An RNA replicon system to investigate promising inhibitors of feline coronavirus.

Feline infectious peritonitis (FIP) is a fatal feline disease, caused by a feline coronavirus (FCoV), namely feline infectious peritonitis virus (FIPV). We produced a baby hamster kidney 21 (BHK) cell line expressing a serotype I FCoV replicon RNA with a green fluorescent protein (GFP) reporter gene (BHK-F-Rep) and used it as an in vitro screening system to test different antiviral compounds. Two inhibitors of the FCoV main protease (Mpro), namely GC376 and Nirmatrelvir, as well as the nucleoside analog Remdesivir proved to be effective in inhibiting the replicon system. Different combinations of these compounds also proved to be potent inhibitors, having an additive effect when combined. Remdesivir, GC376, and Nirmatrelvir all have a 50% cytotoxic concentration (CC50) more than 200 times higher than their half-maximal inhibitory concentrations (IC50), making them important candidates for future in vivo studies as well as clinically implemented drug candidates. In addition, results were acquired with a virus infection system, where Felis catus whole fetus 4 (Fcwf-4) cells were infected with a previously described recombinant GFP-expressing FIPV (based on the laboratory-adapted serotype I FIPV strain Black) and treated with the most promising compounds. Results acquired with the replicon system were comparable to the results acquired with the virus infection system, demonstrating that we successfully implemented the FCoV replicon system for antiviral screening. We expect that this system will greatly facilitate future screens for anti-FIPV compounds and provide a non-infectious system to study and evaluate drug-resistant mutations that may emerge in the FIPV genome.IMPORTANCEFIPV is of great significance in the cat population around the world, causing 0.3%-1.4% of feline deaths in veterinary practices (2). As there are neither effective preventive measures nor approved treatment options available, there is an urgent need to identify antiviral drugs against FIPV. Our FCoV replicon system provides a valuable tool for drug discovery in vitro. Due to the lack of cell culture systems for serotype I FCoVs (the serotype most prevalent in the feline population) (2), a different system is needed to study these viruses. A viral replicon system is a valuable tool for studying FCoVs. Overall, our results demonstrate the utility of the serotype I feline coronavirus replicon system for antiviral screening as well as to study this virus in general. We propose several compounds representing promising candidates for future clinical trials and ultimately with the potential to save cats suffering from FIP.

FeLIX is a restriction factor for mammalian retrovirus infection.

Endogenous retroviruses (ERVs) are remnants of ancestral viral infections. Feline leukemia virus (FeLV) is an exogenous and endogenous retrovirus in domestic cats. It is classified into several subgroups (A, B, C, D, E, and T) based on viral receptor interference properties or receptor usage. ERV-derived molecules benefit animals, conferring resistance to infectious diseases. However, the soluble protein encoded by the defective envelope (env) gene of endogenous FeLV (enFeLV) functions as a co-factor in FeLV subgroup T infections. Therefore, whether the gene emerged to facilitate viral infection is unclear. Based on the properties of ERV-derived molecules, we hypothesized that the defective env genes possess antiviral activity that would be advantageous to the host because FeLV subgroup B (FeLV-B), a recombinant virus derived from enFeLV env, is restricted to viral transmission among domestic cats. When soluble truncated Env proteins from enFeLV were tested for their inhibitory effects against enFeLV and FeLV-B, they inhibited viral infection. Notably, this antiviral machinery was extended to infection with the Gibbon ape leukemia virus, Koala retrovirus A, and Hervey pteropid gammaretrovirus. Although these viruses used feline phosphate transporter 1 (fePit1) and phosphate transporter 2 as receptors, the inhibitory mechanism involved competitive receptor binding in a fePit1-dependent manner. The shift in receptor usage might have occurred to avoid the inhibitory effect. Overall, these findings highlight the possible emergence of soluble truncated Env proteins from enFeLV as a restriction factor against retroviral infection and will help in developing host immunity and antiviral defense by controlling retroviral spread.IMPORTANCERetroviruses are unique in using reverse transcriptase to convert RNA genomes into DNA, infecting germ cells, and transmitting to offspring. Numerous ancient retroviral sequences are known as endogenous retroviruses (ERVs). The soluble Env protein derived from ERVs functions as a co-factor that assists in FeLV-T infection. However, herein, we show that the soluble Env protein exhibits antiviral activity and provides resistance to mammalian retrovirus infection through competitive receptor binding. In particular, this finding may explain why FeLV-B transmission is not observed among domestic cats. ERV-derived molecules can benefit animals in an evolutionary arms race, highlighting the double-edged-sword nature of ERVs.

Multiple recombination events between endogenous retroviral elements and feline leukemia virus.

Feline leukemia virus (FeLV) is an exogenous retrovirus that causes malignant hematopoietic disorders in domestic cats, and its virulence may be closely associated with viral sequences. FeLV is classified into several subgroups, including A, B, C, D, E, and T, based on viral receptor interference properties or receptor usage. However, the transmission manner and disease specificity of the recombinant viruses FeLV-D and FeLV-B remain unclear. The aim of this study was to understand recombination events between exogenous and endogenous retroviruses within a host and elucidate the emergence and transmission of recombinant viruses. We observed multiple recombination events involving endogenous retroviruses (ERVs) in FeLV from a family of domestic cats kept in one house; two of these cats (ON-T and ON-C) presented with lymphoma and leukemia, respectively. Clonal integration of FeLV-D was observed in the ON-T case, suggesting an association with FeLV-D pathogenesis. Notably, the receptor usage of FeLV-B observed in ON-T was mediated by feline Pit1 and feline Pit2, whereas only feline Pit1 was used in ON-C. Furthermore, XR-FeLV, a recombinant FeLV containing an unrelated sequence referred to the X-region, which is homologous to a portion of the 5'-leader sequence of Felis catus endogenous gammaretrovirus 4 (FcERV-gamma4), was isolated. Genetic analysis suggested that most recombinant viruses occurred de novo; however, the possibility of FeLV-B transmission was also recognized in the family. This study demonstrated the occurrence of multiple recombination events between exogenous and endogenous retroviruses in domestic cats, highlighting the contribution of ERVs to pathogenic recombinant viruses.IMPORTANCEFeline leukemia virus subgroup A (FeLV-A) is primarily transmitted among cats. During viral transmission, genetic changes in the viral genome lead to the emergence of novel FeLV subgroups or variants with altered virulence. We isolated three FeLV subgroups (A, B, and D) and XR-FeLV from two cats and identified multiple recombination events in feline endogenous retroviruses (ERVs), such as enFeLV, ERV-DC, and FcERV-gamma4, which are present in the cat genome. This study highlights the pathogenic contribution of ERVs in the emergence of FeLV-B, FeLV-D, and XR-FeLV in a feline population.

Novel mutation N588 residue in the NS1 protein of feline parvovirus greatly augments viral replication.

Feline parvovirus (FPV) infection is highly fatal in felines. NS1, which is a key nonstructural protein of FPV, can inhibit host innate immunity and promote viral replication, which is the main reason for the severe pathogenicity of FPV. However, the mechanism by which the NS1 protein disrupts host immunity and regulates viral replication is still unclear. Here, we identified an FPV M1 strain that is regulated by the NS1 protein and has more pronounced suppression of innate immunity, resulting in robust replication. We found that the neutralization titer of the FPV M1 strain was significantly lower than that of the other strains. Moreover, FPV M1 had powerful replication ability, and the FPV M1-NS1 protein had heightened efficacy in repressing interferon-stimulated genes (ISGs) expression. Subsequently, we constructed an FPV reverse genetic system, which confirmed that the N588 residue of FPV M1-NS1 protein is a key amino acid that bolsters viral proliferation. Recombinant virus containing N588 also had stronger ability to inhibit ISGs, and lower ISGs levels promoted viral replication and reduced the neutralization titer of the positive control serum. Finally, we confirmed that the difference in viral replication was abolished in type I IFN receptor knockout cell lines. In conclusion, our results demonstrate that the N588 residue of the NS1 protein is a critical amino acid that promotes viral proliferation by increasing the inhibition of ISGs expression. These insights provide a reference for studying the relationship between parvovirus-mediated inhibition of host innate immunity and viral replication while facilitating improved FPV vaccine production.IMPORTANCEFPV infection is a viral infectious disease with the highest mortality rate in felines. A universal feature of parvovirus is its ability to inhibit host innate immunity, and its ability to suppress innate immunity is mainly accomplished by the NS1 protein. In the present study, FPV was used as a viral model to explore the mechanism by which the NS1 protein inhibits innate immunity and regulates viral replication. Studies have shown that the FPV-NS1 protein containing the N588 residue strongly inhibits the expression of host ISGs, thereby increasing the viral proliferation titer. In addition, the presence of the N588 residue can increase the proliferation titer of the strain 5- to 10-fold without affecting its virulence and immunogenicity. In conclusion, our findings provide new insights and guidance for studying the mechanisms by which parvoviruses suppress innate immunity and for developing high-yielding FPV vaccines.

Endogenous feline leukemia virus long terminal repeat integration site diversity is highly variable in related and unrelated domestic cats.

Endogenous retroviruses (ERV) are indicators of vertebrate evolutionary history and play important roles as homeostatic regulators. ERV long terminal repeat (LTR) elements may act as cis-activating promoters or trans-activating enhancer elements modifying gene transcription distant from LTR insertion sites. We previously documented that endogenous feline leukemia virus (FeLV)-LTR copy number variation in individual cats tracks inversely with susceptibility to virulent FeLV disease. To evaluate FeLV-LTR insertion characteristics, we assessed enFeLV-LTR integration site diversity in 20 cats from three genetically distinct populations using a baited linker-mediated PCR approach. We documented 765 individual integration sites unequally represented among individuals. Only three LTR integration sites were shared among all individuals, while 412 sites were unique to a single individual. When primary fibroblast cultures were challenged with exogenous FeLV, we found significantly increased expression of both exogenous and endogenous FeLV orthologs, supporting previous findings of potential exFeLV-enFeLV interactions; however, viral challenge did not elicit transcriptional changes in genes associated with the vast majority of integration sites. This study assesses FeLV-LTR integration sites in individual animals, providing unique transposome genotypes. Further, we document substantial individual variation in LTR integration site locations, even in a highly inbred population, and provide a framework for understanding potential endogenous retroviral element position influence on host gene transcription.

Drug screening identified that handelin inhibits feline calicivirus infection by inhibiting HSP70 expression in vitro.

Feline calicivirus (FCV) is considered one of the major pathogens of cats worldwide and causes upper respiratory tract disease in all cats. In some cats, infection is by a highly virulent strain of FCV (vs.-FCV), which can cause severe and fatal systemic disease symptoms. At present, few antiviral drugs are approved for clinical treatment against FCV. Therefore, there is an imminent need for effective FCV antiviral agents. Here, we used observed a cytopathic effect (CPE) assay to screen 1746 traditional Chinese medicine monomer compounds and found one that can effectively inhibit FCV replication, namely, handelin, with an effective concentration (EC50) value of approximately 2.5 µM. Further study showed that handelin inhibits FCV replication via interference with heat shock protein 70 (HSP70), which is a crucial host factor and plays a positive role in regulating viral replication. Moreover, handelin and HSP70 inhibitors have broad-spectrum antiviral activity. These findings indicate that handelin is a potential candidate for the treatment of FCV infection and that HSP70 may be an important drug target.

Efficacy of three EPA-registered antimicrobials and steam against two human norovirus surrogates on nylon carpets with two backing types.

Carpet cleaning guidelines currently do not include the use of an antimicrobial, except after a bodily fluid event. To address this gap, we compared the efficacy of three antimicrobials-two hydrogen peroxide-based (H2O2) products (A and B) and one chlorine-based product (C)-and a steam treatment against two norovirus surrogates, specifically feline calicivirus (FCV) and Tulane virus (TuV). These tests were performed on nylon carpets with either water-permeable or waterproof backing types. The effect of repeated antimicrobial use on carpet properties was also evaluated. For a carpet with water-permeable backing, products A, B, and C achieved a 0.8, 3.1, and 0.9 log10 PFU/coupon reduction of FCV and 0.3, 2.5, and 0.4 log10 TCID50/coupon reduction of TuV, respectively, following a 30 min contact time. For carpet with waterproof backing, only product B achieved a 5.0 log10 PFU/coupon reduction of FCV and >3.0 log10 TCID50/coupon reduction of TuV, whereas products A and C achieved a 2.4 and 1.6 log10 PFU/coupon reduction of FCV and a 1.2 and 1.2 log10 TCID50/coupon reduction of TuV, respectively. Steam treatment achieved a ≥ 5.2 log10 PFU/coupon reduction of FCV and a > 3.2 log10 TCID50/coupon reduction of TuV in 15 seconds on the carpet with both backing types. The repeated use of products A and B decreased the tensile strength of the carpet backing, while use of product B resulted in cracks on carpet fibers. Overall, steam treatment for 15 seconds was efficacious on both carpet types, but only product B achieved efficacy after a 30-minute exposure on the carpet with waterproof backing.IMPORTANCECarpets are common in long-term care facilities, despite its potential as a vehicle for transmission of agents associated with healthcare-associated infections, including human norovirus (NoV). Presently, our understanding of carpet disinfection is limited; hence, there are no commercial antimicrobials against norovirus available for use on carpets. Our findings showed that steam treatment, which minimally affected the properties of carpet fibers and backing, was more efficacious against human norovirus surrogates on carpets compared to the three chemical antimicrobials tested. Additionally, the two surrogates were more sensitive to chemical antimicrobials on the carpet with waterproof backing compared to carpets with water-permeable backing. These findings can inform development of antimicrobials for use on carpets contaminated with human norovirus.

Feline hypertrophic cardiomyopathy: Does the microRNA-mRNA regulatory network contribute to heart sarcomeric protein remodelling?

Feline primary hypertrophic cardiomyopathy (HCM) is an intrinsic myocardial disease characterized by concentric hypertrophy of the left ventricle. In the present study, we investigated the microRNA-mRNA regulatory network in feline myocardial tissue affected by primary (HCMI) and secondary HCM (HCMII). MRNA expression levels of sarcomeric genes, including, TNNT2, TNNI3, MYH7, MYBPC3, TPM1 and ACTC1 were assessed in the FFPE myocardial tissues. FFPE tissues from healthy cats were sequenced by the NGS, to explore, in the entire non-deposited miRNome, the expression level of microRNAs targeting the complementary sequences of selected sarcomeric mRNAs. The sarcomeric genes TNNT2, MYH7, MYBPC3 and TPM1 showed a statistically significant upregulation in HCMI compared to HCMII (p < .01), except ACTC1 which was downregulated (p < .01); TNNI3 showed no statistically significant difference. In HCMII miR-122-5p, miR-338-3p, miR-484, miR-370-3p, miR-92b-3p, miR-375 and miR-370-3p showed a significant upregulation (p < .01) compared to control. The exception was miR-30a-5p which showed downregulation. Worthy of note is the 4-fold higher expression of miR-370-3p, a key regulator of MYBPC3, in HMCI compared to HMCII. This research does not solve the aetiological mystery of HCM, but it may help to find a way to help diagnose and define the prognosis of HCM in cats.

Human transmission and outbreaks of feline-like G6 rotavirus revealed with whole-genome analysis of G6P[9] feline rotavirus.

Group A rotaviruses (RVAs) are generally highly species-specific; however, some strains infect across species. Feline RVAs sporadically infect humans, causing gastroenteritis. In 2012 and 2013, rectal swab samples were collected from 61 asymptomatic shelter cats at a public health center in Mie Prefecture, Japan, to investigate the presence of RVA and any association with human infections. The analysis identified G6P[9] strains in three cats and G3P[9] strains in two cats, although no feline RVA sequence data were available for the former. A whole-genome analysis of these G6P[9] strains identified the genotype constellation G6-P[9]-I2-R2-C2-M2-A3-N2-T3-E3-H3. The nucleotide identity among these G6P[9] strains exceeded 99.5% across all 11 gene segments, indicating the circulation of this G6P[9] strain among cats. Notably, strain RVA/Human-wt/JPN/KF17/2010/G6P[9], previously detected in a 3-year-old child with gastroenteritis, shares high nucleotide identity (>98%) with Mie20120017f, the representative G6P[9] strain in this study, across all 11 gene segments, confirming feline RVA infection and symptomatic presentation in this child. The VP7 gene of strain Mie20120017f also shares high nucleotide identity with other sporadically reported G6 RVA strains in humans. This suggests that feline-origin G6 strains as the probable source of these sporadic G6 RVA strains causing gastroenteritis in humans globally. Moreover, a feline-like human G6P[8] strain circulating in Brazil in 2022 was identified, emphasizing the importance of ongoing surveillance to monitor potential global human outbreaks of RVA.

Preparation of a novel type I feline coronavirus virus-like particle vaccine and its immunogenicity in mice and cats.

Feline coronavirus (FCoV) infection is a leading cause of death in cats. In this study, we produced FCoV-I virus-like particles (VLPs) containing E, M, N, and S proteins using a baculovirus expression system and mixed VLPs with the adjuvants MF59 and CpG 55.2 to prepare an VLP/MF59/CpG vaccine. After immunization of mice with the vaccine, IgG specific antibodies titers against S and N proteins increased to 1:12,800, and IFN-γ+ and IL-4+ splenocytes were significantly increased. Following immunization of FCoV-negative cats, the S protein antibodies in immunized cats (5/5) increased significantly, with a peak of 1:12,800. Notably, after booster vaccination in FCoV-positive cats, a significant reduction in viral load was observed in the feces of partial cats (4/5), and the FCoV-I negative conversion was found in two immunized cats (2/5). Therefore, the VLP/MF59/CpG vaccine is a promising candidate vaccine to prevent the FCoV infection.

The polymicrobial nature of the oral cavity and claws of cats diagnosed by mass spectrometry and next-generation sequencing.

Close contact between cats and humans increases the risk of transmission of zoonotic pathogens, through bites and scratches due to the complexity of microorganisms in the oral and nail microbiotas of felines. This study investigated the presence of bacteria and fungi in the oral cavity and claws of 100 apparently healthy cats using conventional and selective microbiological culture media, and next-generation sequencing (NGS) and mass spectrometry (MALDI-TOF MS). Furthermore, antimicrobial susceptibility testing of bacteria isolates was performed by disc diffusion method. In total, 671 bacteria and 33 yeasts were identified by MALDI-TOF MS. Neisseria animaloris (10.8 %), Staphylococcus felis (8.5 %), and Pasteurella multocida (7 %) were the most prevalent bacteria in oral cavity samples (n = 343), while the most common yeast (n = 19) was Candida albicans (68.4 %). Staphylococcus pettenkoferi (13.4 %), Staphylococcus felis (6.4 %), and Staphylococcus simulans (5.8 %) were the prevalent bacteria identified in the claw samples (n = 328), while Rhodotorula mucilaginosa (57.2 %) was the most common yeast (n = 14). NGS predominantly identified the genera Moraxella, Neisseria, Pasteurella, and Fusobacterium in oral cavity samples, whereas enterobacteria and staphylococci were prevalent in nail bed samples. In addition, the genera Capnocytophaga and Bartonella were identified, which have been described in serious human infections secondary to feline aggressions. Levofloxacin, marbofloxacin, and amoxicillin/clavulanic acid were the most effective drugs against the main groups of bacteria identified. Multidrug resistance was observed in 17 % of the bacterial isolates. Furthermore, three staphylococci harboring the methicillin resistance gene mecA were identified. We highlight the complexity of microorganisms inhabiting the oral/claw microbiotas of cats, the high resistance rate of the isolates to conventional antimicrobial agents, and the zoonotic risk of aggressions caused by bites and scratches from domestic cats.

Molecular epidemiology and phylogenetic analysis of feline calicivirus in Kunshan, China.

Feline calicivirus (FCV) is a highly contagious virus in cats, which typically causes respiratory tract and oral infections. Despite vaccination against FCV being a regular practice in China, new FCV cases still occur. Antigenic diversity of FCV hinders the effective control by vaccination. This is first report which aims to investigate the molecular epidemiology and molecular characteristics of FCV in Kunshan, China. The nasopharyngeal swabs were collected from cats showing variable clinical signs from different animal clinics in Kunshan from 2022 to 2023. Preliminary detection and sequencing of the FCV capsid gene were performed to study genetic diversity and evolutionary characteristics. FCV-RNA was identified in 52 (26%) of the samples using RT-PCR. A significant association was found between FCV-positive detection rate, age, gender, vaccination status and living environment, while a non-significant association was found with breed of cats. Nucleotide analysis revealed two genotypes, GI and GII. GII predominated in Kunshan, with diverse strains and amino acid variations potentially affecting vaccination efficacy and FCV detection. Notably, analysis pinpointed certain strains' association with FCV-virulent systemic disease pathotypes. This investigation sheds light on FCV dynamics, which may aid in developing better prevention strategies and future vaccine designs against circulating FCV genotypes.

Prevalence of natural feline coronavirus infection in domestic cats in Fujian, China.

Only few studies have investigated the prevalence of feline coronavirus (FCoV) infection in domestic cats in Fujian, China. This is the first study to report the prevalence rate of FCoV infection in domestic cats in Fujian, China, and to analyse the epidemiological characteristics of FCoV infection in the region. A total of 112 cat faecal samples were collected from animal hospitals and catteries in the Fujian Province. RNA was extracted from faecal material for reverse transcription polymerase chain reaction (RT-PCR). The prevalence rate of FCoV infection was determined, and its epidemiological risk factors were analysed. The overall prevalence of FCoV infection in the cats, was 67.9%. We did not observe a significant association between the age, sex, or breed of the cats included in the study and the prevalence rate of the viral infection. Phylogenetic analysis showed that the four strains from Fujian were all type I FCoV. This is the first study to analyse the prevalence and epidemiological characteristics of FCoV infection in domestic cats in Fujian, China, using faecal samples. The results of this study provide preliminary data regarding the prevalence of FCoV infection in the Fujian Province for epidemiological studies on FCoV in China and worldwide. Future studies should perform systematic and comprehensive epidemiological investigations to determine the prevalence of FCoV infection in the region.

First report of molecular epidemiology and phylogenetic characteristics of feline herpesvirus (FHV-1) from naturally infected cats in Kunshan, China.

BACKGROUND: Feline herpesvirus type 1 (FHV-1) is a life threatening highly contagious virus in cats and typically causes upper respiratory tract infections as well as conjunctival and corneal ulcers. Genetic variability could alter the severity of diseases and clinical signs. Despite regular vaccine practices against FHV-1 in China, new FHV-1 cases still commonly occur. The genetic and phylogenetic characteristics of FHV-1 in Kunshan city of China has not been studied yet. Therefore, this study was planned to investigate the prevalence, molecular characteristics of circulating strains, and phylogenetic analyses of FHV-1. This is the first report of molecular epidemiology and phylogenetic characteristics of FHV-1 from naturally infected cats in Kunshan, China. METHODS: The occulo-nasal swabs were collected from diseased cats showing respiratory distress, conjunctivitis, and corneal ulcers at different veterinary clinics in Kunshan from 2022 to 2023. Clinical data and general information were recorded. Swab samples were processed for preliminary detection of FHV-1. Thymidine kinase (TK), glycoprotein B (gB) and glycoprotein D (gD) genes were sequenced and analyzed to investigate genetic diversity and evolution of FHV-1. RESULTS: The FHV-1 genome was detected in 43 (43/200, 21.5%) samples using RT-PCR targeting the TK gene. Statistical analysis showed a significant correlation between age, vaccination status and living environment (p < 0.05) with FHV-1 positivity, while a non-significant correlation was observed for FHV-1 positivity and sex of cats (p > 0.05). Additionally, eight FHV-1 positive cats were co-infected with feline calicivirus (8/43,18.6%). FHV-1 identified in the present study was confirmed as FHV-1 based on phylogenetic analyses. The sequence analyses revealed that 43 FHV-1 strains identified in the present study did not differ much with reference strains within China and worldwide. A nucleotide homology of 99-100% was determined among gB, TK and gD genes nucleotide sequences when compared with standard strain C-27 and vaccine strains. Amino acid analysis showed some amino acid substitutions in TK, gB and gD protein sequences. A potential N-linked glycosylation site was observed in all TK protein sequences. Phylogenetic analyses revealed minor variations and short evolutionary distance among FHV-1 strains detected in this study. CONCLUSIONS: Our findings indicate that genomes of 43 FHV-1 strains are highly homogenous and antigenically similar, and the degree of variation in major envelope proteins between strains is low. This study demonstrated some useful data about prevalence, genetic characteristics, and evolution of FHV-1 in Kunshan, which may aid in future vaccine development.

Molecular epidemiological investigation of Cryptococcus spp. Isolated from cats in Japan using multi-locus sequence typing.

Cryptococcosis is an important fungal infection for both humans and cats, but molecular epidemiological studies on strains isolated from cats are limited. We conducted multi-locus sequence typing (MLST) analysis and antifungal susceptibility testing of 14 Cryptococcus spp. strains from domestic cats in Japan and one strain isolated from a cat in Singapore. All 14 strains from domestic cats in Japan were identified as Cryptococcus neoformans molecular type VNI. The sequence types (STs) included eight cases of ST5, five cases of ST31, and one novel ST. VNI ST5 is the most frequently isolated strain in Japanese patients as well, while there are no records of VNI ST31 being isolated from Japanese patients. The Singaporean cat strain was identified as C. gattii VGIIb (Cryptococcus deuterogattii), ST7. We compared these results with strains previously reported to have been isolated from cats. This comparison suggested that molecular types of Cryptococcus spp. isolated from cats may differ depending on the country. In the antifungal susceptibility testing of C. neoformans, one strain each exceeded the epidemiological cutoff value (ECV) for amphotericin B and 5-fluorocytosine, while two strains exceeded the ECV for fluconazole. This study reveals the molecular epidemiology of Cryptococcus spp. isolated from cats with cryptococcosis in Japan. It suggests that investigating Cryptococcus spp. carried by cats, which share close living environments with humans, may contribute to the health of both cats and human populations.

Cryptococcosis is an important fungal disease in both humans and cats. We genotyped strains isolated from cats with cryptococcosis in Japan. Our findings revealed that the most common genotype infecting both cats and humans in Japan is identical.

Glycine supplementation can partially restore oxidative stress-associated glutathione deficiency in ageing cats.

Intracellular levels of glutathione, the major mammalian antioxidant, are reported to decline with age in several species. To understand whether ageing affects circulating glutathione levels in cats, blood was sampled from two age groups, < 3 years and > 9 years. Further, to determine whether dietary supplementation with glutathione precursor glycine (GLY) affects glutathione concentrations in senior cats (> 8 years), a series of free GLY inclusion level dry diets were fed. Subsequently, a 16-week GLY feeding study was conducted in senior cats (> 7 years), measuring glutathione, and markers of oxidative stress. Whole blood and erythrocyte total, oxidised and reduced glutathione levels were significantly decreased in senior cats, compared with their younger counterparts (P ≤ 0·02). The inclusion level study identified 1·5 % free GLY for the subsequent dry diet feeding study. Significant increases in erythrocyte total and reduced glutathione were observed between senior cats fed supplemented and control diets at 4 weeks (P ≤ 0·03; maximum difference of 1·23 µM). Oxidative stress markers were also significantly different between groups at 8 (P = 0·004; difference of 0·68 nG/ml in 8-hydroxy-2'-deoxyguanosine) and 12 weeks (P ≤ 0·049; maximum difference of 0·62 nG/mG Cr in F2-isoprostane PGF2α). Senior cats have lower circulating glutathione levels compared with younger cats. Feeding senior cats a complete and balanced dry diet supplemented with 1·5 % free GLY for 12 weeks elevated initial erythrocyte glutathione and altered markers of oxidative stress. Dietary supplementation with free GLY provides a potential opportunity to restore age-associated reduction in glutathione in cats.

High starch intake favours body weight control in neutered and spayed cats living in homes fed ad libitum.

Different starch-to-protein ratios were compared among neutered and spayed domiciled cats. Male and female obese and non-obese cats were fed kibble diets ad libitum for 4 months high in starch (HS (38 % crude protein (CP)): starch 32 %, protein 38 %; DM basis) or high in protein (HP (55 % CP): starch 19 %, protein 55 %) but similar in energy and fat in a crossover design. Physical activity was evaluated using an accelerometer, and body composition (BC), energy expenditure (EE) and water turnover (WT) using the doubly labelled water method. Results were compared in a 2 diet × 2 sex × 2 body condition factorial arrangement. Cats fed the HS (38 % CP) diet maintained a constant body weight, but lean mass (LM) tended to be reduced in female obese but to be increased in male non-obese (P < 0·08) and increased in female non-obese cats (P = 0·01). The HP (55 % CP) diet induced an increase in cat body weight and LM (P < 0·05) without altering BC proportion. EE tended to be higher in males (351 (se 8) kJ/kg0·67/d) than females (330 (se 8) kJ/kg0·67/d; P = 0·06), was unaffected by diet or BC, decreased as age increased (R 2 0·44; P < 0·01) and increased as physical activity increased (R 2 0·58; P < 0·01). WT was higher for the HP (55 % CP) diet (P < 0·01) and increased with EE (R 2 0·65; P < 0·01). The HS (38 % CP) diet favoured body weight control during 4 months of ad libitum feeding. Caution is necessary to balance protein in diets of female obese cats over 5 years, as they may have low energy and food intake, with LM loss.

Conformational analysis of a new peptide derived from feline immunodeficiency virus gp36 in SDS micelles: An NMR-MD based investigation.

Feline immunodeficiency virus (FIV) shares structural similarities with human immunodeficiency virus (HIV): the surface glycoprotein gp36 corresponds to the HIV gp41, which drives virus-host cell interactions and is targeted by the peptide entry inhibitor enfuvirtide. Following a similar drug design strategy for the development of an anti-FIV therapy, the present study investigates 627-646gp36 NHR, a peptide sequence derived from a region of gp36 that was previously found to interfere with the antiviral activity of the peptide C8, which instead derives from the gp36 MPER. CD, NMR, and MD simulations were employed to probe the conformational characteristics of 627-646gp36 NHR in the membrane-mimicking environment of SDS micelles. Our data show that 627-646gp36 NHR is characterized by three dynamic helix structures. MD simulations involving 627-646gp36 NHR, C8, and a larger protein, including the CHR and MPER regions, suggest that the interaction of C8 with the MPER region, the origin of the antiviral activity of C8, is disfavored in the presence of 627-646gp36 NHR in the simulation. This evidence can be useful for interpreting the molecular mechanism that leads to interference with the activity of C8, providing information on the folding/unfolding mechanism of the viral glycoprotein to design new strategies to inhibit viral entry.

Drug release profile of a novel exenatide long-term drug delivery system (OKV-119) administered to cats.

Beneficial weight-loss properties of glucagon-like peptide-1 receptor agonists (GLP-1RA) in obese people, with corresponding improvements in cardiometabolic risk factors, are well established. OKV-119 is an investigational drug delivery system that is being developed for the long-term delivery of the GLP-1RA exenatide to feline patients. The purpose of this study was to evaluate the drug release characteristics of subcutaneous OKV-119 implants configured to release exenatide for 84 days. Following a 7-day acclimation period, five purpose-bred cats were implanted with OKV-119 protypes and observed for a 112-day study period. Food intake, weekly plasma exenatide concentrations and body weight were measured. Exenatide plasma concentrations were detected at the first measured timepoint (Day 7) and maintained above baseline for over 84 Days. Over the first 28 days, reduced caloric intake and a reduction in body weight were observed in four of five cats. In these cats, a body weight reduction of at least 5% was maintained throughout the 112-day study period. This study demonstrates that a single OKV-119 implant can deliver the GLP-1RA exenatide for a months long duration. Results suggest that exposure to exenatide plasma concentrations ranging from 1.5 ng/ml to 4 ng/ml are sufficient for inducing weight loss in cats.

Characterization of feline-originated probiotics Lactobacillus rhamnosus CACC612 and Bifidobacterium animalis subsp. lactis CACC789 and and evaluation of their host response.

BACKGROUND: Probiotics are beneficial for animal health and new potential probiotics need to be characterized for their prospective use in improving animal health. In this study, 32 bacterial strains were isolated from a Norwegian forest cat (castrated, 12 years old) and a Persian cat (castrated, 10 years old), which were privately owned and had indoor access. RESULTS: Lactobacillus rhamnosus CACC612 (CACC612) and Bifidobacterium animalis subsp. lactis CACC789 (CACC789) were selected as potential probiotics; characterization of the two strains showed equivalent acid tolerance, similar cell adhesion rates on the HT-29 monolayer cell line, and superior bile tolerance compared to Lactobacillus rhamnosus GG (LGG). Subsequently, they exhibited inhibitory effects against a broad spectrum of pathogenic bacteria, including E. coli (KCTC 2617), Salmonella Derby (NCCP 12,238), Salmonella Enteritidis (NCCP 14,546), Salmonella Typhimurium (NCCP 10,328), Clostridium difficile JCM 1296T. From evaluating host effects, the viability of the feline macrophage cell line (Fcwf-4) increased with the treatment of CACC612 or CACC789 (P < 0.05). The induced expression of immune-related genes such as IFN-γ, IL1ß, IL2, IL4, and TNF-α by immune stimulation was significantly attenuated by the treatment of CACC612 or CACC789 (P < 0.05). When 52 clinical factors of sera from 21 healthy cats were analyzed using partial least squares discriminant analysis (PLS-DA), the animals were obviously clustered before and after feeding with CACC612 or CACC789. In addition, hemoglobin and mean corpuscular hemoglobin concentration (MCHC) significantly increased after CACC612 feeding (P < 0.05). CONCLUSIONS: In this study, feline-originated probiotics were newly characterized and their potentially probiotic effects were evaluated. These results contribute to our understanding of the functional effects of feline-derived probiotics and support their industrial applications.