Diagnosing Feline Calicivirus: Interpreting RT-PCR, Sampling and Virus Isolation

A positive feline calicivirus test does not always mean FCV is responsible for a cat’s clinical signs. Asymptomatic cats and cats recently vaccinated with modified-live FCV vaccines can occasionally shed the virus, creating a poor correlation between viral RNA detection and clinical disease^[1],[2]. 

For clinicians, FCV diagnosis therefore depends not only on the test selected, but also on the sampling site, timing, assay limitations, and clinical context. 

RT-PCR: Sensitive, but Not Infallible 

FCV RNA can be detected by conventional, nested or real-time RT-PCR from samples including conjunctival and oral swabs, blood, cutaneous scrapings and lung tissue, depending on the clinical presentation. However, the high variability of the FCV genome means that diagnostic sensitivity depends on both the primers used and the strain being tested. Molecular assays should therefore be optimised against a broad panel of FCV strains to reduce false-negative results^[2]. 

Even established assays may miss some field isolates. Two well-established FCV RT-PCR systems, for example, failed to amplify all isolates examined in a Swiss field study. 

Real-time RT-PCR is generally preferred for diagnosis because of its higher sensitivity. Where available, quantitative assays can additionally provide information about viral load. Multiplex PCR/RT-PCR assays can detect FCV alongside FHV and sometimes Chlamydia felis, although their sensitivity may be lower^[2].

The RT-PCR result should always be interpreted alongside the clinical signs. Sampling time, storage, and transport can also influence the result^[2]. 

Sampling Site Matters^[2] 

Not all swabs have the same likelihood of detecting FCV. Detection by RT-PCR is significantly more likely from the oropharynx and tongue than from conjunctival swabs. 

When FCV, FHV and Chlamydia felis are being investigated together, the oropharynx is recommended as the preferred sampling site because detection of FHV and Chlamydia felis did not differ significantly among the sampling sites assessed. Ideally, multiple swabs or multiple sites should be sampled; when using the same swab sequentially, sampling can begin with the conjunctiva to maximise pathogen detection. 

What Does Virus Isolation Add?^[2] 

Virus isolation offers a different diagnostic advantage: it demonstrates the presence of replicating FCV and is less affected by viral strain variation than RT-PCR. FCV can be isolated from nasal, conjunctival, and oropharyngeal swabs. 

However, isolation is not fail-safe. Low numbers of virions, virus inactivation during transport, and antibodies in extracellular fluids can prevent successful replication in vitro. Collecting both conjunctival and oropharyngeal swabs can maximise the likelihood of successful isolation. 

Combining RT-PCR directly from swabs with detection after virus isolation can further increase sensitivity. Neither RT-PCR nor virus isolation alone detected all FCV-positive samples in the Swiss field study. Consequently, a negative FCV test does not completely exclude infection when clinical suspicion remains. 

Antibody Detection is Not a Diagnostic Test^[2] 

FCV antibodies can be measured using virus neutralisation or ELISA. However, antibody prevalence is generally high because of both natural infection and vaccination. Therefore, detecting FCV-specific antibodies is not useful for diagnosing current infection. 

Virus-neutralising antibody levels may help predict whether a cat is protected, but interpretation requires caution. False-negative results can occur when antibodies do not cross-react with the laboratory strain used for testing. Titres can also differ depending on whether homologous or heterologous virus-antibody pairs are used, and an undefined test strain makes interpretation more difficult. 

Diagnosing VS-FCV Requires More Than Sequencing^[2] 

VS-FCV presents an additional diagnostic challenge. No consistent genetic markers of virulence have been established, and there are currently no diagnostic assays that specifically identify VS-FCV. Although particular capsid mutations have been proposed as markers of the VS-FCV pathotype, findings from subsequent outbreaks have not consistently supported this association. 

A single negative FCV RT-PCR result also does not rule out VS-FCV infection. When clinical signs compatible with VS-FCV are observed, appropriate hygiene measures should be implemented promptly. This is particularly important because severe systemic but non-epizootic FCV forms, including paw-and-mouth disease, can initially resemble VS-FCV-associated disease. 

Clinical Interpretation Remains Central 

FCV diagnosis is therefore not a matter of treating a laboratory result in isolation. A positive RT-PCR may reflect viral shedding rather than clinical disease, while a negative result may occur because of strain variation, sampling factors, or limitations of the detection method. 

For suspected FCV infection, clinicians should consider the clinical presentation, sampling site, timing and handling of specimens, assay limitations, and whether replicating virus has been demonstrated. When VS-FCV is suspected, the absence of a specific molecular marker means that clinical and epidemiological assessment remains essential alongside laboratory investigation.

References:

1. Ruch-Gallie RA, Veir JK, Hawley JR, Lappin MR. Results of molecular diagnostic assays targeting feline herpesvirus-1 and feline calicivirus in adult cats administered modified live vaccines. Journal of Feline Medicine and Surgery. 2011 Aug;13(8):541-5. https://journals.sagepub.com/doi/pdf/10.1016/j.jfms.2010.12.010
2. Hofmann-Lehmann R, Hosie MJ, Hartmann K, Egberink H, Truyen U, Tasker S, Belák S, Boucraut-Baralon C, Frymus T, Lloret A, Marsilio F. Calicivirus infection in cats. Viruses. 2022 Apr 29;14(5):937. https://www.mdpi.com/1999-4915/14/5/937
3. Schulz C, Hartmann K, Mueller RS, Helps C, Schulz BS. Sampling sites for detection of feline herpesvirus-1, feline calicivirus and Chlamydia felis in cats with feline upper respiratory tract disease. Journal of feline medicine and surgery. 2015 Dec;17(12):1012-9. https://journals.sagepub.com/doi/pdf/10.1177/1098612X15569615