infections in chinchillas are extremely scarce, and only two reports about the herpesvirus in chinchillas are available at present. OBJECTIVES: The aim of this study was to present the first report of FHV-1 infection in chinchillas. METHODS: A total of 130 nasopharyngeal swab samples of chinchillas and three nasopharyngeal swabs of domestic cats collected from a chinchillas farm were investigated by nested PCR for FHV-1. RESULTS: Four chinchillas were infected with FHV-1, the positive rate was 3.08% (4/130), and two domestic cats were FHV-1 positive (2/3). The 253 bp fragments of FHV-1 gD gene from four chinchillas and two domestic cats were 100% identical, respectively, and the homology between chinchillas and domestic cat was 99.21%, but they all shared nearly 98.81% homology with the reference strain sequences. Phylogenetic tree analysis showed that these four chinchillas strains were clustered together with FHV-1. CONCLUSIONS: This is the first time that FHV-1 was detected in chinchillas and suggested chinchillas are susceptible to FHV-1 and may play a role as a temporary reservoir for FHV-1.

Prevalence of four viruses in captive Siberian tigers from Northeastern China.

Our study investigated the prevalence of feline herpesvirus-1 (FHV-1), feline calicivirus (FCV), feline immunodeficiency virus (FIV), and feline leukemia virus (FeLV) in captive Siberian tigers in Northeastern China. A total of 324 blood samples and 33 nasopharyngeal swab samples of Siberian tigers collected from 2019 to 2021 in three cities were investigated by nested PCR. The results showed that 28.1% (91/324) tigers were infected with at least one virus; the positive rates of FHV-1, FCV, and FIV were 17.3%, 13.6%, and 0.9%, respectively; and the coinfection prevalence was 13.2%. No FeLV-positive sample was detected. And we found that the blood is the best for FCV, FIV, and FeLV detection, but nasopharyngeal swabs for FHV-1. By comparing the gD genes, TK gene, and gI gene of FHV-1, the homology of the three FHV-1 positive strains in this study was found to be 91.5%-99.9% shared with tigers and domestic cats. Based on a comparison of the nucleic acid sequences of 13 FCV strains, we found that the homology of strain HB-1926 with the other strains in this study was only about 77.7%, but shared 99.3% and 98.6% homology with Urnaba strain in American cat and TG1 strain in Chinese tiger, respectively. However, the other 12 FCV strains shared 87.1%-87.5% homology compared with the Chinese domestic cats. Phylogenetic tree analysis showed that the HB-1926 strain was not in the same clade as other strains. The fragments gag-p26, pol-RT, and pol-RNAse of Siberian tiger FIV shared more than 99% homology than domestic cats FIV subtype A. This study demonstrated that captive Siberian tigers in Northeastern China were exposed to FHV-1, FCV, and FIV, and it is necessary to develop more effective vaccines and improve daily management measures.

Variable accuracy, precision, and consistency of compounded famciclovir formulated for management of feline herpesvirus-1 in cats.

OBJECTIVE: To evaluate compounded famciclovir suspensions for accuracy, precision, and consistency in drug content. PROCEDURES: Two compounded famciclovir concentrations were evaluated (250 and 400 mg/mL, 30 preparations total from nine 503A compounding pharmacies) with U.S. Food and Drug Administration (FDA)-approved famciclovir tablets as control. Drug quantification via high-performance liquid chromatography (with famciclovir reference standard and pramipexole internal standard) was performed at 0, 14, and 28 days with concentrations of 90%-110% of labeled dose considered acceptable (US Pharmacopoeia standards). RESULTS: FDA-approved tablets from three different manufacturers were found to be accurate and precise with acceptable drug content. A significantly greater mean deviation from labeled content was noted for 400 mg/mL suspensions (-52.9%) compared to 250 mg/mL suspensions (-18.0%). When assessing time points separately, 15/63 (24%) samples of 250 mg/mL and 0/27 (0%) samples of 400 mg/mL suspensions met the acceptance standards. Coefficients of variation (CV) in drug content among pharmacy batches ranged from 0.5% to 29%, with 5/10 formulations having significantly lower CV% compared to control (decreased precision). Similarly, drug content changed over time (0-28 days) in all compounded formulations, with both downward and upward trends observed (variable consistency). CONCLUSIONS: Most compounded famciclovir formulations were inaccurate, imprecise, and inconsistent. FDA-approved famciclovir tablets may be preferred over compounded famciclovir formulations for the management of feline herpesvirus-1. If compounded famciclovir is used in practice, a concentration of 250 mg/mL is preferred over 400 mg/mL given the lower accuracy of the higher concentration.

Development and Application of a Triplex TaqMan Quantitative Real-Time PCR Assay for Simultaneous Detection of Feline Calicivirus, Feline Parvovirus, and Feline Herpesvirus 1.

As companion animals, felines play an important role in human's family life, and their healthcare has attracted great attention. Viruses such as feline calicivirus (FCV), feline herpesvirus 1 (FHV-1), and feline parvovirus virus (FPV) are the most common pathogens that cause severe infectious disease in baby cats. Thus, preclinical detection and intervention of these three viruses is an effective means to prevent diseases and minimize their danger condition. In this study, a triplex TaqMan quantitative real-time polymerase chain reaction (qRT-PCR) assay was developed to detect these three viruses simultaneously. The detection limit of FPV, FCV, and FHV-1 was 5 × 101 copies/assay, which exhibited higher sensitivity (about 10- to100-fold) than conventional PCR. The coefficients of variation (CVs) of the intra-assay variability were lower than 1.86%, and that of inter-assay variability were lower than 3.19%, indicating the excellent repeatability and reproducibility of the triplex assay. Additionally, the assay showed good specificity. Finally, samples from 48 cats were analyzed using the established assay and commercial kits. As a result, the total positive rates for these viruses were 70.83 or 62.5%, respectively, which demonstrated that the developed qRT-PCR assay was more accurate than the commercial kits and could be used in clinical diagnosis.

Antibody response to feline herpesvirus-1 vaccination in healthy adult cats.

OBJECTIVES: Vaccination against feline herpesvirus-1 (FHV-1) is recommended for all cats. However, it is unknown how adult healthy cats with different pre-vaccination antibodies respond to FHV-1 vaccination in the field. The aim of the study was to determine the prevalence of neutralising antibodies against FHV-1 in healthy adult cats and the response to FHV-1 vaccination within 28 days of vaccination. METHODS: One hundred and ten cats (⩾1 year of age) that had not received a vaccination within the past 12 months were vaccinated with a combined FHV-1 vaccine. Antibodies against FHV-1 were determined before vaccination (day 0), on day 7 and day 28 by serum neutralisation test. Uni- and multivariate statistical analyses were used to determine factors associated with the presence of pre-vaccination antibodies and response to vaccination. RESULTS: Pre-vaccination neutralising antibody titres (⩾10) were present in 40.9% of cats (45/110; 95% confidence interval [CI] 32.2-50.3); titres were generally low (range 10-640). Antibody response to vaccination (⩾four-fold titre increase) was observed in 8.3% (9/109; 95% CI 4.2-15.1). Cats ⩾2 years of age were more likely to have pre-vaccination neutralising antibodies than cats aged between 1 and 2 years (odds ratio [OR] 24.619; P = 0.005). Cats from breeders were more likely to have pre-vaccination neutralising antibodies than privately owned cats (OR 7.070; P = 0.007). Domestic shorthair cats were more likely to have an at least four-fold titre increase vs purebred cats (OR 11.22; P = 0.027). CONCLUSIONS AND RELEVANCE: Many cats have no detectable neutralising antibodies against FHV-1 despite previous vaccinations and fail to develop a ⩾four-fold titre increase after vaccination. This is likely because older cats and cats with a higher FHV-1 exposure risk are more likely to get infected with FHV-1 and thus to have FHV-1 neutralizing antibodies. Purebred cats more often fail to develop a ⩾four-fold titre increase after vaccination.

Cat flu: Broad spectrum polymeric antivirals.

Feline herpesvirus type 1 (FHV-1) and feline calicivirus (FCV) are considered as main causes of feline upper respiratory tract disease and the most common clinical manifestations include rhinotracheitis, conjunctivitis, and nasal/facial ulcerations. While the primary infection is relatively mild, secondary infections pose a threat to young or immunocompromised cats and may result in a fatal outcome. In this study, we made an effort to evaluate antiviral potency of poly(sodium 4-styrenesulfonates) (PSSNa) as potent FHV-1 and FCV inhibitors for topical use. Mechanistic studies showed that PSSNa exhibits a different mechanism of action depending on target species. While PSSNa acts directly on FHV-1 particles blocking their interaction with the host's cell and preventing the infection, the antiviral potency against FCV is based on inhibition at late stages of the viral replication cycle. Altogether, PSSNa polymers are promising drug candidates to be used in the treatment and prevention of the viral upper respiratory tract disease (URTD), regardless of the cause.

Feline herpesvirus 1 viral load related to environmental factors in sheltered cats / Relação entre carga viral de herpesvirus felino 1 e fatores causadores de estresse em felinos de abrigo

ABSTRACT: Shelter environment stress factors are related to FHV-1 viral reactivation. However, comparisons between conjunctival viral load and environmental factors have not been commonly evaluated. The aim of this study was to correlate FHV-1 viral load in domestic cats with and without clinical signs of conjunctivitis to shelter design in order to use FHV-1 viral load as a parameter of "health management". Cats from four different shelters underwent an ophthalmological examination. Samples were collected by rolling a DNA/RNAse-free cytobrush over the ventral conjunctival fornix and were stored in 1.5 mL sterile microtubes in 500 μL of Eagle's minimum essential medium and kept at 4 ºC. Molecular procedures were performed up to 48 hours after collection. Different routines regarding new arrivals were directly related to FHV-1 viral load. Shelters where new arrivals occurred on daily basis had the highest viral load (2.69x108 copies/µL), while those shelters where new arrivals had not occurred in the few months prior to the beginning of the study had the lowest rate (1.63x103 copies/µL). Environmental factors directly influenced FHV-1 DNA viral load. This study highlighted the need to improve the management approach in the animal shelter environment to reduce stressful situations responsible for FHV-1 reactivation and higher viral load quantification.

RESUMO: No ambiente do abrigo encontram-se fatores que geram estresse nos animais que ali residem. Esses fatores acabam por provocar a reativação do FHV-1. No entanto, comparações entre carga viral conjuntival e fatores ambientais não foram ainda avaliadas. Objetivo deste estudo foi correlacionar a carga viral de FHV-1 em felinos domésticos com e sem sinais clínicos de conjuntivite com as características dos abrigos. Assim, pode-se usar carga viral de FHV-1 como parâmetro de sanidade. Todos os gatos foram submetidos a exame clínico oftalmológico. Amostras foram coletadas com uso de escova citológica, acondicionadas em microtubos estéreis de 1,5mL contendo 500 μL de meio Eagle essencial mínimo e mantidas em 4 ºC. Análises moleculares foram realizadas no prazo de 48 horas após coleta. A rotina de entrada de novos animais estava diretamente relacionada a carga viral de FHV-1. Abrigos com entrada diária apresentaram carga viral maior (2.69x108 cópias/µL), do que abrigo onde novos animais não chegaram nos meses que antecederam a coleta (1.63x103 cópias/µL). Fatores ambientais influenciam diretamente carga viral de FHV-1. Esse estudo evidencia a necessidade de aprimorar o sistema de manejo dos abrigos de forma a reduzir situações de estresse responsáveis pela reativação de FHV-1 e consequente aumento na carga viral.

Transcriptome profiling of alphaherpesvirus-infected cells treated with the HIV-integrase inhibitor raltegravir reveals profound and specific alterations in host transcription.

Anti-microbial compounds typically exert their action by directly interfering with one or more stages of the pathogen's life cycle. However, some compounds also have secondary effects on the host that aid in pathogen clearance. Raltegravir is a human immunodeficiency virus (HIV)-integrase inhibitor that has been shown to alter the host immune response to HIV in addition to its direct antiviral effect. Interestingly, raltegravir can also directly inhibit the replication of various herpesviruses. However, the host-targeted effects of this drug in the context of a herpesvirus infection have not been explored. Here, we used felid alphaherpesvirus 1 (FHV-1), a close relative of human alphaherpesvirus 1 (HHV-1) that similarly causes ocular herpes, to characterize the host-targeted effects of raltegravir on corneal epithelial cells during an alphaherpesvirus infection. Using RNA deep sequencing, we found that raltegravir specifically boosts the expression of anti-angiogenic factors and promotes metabolic homeostasis in FHV-1-infected cells. In contrast, few changes in host gene transcription were found in uninfected cells. Importantly, we were able to demonstrate that these effects were specific to raltegravir and independent of the direct-acting antiviral effect of the drug, since treatment with the DNA polymerase inhibitor phosphonoacetic acid did not induce these host-targeted effects. Taken together, these results indicate that raltegravir has profound and specific effects on the host transcription profile of herpesvirus-infected cells that may contribute to the overall antiviral activity of the drug and could provide therapeutic benefits in vivo. Furthermore, this study provides a framework for future efforts evaluating the host-targeted effects of anti-microbial compounds.

Feline Herpesvirus 1 US3 Blocks the Type I Interferon Signal Pathway by Targeting Interferon Regulatory Factor 3 Dimerization in a Kinase-Independent Manner.

As a prevalent agent in cats, feline herpesvirus 1 (FHV-1) infection contributes to feline respiratory disease and acute and chronic conjunctivitis. FHV-1 can successfully evade the host innate immune response and persist for the lifetime of the cat. Several mechanisms of immune evasion by human herpesviruses have been elucidated, but the mechanism of immune evasion by FHV-1 remains unknown. In this study, we screened for FHV-1 open reading frames (ORFs) responsible for inhibiting the type I interferon (IFN) pathway with an IFN-ß promoter reporter and analysis of IFN-ß mRNA levels in HEK 293T cells and the Crandell-Reese feline kidney (CRFK) cell line, and we identified the Ser/Thr kinase US3 as the most powerful inhibitor. Furthermore, we found that the anti-IFN activity of US3 depended on its N terminus (amino acids 1 to 75) and was independent of its kinase activity. Mechanistically, the ectopic expression of US3 selectively inhibited IFN regulatory factor 3 (IRF3) promoter activation. Furthermore, US3 bound to the IRF association domain (IAD) of IRF3 and prevented IRF3 dimerization. Finally, US3-deleted recombinant FHV-1 and US3-repaired recombinant FHV-1 (rFHV-dUS3 and rFHV-rUS3, respectively) were constructed. Compared with wild-type FHV-1 and rFHV-rUS3, infection with rFHV-dUS3 induced large amounts of IFN-ß in vitro and in vivo More importantly, US3 deletion significantly attenuated virulence, reduced virus shedding, and blocked the invasion of trigeminal ganglia. These results indicate that FHV-1 US3 efficiently inhibits IFN induction by using a novel immune evasion mechanism and that FHV-1 US3 is a potential regulator of neurovirulence.IMPORTANCE Despite widespread vaccination, the prevalence of FHV-1 remains high, suggesting that it can successfully evade the host innate immune response and infect cats. In this study, we screened viral proteins for inhibiting the IFN pathway and identified the Ser/Thr kinase US3 as the most powerful inhibitor. In contrast to other members of the alphaherpesviruses, FHV-1 US3 blocked the host type I IFN pathway in a kinase-independent manner and via binding to the IRF3 IAD and preventing IRF3 dimerization. More importantly, the depletion of US3 attenuated the anti-IFN activity of FHV-1 and prevented efficient viral replication in vitro and in vivo Also, US3 deletion significantly attenuated virulence and blocked the invasion of trigeminal ganglia. We believe that these findings not only will help us to better understand the mechanism of how FHV-1 manipulates the host IFN response but also highlight the potential role of US3 in the establishment of latent infection in vivo.

Effect of a Pheromone on Stress-Associated Reactivation of Feline Herpesvirus-1 in Experimentally Inoculated Kittens.

BACKGROUND: Stress contributes to reactivation of feline herpesvirus-1 (FHV-1). The usage of pheromones to decrease stress in FHV-1 experimentally inoculated kittens has not previously been investigated. HYPOTHESIS/OBJECTIVES: To determine whether a feline pheromone would lessen stress, resulting in decreased recurrence of FHV-1-associated illness in kittens. ANIMALS: Twelve 5-month-old, purpose-bred kittens. METHODS: Randomized, double-blind, placebo-controlled clinical trial. Kittens previously infected with the same dose of FHV-1 were randomized into 2 separate but identical group rooms. After a 2-week equilibration period, a diffuser containing either the pheromone or placebo was placed in each of the rooms, and the kittens acclimated for an additional 2 weeks. Every 2 weeks thereafter, for the 8-week study period, housing was alternated between kennel- and group housing. Blinded observers applied a standardized clinical and behavioral scoring rubric daily. After each 2-week period, serum cortisol concentrations and quantitative PCR for FHV-1 and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) ratios were evaluated. Clinical, behavioral, and laboratory test results were compared between groups within individual and combined study periods. RESULTS: Sneezing occurred more frequently in the placebo group during individual (P = 0.006) and combined study periods (P = 0.001). Sleep at the end of observation periods occurred more frequently in the pheromone group during individual (P = 0.006) and combined study periods (P < 0.001). CONCLUSIONS AND CLINICAL IMPORTANCE: The findings suggest that the pheromone decreased stress, and the decrease in stress response may have resulted in decreased sneezing associated with FHV-1.

New Paradigms for the Study of Ocular Alphaherpesvirus Infections: Insights into the Use of Non-Traditional Host Model Systems.

Ocular herpesviruses, most notably human alphaherpesvirus 1 (HSV-1), canid alphaherpesvirus 1 (CHV-1) and felid alphaherpesvirus 1 (FHV-1), infect and cause severe disease that may lead to blindness. CHV-1 and FHV-1 have a pathogenesis and induce clinical disease in their hosts that is similar to HSV-1 ocular infections in humans, suggesting that infection of dogs and cats with CHV-1 and FHV-1, respectively, can be used as a comparative natural host model of herpesvirus-induced ocular disease. In this review, we discuss both strengths and limitations of the various available model systems to study ocular herpesvirus infection, with a focus on the use of these non-traditional virus-natural host models. Recent work has demonstrated the robustness and reproducibility of experimental ocular herpesvirus infections in dogs and cats, and, therefore, these non-traditional models can provide additional insights into the pathogenesis of ocular herpesvirus infections.

The domestic cat antibody response to feline herpesvirus-1 increases with age.

Herpesviruses establish lifelong infections, normally characterized by prolonged periods of latency with intermittent episodes of viral reactivation. Feline herpesvirus-1 (FHV-1) infects domestic cats, and epidemiological studies indicate that many or most domestic cats are exposed to FHV-1, but the strength and longevity of the antibody response to FHV-1 is not fully characterized. Here we describe development of an ELISA, using lysates of cat cells infected with FHV-1, that measure feline antibodies against FHV-1. The assay is sensitive, quantitative and has a large dynamic range. We found that serum anti-FHV-1 antibodies primarily recognize FHV-1 proteins of the Late (L) class and are primarily of the IgG isotype. We then analyzed serum from a cross-sectional cohort of 100 client-owned cats that differed in age, sex and vaccination history. While there was no difference in FHV-1 antibody responses between females and males, antibody levels were significantly increased in older cats in comparison with younger animals (p=0.01). Surprisingly, as the length of time since the most recent vaccination increased, there was no corresponding drop in serum anti-FHV-1 antibody. These data suggest that FHV-1 immunity is very long-lived and support the current recommendation that many cats do not require revaccination against FHV-1 annually.

TREATMENT OF CHRONIC HERPESVIRAL DERMATITIS IN A CAPTIVE CHEETAH (ACINONYX JUBATUS) IN NAMIBIA.

A 9-yr-old male cheetah (Acinonyx jubatus) housed at the Cheetah Conservation Fund in Namibia developed cutaneous lesions consisting of alopecia, erythema, ulceration, and crusting on the left fore and hind limbs. Histopathology of skin biopsies in conjunction with indirect fluorescent antibody and polymerase chain reaction testing confirmed a diagnosis of feline herpesvirus-1 dermatitis; microbial culture indicated secondary bacterial infection. Therapy included targeted systemic antimicrobial and antiviral treatment, topical medications, and repeated cryotherapy. Lesions exhibited varying degrees of clinical improvement but, overall, progressed in extent, size, and severity during the subsequent 2.5 yr of intense treatment. The cheetah was ultimately euthanized due to a guarded prognosis and concerns about poor quality of life. Potential factors initiating or contributing (or both) to the severity and nonhealing nature of the cutaneous lesions include chronic unidentified stress, altered immune system function, and other environmental influences.

Isolation and identification of feline calicivirus and feline herpesvirus in Southern Brazil

Feline calicivirus (FCV) and feline herpesvirus type 1 (FHV-1) are the two primary causes of upper respiratory tract disease in cats. The aim of this study was to demonstrate the distribution of FCV and FHV-1 among the feline population of several counties in Rio Grande do Sul State, Brazil. To this end, conjunctival and nasal swabs were collected from 302 cats from different locations, including households, breeding catteries, veterinary clinics, animal hospitals and experimental research facilities. The samples were collected between July 2006 to June 2009. The virus isolation was performed in CRFK cells and, subsequently, the identification was confirmed by PCR. FCV, FHV-1, or both were isolated from 55 cats from 28 different locations. FCV alone was isolated from 52.7% (29/55) of the animals that tested positively, FHV-1 alone was isolated from 38.2% (21/55) of the animals that tested positively, and co-infection were detected in 9.1% (5/55) of the animals that tested positively. Virus detection was more prevalent in cats that were less than 1 year old, among animals that shared a living space with other cats, and females. FCV and FHV-1 were isolated from vaccinated cats. In addition, both viruses were isolated from cats that showed no signs of disease. The results suggest that a carrier state is common for both viruses in the evaluated population. A search for other causes of respiratory disease in that population is necessary; and further studies relating to the molecular characterization of viruses and vaccine efficacy are also necessary.