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.

Simian varicella virus infection and reactivation in rhesus macaques trigger cytokine and Aß40/42 alterations in serum and cerebrospinal fluid.

Simian varicella virus (SVV) produces peripheral inflammatory responses during varicella (primary infection) and zoster (reactivation) in rhesus macaques (RM). However, it is unclear if peripheral measures are accurate proxies for central nervous system (CNS) responses. Thus, we analyzed cytokine and Aß42/Aß40 changes in paired serum and cerebrospinal fluid (CSF) during the course of infection. During varicella and zoster, every RM had variable changes in serum and CSF cytokine and Aß42/Aß40 levels compared to pre-inoculation levels. Overall, peripheral infection appears to affect CNS cytokine and Aß42/Aß40 levels independent of serum responses, suggesting that peripheral disease may contribute to CNS disease.

Identification of neuropathogenic Varicellovirus equidalpha1 as a potential cause of respiratory disease outbreaks among horses in North Xinjiang, China, from 2021-2023.

BACKGROUND: Varicellovirus equidalpha1 (formerly Equid alphaherpesvirus 1, EqAHV-1) is among the most important viruses responsible for respiratory disease outbreaks among horses throughout the world. No reports to date have detailed the association between EqAHV-1 and respiratory disease among horses in China. This study described one such outbreak among a population of horses in north Xinjiang that occurred from April 2021 - May 2023. RESULTS: qPCR revealed that EqAHV-1 was detectable in all samples and this virus was identified as a possible source of respiratory disease, although a limited subset of these samples were also positive for EqAHV-2, EqAHV-4, and EqAHV-5. In total, three EqAHV-1 strains responsible for causing respiratory illness in horses were isolated successfully, and full-length ORF33 sequence comparisonsand phylogenetic analyses indicated that these isolates may have originated from EqAHV-1 strains detected in Yili horse abortions. ORF30 sequence data additionally suggested that these strains were neuropathic, as evidenced by the presence of a guanine residue at nucleotide position 2254 corresponding to the aspartic acid present at position 752 in the DNA polymerase encoded by this virus. CONCLUSION: This study is the first report of an outbreak of respiratory disease among horses in China caused by EqAHV-1. ORF30 sequence characterization revealed that these EqAHV-1 strains harbored a neuropathogenic genotype. Given the detection of this virus in horses suffering from respiratory disease, concern is warranted with respect to this neuropathogenic EqAHV-1 outbreak.

Saikosaponin B2, Punicalin, and Punicalagin in Vitro Block Cellular Entry of Feline Herpesvirus-1.

In the realm of clinical practice, nucleoside analogs are the prevailing antiviral drugs employed to combat feline herpesvirus-1 (FHV-1) infections. However, these drugs, initially formulated for herpes simplex virus (HSV) infections, operate through a singular mechanism and are susceptible to the emergence of drug resistance. These challenges underscore the imperative to innovate and develop alternative antiviral medications featuring unique mechanisms of action, such as viral entry inhibitors. This research endeavors to address this pressing need. Utilizing Bio-layer interferometry (BLI), we meticulously screened drugs to identify natural compounds exhibiting high binding affinity for the herpesvirus functional protein envelope glycoprotein B (gB). The selected drugs underwent a rigorous assessment to gauge their antiviral activity against feline herpesvirus-1 (FHV-1) and to elucidate their mode of action. Our findings unequivocally demonstrated that Saikosaponin B2, Punicalin, and Punicalagin displayed robust antiviral efficacy against FHV-1 at concentrations devoid of cytotoxicity. Specifically, these compounds, Saikosaponin B2, Punicalin, and Punicalagin, are effective in exerting their antiviral effects in the early stages of viral infection without compromising the integrity of the viral particle. Considering the potency and efficacy exhibited by Saikosaponin B2, Punicalin, and Punicalagin in impeding the early entry of FHV-1, it is foreseeable that their chemical structures will be further explored and developed as promising antiviral agents against FHV-1 infection.

Ivermectin antiviral activity against Varicellovirus bovinealpha 1: assessment of intracellular drug accumulation in virus-infected cells.

Varicellovirus bovinealpha 1 (formerly bovine alphaherpesvirus type 1, BoAHV-1) is associated with several syndromes in cattle, including respiratory disease and is one of the main agents involved in the bovine respiratory disease complex (BRDC). Its infectious cycle is characterized by latent infections with sporadic virus reactivation and transmission. Although the acute disease can be prevented by the use of vaccines, specific therapeutic measures are not available. Ivermectin (IVM) is a semi-synthetic avermectin with a broad-spectrum antiparasitic activity, which has previously shown to have potential as an antiviral drug. In this study, IVM antiviral activity against BoAHV-1 was characterized in two cell lines (MDBK [Madin Darby bovine kidney] and BT [bovine turbinate]), including the measurement of intracellular drug accumulation within virus-infected cells. IVM antiviral activity was assessed at three different drug concentrations (1.25, 2.5 and 5 µM) after incubation for 24, 48 and 72 h. Slight cytotoxicity was only observed with 5 µM IVM. Even the lowest IVM dose was able to induce a significant reduction in virus titers in both cell lines. These findings indicate that the antiviral effects of IVM were evident in our experimental model within the range of concentrations achievable through therapeutic in vivo administration. Consequently, additional in vivo trials are necessary to validate the potential utility of these results in effectively managing BoAHV-1 in infected cattle.

A potential dual protection vaccine: Recombinant feline herpesvirus-1 expressing feline parvovirus VP2 antigen.

Recently, herpesvirus viral vectors that stimulate strong humoral and cellular immunity have been demonstrated to be the most promising platforms for the development of multivalent vaccines, because they contain various nonessential genes and exhibit long-life latency characteristics. Previously, we showed that the feline herpesvirus-1 (FHV-1) mutant WH2020-ΔTK/gI/gE, which was safe for felines and provided efficacious protection against FHV-1 challenge, can be used as a vaccine vector. Moreover, previous studies have shown that the major neutralizing epitope VP2 protein of feline parvovirus (FPV) can elicit high levels of neutralizing antibodies. Therefore, to develop a bivalent vaccine against FPV and FHV-1, we first generated a novel recombinant virus by CRISPR/Cas9-mediated homologous recombination, WH2020-ΔTK/gI/gE-VP2, which expresses the VP2 protein of FPV. The growth characteristics of WH2020-ΔTK/gI/gE-VP2 were similar to those of WH2020-ΔTK/gI/gE, and WH2020-ΔTK/gI/gE-VP2 was stable for at least 30 generations in CRFK cells. As expected, we found that the felines immunized with WH2020-ΔTK/gI/gE-VP2 produced FPV-neutralizing antibody titers (27.5) above the positive cutoff (26) on day 14 after single inoculation. More importantly, recombinant WH2020-ΔTK/gI/gE-VP2 exhibited severely impaired pathogenicity in inoculated and cohabiting cats. The kittens immunized with WH2020-ΔTK/gI/gE and WH2020-ΔTK/gI/gE-VP2 produced similar levels of FHV-specific antibodies and IFN-ß. Furthermore, felines immunized with WH2020-ΔTK/gI/gE-VP2 were protected against challenge with FPV and FHV-1. These data showed that WH2020-ΔTK/gI/gE-VP2 appears to be a potentially safe, effective, and economical bivalent vaccine against FPV and FHV-1 and that WH2020-ΔTK/gI/gE can be used as a viral vector to develop feline multivalent vaccines.

Clinical evaluation of antiviral combination treatment in cats with feline herpesvirus-1 infection.

Feline herpesvirus-1 (FHV-1) can cause lifelong problems such as rhinotracheitis and ocular disease due to latency and reactivation in affected cats. The particular effects of antiviral drugs have been separately investigated in previous studies for decades and little is known about the combination treatment in active FHV-1 infection. Therefore, we aimed to evaluate the effects of antiviral combination on clinical effectiveness in cats with naturally occurring FHV-1 infection. 28 cats suffering from clinical signs of sneezing, nasal congestion, conjunctivitis, and eye/nose discharge were involved in this study following FHV-1 DNA detection by PCR assay in oculo-oropharyngeal samples. The treatment protocol was as follows: oral famciclovir and L-lysine, ophthalmic acyclovir, and subcutaneous amoxicillin plus clavulanic acid. The symptoms improved each day and total recovery success rate was 80% reduction in clinical scores at the end of the treatment on day 10 (p<0.001). Additionally, PCR was found to be negative for FHV-1 DNA in 82.1% of the samples after the treatment. There were mild decreases in neutrophil and monocyte counts (p>0.05). The arginine to lysine ratio decreased in favour of lysine (p<0.01). As a result, the antiviral combination treatment with famciclovir, L-lysine and ophthalmic acyclovir, and antibacterial drug appears to be clinically effective for the treatment of naturally occurring active FHV-1 infection in cats. In addition, any adverse clinical effect has not been determined associated with the antiviral combination during the study.

Frequency of feline herpesvirus 1 (FHV-1) in domestic cats from Campo Grande, MS, Brazil.

Feline herpesvirus type 1 (HVF-1) is the infectious agent of feline viral rhinotracheitis. The main clinical signs are cough, nasal and eye discharge, fever, conjunctivitis and sneezing. Although the occurrence of the virus is known in some regions of Brazil, in Campo Grande, Mato Grosso do Sul (MS), there is no epidemiological information about its frequency. Thus, this study aimed to determine the frequency of feline herpesvirus type 1 in the region, and to evaluate its possible association with clinical and epidemiological factors. Ocular, nasal and oropharyngeal swabs, and blood were collected from 152 animals and analyzed through PCR and sequencing. In addition, epidemiological and clinical data were obtained through clinical examination and anamnesis. FHV-1 was detected in samples from 84 (55.26%) animals. There was no association between infection and age or sex. However, there was a significant association between infection and nasal (p < 0.0001) and ocular (p = 0.014) discharge and sneezing (p = 0.001). The results demonstrate the occurrence of the virus in domestic cats in the region with a high frequency of infection. Thus, FHV-1 should be considered as a potential causal agent of upper respiratory tract disease in domestic cats from Campo Grande, MS, Brazil.

Feline herpesvirus 1 (FHV-1) enters the cell by receptor-mediated endocytosis.

Feline herpesvirus type 1 (FHV-1) is an enveloped dsDNA virus belonging to the Herpesviridae family and is considered one of the two primary viral etiological factors of feline upper respiratory tract disease. In this study, we investigated the entry of FHV-1 into host cells using two models: the AK-D cell line and primary feline skin fibroblasts (FSFs). We employed confocal microscopy, siRNA silencing, and selective inhibitors of various entry pathways. Our observations revealed that the virus enters cells via pH and dynamin-dependent endocytosis, as the infection was significantly inhibited by NH4Cl, bafilomycin A1, dynasore, and mitmab. Additionally, genistein, nystatin, and filipin treatments, siRNA knock-down of caveolin-1, as well as FHV-1 and caveolin-1 colocalization suggest the involvement of caveolin-mediated endocytosis during the entry process. siRNA knock-down of clathrin heavy chain and analysis of virus particle colocalization with clathrin indicated that clathrin-mediated endocytosis also takes part in the primary cells. This is the first study to systematically examine FHV-1 entry into host cells, and for the first time, we describe FHV-1 replication in AK-D and FSFs. IMPORTANCE Feline herpesvirus 1 (FHV-1) is one of the most prevalent viruses in cats, causing feline viral rhinotracheitis, which is responsible for over half of viral upper respiratory diseases in cats and can lead to ocular lesions resulting in loss of sight. Although the available vaccine reduces the severity of the disease, it does not prevent infection or limit virus shedding. Despite the clinical relevance, the entry mechanisms of FHV-1 have not been thoroughly studied. Considering the limitations of commonly used models based on immortalized cells, we sought to verify our findings using primary feline skin fibroblasts, the natural target for infection in cats.

[Circulation of bovine herpesvirus (Herpesviridae: Varicellovirus) and bovine viral diarrhea virus (Flaviviridae: Pestivirus) among wild artiodactyls of the Moscow region].

INTRODUCTION: Pestiviruses and viruses of the Herpesviridae family are widely distributed among different species of ungulates, but the main information about these pathogens is related to their effect on farm animals. Data on detection of bovine viral diarrhea virus (BVDV) and bovine herpes virus (BoHV) in wild ungulates reported from different countries in recent years raises the question of the role of wild animals in the epidemiology of cattle diseases. AIM OF WORK: To study the prevalence of herpesviruses and pestiviruses in the population of wild artiodactyls of the Moscow region. MATERIALS AND METHODS: Samples of parenchymal organs and mucosal swabs from 124 wild deer (moose and roe deer) shot during hunting seasons 20192022 in Moscow Region were examined by PCR, virological and serological methods for the presence of genetic material and antibodies to bovine infectious rhinotracheitis and viral diarrhea. RESULTS: BVDV RNA was found in a sample from one moose, BoHV DNA was detected in samples from three roe deer and two moose shot in the Moscow region. Seropositive animals were of different sex and age, the total BoHVs and BVDV seroprevalence rates in wild artiodactyls were 46 and 29%, respectively. CONCLUSION: Wild ruminant artiodactyls of the Moscow Region can be a natural reservoir of BoHV-1, and this must be taken into account when planning and organizing measures to control the infectious bovine rhinotracheitis. Cases of BVDV infection in wild artiodactyls are less common, so more research is needed to definitively establish their role in the epidemiology of this disease in cattle.

Pathogenicity and immunogenicity of gI/gE/TK-gene-deleted Felid herpesvirus 1 variants in cats.

BACKGROUND: Felid herpesvirus 1 (FHV-1) is a major pathogenic agent of upper respiratory tract infections and eye damage in felines worldwide. Current FHV-1 vaccines offer limited protection of short duration, and therefore, do not reduce the development of clinical signs or the latency of FHV-1. METHODS: To address these shortcomings, we constructed FHV ∆gIgE-eGFP, FHV ∆TK mCherry, and FHV ∆gIgE/TK eGFP-mCherry deletion mutants (ΔgI/gE, ΔTK, and ΔgIgE/TK, respectively) using the clustered regularly interspaced palindromic repeats (CRISPR)/CRISP-associated protein 9 (Cas9) system (CRISPR/Cas9), which showed safety and immunogenicity in vitro. We evaluated the safety and efficacy of the deletion mutants administered with intranasal (IN) and IN + subcutaneous (SC) vaccination protocols. Cats in the vaccination group were vaccinated twice at a 4-week interval, and all cats were challenged with infection 3 weeks after the last vaccination. The cats were assessed for clinical signs, nasal shedding, and virus-neutralizing antibodies (VN), and with postmortem histological testing. RESULTS: Vaccination with the gI/gE-deleted and gI/gE/TK-deleted mutants was safe and resulted in significantly lower clinical disease scores, fewer pathological changes, and less nasal virus shedding after infection. All three mutants induced virus-neutralizing antibodies after immunization. CONCLUSIONS: In conclusion, this study demonstrates the advantages of FHV-1 deletion mutants in preventing 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.

Safety and immunogenicity of a TK/ gI/gE gene-deleted feline herpesvirus-1 mutant constructed via CRISPR/Cas9 in feline.

Feline herpesvirus-1 (FHV-1) is the aetiological agent of feline viral rhinotracheitis, which accounts for approximately 50 % of all viral upper respiratory diseases in cats. Commercially available modified live vaccines containing FHV-1 are generally safe and effective, but these FHV-1 vaccines retain full virulence genes and can establish latency and reactivate to cause infectious rhinotracheitis in vaccine recipients, raising safety concerns. To address this shortcoming, we constructed a novel TK/gI/gE -gene-deleted recombinant FHV-1 (WH2020-ΔTK/gI/gE) through CRISPR/Cas9-mediated homologous recombination. The growth kinetics of WH2020-ΔTK/gI/gE were slightly delayed compared to those of the parent strain WH2020. Recombinant FHV-1 had severely impaired pathogenicity in cats. Felines immunized with WH2020-ΔTK/gI/gE produced high levels of gB-specific antibodies, neutralizing antibodies and IFN-ß. Additionally, WH2020-ΔTK/gI/gE provided greater protection against challenge with FHV-1 field strain WH2020 than did the commercial modified live vaccine. After challenge, the cats vaccinated with WH2020-ΔTK/gI/gE showed significantly fewer clinical signs, pathological changes, viral shedding, and viral loads in the lung and trigeminal ganglia than those vaccinated with the commercial vaccine or unvaccinated. Our results suggest that WH2020-ΔTK/gI/gE is a promising candidate as a safer and more efficacious live FHV-1 vaccine, with a decreased risk of vaccine-related complications, and could inform the design of other herpesvirus vaccines.

POSTVACCINAL VIRAL DISEASE PRESENTATION IN TWO LITTERS OF CHEETAHS (ACINONYX JUBATUS).

Protective antibody titers against core vaccines have not been standardized for cheetahs (Acinonyx jubatus) under human care. Vaccine-induced disease has been suspected after administration of modified live virus vaccine (MLVV), but it has not been confirmed as the causative agent. MLVV and killed virus vaccines (KVV) elicit humoral response in cheetahs; however, the use of both vaccines for initial immunization in cheetah cubs <6 months old within the same population has not been reported. The current case series describes viral disease presentation in two cheetah litters after using both vaccines and presents results for serum neutralization titers against feline calicivirus (FCV) and feline herpesvirus-1 (FHV-1) and hemagglutination inhibition titers against feline panleukopenia virus (FPV). For Litter 1, MLVV was administered at 6 and 9 wk old. On week 11, one male developed ocular, oral, and dermal lesions. Viral isolation recovered FCV. Because of suspected vaccine-induced FCV, KVV was administered on weeks 13 and 16. Litter 2 was vaccinated with KVV via the same vaccination schedule. Fifty-three days after the last booster, two cubs presented with ocular, respiratory, and oral clinical signs; both were PCR positive for FHV-1. Serology reported a better anamnestic response and protective titers against FCV and FPV with the protocol used with Litter 1. In Litter 2, FCV and FHV-1 titer measurement failed in three of four cubs, limiting comparison of titers between litters. In spite of limited measurements, absence of a statistical evaluation, and presence of infection, serology showed a better humoral response when MLVV was used.

Characteristics and epidemiological investigation of equid herpesvirus 8 in donkeys in Shandong, China.

Equid herpesvirus 8 (EHV-8), also known as asinine herpesvirus type 3 (AHV-3), can cause severe respiratory disease, abortion in mares, and neurological disorders. There is limited information on the prevalence of EHV-8 in donkeys in China. In this study, we investigated EHV-8 infection in donkeys using PCR, resulting in the identification of a field strain, termed EHV-8 SD2020113, which was isolated using RK-13 cells and characterized by high-throughput sequencing and transmission electron microscopy. Our data indicated that 38.7% (457/1180) of donkeys showed the presence of EHV-8 in blood samples. Analysis of the ORF70 gene showed the highest similarity (99.8-99.9% identity) to EHV-8 IR/2015/40 (MF431614.1) and SDLC66 (MW816102), and, in phylogenetic analysis, it clustered with EHV-8 SDLC66 from China. The findings of this study indicate that EHV-8 is likely to represent a threat to the donkey industry, and breeders and veterinarians who care for donkey farms should be aware of this.

Development of immortalized feline respiratory epithelial cells in an air-liquid-interface culture system for feline herpesvirus-1 study.

Feline herpesvirus-1 (FHV-1) is responsible for approximately 50% of diagnosed viral upper respiratory tract disease in cats. The virus infects and replicates in the epithelial cells located in upper respiratory tract. Commercial vaccines do not protect cats from the infection itself or development of latency. Previously, our lab developed a cell culture model using primary feline respiratory epithelial cells (pFRECs) to study respiratory innate immunity to FHV-1 and FHV-1 deletion mutants. However, the numbers of pFRECs that can be obtained per cat is limited. To improve the usage of respiratory epithelial 3D cultures in FHV-1 research, the present study immortalized feline respiratory epithelial cells (iFRECs) and characterized them morphologically and immunologically and evaluated the response to FHV-1 infection. Immortalization was achieved by transduction with Lenti-SV40T and Lenti-HPV E6/E7. Immortalized FRECs could be successfully subcultured for >20 passages, with positive gene expression of SV40T and HPV E6/E7. Immortalized FRECs expressed similar innate immunity-associated genes compared to pFRECs, including genes of Toll-like receptors (TLR1-9), interferon induced genes (OAS1, OAS3, IFI44, IFITM1, IFIT1), chemokines (CCL2, CCL3, CXCL8), pro-inflammatory and regulatory cytokines (IL-6, IL-4, IL-5, IL-12, and IL-18), and antimicrobials (DEFß10, DEFß4B). Finally, FHV-1 inoculation resulted in characteristic cytopathic effects starting at 24 hpi, with more than 80% cells detached and lysed by 72 hpi. Overall FHV-1 growth kinetics in iFRECs resembled the kinetics observed in pFRECs. In conclusion, we demonstrated that iFRECs are a useful tool to study feline respiratory disease including but not limited to FHV-1.

Evaluation of compounded cidofovir, famciclovir, and ganciclovir for the treatment of feline herpesvirus ocular surface disease in shelter-housed cats.

OBJECTIVE: To assess the efficacy of compounded cidofovir, famciclovir, and ganciclovir for the treatment of feline herpesvirus type 1 (FHV-1) ocular surface disease. ANIMALS STUDIED: 132 shelter-housed cats qPCR positive for FHV-1. PROCEDURES: A masked placebo-controlled study design was utilized. Animals were enrolled in one of four treatment groups: topical ocular placebo + oral placebo (n = 32), compounded cidofovir 0.5% ophthalmic solution + oral placebo (n = 32), compounded famciclovir oral solution (90 mg/kg) + topical ocular placebo (n = 32), and compounded ganciclovir 0.15% ophthalmic solution + oral placebo (n = 36). Cats were treated with each medication twice daily for 7 days and were evaluated on Day 1 and Day 8 using an ocular scoring system, body weight, and qPCR for FHV-1 viral load. RESULTS: Cidofovir significantly decreased viral load from Day 1 to Day 8 compared with placebo (p = .024). Neither famciclovir nor ganciclovir decreased viral load compared with placebo (p = .14, p = .41). There was no significant improvement of ocular scores for any drug group compared with placebo (p = .62). In all groups, 65%-75% of cats improved from Day 1 to Day 8. Juvenile cats had a significant increase in weight gain compared with placebo for cidofovir (p = .025) and ganciclovir (p = .023). All corneal ulcers in placebo animals failed to heal whereas 77% of ulcers in antiviral group animals healed. CONCLUSIONS: Topical ophthalmic cidofovir significantly decreased ocular FHV-1 viral shedding and increased weight gain in juvenile cats. Ganciclovir increased weight gain in juvenile cats. Compounded famciclovir demonstrated limited efficacy for the treatment of FHV-1 ocular surface disease in shelter-housed cats.

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.

Effect of repeated administration of a parenteral feline herpesvirus-1, calicivirus, and panleukopenia virus vaccine on select clinicopathologic, immunological, renal histologic, and immunohistochemical parameters in healthy adult cats.

OBJECTIVE: To assess whether hyperinoculation of cats with a feline herpesvirus-1, calicivirus, and panleukopenia virus (FVRCP) vaccine could be used as a model to study interstitial nephritis and to assess humoral and cell-mediated immune responses toward vaccinal α-enolase. ANIMALS: 6 healthy young adult purpose-bred research cats. PROCEDURES: Baseline renal cortical biopsies, whole blood, serum, and urine were collected prior to administration of a commercial FVRCP parenteral vaccine. Vaccine hyperinoculation was defined as a total of 8 vaccinations given at 2-week intervals over a 14-week period. Blood samples were collected immediately prior to each vaccination, and a second renal biopsy was performed 2 weeks after hyperinoculation (week 16). Renal histopathology, renal α-enolase immunohistochemistry, and assays to detect humoral and cell-mediated immune reactions against Crandell-Rees feline kidney (CRFK) cell lysates and α-enolase were performed. An α-enolase immunoreactivity score for renal tubules and glomeruli based on signal intensity was determined by a blinded pathologist. RESULTS: Hyperinoculation with the vaccine was not associated with clinicopathologic evidence of renal dysfunction, and interstitial nephritis was not recognized by light microscopy in the time studied. The mean serum absorbance values for antibodies against CRFK antigen and α-enolase were significantly (P < 0.001) higher at weeks 4, 8, and 16 versus week 0. Renal tubular and glomerular α-enolase immunoreactivity scores were higher at week 16 compared to baseline. CLINICAL RELEVANCE: Findings suggested that systemic immunological reactions occurred and renal tissues were affected by vaccine hyperinoculation; however, short-term FVRCP vaccine hyperinoculation cannot be used to study interstitial nephritis in cats.