Feline Adenovirus Isolate Shows Silent Nucleotide Alterations, Alternative Receptor/Coreceptor Binding, High Resistance to Disinfectants and Antiviral Drugs, as Well as Immunomodulation

Abstract

Adenovirus (AdV) infection has been rarely documented in cats and other felids. Partial sequences of the hexon and fiber genes of a Hungarian feline adenovirus isolate (FeAdV isolate) showed a close relationship to human AdV (HAdV) type C1. Further molecular and biological characterization is reported here. Whole-genome sequencing revealed two silent mutations in the genome of the FeAdV isolate compared to a HAdV-C1 reference strain (at positions 14,096 and 15,082). Competitive antibody binding to the Coxsackie-adenovirus receptor and α_vβ₃ and α_vβ₅ integrin coreceptors inhibited the binding of the FeAdV isolate in different cell lines, but residual infections suggested alternative entry routes. The FeAdV isolate was found to be more sensitive to heat, low pH and detergents, but more resistant to alkaline and free chlorine treatments, as well as to ribavirin, stavudine and cidofovir treatments, than other human AdV types. We observed a suppression of IL-10 and TGF-β1 production during the entire course of viral replication. This immunomodulation may restore intratumoral immunity; thus, the FeAdV isolate could serve as an alternative oncolytic vector. Collectively, our results support that the Hungarian FeAdV isolate is a variant of common HAdV-C1. The cohabitation of cats with humans might result in reverse zoonotic infection. Felids appear to be susceptible to persistent and productive adenovirus infection, but further studies are needed to better understand the clinical and epidemiological implications.

Keywords: antiviral therapy; cytokine production; feline adenovirus; physico-chemical effects; receptors; whole-genome sequencing.

Figure 1

Sequence alignment showing SNPs at positions 14,096 and 15,081 in FeAdV isolate. For alignment purposes, FeAdV isolate nucleotide 15,082 was compared to HAdV-C1 nucleotide 15,081. Red: a nucleotide insertion, green to blue: a nucleotide change.

Figure 2

Uninfected ongjos cells. (a) Subconfluent monolayer with several elongated cells. Nuclei contain one or multiple nucleoli, day 3. (b) Higher magnification of the confluent monolayer. Nuclei contain dark nucleoli, day 7. (c) Torn monolayer. Apoptotic granules are in the cytoplasm of dying, non-colored cells, day 9. (d) Disintegrating cells with very large nucleoli, apoptotic granules in the cytoplasm, day 12. (a–d) Original magnification is 400× plus picture magnification. (a,b) Methylene blue staining).

Figure 3

FeAdV isolate replication in ongjos cells. (a) Cytopathic effect of FeAdV isolate on day 5 (original magnification 400× plus picture magnification). (b) FeAdV isolate particles dispersed in the cytoplasm (original magnification 7000× plus picture magnification). (c) A pseudoarray of viral particles in the cytoplasm (original magnification 14,000× plus picture magnification). (d) Negatively stained viral particles from the supernatant of ongjos cells. Fibers could not be visualized (original magnification 50,000× plus picture magnification). Arrowheads point out virions (b,d) and a pseudoarray (c).

Figure 4

The effect of anti-Coxsackie–adenovirus receptor (CAR) and anti-α_vβ₃ and anti-α_vβ₅ antibodies on the relative number of virus-infected cells compared to the virus control (VC). (a) FeAdV isolate infection (moi 1) of Hela cells. (b) Combined antibody treatment of infected Hela cells (moi 0.1 and moi 1). (c) Combined antibody treatment of infected HEK cells (moi 0.1 and moi 1). (d) Combined antibody treatment of infected CRFK cells (moi 1 and moi 10). n = 3; * p < 0.05, ** p < 0.001.

Figure 5

The effect of physico-chemical agents on residual virus infectivity assayed in HeLa cells. (a) Heat treatment at 56 °C. (b) UV treatment. (c) FeAdV isolate exposed to a pH range. n = 3; * p < 0.05, ** p < 0.001.

Figure 6

(a) The pattern of IL-10 secretion by mock-infected (blue) and FeAdV isolate-infected (red) HeLa cells at different time points. (b) Summary of IL-10 secretion after FeAdV isolate infection compared to mock-infected HeLa cells. All dots represent a different time point (n = 10). (c) The pattern of TGF-β₁ secretion by mock-infected (blue) and FeAdV isolate-infected (red) HeLa cells at different time points. (d) Summary of TGF-β₁ secretion after FeAdV infection compared to mock-infected HeLa cells. All dots represent a different time point (n = 10). Non-parametric t-test was used. All data are shown as means ± standard errors of means (SEMs). ** p < 0.001.