Molecular detection and characterisation of Domestic Cat Hepadnavirus (DCH) from blood and liver tissues of cats in Malaysia.

BACKGROUND: A new domestic cat hepadnavirus (DCH, family Hepadnaviridae) was first reported from whole blood samples of domestic cats in Australia in 2018, and from cat serum samples in Italy in 2019. The pathogenesis of DCH is unknown, but it was reported in cats with viraemia (6.5-10.8%), chronic hepatitis (43%) and hepatocellular carcinoma (28%). Recent reports suggest that DCH resembles the human hepatitis B virus (HBV) and its related hepatopathies. This study aims to detect and characterize DCH among domestic cats in Malaysia. A cross-sectional study was performed on 253 cats, of which 87 had paired blood and liver samples, entailing whole-genome sequencing and phylogenetic analysis of DCH from a liver tissue sample. RESULTS: Among the 253 cats included in this study, 12.3% of the whole blood samples tested positive for DCH. The detection rate was significantly higher in pet cats (16.6%, n = 24/145) compared to shelter cats (6.5%, n = 7/108). Liver tissues showed higher a DCH detection rate (14.9%, n = 13/87) compared to blood; 5 out of these 13 cats tested positive for DCH in their paired liver and blood samples. Serum alanine transaminase (ALT) was elevated (> 95 units/L) in 12 out of the 23 DCH-positive cats (52.2%, p = 0.012). Whole-genome sequence analysis revealed that the Malaysian DCH strain, with a genome size of 3184 bp, had 98.3% and 97.5% nucleotide identities to the Australian and Italian strains, respectively. The phylogenetic analysis demonstrated that the Malaysian DCH genome was clustered closely to the Australian strain, suggesting that they belong to the same geographically-determined genetic pool (Australasia). CONCLUSIONS: This study provided insights into a Malaysian DCH strain that was detected from a liver tissue. Interestingly, pet cats or cats with elevated ALT were significantly more likely to be DCH positive. Cats with positive DCH detection from liver tissues may not necessarily have viraemia. The impact of this virus on inducing liver diseases in felines warrants further investigation.

In Vitro Evaluation of Curcumin-Encapsulated Chitosan Nanoparticles against Feline Infectious Peritonitis Virus and Pharmacokinetics Study in Cats.

Feline infectious peritonitis (FIP) is an important feline viral disease, causing an overridden inflammatory response that results in a high mortality rate, primarily in young cats. Curcumin is notable for its biological activities against various viral diseases; however, its poor bioavailability has hindered its potential in therapeutic application. In this study, curcumin was encapsulated in chitosan nanoparticles to improve its bioavailability. Curcumin-encapsulated chitosan (Cur-CS) nanoparticles were synthesised based on the ionic gelation technique and were spherical and cuboidal in shape, with an average particle size of 330 nm and +42 mV in zeta potential. The nanoparticles exerted lower toxicity in Crandell-Rees feline kidney (CrFK) cells and enhanced antiviral activities with a selective index (SI) value three times higher than that of curcumin. Feline-specific bead-based multiplex immunoassay and qPCR were used to examine their modulatory effects on proinflammatory cytokines, including tumour necrosis factor (TNF)α, interleukin- (IL-) 6, and IL-1ß. There were significant decrements in IL-1ß, IL-6, and TNFα expression in both curcumin and Cur-CS nanoparticles. Based on the multiplex immunoassay, curcumin and the Cur-CS nanoparticles could lower the immune-related proteins in FIP virus (FIPV) infection. The single- and multiple-dose pharmacokinetics profiles of curcumin and the Cur-CS nanoparticles were determined by high-performance liquid chromatography (HPLC). Oral delivery of the Cur-CS nanoparticles to cats showed enhanced bioavailability with a maximum plasma concentration (C max) value of 621.5 ng/mL. Incorporating chitosan nanoparticles to deliver curcumin improved the oral bioavailability and antiviral effects of curcumin against FIPV infection. This study provides evidence for the potential of Cur-CS nanoparticles as a supplementary treatment of FIP.

Cellular Metabolic Profiling of CrFK Cells Infected with Feline Infectious Peritonitis Virus Using Phenotype Microarrays.

Feline infectious peritonitis (FIP) is a fatal feline immune-mediated disease caused by feline infectious peritonitis virus (FIPV). Little is known about the biological pathways associated in FIP pathogenesis. This is the first study aiming to determine the phenotypic characteristics on the cellular level in relation to specific metabolic pathways of importance to FIP pathogenesis. METHODS: The internalization of type II FIPV WSU 79-1146 in Crandell-Rees Feline Kidney (CrFK) cells was visualized using a fluorescence microscope, and optimization prior to phenotype microarray (PM) study was performed. Then, four types of Biolog Phenotype MicroArray™ plates (PM-M1 to PM-M4) precoated with different carbon and nitrogen sources were used to determine the metabolic profiles in FIPV-infected cells. RESULTS: The utilization of palatinose was significantly low in FIPV-infected cells; however, there were significant increases in utilizing melibionic acid, L-glutamine, L-glutamic acid and alanyl-glutamine (Ala-Gln) compared to non-infected cells. CONCLUSION: This study has provided the first insights into the metabolic profiling of a feline coronavirus infection in vitro using PMs and deduced that glutamine metabolism is one of the essential metabolic pathways for FIPV infection and replication. Further studies are necessary to develop strategies to target the glutamine metabolic pathway in FIPV infection.

Gamification, a Successful Method to Foster Leptospirosis Knowledge among University Students: A Pilot Study.

Leptospirosis is a zoonotic disease that has been reported in Malaysia and has been associated with a recent trend of recreational activities among the youth. Thus, efforts such as educational interventions among high-risk populations, especially the youth, are key to increasing public awareness regarding leptospirosis. This paper presents the findings of a pilot study wherein an educational intervention using a gamification intervention method was used to determine changes in leptospirosis knowledge among youth. On this note, students from a public university in Seremban district, Malaysia, were recruited and were asked to complete questionnaires before and after gamification activities. Baseline and immediate post-intervention data on leptospirosis knowledge were obtained. The total knowledge score was calculated, and differences in the mean pre- and post-intervention knowledge score were determined. Of the total 185 questionnaires that were completed at baseline and immediately post-intervention, only 168 that belonged to respondents who had heard of leptospirosis were analysed in this paper. A significant increase in leptospirosis knowledge was observed for the students following health education by gamification (p < 0.01). The results demonstrate the effectiveness of an educational intervention using gamification in improving leptospirosis knowledge among youth and suggest that gamification could become an efficient tool to prevent the disease within university-age demographics.

Expression profiles of immune mediators in feline Coronavirus-infected cells and clinical samples of feline Coronavirus-positive cats.

BACKGROUND: There are two biotypes of feline coronavirus (FCoV): the self-limiting feline enteric coronavirus (FECV) and the feline infectious peritonitis virus (FIPV), which causes feline infectious peritonitis (FIP), a fatal disease associated with cats living in multi-cat environments. This study provides an insight on the various immune mediators detected in FCoV-positive cats which may be responsible for the development of FIP. RESULTS: In this study, using real-time PCR and multiplex bead-based immunoassay, the expression profiles of several immune mediators were examined in Crandell-Reese feline kidney (CRFK) cells infected with the feline coronavirus (FCoV) strain FIPV 79-1146 and in samples obtained from FCoV-positive cats. CRFK cells infected with FIPV 79-1146 showed an increase in the expression of interferon-related genes and pro-inflammatory cytokines such as MX1, viperin, CXCL10, CCL8, RANTES, KC, MCP1, and IL8. In addition, an increase in the expression of the above cytokines as well as GM-CSF and IFNγ was also detected in the PBMC, serum, and peritoneal effusions of FCoV-positive cats. Although the expression of MX1 and viperin genes was variable between cats, the expression of these two genes was relatively higher in cats having peritoneal effusion compared to cats without clinically obvious effusion. Higher viral load was also detected in the supernatant of peritoneal effusions compared to in the plasma of FCoV-positive cats. As expected, the secretion of IL1ß, IL6 and TNFα was readily detected in the supernatant of peritoneal effusions of the FCoV-positive cats. CONCLUSIONS: This study has identified various pro-inflammatory cytokines and interferon-related genes such as MX1, viperin, CXCL10, CCL8, RANTES, KC, MCP1, IL8, GM-CSF and IFNγ in FCoV-positive cats. With the exception of MX1 and viperin, no distinct pattern of immune mediators was observed that distinguished between FCoV-positive cats with and without peritoneal effusion. Further studies based on definitive diagnosis of FIP need to be performed to confirm the clinical importance of this study.