Antiviral Research 170 (2019) 104563

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Antiviral Research
journal homepage: www.elsevier.com/locate/antiviral

Cat flu: Broad spectrum polymeric antivirals T

a,b c a,b b d
Aleksandra Synowiec , Irma Gryniuk , Magdalena Pachota , Łukasz Strzelec , Olga Roman ,
Katarzyna Kłysik-Trzciańskae, Mateusz Zające, Inga Drebota, Katarzyna Gulaa,
Aleksandra Andruchowiczf, Zenon Rajfurg, Krzysztof Szczubiałkae, Maria Nowakowskae,**,
Krzysztof Pyrca,*
a
Virogenetics Laboratory of Virology, Malopolska Centre of Biotechnology, Jagiellonian University, Gronostajowa 7a, 30-387, Krakow, Poland
b
Microbiology Department, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Gronostajowa 7, 30-387, Krakow, Poland
c
Department of Cell Biophysics, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Gronostajowa 7, 30-387, Krakow, Poland
d
Department of Cell Biology, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Gronostajowa 7, 30-387, Krakow, Poland
e
Department of Physical Chemistry, Faculty of Chemistry, Jagiellonian University, Gronostajowa 2, 30-387, Krakow, Poland
f
Veterinary Practice AMBUVET, Pod Fortem 2E, 31-302, Krakow, Poland
g
Institute of Physics, Faculty of Physics, Astronomy and Applied Computer Sciences, Jagiellonian University, Lojasiewicza 11, 30-348, Krakow, Poland

ARTICLE INFO ABSTRACT

Keywords: Feline herpesvirus type 1 (FHV-1) and feline calicivirus (FCV) are considered as main causes of feline upper
Synthetic polymers respiratory tract disease and the most common clinical manifestations include rhinotracheitis, conjunctivitis,
poly(sodium 4-styrenesulfonates) and nasal/facial ulcerations. While the primary infection is relatively mild, secondary infections pose a threat to
PSSNa young or immunocompromised cats and may result in a fatal outcome. In this study, we made an effort to
Antivirals
evaluate antiviral potency of poly(sodium 4-styrenesulfonates) (PSSNa) as potent FHV-1 and FCV inhibitors for
Feline herpesvirus type 1
FHV-1
topical use. Mechanistic studies showed that PSSNa exhibits a different mechanism of action depending on target
Feline calicivirus species. While PSSNa acts directly on FHV-1 particles blocking their interaction with the host's cell and pre-
FCV venting the infection, the antiviral potency against FCV is based on inhibition at late stages of the viral re-
Cat flu plication cycle. Altogether, PSSNa polymers are promising drug candidates to be used in the treatment and
prevention of the viral upper respiratory tract disease (URTD), regardless of the cause.

1. Introduction salivation, chronic stomatitis, mild respiratory or conjunctival disorders

and limping syndrome (Dawson et al., 1994; Reubel et al., 1992;
Feline upper respiratory tract disease (URTD), colloquially termed TerWee et al., 1997). It has been, however, noted that some FCV iso-
as ‘cat flu’, is a relatively mild condition, manifested by oral or nasal lates cause outbreaks with mortality rates reaching 40–60% (Hurley
discharges, lethargy, pyrexia, conjunctivitis and sneezing (Helps et al., et al., 2004; Coyne et al., 2006; Pedersen et al., 2000; Reynolds et al.,
2005; Binns et al., 2000). The disease, however, may progress to the 2009; Schulz et al., 2011). No adequate antiviral treatment is available.
severe sequelae, with potentially fatal outcome (Bannasch and Foley, Previous reports show that FCV infection may be inhibited in vitro
2005). Feline herpesvirus type 1 (FHV-1) and feline calicivirus (FCV) (McDonagh et al., 2015; Wu et al., 2015; Fumian et al., 2018), but these
are considered to be the two major causes of URTDs (Helps et al., 2005; findings were not confirmed in clinic.
Bannasch and Foley, 2005; Fernandez et al., 2017; Cohn, 2011). Herpesviridae family encompasses enveloped, double-stranded DNA
Caliciviridae family encompasses non-enveloped, single-stranded, viruses associated with incurable, lifelong lasting herpetic infections.
positive-sense RNA viruses (Fields BN et al., 2013) that can infect both Feline herpesvirus type 1 (FHV-1, felid herpesvirus 1) is classified under
humans and animals (Ohlinger et al., 1993; Bank-Wolf et al., 2010; the genus Varicellovirus and within the Alphaherpesvirinae subfamily
Hurley et al., 2004). Feline calicivirus (FCV) (Vesivirus (Fields BN et al., (Davison et al., 2009). The virus is present in the domestic cat popu-
2013)) is prevalent worldwide in the domestic cat population (Radford lation worldwide, causing viral rhinotracheitis (FVR) and ocular disease
et al., 2009), and is commonly associated with oral ulceration, (Gould, 2011; Hartley, 2010; Bistner et al., 1971; Stiles, 2003). It is

*
Corresponding author.
**
Corresponding author.
E-mail addresses: nowakows@chemia.uj.edu.pl (M. Nowakowska), k.a.pyrc@uj.edu.pl (K. Pyrc).
URL: http://virogenetics.info (K. Pyrc).

https://doi.org/10.1016/j.antiviral.2019.104563
Received 15 February 2019; Received in revised form 15 July 2019; Accepted 17 July 2019
Available online 17 July 2019
0166-3542/ © 2019 Elsevier B.V. All rights reserved.
A. Synowiec, et al. Antiviral Research 170 (2019) 104563

estimated that 90% of cats are seropositive to this virus, whereas about obtained from cats with clinical signs of URTD. Briefly, oropharyngeal
80% of infected animals establish lifelong latency and ~45% of these swabs were collected with virus transport plastic sticks (COPAN, Italy),
shed the virus (Gaskell and Povey, 1977; Maggs et al., 1999). The resuspended in medium, filtered (0.2 μm) and overlaid on fully con-
ocular herpetic disease is commonly manifested as acute conjunctivitis fluent CrFK cells. Plates were incubated up to 72 h and, if cytopathic
or chronic stromal keratitis and may lead to permanent eye damage or effect (CPE) occurred, the supernatants were collected. Supernatants
blindness (Gould, 2011; Hartley, 2010; Bistner et al., 1971; Stiles, were further subjected to the plaque assay, and 24 h–48 h post-infection
2003). Following the acute phase, the virus is transported retrogradely (p.i.) single plaques were collected. Viruses were typed by qPCR and
to sensory neurons, to establish incurable, life-long lasting latency in sequencing of thymidine kinase gene fragment (FHV-1 K7) and major
trigeminal ganglia. Recovery from latency results in recurrent disease capsid protein, VP1 gene fragment (FCV K3).
(Gaskell and Povey, 1973, 1977; Nasisse et al., 1992). Treatment op- Virus stocks and mock-infected samples were generated by in-
tions for human herpetic infections often involve the use of specific oculation of fully confluent CrFK cells with virus or control sample. 24 h
nucleoside analogs (NA). These compounds are initially inactive and (FCV) or 48 h (FHV-1) p.i. cells were lysed by two freeze-thaw cycles.
gain the function in infected cells due to phosphorylation by virus-en- Lysates were aliquoted and stored at −80 °C. Infectious samples were
coded thymidine kinase (TK) and subsequently host GMP kinases titrated according to the Reed and Muench protocol (Reed, 1938).
(Miller and Miller, 1980, 1982) and act as effective viral polymerase
inhibitors (Elion, 1983). While some of the compounds used in humans 2.4. XTT cytotoxicity assay
appear to be toxic in cats (Maggs, 2005; Maggs and Clarke, 2004;
Nasisse et al., 1989), penciclovir (PCV) appears to be a potent and safe CrFK cells were cultured on 96-well plate for 24 h. Subsequently,
inhibitor of FHV-1 replication in vitro (Maggs and Clarke, 2004; Hussein supernatants were discarded and 100 μl of fresh medium with polymer
et al., 2008; Groth et al., 2014); its prodrug famciclovir administered to or with the mock sample was added to each well. After 48 h at 37 °C,
cats is reported to be a safe and effective antiviral (Thomasy et al., cell viability was evaluated using XTT Viability Assay Kit (Biological
2011; Malik et al., 2009). Industries, Israel), according to the manufacturer's protocol. Following
In this study, we evaluated the antiviral potency of poly(sodium 4- 2 h incubation at 37 °C the supernatants were transferred into the
styrenesulfonates) (PSSNa) against FHV-1 and FCV. Obtained results transparent 96-well plate and absorbance (λ = 480 nm) was measured.
show that these polymers inhibit replication of both viruses at nontoxic Obtained results were normalized to the absorbance of the control
concentrations but their mode of action differs for the two tested pa- sample (untreated cells).
thogens. While PSSNa interacts with the FHV-1 virion and blocks its
entry to the cell, it restricts FCV infection during later stages of the 2.5. Quantitative real-time PCR (qPCR)
replication cycle. Our observations were confirmed with clinical strains.
Additionally, what is important, PSSNa was previously used in clinic for 2.5 μl of isolated DNA or cDNA was amplified in a reaction of 10 μl
other indications such as treatment of hyperkalemia (Mistry et al., containing 1 × Kapa Probe Fast qPCR MasterMix (Sigma-Aldrich,
2016; Hollander-Rodriguez and Calvert, 2006; (FDA), 2017). However, Poland), 100 nM of specific probe labeled with 6-carboxyfluorescein
given the above, we tested transdermal toxicity of concentrated poly- (FAM) and 6-carboxytetramethylrhodamine (TAMRA) and 450 nM of
mers in a mouse model as it would be a beneficial route of adminis- each sense and antisense primer. The sequences of oligonucleotides are
tration in the treatment of URTD. As no toxicity was observed thus far, listed in Supplementary Table 1.
we believe that PSSNa is a safe and effective drug candidate to be used Specific probe and primers were used to amplify 81 bp sequence
in the treatment of viral infections in domestic cats. fragment of the glycoprotein B (gB) gene of FHV-1 (Vogtlin et al., 2002)
or 151 bp region of the FCV genome (nt 5321 to 5471) (Chander Y
2. Materials and methods et al., 2007). The reaction was carried out in thermocycler (CFX96
Touch™ Real-Time PCR Detection System, Bio-Rad), according to the
2.1. Inhibitors scheme: 3 min at 95 °C, followed by 39 cycles of 15 s at 95 °C and 30 s at
58 °C (FHV-1) or 51 °C (FCV). In order to quantify the nucleic acids,
Standards of poly(sodium 4-styrenesulfonate) (PSSNa), Mw of 1.5, DNA standards were prepared as described previously (Pachota et al.,
5.4, 8, 19.3, 35, 46, 93.5, 200, 400, 780 and 1200 kDa (PSSNaMwkDa), 2017). For FCV linearized DNA standard was used as a template for in
were purchased from Pressure Chemical. PSSNa70kDa and PSSNa1000kDa vitro transcription with T7 RiboMAX™ Express Large Scale RNA Pro-
were purchased from Sigma-Aldrich, Poland. Stock solutions were duction System (Promega). Transcribed RNA was DNase treated, and
prepared in distilled water (dH2O) supplemented with penicillin (100 RNA was purified with lithium chloride (Invitrogen) precipitation.
U/ml) and streptomycin (100 μg/ml) and stored at 4 °C. Subsequently, the concentration of RNA was assessed using a spectro-
photometer, then the number of RNA copies per milliliter was calcu-
2.2. Cell culture lated using the approximate molecular weight of ribonucleotide (340 g/
mol) and Avogadro's constant. Eight 10-fold serial dilutions were used
Crandell Rees Feline Kidney cells (CrFK, ATCC: CCL-94) were as qPCR template to develop a standard curve.
maintained in Dulbecco-modified Eagle's medium (DMEM, high glu-
cose, Life Technologies, Poland) supplemented with 3% heat-in- 2.6. Plaque assay
activated fetal bovine serum (FBS, Life Technologies, Poland) (3%
DMEM). Medium was also supplemented with penicillin (100 U/ml), CrFK cells were seeded in 24-well plates 24 h prior to the inocula-
streptomycin (100 μg/ml), gentamycin (5 μg/ml) and ciprofloxacin tion. At the time of the infection, 80–90% confluent cells were overlaid
(0.5 μg/ml). Cells were cultured at 37 °C in an atmosphere containing with 250 μl of serial (10 fold) dilutions of infectious samples. Cells were
5% CO2 and humidity of 95%. incubated for 1 h at 37 °C, washed once with PBS and overlaid with
0.5 ml of DMEM supplemented with 10% heat-inactivated fetal bovine
2.3. Viruses serum (FBS, Life Technologies, Poland), penicillin (100 U/ml), strep-
tomycin (100 μg/ml) and 1% methylcellulose (FHV-1; Sigma-Aldrich,
Feline herpesvirus type 1 strain C-27 (FHV-1 C-27; ATCC: VR-636), Poland) or 0.6% UltraPure™ Low Melting Point Agarose (FCV; Thermo
feline calicivirus strain F9 (FCV F9; ATCC VR-782™) and clinical iso- Scientific, Poland). 72 h (FHV-1) or 24 h (FCV) p.i. For visualization of
lates FHV-1 K7 (Accession number: MK820647) and FCV K3 (Accession FHV-1 plaques cells were incubated with dH2O:methanol (1:1) with
number: MK820648) were used. FHV-1 K7 and FCV K3 strains were 0.1% crystal violet (20 min, room temperature) and washed once with

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A. Synowiec, et al. Antiviral Research 170 (2019) 104563

Scheme 1. Synthesis of fluorescently labeled PSSNa70kDa.

dH2O. For visualization of FCV plaques, cells were incubated with 1 ml/ receptors is possible. After the incubation, cells were washed twice with
well of 4% paraformaldehyde (PFA) in dH2O for at least 6 h at room ice-cold PBS, fresh medium was added to each well and cells were in-
temperature. Next cells were washed, incubated in dH2O:methanol cubated for 18 h (FCV) or 48 h (FHV-1) at 37 °C. After incubation, the
(1:1) with 0.1% crystal violet for 20 min at room temperature, and virus was quantified in the supernatants by qPCR and plaque assay.
washed once with dH2O.
2.8.4. Assay IV. Inhibition of virus replication, assembly, and egress
2.7. Virus replication assay This assay allows determining whether polymer inhibits the virus
replication, assembly, or release. CrFK cells were seeded on 96-wells
Fully confluent CrFK cells were seeded on 96-wells plates 24 h prior plates 24 h prior to the inoculation. Confluent CrFK cells were overlaid
to the inoculation. At the moment of infection, the supernatant was with fresh medium containing mock or virus (400 TCID50/ml), and
discarded and 20 μl of fresh medium supplemented with the polymer plates were incubated for 1.5 h (FCV) or 2 h (FHV-1) at 37 °C. Following
was added. Plates were incubated for 30 min at 37 °C and subsequently incubation, cells were washed twice with PBS and overlaid with fresh
medium with polymer was discarded and 50 μl of polymer solution or medium containing the polymer. Plates were incubated for 18 h (FCV)
control sample in 3% DMEM with mock or virus (800 TCID50/ml) was or 48 h (FHV-1) at 37 °C. After incubation, supernatants were collected,
added. Plates were incubated for 1.5 h (FCV) or 2 h (FHV-1) at 37 °C, and cells were washed twice with PBS, lysed with two freeze-thaw
supernatants were discarded and cells were washed twice with PBS. cycles and collected. The virus was quantified in supernatants and cell
Finally, 100 μl of polymer solution or control sample in 3% DMEM was lysates by qPCR and plaque assay.
added to each well and cells were incubated for 18 h (FCV) or 48 h
(FHV-1). After that time virus was quantified by qPCR and plaque 2.9. Synthesis of fluorescently labeled PSSNa70kDa
assay.
Fluorescently labeled PSSNa (PSSNa70kDa−F) was synthesized as
2.8. Mechanism of action described previously (Sohn et al., 1996), the details are described in
Supplementary Materials and Methods. The synthesis is schematically
Functional assays aimed to reveal the mechanism of polymer action presented in Scheme 1.
were carried out as described below.
2.10. Synergistic effect
2.8.1. Assay I. Inactivation of virions
This assay allows determining whether the tested compound affects Synergistic effect of antiviral drugs was assessed as previously de-
directly the viral particle. Briefly, concentrated virus suspension was scribed (Benzekri et al., 2018), with some modifications. All dilutions
incubated at 22 °C with polymers for 1 h. Subsequently, samples were were prepared using DMEM medium. Virus replication assay on fully
diluted to decrease polymer concentration below the active range and confluent CrFK cells was performed. After 48 h the supernatants were
virus titer is evaluated by plaque assay. collected, and virus yields and titers were assessed by qPCR and plaque
assay, respectively. The synergy was evaluated by calculating the
2.8.2. Assay II. Cell protection combination index (CI) (Benzekri et al., 2018).
This assay allows determining whether the polymer protects the cell
from viral infection. CrFK cells were seeded on 96-wells plates 24 h 2.11. Statistical analysis
prior to the inoculation. Fully confluent cells were incubated in the
presence or absence of polymer for 1 h at 37 °C. Subsequently, plates Statistical analyses were performed with GraphPad Prism 7: one-
were washed twice with PBS, then fresh medium containing mock or way ANOVA with Tukey HSD post hoc test or non-parametric Kruskal-
virus (400 TCID50/ml) was added to each well and plates were in- Wallis one-way variance analysis with Dunn's post hoc test. Data are
cubated for 1.5 h or 2 h at 37 °C for FCV and FHV-1, respectively. presented as the mean ± SD (standard deviation), mean ± SEM
Subsequently, plates were washed twice with PBS, overlaid with fresh (standard error of the mean) or medians ± interquartile range (when
medium and incubated for 18 h (FCV) or 48 h (FHV-1) at 37 °C. After non-parametric tests were applied) from at least three independent
that time, supernatants were collected, and the virus was quantified by experiments. p-value < 0.05 was considered as statistically significant.
qPCR and plaque assay.
3. Results
2.8.3. Assay III. Virus attachment
This assay allows determining if polymers are able to block the at- 3.1. PSSNa inhibits FHV-1 and FCV replication in CrFK cell line at non-
tachment of virus particles to cellular receptors. CrFK cells were seeded toxic concentration
on 96-wells plates 24 h prior to the inoculation. Fully confluent CrFK
cells were cooled at 4 °C for 20 min. Subsequently, cells were overlaid XTT (2,3-bis-(2-methoxy-4-nitro-5-sulphenyl)-(2H)-tetrazolium-5-
with ice-cold medium containing mock or virus (400 TCID50/ml) with carboxanilide) assay was performed on CrFK cells to examine the cy-
or without the polymer. Plates were incubated for 1 h at 4 °C. While totoxicity of eleven PSSNa polymers that differed in molecular weight
intracellular transport at 4 °C is hampered, viral adsorption to cellular (Mw). The assay relies on the ability of viable cells to convert XTT into

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A. Synowiec, et al. Antiviral Research 170 (2019) 104563

Fig. 1. Cytotoxicity of PSSNa polymers in CrFK cells. Cell viability was determined using XTT assay. PSSNa polymers (200 μg/ml) of different Mw [kDa] were used
(A), for PSSNa93.5kDa and PSSNa780kDa higher concentrations of 500, 1000 and 2000 μg/ml were tested (B). The data are presented as the percentage of the reference,
untreated sample for which viability was set to 100%. Results are presented as mean ± SEM from three independent experiments.

Fig. 2. PSSNa inhibits FCV and FHV-1 replication at non-toxic concentration. Experiments were carried out for each PSSNa polymer of different Mw at a
concentration of 200 μg/ml (FCV) or 20 μg/ml (FHV-1). Inhibition of viral infection was assessed by qPCR and presented as log change of virus RNA/DNA copies per
milliliter (A, C) or plaque assay and presented as PFU log change (B, D). Results were normalized to the values of untreated, infected cells and are presented as
mean ± SEM from three independent experiments. To determine the significance of differences between compared groups, one-way ANOVA with post hoc Tukey
HSD test was used. Values statistically significant are indicated by asterisks: *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.001, values statistically non-
significant (p > 0.05) are indicated by “ns”.

the colored derivative, which correlates with cell viability. No cyto- Furthermore, the dermal toxicity of PSSNa1000kDa was tested in vivo
toxicity was observed following the 48 h of incubation at tested con- (BALB/C mice model). Firstly, XTT assay was performed on CrFK cells
centrations (Fig. 1). to determine the highest non-toxic in vitro concentration of PEG400 that

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Fig. 3. Antiviral activity of PSSNa against FCV and FHV-1 is dose dependent. Virus replication assay was carried out for each PSSNa polymer. Inhibition of FCV
and FHV-1 infection by PSSNa polymers was evaluated using qPCR and presented as log change of virus RNA/DNA copies per milliliter (A, B, E, F) or plaque assay
and presented as PFU (plaque forming unit) log change (C, D, G, H). Results were normalized to the values of untreated, infected cells and are presented as
mean ± SEM from three independent experiments.

was 30 mg/ml (Supplementary Fig. 1A). Subsequently, antiviral activity Necropsy did not reveal any alteration of the internal organs and the
of the hydrogel containing PEG400 (30 mg/ml) and PSSNa1000kDa biochemical blood analysis did not reveal any abnormalities (data not
(200 μg/ml) against FCV and FHV-1 was confirmed in vitro shown). The details regarding the methodology of dermal toxicity stu-
(Supplementary Figs. 1B–E). The hydrogel with polymers or saline were dies are given in supplementary materials and methods.
administered daily for 7 days and no toxicity was observed at 50, 75, To examine antiviral potency of PSSNa Virus Replication Assay was
and 100 mg/ml. Mice were monitored each day and skin redness, le- performed for each polymer at 20 μg/ml for FHV-1 and at 200 μg/ml for
sions or other alterations were not observed during the experiment. FCV. Obtained results are presented in Fig. 2 and indicate that almost

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Table 1 For further studies, PSSNa93.5kDa and PSSNa780kDa were selected due
Determined IC50 values. to their high antiviral activity and significant difference in their Mw.
Target species Polymer IC50 ± SD [μg/ml] The IC50 values for PSSNa93.5kDa and PSSNa780kDa qPCR and plaque
assays were used. The results are presented in Fig. 3 and calculated IC50
qPCR Plaque Assay values are listed in Table 1.
93.5kDa
FHV-1 C-27 PSSNa 2.25 ± 1.01 5.74 ± 1.32
PSSNa780kDa 2.28 ± 1.01 5.06 ± 1.33 3.2. Internalization of PSSNa to the cell
FCV F9 PSSNa93.5kDa 42.75 ± 2.46 49.51 ± 0.14
PSSNa780kDa 9.72 ± 1.05 10.47 ± 1.47
To determine whether PSSNa polymers are internalized to the cell,
fluorescently labeled PSSNa with Mw = 70 kDa (PSSNa70kDa−F) was
Table 2 synthesized. Elemental analysis revealed the presence of nitrogen in the
Elemental composition of the non-labeled and labeled PSSNa70kDa. obtained polymer that unambiguously confirmed the substitution of
PSSNa70kDa with fluoresceinamine. The elemental composition of the
Polymer Elemental composition [%]
polymer before and after substitution is presented in Table 2. In the
N C H S FTIR spectra of PSSNa70kDa–F (Nicolet iS10 FT-IR spectrometer
equipped with an ATR accessory (SMART iTX); Supplementary Fig. 2),
PSSNa70kDa – 37.69 3.778 14.50 a weak band at 1318 cm−1 appeared that is characteristic for sulfona-
PSSNa70kDa−F 0.29 37.93 4.895 15.54
mides (-SO2-NH-). In comparison with the spectrum of PSSNa70kDa, an
additional band appeared at 947 cm−1 in the spectrum of PSSNa70kDa–F.
all PSSNa polymers with Mw above 8 kDa block FHV-1 replication at The electronic absorption spectra recorded in the visible spectral region
20 μg/ml, but there is no relationship between the Mw and the antiviral for the labeled polymers resemble that of fluoresceinamine in water
activity (Fig. 2A and B). On the other hand, the correlation between the (Supplementary Fig. 3).
Mw and the antiviral activity against FCV was observed, as PSSNa with Localization of polymer after the incubation was analyzed with
higher Mw inhibited FCV replication more efficiently (Fig. 2C and D). confocal microscopy after 2, 6 and 24 h. Presented images show that
PSSNa70kDa−F is slowly absorbed and accumulated in the cell

Fig. 4. PSSNa polymers inhibit FHV-1 replication mainly at early stages of the infection. Inhibition of viral replication in CrFK cells by PSSNa polymers at a
concentration of 20 μg/ml was evaluated with qPCR and presented as log change of virus DNA copies per milliliter (A) or plaque assay and presented as PFU (plaque
forming unit) log change (B). Assay I: inactivation; Assay II: cell protection; Assay III: virus attachment; Assay IV: late stages. The decrease in virus titers in Assay I
(Inactivation) for increasing concentrations of PSSNa93.5kDa (C) and PSSNa780kDa (D) was presented. Results were normalized to the values of untreated, infected cells
and are presented as mean ± SEM from three independent experiments. To determine the significance of differences between compared groups, one-way ANOVA
with post hoc Tukey HSD test was used. Values statistically significant are indicated by asterisks: *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.001, values
statistically nonsignificant (p > 0.05) are indicated by “ns”.

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Fig. 5. PSSNa inhibits FHV-1 adhesion to the cell surface. Two functional assays: Cell Protection Assay (Assay II) and Attachment Assay (Assay III) were
performed. CrFK cells were incubated in media supplemented with 200 μg/ml or in the absence of PSSNa polymer. (A, E) mock-infected cells (B, F) virus-infected
cells (C, G) virus-infected cells in the presence of PSSNa93.5kDa (D, H) virus-infected cells in the presence of PSSNa780kDa. Immunostaining was performed as described
in the Supplementary information section. Blue color denotes nuclei, red f-actin, and green FHV-1 virions. Representative images are shown as maximum projections
of XY stacks. Scale bar: 10 μm. The quantitative analysis of images was performed. The number of counts per cell was assessed using ImageJ FiJi and is presented as %
of the virus control mean (I, J). The data is presented as mean ± SEM from two independent experiments and was calculated from at least 10 different cells. To
determine the significance of differences between compared groups, one-way ANOVA with post hoc Tukey HSD test was used. Values statistically significant are
indicated by asterisks: ****p < 0.001, values statistically nonsignificant (p > 0.05) are indicated by “ns”.

(Supplementary Fig. 4). surface. Further, to ensure the unbiased results, virions were scored and
numerical results are presented in Fig. 5I and J.
The hypothesis that the polymer binds to virus particles and blocks
3.3. PSSNa inhibits FHV-1 binding to the cell by direct interaction with their interaction with cellular receptors was also tested. Inactivation of
virions virions assay (Assay I) was performed using PSSNa93.5kDa and
PSSNa780kDa at six different concentrations (10, 20, 50, 100, 200 and
To determine, whether PSSNa affects FHV-1 attachment to the cell 500 μg/ml). Virus stock was pre-incubated in the presence of polymer
surface virus, Assay III was performed. Plaque assay and qPCR showed and the virus’ infectivity was tested afterward using the plaque assay.
complete inhibition of FHV-1 replication by both PSSNa (Fig. 4A and Results are presented in Fig. 4C and D. Significant decrease in viral
B). Assay II was performed to determine whether PSSNa polymers in- yield was observed for concentrations exceeding 50 μg/ml. Further, a
teract with the cell and may protect it from viral infection. No sig- surface binding assay was performed to find out if FHV-1 virion binding
nificant decrease in virus yields or titers was detected (Fig. 4A and B). to the surface coated with the polymer occurs. Confocal visualization of
To confirm that PSSNa interacts with FHV-1 and blocks its adhesion viruses on the surfaces coated with fluorescently labeled PSSNa70kDa−F
to the cells, virions bound to the cell were visualized with confocal allowed to determine that virions bind to the polymer-coated surface
microscopy (Fig. 5A–H). It is clearly visible that virus–cell binding is more effectively than to non-coated surfaces (Fig. 6A). To confirm the
abolished in the presence of polymers. However, pre-incubation of cells obtained results, virions bound to the surfaces were scored and
with the polymer did not affect the number of virions bound to the cell

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Fig. 6. FHV-1 virions interact with PSSNa polymers. In surface binding assay coverslips were coated with fluorescently labeled PSSNa polymer and nonspecific
binding sites were blocked with bovine collagen solution (Purecoll) or fetal bovine serum (FBS) or not blocked (control). The red color denotes FHV-1 virions, while
the green color corresponds to the PSSNakDa70−F. Selected images are presented as maximum projections of XZ stacks. Scale bar: 10 μm. To determine if the increase
in the number of bound viruses is statistically significant, the number of virions was counted from 12 different focal planes obtained in two independent experiments
and the results are presented in panel (B). Kruskal-Wallis one-way variance analysis with Dunn's post hoc test was applied. The data are presented as medians ±
interquartile range. Values statistically significant are indicated by asterisks (****, p < 0.0001, ***, p < 0.001, **, p < 0.01), values statistically nonsignificant
(p > 0.05) are indicated by “ns”.

numerical results are presented in Fig. 6B. fluorescently labeled PSSNa of higher Mw was not water-soluble.
It was shown that the polymer interacts directly with the viral Nonetheless, confocal visualization of viruses on the surfaces coated
particle and blocks its attachment and entry to the cell. It is worth to with PSSNa780kDa allowed to determine that also FCV virions bind to the
note that also in Assay IV that aimed to determine whether polymers polymer-coated surface more effectively than to non-coated surfaces
may interfere with virus replication, assembly or egress, mild, but sig- (Fig. 9A). Further, to ensure the unbiased results, virions bound to the
nificant decrease in virus replication was noted (Fig. 4A and B). How- surfaces were scored and numerical results are presented in Fig. 9B.
ever, an additional mechanism of action at later stages of infection was We observed that only PSSNa of high Mw blocks FCV adhesion to the
excluded after performing the experiment where samples were col- cell, but the antiviral effect of PSSNa of lower Mw was also observed.
lected right after one round of FHV-1 replication cycle ~16 h (data not Assay IV was performed to determine whether PSSNa interferes with
shown). viral replication, assembly, and egress of FCV. Significant inhibition
was observed for both tested polymers, as presented in Fig. 7C and D.
3.4. PSSNa780kDa inhibits FCV replication and, to a lower extent, also early As virus titers and yields were decreased in supernatants and in cell
stages of the infection lysates, we believe that the polymers inhibit virus replication stage.

Mechanism of PSSNa action was also delineated for FCV. First, we 3.5. Clinical strains
made an effort to determine whether viral particles are able to attach to
the cell surface in the presence of polymer (Assay III). Pre-cooled cells To ensure that the inhibition is not limited to cell culture-adapted
were infected with FCV (400 TCID50/ml) in the presence of strains, we recovered clinical isolates for both species from the clinical
PSSNa93.5kDa or PSSNa780kDa at 200, 500 and 1000 μg/ml and incubated material collected in fall of 2018 (FHV-1 K7 and FCV K3) from cats with
for 1 h at 4 °C. 18 h p.i. virus yields and titers were assessed. symptoms of URTD and tested PSSNa activity against isolated viruses.
PSSNa780kDa but not PSSNa93.5kDa inhibited FCV infection at 500 μg/ml CrFK cells were incubated in the presence of PSSNa93.5kDa and
and higher (Fig. 7A and B). Assay II revealed that pre-incubation of cells PSSNa780kDa polymers at 20, 200, and 500 μg/ml (FHV-1 K7); or 200,
with PSSNa polymers did not affect the FCV infection (Fig. 7C and D), 500, and 1000 μg/ml (FCV K3) through the whole infection process.
showing that the polymers do not protect the cells from the infection. Viral titers and yields were evaluated after the infection. Obtained re-
Confocal analysis confirmed these observations, as FCV adhesion to sults, presented in Fig. 10 A, B, indicate that FHV-1 K7 was fully in-
the cells in the presence of PSSNa780kDa was limited, while no alteration hibited by both PSSNa polymers. Inhibition of FCV K3 virus was weaker
in virus adhesion was visible for PSSNa93.5kDa (Fig. 8A–H). To confirm but still significant and PSSNa780kDa restricted viral infection more ef-
these observations, virions bound to the cell surface were scored and ficiently (Fig. 10 C, D).
numerical results are presented in Fig. 8I and J.
The surface binding assay was performed, where FCV virion binding 3.6. Synergistic effect of PSSNa780kDa and ACV
to the surface coated with the polymer was evaluated. Unfortunately,
we were not able to visualize the PSSNa70kDa−F polymer, as FCV does To determine whether PSSNa and nucleoside analogs used in the
not bind to PSSNa of Mw as low as that of PSSNa70kDa−F, while treatment of herpetic infections show synergistic, additive or

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A. Synowiec, et al. Antiviral Research 170 (2019) 104563

Fig. 7. PSSNa polymers inhibit FCV replication and adhesion. Inhibition of viral replication in CrFK cells by PSSNa polymers was evaluated with qPCR and
presented as log change of virus DNA copies per milliliter (A, C) or plaque assay and presented as PFU (plaque forming unit) log change (B, D). Assay I: Inactivation;
Assay II: Cell Protection; Assay III: Virus Attachment; Assay IV: Late Stages. The decrease in virus titers in Assay III for increasing concentrations of PSSNa93.5kDa and
PSSNa780kDa (A, B) was presented. Results were normalized to the values of untreated, infected cells and are presented as mean ± SEM from three independent
experiments. To determine the significance of differences between compared groups, one-way ANOVA with post hoc Tukey HSD test was used. Values statistically
significant are indicated by asterisks: *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.001, values statistically nonsignificant (p > 0.05) are indicated by “ns”.

antagonistic effect, combined drug effect assay was performed. 4. Discussion

Referring to literature, IC50 value for ACV varies from 13.5 to 56 μg/ml
(Maggs and Clarke, 2004; van der Meulen et al., 2006) and from 0.3 to Feline upper respiratory tract disease (URTD) is a common disease,
32.9 μg/ml for PCV (Maggs and Clarke, 2004; Hussein et al., 2008; caused mainly by feline herpesvirus type 1 (FHV-1) and/or feline ca-
Williams et al., 2004). First, XTT assay was performed to exclude drug- licivirus (FCV), which may have fatal sequelae if not managed appro-
related toxicity. Second, virus replication assay was performed. Calcu- priately. Available prophylactic measures – a trivalent vaccine (FVRCP)
lated IC50 for FHV-1 C-27 at 400 TCID50/ml equaled to containing feline herpesvirus (FHV), feline calicivirus (FCV) and feline
12.1 ± 1.37 μg/ml for ACV and 1.37 ± 1.11 μg/ml for PCV, as de- panleukopenia virus (FPV) antigens is not fully protective and the ex-
termined by qPCR (Supplementary Fig. 5). posure still leads to the infection and virus transmission (Scott and
Subsequently, the synergistic effect of ACV/PCV and PSSNa780kDa Geissinger, 1999). Current FHV-1 therapy is based on virostatic agents,
was investigated. As shown in Table 3, calculated CI values were 0.92 mainly nucleoside analogs (NA) and concomitant antibiotic therapy to
for PSSNa780kDa/ACV and 0.46 for PSSNa780kDa/PCV. Obtained results manage secondary bacterial infections. No antiviral therapy is available
indicate that ACV and PCV exhibit synergistic effect while combined for FCV.
with PSSNa780kDa, and that this effect is more pronounced for PCV, In order to identify the active compounds, we screened a wide li-
which is used in the treatment of FHV-1 infection. brary of polymers. Some compounds were active against other her-
pesviruses, including dextran substituted with GTMAC (Pachota et al.,
2017), poly(sodium 4-styrenesulfonates) (PSSNa) (Herold et al., 2000)
and cationic block polymers (Pachota et al., 2019). Unexpectedly,

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A. Synowiec, et al. Antiviral Research 170 (2019) 104563

Fig. 8. PSSNa inhibits viral adhesion to the cell surface. Two functional assays: Cell Protection Assay (Assay II) and Attachment Assay (Assay III) were performed.
CrFK cells were incubated in media supplemented with 200 μg/ml (Assay II), 1000 μg/ml (Assay III) or in the absence of PSSNa polymer. (A, E) mock-infected cells
(B, F) virus-infected cells (C, G) virus-infected cells in the presence of PSSNa93.5kDa (D, H) virus-infected cells in the presence of PSSNa780kDa. Immunostaining was
performed as described in the Supplementary information section. Blue color denotes nuclei, red f-actin, and green FCV virions. Representative images are shown as
maximum projections of XY stacks. Scale bar: 10 μm. The quantitative analysis of images was performed. The number of counts per cell was assessed using ImageJ
FiJi and is presented as % of the virus control mean (I, J). The data are presented as mean ± SEM from two independent experiments and were calculated from at
least 10 different cells. To determine the significance of differences between compared groups, one-way ANOVA with post hoc Tukey HSD test was used. Values
statistically significant are indicated by asterisks: ****p < 0.001, values statistically nonsignificant (p > 0.05) are indicated by “ns”.

dextran derivatives and cationic block polymers did not exhibit anti- with PSSNa reduced the number of virions attached to the cells and
FHV activity (data not shown), but PSSNa effectively inhibited viral consequently limited virus replication. Subsequently, we confirmed
replication and was selected for further studies. Surprisingly, PSSNa that PSSNa interacts with FHV-1 virions and blocks their attachment to
exhibited antiviral potency also against FCV and to the authors’ the cells. Here, the inhibitory effect was not dependent on the polymer
knowledge it is the first report where one compound is able to inhibit size and we believe that the polymer binds to the well-defined GAG-
two viral agents associated with feline URTD. What is more, the poly- binding site on the viral surface and blocks its attachment. The local
mers were able to abolish replication of laboratory (FHV-1 C-27, FCV charge distribution seems to be more important here, while the poly-
F9) and clinical strains (FHV-1 K7, FCV K3). meric chain conformation is less relevant. Of note, if the polymer was
To select the optimal compound, we tested a series of polymers present at later stages of the infection, it also abolished replication of
varying in Mw. In this study, we show that PSSNa exhibit promising the virus, but this resulted from an inhibition of multi-cycle infection
antiviral activity against FHV-1 and FCV. While no association between and virus transmission instead of an additional mechanism of action.
polymer Mw and antiviral activity was noted for FHV-1, such associa- PSSNa is a sulfonated anionic derivative of polystyrene. It is known for
tion was observed for FCV. its broad-spectrum antimicrobial activity, as it was reported to inhibit
To resolve the mechanism of PSSNa action we performed an array of bacteria and viruses (Herold et al., 2000; Anderson et al., 2000;
functional assays. While pre-incubation of the polymer with cells did Christensen et al., 2001; Simoes et al., 2002). Considering that anti-
not affect virus attachment and entry, pre-incubation of FHV-1 virions microbial potency of PSSNa is mainly observed during early stages of

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A. Synowiec, et al. Antiviral Research 170 (2019) 104563

Fig. 9. FCV interacts with high molecular

weight PSSNa. In surface binding assay
coverslips were coated with PSSNa93.5kDa
and PSSNa780kDa polymer and nonspecific
binding sites were blocked with bovine
collagen solution (Purecoll) or fetal bovine
serum (FBS) or not blocked (control). The
red color denotes FCV virions. Selected
images are presented as maximum projec-
tions of XZ stacks (A). Scale bar: 10 μm. To
determine if the increase in the number of
bound viruses is statistically significant, the
number of virions was counted in 12 dif-
ferent focal planes obtained in two in-
dependent experiments and the results are
presented in panel (B). Kruskal-Wallis one-
way variance analysis with Dunn's post hoc
test was applied. The data are presented as
medians ± interquartile range. Values
statistically significant are indicated by as-
terisks (****, p < 0.0001, **, p < 0.01),
values statistically nonsignificant
(p > 0.05) are indicated by “ns”.

Fig. 10. Antiviral activity of PSSNa against clinical strains of FCV and FHV-1 is dose dependent. Virus replication assay was performed as described. Two
veterinary strains FCV K3 (A, B) and FHV-1 K7 (C, D) were used, antiviral activity was evaluated with qPCR and presented as log change of virus RNA/DNA copies
per milliliter (A, C) or plaque assay and presented as PFU (plaque forming unit) log change (B, D). Experiments were carried out for two PSSNa polymers of different
Mw (PSSNa93.5kDa and PSSNa780kDa) at 200, 500 and 1000 μg/ml for FCV K3 or at 20, 200 and 500 μg/ml for FHV K7. Results were normalized to the values of
untreated, infected cells are presented as mean ± SEM from three independent experiments.

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A. Synowiec, et al. Antiviral Research 170 (2019) 104563

Table 3 Funding sources

ACV, PCV and PSSNa780kDa effect on FHV-1 replication.
IC50 [μg/ml] CI This work was supported by the Leading National Research Center,
ACV ACV (PSSNa780kDa) PSSNa780kDa PSSNa780kDa (ACV) Poland (Grant No. 6/SPB/2017 to A.S.) and National Science Center,
12.1 2.96 2.45 1.64 0.92 Poland (UMO-2014/13/B/ST5/04510 to M.N.; UMO-2012/07/E/NZ6/
PCV PCV (PSSNa780kDa) PSSNa780kDa PSSNa780kDa (PCV) 01712 to K.P.). K.P. acknowledges networking contribution by the
1.37 0.31 2.45 0.55 0.46
COST Action CM1407 “Challenging organic syntheses inspired by
IC50 – concentration required to obtain 50% of inhibitory concentration in nature - from natural products chemistry to drug discovery”. Faculty of
presence of compound [μg/ml], IC50 “Compound X (Compound Y)” – con- Biochemistry, Biophysics and Biotechnology and Faculty of Chemistry
centration required to obtain 50% of inhibitory concentration in presence of of Jagiellonian University are partners of the Leading National Research
compound X [μg/ml] when the concentration of compound Y is constant and Center (KNOW) supported by the Ministry of Science and Higher
equal to IC50 , CI – combination index.
2
Education.
The funders had no role in study design, data collection, and ana-
lysis, decision to publish, or preparation of the manuscript.
the infection, it was proposed that it mimics negatively charged gly-
cosaminoglycans (GAGs; e.g., heparan sulfate) and binds to virions or Author contributions
microbes at the GAGs binding sites, consequently blocking their at-
tachment to the cell (Ito et al., 1989; Baba et al., 1988; Mohan et al., The manuscript was written through the contributions of all au-
1992; Zacharopoulos and Phillips, 1997). Considering this property it thors. All authors have given approval to the final version of the
was surprising to observe that PSSNa also inhibits FCV infection, as it manuscript.
was reported that FCV does not require GAGs to enter the cell (Stuart
and Brown, 2007). Functional assays allowed us to determine that the Acknowledgments
mechanism of polymer action was somewhat different in this case.
Polymers inhibited the viral replication and we assume that they di- Authors would like to thank veterinarians from The Shelter for
rectly or indirectly affect essential steps of the viral replication cycle, as Homeless Animals in Cracow, directed by The Animal Welfare
viral enzymes or their cellular partners. However, further studies are Organization in Cracow (KTOZ) for taking and providing swabs from
necessary to elucidate the detailed mechanism of action. Interestingly, cats. Authors would also like to thank Artur Szczepanski M.Sc. and
polymers with higher Mw were more potent against FCV due to their Zbigniew Baster M.Sc. for technical support and helpful discussions.
ability to inhibit also the attachment of viruses to susceptible cells.
Intriguingly, FCV does not bind to GAGs and they do not have a well- Appendix A. Supplementary data
defined binding site that may be blocked with the negatively charged
PSSNa, as it was proposed for FHV-1. We believe that in this case, Supplementary data to this article can be found online at https://
PSSNa binds to the surface of the virion and forms a protective shell doi.org/10.1016/j.antiviral.2019.104563.
that sterically blocks its interaction with the cell. To do so, it must be of
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