Research Article

Phylogenetic Evidence for a Distinct Feline calicivirus lineage in Indonesian Cats

Berlian Ramadhanti1, Farrel Arif Muhammad1, Yulianna Puspitasari2, Jola Rahmahani2, Iwan Sahrial Hamid3, Sri Agus Sudjarwo3, Fedik Abdul Rantam2*

1Vaccinology and Immunotherapeutics Program, Faculty of Veterinary Medicine, Universitas Airlangga, Surabaya, Indonesia, Zip Code 60115; 2Department of Veterinary Microbiology, Faculty of Veterinary Medicine, Universitas Airlangga, Surabaya, Indonesia, Zip Code 60115; 3Department of Basic Veterinary Medicine, Faculty of Veterinary Medicine, Universitas Airlangga, Surabaya, Indonesia, Zip Code 60115.

Received | November 08, 2025; Accepted | December 17, 2025; Published | February 04, 2026

*Correspondence | Fedik Abdul Rantam, Division of Veterinary Microbiology, Faculty of Veterinary Medicine, Universitas Airlangga, Surabaya, Indonesia, Zip Code 60115; Email: [email protected]

Citation | Ramadhanti B, Muhammad FA, Puspitasari Y, Rahmahani J, Hamid IS, Sudjarwo SA, Rantam FA (2026). Phylogenetic evidence for a distinct Feline calicivirus lineage in Indonesian cats. Adv. Anim. Vet. Sci., 14(2):389-394.

DOI | https://dx.doi.org/10.17582/journal.aavs/2026/14.2.389.394

ISSN (Online) | 2307-8316

Copyright: 2026 by the authors. Licensee ResearchersLinks Ltd, England, UK.

This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).

INTRODUCTION

Feline calicivirus (FCV) is a highly contagious viral pathogen that predominantly impacts cats. FCV infection leading to various clinical presentations ranging from mild respiratory issues to severe systemic infections and is closely linked to the immune response that causes feline chronic gingivostomatitis (Nafi’ah et al., 2025). FCV is a positive-sense, single-stranded, non-enveloped RNA virus which belongs to the genus Vesivirus in the family Caliciviridae, with a size of approximately 7.6-7.7 kb with considerable genetic variability and mutability (Tyasningsih et al., 2024; Coyne et al., 2007). The diagnosis, treatment, and prevention of FCV is severely hampered by the similarity of its clinical signs to those of FHV-1 and respiratory bacteria infection (Al Hafid et al., 2022).

The genome of Feline calicivirus is quite similar with the other members of Caliciviridae. Three open reading frames (ORFs) configurate FCV genome. A nonstructural protein is encoded by ORF1, the primary antigenic protein VP1 is encoded by ORF2, and VP2 is encoded by ORF3 (Yang et al., 2023). Research has demonstrated that the variable regions of the capsid protein gene (VP1) of FCV, particularly C and E region, undergoes rapid molecular evolution, making it an important target for phylogenetic and genetic investigations. However, this high hypervariability poses a challenge for initial screening in uncharacterized populations, as primer mismatches in the E region can lead to detection failure. Therefore, to ensure robust detection and broad lineage identification, the more conserved region of the VP1 gene (regions A and B) is preferable as the target (Spiri et al., 2016). Significant genetic variety among FCV strains has been found through phylogenetic analyses, with different genotypes and lineages found in various geographical locations. FCV is commonly referred as single serotype and divided into two primary genotypes of FCV strains, GI and GII. GI strains are more common globally, whereas GII strains are mostly found in Asia (Yang et al., 2023).

Investigations in Thailand have demonstrated high genetic diversity among FCV strains, with amino acid sequence homology ranging from 0 to 86.6% when compared to worldwide strains. In studies conducted in Thailand, the genetic diversity of FCV strains was found to be significant, with amino acid sequence homology ranging from 0 to 86.6% compared to global strains (Phongroop et al., 2024). Similarly, phylogenetic analysis of FCV strains from China revealed a high genetic diversity, with distinct genotypes and potential virulence-related amino acid loci discovered. The results of research by Lu et al. (2023) stated that 17 FCV isolates spread across China had homology relationship ranging from 75–85%. Two isolates isolated from the same region, namely SH (KP987265) and SH2014 (KT000003), showed 76.3% for nucleotide homology rate. The intrinsic mutation rate of FCV, along with Indonesia geographical characteristics as an archipelagic country, renders Indonesia theoretically capable of exhibiting a significantly high variety of FCV strains.

The high mutation rate highlight the significance of ongoing observation and molecular characterization of FCV strains in order to provide a commencement for developing disease control strategies. Feline upper respiratory disease is a substantial clinical burden, the diagnosis remains largely symptomatic. An animal clinic in Indonesia reported high intensity of FCV infection cases, with over 60 cases occurred in just two months (De Lucas, 2021). While other feline viruses been molecularly studied, which are Feline Panleukopenia virus (Munawaroh et al., 2020) and Feline Coronavirus (Aksono et al., 2023; Al Hafid et al., 2022), there have been no prior molecular studies regarding FCV in Indonesia. Molecular characterization studies of FCV in Indonesia, which include homology and phylogeny analyses, can provide insight into the genetic diversity and evolutionary dynamics of the virus. This study is intended to be the preliminary molecular study of FCV strains circulating in Indonesia. The resulting data is crucial for the development of more specific prevention and treatment strategies.

MATERIALS AND METHODS

Sampling

This investigation included samples from ten cats and was carried out between December 2024 and January 2025. The inclusion criteria were based on the presence of clinical signs associated with the FURD complex. Observed symptoms included fever, nasal discharge, swollen submandibular lymph nodes, conjunctivitis, oral ulcerations, and inappetence (Baksi and Simsek, 2021; Litster, 2021). While these signs can overlap with other pathogens, samples were collected from symptomatic cases to screen for FCV presence. Oropharynx swabs were collected and homogenized in tube containing 2 mL viral transport medium with the consent of owners. Samples were subsequently stored at -80°C freezer until PCR analysis was performed (Wulandari et al., 2021). All sample analysis processes were conducted at Institute of Tropical Disease University of Airlangga, Surabaya, Indonesia.

Reverse transcription-polymerase chain reaction and DNA visualization for Feline calicivirus

Prior to RNA extraction, stored samples were thawed and centrifuged at 2000 rpm for 10 minutes. Supernatants were filtered with Nest® filter syringe 0.22 µm. Viral nucleic acid was extracted using Viral Nucleic Acid Kit II (Geneaid, Taipei) following the manufacturer protocol. The concentration and quality of the extraction yield were quantified using a NanoDrop Lite Spectrophotometer. The extracted nucleic acid samples were then kept at -80 °C until further use.

The reverse-transcription reaction followed the provided protocols of SensiFAST™ cDNA Synthesis Kit (Meridian Bioscience, London, UK). The reverse transcription master mix was prepared on an ice rack to maintain the temperature low. RNA template, 5x TransAmp Buffer, and reverse transcriptase were briefly centrifuged. The total reaction volume for one reaction was 20µl, consisting 4µl of buffer, 1µl of reverse transcriptase, 10µl of nuclease free water, and 5µl of RNA sample. The mixture was homogenized, then incubated using a Takara PCR Thermal Cycle Dice TP600 with following conditions: annealing at 25°C for 10 minutes; reverse transcription at 42°C for 15 minutes; inactivation at 85°C for 10 minutes, followed by a cooling step at 4°C. The resulting cDNA samples were stored at -20°C until PCR analysis.

The polymerase chain reaction utilized forward primer Cali1(5’-AACCTGCGCTAACGTGCTTA-3’) and reverse primer Cali2 (5’-CAGTGACAATACACCCAGAAG-3’) which targeted the fragment of A - B region in VP1 gene (Marsilio et al., 2005). The PCR reactions were carried out using MyTaq™ HS Red Mix (Meridian Bioscience, London, UK) following manufacturer instruction. The cycles conditions comprised an initial denaturation at 94°C for 4 min, followed by 40 cycles at 94°C for 30 s, 54°C for 40 s, and 72°C for 45 s followed by last extension at 72°C for 7 minutes. Comercially available vaccine (Felocell 3®, Zoetis) was included as positive control (Özkanlar et al., 2023). Otherwise, nuclease free water used as negative control. PCR products were stored in a refrigerator until electrophoresis was run (Rahmahani et al., 2022).

The PCR output was subjected to electrophoresis. The PCR products were loaded into a 2% agarose gel, which was prepared using 1x TBE buffer and ethidium bromide. The agarose gel was inserted in MUPID-2 Plus electrophoresis system. First well filled with 2 µl of DNA ladder. Samples and controls are inserted into the other wells as much as 5 µl. Electrophoresis process was performed in constant voltage 100 volts for 30 minutes (Tyasningsih et al., 2024). The gel was visualized through Gel Doc UV transilluminator (Axygen®, Corning, USA) to observe the amplified DNA bands.

Phylogenetic analysis

Sequences were directly obtained from positive RT-PCR results utilizing the BigDye Terminator v3.1 cycle sequencing kit and an Applied Biosystems 3500xL Genetic Analyzer (Applied Biosystems, Waltham, MA, USA). Sequence accuracy was ensured by generating consensus sequences from both forward and reverse reads. Sequences were manually inspected through examining the chromatograms in UGENE. Ambiguous sites were resolved and sequences were trimmed to exclude low-quality ends, ensuring that only robust data were included in the alignment. Nucleotide sequences were aligned with default parameters and manually inspected as needed. Phylogenetic trees were constructed using the maximum composite likeli­hood method with the neighbor-joining method with 1000 bootstrap replicates, and sequences were compared to global FCV reference strains from GenBank to assess evolutionary relationships and potential geographic origins (Yesica et al., 2024). All strain sequence references obtained from GenBank are below: LC842011; LC859024; LC836032 (Japan), AY560117; L40021; AY560118 (United States), AF479590 (Germany), KJ495729; KJ495728; KJ495730 (China), KJ572400; KJ572401 (South Korea), MZ542330 (Thailand), MW880771 (Australia), ON360073, M86379 (vaccine strain), and MF677852 (Mink Calicivirus) as an outgroup.

RESULTS AND DISCUSSION

Ten FCV-suspected cats data stated in Table 1. Positive samples were indicated by expected band with size of 924

bp in Figure 1 and 2. The visualization of sample amplification products on agarose gel which had been run through electrophoresis showed two positive samples (20%). The low prevalence rate of FCV observed in the samples might be attributed to the clinical symptoms of FCV infection in cats overlap with other pathogens, such as FHV-1, Feline Parvovirus, Retrovirus, Bordetella bronchiseptica, and Mycoplasma (Al-Hafid et al., 2022). However, the other infectious pathogens probability in the symptomatic cat samples had not been identified yet due to the limited scope of the study.

 

Table 1: The clinical signs and RT-PCR examination results from samples of cats with upper respiratory disease signs in Surabaya

Cat	Temperature (°C)	Clinical signs	Specimen	RT-PCR result
Cat 1	39.4	Pyrexia, dyspnea, nasal discharge, conjunctivitis, gingivitis, inappetence	Oropharynx swab	Positive
Cat 2	40.5	Pyrexia, dyspnea, nasal discharge, ocular discharge, gingivitis, lingua ulceration, conjunctivitis, inappetence	Oropharynx swab	Positive
Cat 3	39	Pyrexia, dyspnea, conjunctivitis, inappetence	Oropharynx swab	Negative
Cat 4	39.8	Pyrexia, dyspnea, sneezing, inappetence	Oropharynx swab	Negative
Cat 5	39.1	Pyrexia, dyspnea, nasal discharge, conjunctivitis, inappetence	Oropharynx swab	Negative
Cat 6	38.9	Pyrexia, inappetence	Oropharynx swab	Negative
Cat 7	39.1	Pyrexia, dyspnea, nasal discharge, inappetence	Oropharynx swab	Negative
Cat 8	40.2	Pyrexia, dyspnea, nasal discharge, ocular discharge, pale mucous membrane	Oropharynx swab	Negative
Cat 9	39.2	Pyrexia, inappetence	Oropharynx swab	Negative
Cat 10	39.3	Pyrexia, dyspnea, nasal discharge, ocular discharge	Oropharynx swab	Negative

 

 

 

Clinical signs of FCV infection are highly variable. Upper respiratory tract disease, limping syndrome, gingivostomatitis, lingual ulcerations, and in severe condition, virulent systemic FCV infection able to result in alopecia, skin ulceration, oral cavity ulceration, pinnae, nares, and necrotizing pododermatitis with serocellular crusts. Numerous symptoms, including subcutaneous oedema, bronchointerstitial pneumonia, as well as necrosis of the pancreas, liver, and spleen, can be brought on by feline infections, all of which increase death rates (Spiri et al., 2022). All FCV positive-identified cats undergo fever, gingivitis, and emit nasal discharge. Pyrexia (fever) in cats probably occurs due to disruption in thermoregulation, which begins when either endogenous or exogenous pyrogens enter the body (Syarifuddin et al., 2025). The main site of replication for FCV is oropharynx, which enters the host by the nasal, oral, and conjunctival pathways. The ability of FCV to elude the immune response might result in recurring infections or cycles of reinfection. The virus causes inflammation and necrosis of the nasal passages epithelial cells, which increases the production of mucus and causes nasal discharge (Baksi and Simsek, 2021; Asif et al., 2025).

The capsid (VP1) gene of FCV has one of the highest recorded rates of molecular evolution, leading to substantial genetic diversity among strains. The greatest rate of nucleotide substitution among RNA viruses is observed in FCV. The substitutions are estimated to range from 1.32 × 10-2 to 2.64 × 10-2 substitutions per nucleotide per year in the same host and from 3.84 × 10-2 to 4.56 × 10-2 substitutions per nucleotide per year in a population (Coyne et al., 2007). This genetic drift results in the emergence of new variants. Consistent with this high evolutionary rate, the homology analysis revealed that FCV IDN SBY 1 and FCV IDN SBY 2 respectively shared 65.2% and 65.4% homology, with the commercial FCV vaccine strain (F9). This is notably lower than the typical homology range observed between other global reference strains and F9 (69.1-78.4%). While the correlation between genetic distance and antigenic difference is not absolute without serological confirmation, the marked divergence between the local strains and the F9 vaccine strain raises valid concerns regarding protective immunity. Previous reports have documented the variable performance of F9-based vaccines, with neutralization ranges spanning from 20.5% to 97% depending on the circulating field strains (Wensman et al., 2015; Afonso et al., 2017). These findings serve as a critical early warning that current vaccination protocols may be insufficient.

A phylogenetic tree of the FCV Indonesia samples along with global FCV strain was con­structed (Figure 3). The positive samples clustered together to form a distinct subclade with a moderate bootstrap value of 77%. The sample lineage demonstrates close phylogenetic proximity to Genogroup I reference strains, including FCV Urbana, FCV 12Q087-5, and FCV 12Q087-1. This suggests a high probability that Indonesia FCV strains belong to Genogroup I. It is possible that with more extensive sampling across the Indonesian archipelago, these isolates might group within broader FCV lineages rather than forming a unique monophyletic group. Mink Calicivirus (MF677852) which shares same genus with Feline calicivirus, was positioned as an outgroup, showing a clearly distinct evolutionary relationship from both the FCV reference strains and the Indonesian samples.

The presence of this FCV subclade highlights the need to monitor the domestic-wildlife interface. While current findings are limited to domestic cats, the unconfined nature of the local feline population creates a theoretical pathway for pathogen spillover into Indonesian wildlife reservoirs. To address this concern, subsequent research should prioritize the sampling of feral cats in high-risk interaction zones. Establishing such a surveillance protocol is vital for the early detection of any viral variants capable of crossing species barriers.

 

CONCLUSIONS

This study suggests a potential vulnerability in current feline health management, specifically highlighting the possibility of suboptimal protection offered by commercial FCV vaccines against the identified local strains. However, these findings must be interpreted within the context of this study limitations, including the small sample size, lack of co-pathogen screening, and the absence of further neutralization assay. Consequently, broader surveillance and further laboratory studies particularly cross-neutralization assays are urgently needed to establish the precise prevalence of this novel cluster and confirm its impact on vaccine efficacy.

ACKNOWLEDGEMENT

The authors would like to show gratitude towards The Ministry of Research and Technology Republic Indonesia for the funding during the study and Research Center for Vaccine Technology and Development of Institute of Tropical Disease Universitas Airlangga for the guidance.

Novelty Statement

This study provides novel insights into the phylogenetic distinctiveness of Feline calicivirus lineages circulating in Indonesia. Molecular evidence in this research fills a vital gap in context-specific virological data for feline health management in Southeast Asia.

AUTHORS CONTRIBUTION

BR and FAM: conceptualization, samples collection, laboratory procedure, analysis, manuscript drafting; SAS and ISH: conceptual and methodological guidance, data interpretation and manuscript supervision; JR and YP: conceptual and methodological guidance, laboratory supervision; FAR: supervision, project-funding administration, validation, manuscript review, and correspondence. All authors have read and approved the final version of the manuscript.

Generative AI and AI-assisted technology statement

The authors acknowledge the use of AI-assisted platforms, specifically DeepL for grammatical refinement. Every AI-generated suggestion was manually verified by the authors, and no AI technology was employed during the data analysis or interpretation of this study.

Conflict of interest

The authors have declared no conflict of interest.

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