# The sulphated polysaccharides extract ulvans from Ulva armoricana limits Marek's disease virus dissemination in vitro and promotes viral reactivation in lymphoid cells

Frédérick Bussy, Sylvie Rémy, Matthieu Le Goff, Pi Nyvall Collén, Laëtitia

Trapp-Fragnet

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Frédérick Bussy, Sylvie Rémy, Matthieu Le Goff, Pi Nyvall Collén, Laëtitia Trapp-Fragnet. The sulphated polysaccharides extract ulvans from Ulva armoricana limits Marek's disease virus dissemination in vitro and promotes viral reactivation in lymphoid cells. BMC Veterinary Research, 2022, 18 (1), 9 p. 10.1186/s12917-022-03247-y. hal-03699467

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# The sulphated polysaccharides extract ulvans from *Ulva armoricana* limits Marek's disease virus dissemination in vitro and promotes viral reactivation in lymphoid cells

Frédérick Bussy¹,², Sylvie Rémy<fnref n="3" />, Matthieu Le Goff¹,², Pi Nyvall Collén¹,² and Laëtitia Trapp-Fragnet<fnref n="3" />*

## Abstract

**Background:** Marek's disease (MD) is a highly contagious lymphoproliferative disease of chickens caused by an alphaherpesvirus, Marek's disease virus (MDV). MD is presently controlled by systematic vaccination of animals, which protects efficiently against the development of clinical disease. However, MDV vaccines do not prevent the multiplication and spread of MDV field strains and may favor the emergence of strains with increased virulence. Therefore, MDV persists to be a major problem for the poultry industry and the development of new alternative strategies to control MDV is needed. Seaweed extracts have previously been shown to exert immunomodulatory and antiviral activities, especially against herpesviruses. The objective of the present study was to explore the effect of *Ulva armoricana* extracts on MDV infection in vitro.

**Results:** We could demonstrate that the ulvan extract as well as its vitamin-enriched formulation reduce the viral load by about 80% at 24 h post-infection in infected chicken fibroblasts at concentrations that are innocuous for the cells. We also observed a substantial decrease in MDV plaque size suggesting that ulvans impede MDV cell-to-cell spread in vitro. Moreover, we showed that ulvan extract could promote MDV reactivation in lymphoid cells.

**Conclusions:** Our data provide the first evidence that the use of the ulvan extract could be a good alternative to limit MDV infection in poultry.

**Keywords:** Marek's disease virus, Seaweed, *Ulva armoricana*, Searup®, Cytotoxicity, Viral replication, Viral dissemination, Viral reactivation

## Background

Marek's disease virus (MDV) is an alphaherpesvirus inducing lethal lymphoma in chickens. MDV infection progresses in 3 major phases: (i) an early cytolytic infection corresponding to the entry of the virus

through the respiratory tract and its transport to the lymphoid organs where it infects B-cells and activated T-lymphocytes; (ii) a latent phase (between 7 to 10 days post-infection (pi)) in CD4+T lymphocytes, which is characterized by the integration of the viral genome into the genome of the host cells; (iii) the tumor phase occurring in CD4+T lymphocytes leading to malignant lymphoma formation and the death of animals from the 3rd week of infection [1, 2].

<footnote>*Correspondence: laetitia.trapp-fragnet@inrae.fr</footnote>

<footnote n="3">³ INRAE, Université de Tours, ISP, F-37380 Nouzilly, France</footnote>

<footnote>Full list of author information is available at the end of the article</footnote>
Concomitantly, a late cytolytic phase resulting from MDV reactivation in latently-infected lymphocytes occurs. The capacity of MDV to reactivate constitutes a perennial source of virus production and contributes to the spread of the virus to the skin, more particularly to the cells of the feather follicle (the only site of production and excretion of free infectious viral particles).

MDV is endemic worldwide and, despite the avail-
ability of vaccines, MDV continues to threaten the
poultry industry with economic losses estimated at
1–2 billion dollars per year [3, 4]. Vaccination protects
infected animals against the development of tumors
induced by MDV, but does not prevent the replication
and the shedding of the virus in poultry flocks [5]. In
addition, the vast vaccination campaigns implemented
since the end of the 1960s have caused the virus to
gradually evolve towards higher pathogenicity allowing
the virus to overcome the protection provided by cur-
rent vaccines [6–8]. To date, no treatment (other than
vaccine prophylaxis) is available to control the replica-
tion of the virus and its dissemination in the environ-
ment. Consequently, the development of alternative
and sustainable strategies to control MDV is needed.

Numerous scientific publications have reported that algae extracts or algae-derived compounds can exhibit inhibitory activities against viral infections (for reviews [9, 10]). The first study demonstrating the anti-viral activity of seaweed extracts against herpesvirus infections was reported in 1974 [11]. Since then, many active compounds in algae have been shown to interfere with the viral life cycle of alphaherpesviruses Herpes Simplex virus type 1 and 2, the betaherpesvirus Human Cytomegalovirus and the gammaherpesviruses Human Herpesvirus type 6 and 7 [12–25]. Algae polysaccharides can exert their anti-viral activities by preventing the entry and multiplication of the virus in its target cell, and/or by modulating the immune response [10].

While research on the antiviral and immunomodu-
latory properties of algae is sparking great interest
in human medicine, studies related to animal health
are still scarce, particularly with regard to herpesvi-
rus infections. We recently demonstrated that ulvans,
the sulfated polysaccharides (SPs) fraction purified
from *Ulva armoricana*, is able to activate chicken het-
erophils and monocytes in vitro and in vivo [26]. This
suggested that ulvans can potentially be used as an
immunostimulant for controlling infectious diseases.
Here, we explored whether the ulvans fraction and its
formulated version, dubbed and commercialised as
Searup® could reduce MDV lytic replication and MDV
reactivation from latently infected cells in vitro.

**Results**

The ulvans SPs fraction does not alter the viability of avian cells

Prior to determining the effect of ulvans and Searup® on MDV replication, we evaluated the cytotoxicity of the extracts on avian cells. Chicken embryo fibroblasts (CEFs) and the lymphoid cell line 3867K were treated with four increasing doses (0.5; 1; 1.5 and 2ml/l) of the vitamin complex (VitComp), Searup® or the ulvans extract. Untreated cells were maintained in culture and used as negative control. In order to exclude any side effects induced by degradation products of the compounds and/or cellular metabolites, the culture media was replaced after 48h by fresh media supplemented (or not for the control condition) with the defined treatment doses. At 24, 48, 72 and 96h post-treatment, the viability of CEFs was monitored by evaluating the level of ATP produced by the cells (CellTiter Glo assay) and the viability of 3867K was assessed by flow cytometry using the Viability Dye eFluor™ 780.

We could show that the purified ulvans extract and the
vitamin complex had virtually no cytotoxic effect on pri-
mary chicken fibroblasts (Fig. 1A). Treatment with the
highest concentration (2 ml/l) of Searup® induced a mild
decrease of viability of the CEFs between 24 to 72 hpt,
but no cytotoxicity was observed at lower doses. This
effect of high-dose Searup® treatment on the metabolic
activity of CEFs (ATP production) appeared to be revers-
able since the cells recovered at 96 hpt and may instead
reflect a delay in cell proliferation.

The MDV transformed T-lymphocytes (3867 K) treated with the different formulations showed a level of viability similar to untreated cells (of about 80%), independently of the doses used (Fig. 1B). A slight decrease in the viability of 3867 K (albeit not statistically significant) was detected from 72 h after treatment with Searup® and the vitamin complex. However, this effect of the Searup® seems to be mainly associated with the vitamin complex contained in the formulated Searup® since the pure SPs extract (ulvans) was completely innocuous for the 3867 K cells. Consequently, our results indicate that the various treatments and in particular the ulvans extract showed a very limited cytotoxicity on avian lymphoid and fibroblast cells.

Ulvans extract reduces MDV dissemination in vitro

To determine whether ulvans treatment can impact
MDV lytic replication, we infected CEFs and treated the
infected cells with increasing doses of the vitamin com-
plex, Searup® and the ulvans extract. The viral load was
quantified at 24, 48, 72 and 96 hpi by qPCR. Our results
showed that all treatments significantly decreased MDV
<figure><img src="image_3.png" /><figcaption>**Fig. 1** Ulvans does not affect avian cell viability. Chicken embryo fibroblasts (CEFs) and MDV transformed T-lymphocytes (3867 K) were treated with vitamin complex (VitComp), Searup® or the ulvans extract at the indicated concentrations or left untreated (NT). At indicated time points (hpt), (A) the viability of the CEFs cells was assessed using the CellTiter-Glo® kit. Results are presented as fold-change of ATP production values assessed for treated cells relative to ATP produced in non-treated cells (NT = 1). (B) The viability of 3867 K cells was estimated by flow cytometry using the Fixable Viability Dye eFluor™ 780. Three independent experiments are represented as means (+/- SEM) of the percentage of viable 3867 K cells (eFluor negative). Statistics analyses did not show any difference (*P* > 0.05)</figcaption></figure>

lytic replication in CEFs (Fig. 2A) in a dose-dependent fashion with the strongest effect observed with concentrations from 1 ml/l. From 24 hpi, the viral load is reduced by about 80% following the treatment with Searup® and the ulvans extract at a concentration of 2 ml/l (the recommended dose in farms corresponding to the administration of 1 ml/l/24 h). This decrease is then maintained over time and reaches 89 and 94% at 96 hpi, respectively. To

determine whether this reduced viral load was related to an activity of the ulvans on the replication of the virus or on its cell-to-cell spread, we counted and measured the plaques of MDV infection obtained after the treatments. We showed that the number of MDV plaques did not differ upon treatment (Fig. 2B). However, we observed a reduction of 2- and 3-fold in the size of MDV plaque upon treatment with the ulvans extract and Searup®,
<figure><img src="image_4.png" /></figure>

respectively (Fig. 2C). These results showed that the SPs fraction of the purified from *Ulva armoricana* as well as the commercialized Searup® formulation can impede MDV dissemination in vitro in the concentration range recommended in poultry farms (1 ml/l/24 h).

### Ulvans promotes viral reactivation in MDV transformed lymphoid cells

We assessed whether the two ulvans formulations and the VitComp control could have an effect on the maintenance of MDV latency and viral reactivation (corresponding to the late cytolytic phase of infection) in vitro. To this end, we used the 3867 K cells, a lymphoblastoid cell line that expresses the green fluorescent protein (GFP) fused to the tegument protein UL47 upon reactivation, as described previously [27, 28]. The 3867 K cells were treated with the vitamin complex, Searup® and ulvans

at different concentrations and the rate of viral reactivation (GFP positive cells within the viable cell population) was evaluated by cytometry upon 96 h of treatment (Fig. 3A and B). In cells treated with the vitamin complex or the ulvan extract, we observed that the proportion of cells in which the virus reactivated (GFP positive cells) increased similarly to the untreated cells from 24 to 96 hpt, independent of the doses used in the assay. At 96 hpt, we detected 7.6, 8.6 and 9.4% of GFP positive cells among the untreated cells, the cells treated with VitComp and the cells treated with the ulvan extract, respectively. Albeit not statistically different, the Searup® tended to induce an increase in viral reactivation since we observed 14% of GFP positive cells after 96 h of treatment at the highest dose (2 ml/l). We then investigated further the effect of the ulvans on viral latency/reactivation by treating the 3867 K cells with the VitComp, Searup® or ulvan
<figure><img src="image_5.png" /></figure>

extract (at 2 ml/l) in combination with sodium butyrate (NaBut 0.5 mM), a histone deacetylase inhibitor known to induce herpesvirus reactivation. The viral reactivation was assessed by cytometry at 24, 48 and 72 hpt (Fig. 3C). As expected, the NaBut treatment substantially increased the proportion of cells in which the MDV reactivated, reaching 9.5% of GFP positive cells at 72 hpt, while in untreated cells only 1.5% were GFP positive. This increase in viral reactivation is also detected upon co-treatment of the cells with NaBut together with the VitComp, Searup® or Ulvan extract. The level of reactivation in cells treated

with NaBut and the vitamin complex is comparable to the reactivation in cells treated with NaBut treatment alone, despite a slight transitory increase at 48 hpt. The ulvan extract showed only a slight effect on the NaBut-induced reactivation at 72 hpt (11% of GFP positive cells). However, in cells treated with Searup® we could clearly observe a higher proportion of cells in which the virus reactivated with 8.9 and 15% of GFP positive cells at 48 and 72 hpt, respectively. These results suggest that ulvans did not prevent a chemically-induced reactivation but
that the ulvans fraction formulated in the Searup® version might rather promote MDV reactivation.

## Discussion

The intensification of chicken farms around the world, the incomplete protection of anti-MDV vaccines and the emergence of new hypervirulent pathotypes of MDV suggest that MDV could become a major problem in the poultry industry if new therapies or vaccines are not developed. Most efforts to control MDV are focused on developing next-generation vaccines. Although many natural products or chemicals have been shown to be effective against many viral infections affecting humans and animals, only a few studies have reported antiviral activity of compounds against MDV infection [29–32]. Here, we have shown that ulvan extract can significantly reduce the replication and dissemination of MDV in vitro with a limited cytotoxicity in avian lymphoid cells.

Several studies reported that algae SPs are able to directly inhibit transcription/replication of alpha and gammaherpesviruses after viral internalization in the host cell [11–13, 15, 16, 18–20, 24, 33]. Our data demonstrate that *Ulva armoricana* SPs also limit MDV replication in chicken fibroblasts since the viral load was strongly reduced while the number of MDV plaques was similar to that of untreated cells. In addition, the significant reduction of MDV plaque size suggests that the ulvans extract prevents MDV spread. Algae SPs have also been shown to be able to hinder the early stages of viral infection (adsorption/attachment and penetration of the virus) by binding directly to viruses or to viral receptors present on the surface of cells [16, 22, 23, 34–37]. MDV is strictly associated to cells and no extracellular viral particles are produced in vitro. Therefore, ulvans SPs cannot exert a direct neutralizing activity on MDV virions but might rather have an effect on cell permissiveness to MDV infection, which in turn reduces MDV spread by cell-to-cell fusion. Previous reports indeed showed that SPs from different sources could be powerful inhibitors of viral cell-cell spread and/or fusion [34, 38–40]. Also, in our previous study we have found that ulvan extract of *Ulva armoricana* can modulate the immune response notably by triggering the expression of a number of pro-inflammatory cytokines (e.g. IL-1β, IFN-α and IFN-γ) [26]. We thus cannot exclude that *Ulva armoricana* SPs extract can also control MDV infection/dissemination through the secretion of factors such as IFN-α which was shown to strongly impede the infectivity of MDV [41].

Aside the effect of the ulvan extract, we showed that the vitamin complex alone also reduced the replication of MDV. Nutrients, notably vitamins A and D, are known to exert antiviral activities and to stimulate

immunity (for review [42]). Likewise, even though the vitamins contained in the algae formulation are low-concentrated, we cannot exclude that they may potentiate the effect of the ulvans extract.

In latently infected lymphoid cells, we observed that treatments with ulvans have a limited effect on viral reactivation when used alone. However, in combination with a reactivation-inducing chemical treatment (sodium butyrate), SPs extract from *Ulva* and especially its formulated version Searup® could promote the reactivation of MDV. We have not currently identified the mechanisms/factors triggered by ulvans that may contribute to MDV reactivation. However, among the many mechanisms identified to induce herpesvirus reactivation, we suspect that ulvans may promote MDV reactivation through the induction of pro-inflammatory cytokines expression, oxidative stress and/or the modulation of key cellular pathways (for review [43]). Interestingly, we previously demonstrated that ulvan extract of *Ulva armoricana* can activate heterophils and monocytes via the activation of Toll-Like receptor 2 and 4, resulting in the release of pro-inflammatory cytokines, including IFN-γ, which is a potent inducer of herpesvirus reactivation [26, 44]. Also in the case of Epstein Barr latent infection, it was shown that lytic cycle can be induced through TLR2 and B-cell receptor activation [45]. In addition, ulvans induced a strong oxidative burst in heterophils and monocytes accompanied by a significant secretion of nitric oxide (NO). Oxidative stress and reactive oxygen species are powerful inducers of herpesvirus reactivation, as we have shown in particular for MDV [46, 47]. Finally, ulvans may promote the re-entry into lytic cycle by modulating several cellular pathways involving notably protein kinase C, p38MAPK, c-Jun N-terminal kinase (JNK), ERK kinase and PI3 kinase, which have been identified to trigger herpesvirus reactivation [43, 48].

In conclusion, we demonstrated that the *Ulva armoricana* SPs extract ulvans and its commercialized Searup® formulation considerably reduce the dissemination of MDV in vitro with limited to no cytotoxicity for avian cells. Clearly the anti-MDV activities of ulvans warrant further investigation in vivo, however, based on our data it can be speculated that the ulvans SPs extract could limit MDV infection at the early stages of infection. Ulvans could indeed exert an antiviral activity by limiting replication/dissemination of MDV and/or by stimulating the innate immune system of chickens [26]. In addition, at a later time of infection, treatment of chickens with ulvans could promote MDV secondary cytolytic infection (viral reactivation) occurring in MDV transformed T-cells. The resulting expression of MDV antigens and viral production could thus help to “purge” the
cells latently infected either by the immune system or by
virus-associated toxicity.

Conclusion

Altogether, our results identify ulvans as a natural sea-
weed extract with potent antiviral activity against MDV,
and potentially other avian viruses, that could be readily
exploited in alternative treatment strategies for control-
ling viral diseases in poultry farming.

Methods

Cells and virus

Chicken embryonic fibroblasts (CEFs) were prepared
from 12-day-old specific pathogen-free White Leghorn
(LD1) embryos and maintained in culture as previously
described [49]. CEFs infection was carried out in P6-well
plates with 225 plaque forming unit/well of the recEGF-
PVP22 recombinant MDV [50]. The 3867K-lympho-
blastoid cell line was established from a renal lymphoma
induced by the highly pathogenic MDV clone vRB-
1B 47EGFP and maintained in culture as previously
described [27].

Treatments

Cells were treated with increasing doses of (i) the *Ulva
armoricana* SPs fraction (ulvans) (ii) Searup® (Olmix
SA) composed of the ulvans fraction formulated with
vitamins A and D3 and (iii) the vitamin solution without
seaweed extracts (VitComp) containing vitamins A and
D3 according to the European legislation (EU 2015/724
and EU 2017/1492). The range of concentration (0; 0.5;
1; 1.5 and 2 ml/l) of SPs extract from *Ulva* corresponds
to 11,5 mg/L to 46 mg/L of active seaweed ingredient. In
the formulated version Searup®, the dose of SPs extract
range between 8 mg/l to 32 mg/L. Of note, the concen-
trations used in the study was defined according to the
recommended dose of Searup® (1 ml/l) used in poultry
farms. Treatments were added to the culture media of
infected CEFs from 1h post-infection (hpi). The 3867 K
cells were cultured at a density of 5.10⁵ cells/P6-well
plates in media containing the different treatments. All
media supplemented with the different treatments doses
were renewed after 48 h of culture. In a second set of
experiment, the 3867 K cells were treated with 2 ml/l of
VitComp, Searup® or ulvans extract in combination with
0.5 mM of sodium butyrate (NaBut; Sigma-Aldrich), a
histone deacetylase inhibitor known to trigger herpesvi-
rus reactivation [28, 51, 52]. Cells were treated for 24 to
96 h depending on the test performed. All media supple-
mented with the different treatments doses were renewed
after 48 h of culture.

Cell viability assays

The viability of CEFs incubated with the different concen-
trations of VitComp, Searup® and ulvans was assessed by
quantification of ATP levels using the CellTiter-Glo® kit
(Promega) according to the manufacturer's instructions.
Cells (5 × 10³) were grown in opaque-walled 96-well
plates (Thermo Scientific) in 100 µl complete media con-
taining the different doses of VitComp, Searup® or ulvan
extract. At 24, 48, 72 and 96 h post-treatment (hpt), cells
were washed in PBS1X and incubated with 100 µl of ATP
detection reagent for 10 min at room temperature in the
dark. Luminescence was measured using a GloMax-Multi
Detection System (Promega). Relative ATP production
values for treated cells were calculated as fold changes
with reference to mean values obtained for non-treated
cells (set to 1). Three independent experiments were per-
formed in triplicate.

The viability of 3867 K lymphoid cells was assessed at indicated time points using the Fixable Viability Dye eFluor™ 780 (eBioscience) according to standard procedures. Viable cells were detected by flow cytometry using a MoFlo AstriosEQ high-speed cell sorter (Beckman Coulter Inc., Brea, CA, USA). A first gating was based on morphological criteria (FSC/SSC) in order to eliminate cellular debris and damaged cells. A second gating was then performed based on the eFluor 780 staining which allowed us to discriminate between dead (eFluor positive) and viable (eFluor negative) cells.

Detection of MDV replication

CEFs infected with the recEGFPVP22 virus were treated
at 1 hpi with increasing doses (0.5, 1, 1.5 and 2 ml/l) of
the different treatments. Infected CEFs left untreated
were used as negative control. MDV replication was
assessed by real-time quantitative PCR (qPCR) as previ-
ously described [53–55]. At 24, 48, 72 and 96 hpi, DNA
was extracted using the QIAamp DNA minikit (Qia-
gen) according to the manufacturer's instructions. MDV
genome copy numbers were quantified by qPCR using
primers and probes targeting the viral ICP4 gene and
normalized to 10⁶ copies of the cellular genome quanti-
fied by detection of the *Gallus gallus* inducible nitric
oxide synthase gene (*iNOS*).

In 3867K cells, MDV reactivation (lytically infected
cells) was detected by flow cytometry based on the
expression of the late antigen UL47-GFP. First, viable
cells were selected on morphological criteria (FSC/SSC)
and the eFluor 780 staining. The eFluor 780-negative
population (viable cells) was then further gated for UL47-
GFP expression to determine the percentage of reacti-
vated cells (eFluor 780 negative/UL47-GFP positive).
## Virus cell-to-cell spread by plaque size assay

At 96 hpi, CEFs infected with MDV and treated with the vitamin complex, Searup® and ulvans were fixed using 4% paraformaldehyde and MDV plaque numbers were counted. MDV dissemination was evaluated by measuring the size of plaques visualized by GFP expression, as previously described [50]. At least 50 plaques were measured for each treatment condition.

## Statistical analyses

All graphs and statistics were performed using the GraphPad Prism software version 5.02 (San Diego, USA). Data are presented as means and standard error of the mean (±SEM) from at least 3 independent experiments. The Kruskal-Wallis test was used to compare differences in multiple groups and the Mann-Whitney (two-tailed) test was used to compare non-parametric variables between two groups. *p* values < 0.05 were considered statistically significant as indicated in the figure legends.

## Abbreviations

MD: Marek's disease; MDV: Marek's disease virus; SPs: Sulfated polysaccharides; CEFs: Chicken embryo fibroblasts; GFP: Green fluorescent protein.

## Acknowledgments

We thank Alix Sausset and Yves Levern (INRAE, Nouzilly, France) for FACS analyses.

## Authors' contributions

FB, MLG and PNC prepared the ulvans extract. LFT and SR designed the study, performed the in vitro experiments, analysed the data. LFT wrote the manuscript. All authors read and approved the manuscript.

## Funding

This study was supported by Amadéite, Bréhan (France). The funders were not involved in the study design, collection, analyses, interpretation of data, the writing of this article or the decision to submit it for publication.

## Availability of data and materials

The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.

## Declarations

### Ethics approval and consent to participate

The preparation of chicken fibroblasts from embryos performed in the present study did not require any authorization from the local ethics committee. The French regulation (Art. R.214-88) states that embryos from oviparous species (including the chicken) before the last third of their normal development (which is clearly the case for the 12-days-old embryos being used in our study) are not submitted to any regulation.

### Consent for publication

Not applicable.

### Competing interests

FB, PNC and MLG are employed by company Amadéite, now Olmix SA. Searup® is a commercialised product sold by Olmix SA. All other authors declare that the research was conducted in the absence of any commercial

or financial relationships that could be construed as a potential conflict of interest.

## Author details

¹Amadeite SAS, 56580 Bréhan, France. ²Olmix, SA Le Lintan, 56580 Bréhan, France. ³INRAE, Université de Tours, ISP, F-37380 Nouzilly, France.

Received: 10 November 2021 Accepted: 31 March 2022

Published online: 27 April 2022

## References

1. Calnek BW. Pathogenesis of Marek's disease virus infection. Curr Top Microbiol Immunol. 2001;255:25-55.

2. McPherson MC, Delany ME. Virus and host genomic, molecular, and cellular interactions during Marek's disease pathogenesis and oncogenesis. Poult Sci. 2016;95(2):412-29.

3. Nair V. Evolution of Marek's disease — a paradigm for incessant race between the pathogen and the host. Vet J. 2005;170(2):175-83.

4. Morrow C, Fehler F. Marek's disease: a worldwide problem. In: Davison F, Nair V, editors. Marek's Disease: An Evolving Problem. San Diego: Elsevier Academic Press; 2004. p. 49-61.

5. Jarosinski KW, Tischer BK, Trapp S, Osterrieder N. Marek's disease virus: lytic replication, oncogenesis and control. Expert Rev Vaccines. 2006;5(6):761-72.

6. Shi MY, Li M, Wang WW, Deng QM, Li QH, Gao YL, et al. The emergence of a vv + MDV can break through the protections provided by the current vaccines. Viruses. 2020;12(9):1048.

7. Witter RL. Increased virulence of Marek's disease virus field isolates. Avian Dis. 1997;41(1):149-63.

8. Witter RL, Calnek BW, Buscaglia C, Gimeno IM, Schat KA. Classification of Marek's disease viruses according to pathotype: philosophy and methodology. Avian Pathol. 2005;34(2):75-90.

9. Pagarete A, Ramos AS, Puntervoll P, Allen MJ, Verdelho V. Antiviral potential of algal metabolites-a comprehensive review. Mar Drugs. 2021;19(2):94.

10. Hans N, Malik A, Naik S. Antiviral activity of sulfated polysaccharides from marine algae and its application in combating COVID-19: Mini review. Bioresour Technol Rep. 2021;13:100623.

11. Deig EF, Ehresmann DW, Hatch MT, Riedling DJ. Inhibition of herpesvirus replication by marine algae extracts. Antimicrob Agents Chemother. 1974;6(4):524-5.

12. Abrantes JL, Barbosa J, Cavalcanti D, Pereira RC, Frederico Fontes CL, Teixeira VL, et al. The effects of the diterpenes isolated from the Brazilian brown algae Dictyota pfaffii and Dictyota menstrualis against the herpes simplex type-1 replicative cycle. Planta Med. 2010;76(4):339-44.

13. Bouhlal R, Haslin C, Chermann JC, Colliec-Jouault S, Sinquin C, Simon G, et al. Antiviral activities of sulfated polysaccharides isolated from Sphaero-coccus coronopifolius (Rhodophyta, Gigartinales) and Boergeseniella thuyoides (Rhodophyta, Ceramiales). Mar Drugs. 2011;9(7):1187-209.

14. de Souza LM, Sassaki GL, Romanos MT, Barreto-Bergter E. Structural characterization and anti-HSV-1 and HSV-2 activity of glycolipids from the marine algae Osmundaria obtusiloba isolated from southeastern Brazilian coast. Mar Drugs. 2012;10(4):918-31.

15. Faral-Tello P, Mirazo S, Dutra C, Perez A, Geis-Asteggiante L, Frabasile S, et al. Cytotoxic, virucidal, and antiviral activity of south American plant and algae extracts. Sci World J. 2012;2012:174837.

16. Harden EA, Falshaw R, Carnachan SM, Kern ER, Prichard MN. Virucidal activity of polysaccharide extracts from four algal species against herpes simplex virus. Antivir Res. 2009;83(3):282-9.

17. Lee JB, Hayashi K, Maeda M, Hayashi T. Antiherpetic activities of sulfated polysaccharides from green algae. Planta Med. 2004;70(9):813-7.

18. Lopes N, Ray S, Espada SF, Bomfim WA, Ray B, Faccin-Galhardi LC, et al. Green seaweed Enteromorpha compressa (Chlorophyta, Ulvaceae) derived sulphated polysaccharides inhibit herpes simplex virus. Int J Biol Macromol. 2017;102:605-12.

19. Naesens L, Bonnafous P, Agut H, De Clercq E. Antiviral activity of diverse classes of broad-acting agents and natural compounds in HHV-6-infected lymphoblasts. J Clin Virol. 2006;37(Suppl 1):S69-75.
20. Naesens L, Stephens CE, Andrei G, Loregian A, De Bolle L, Snoeck R, et al. Antiviral properties of new arylsulfone derivatives with activity against human betaherpesviruses. Antivir Res. 2006;72(1):60–7.

21. Park HJ, Kurokawa M, Shiraki K, Nakamura N, Choi JS, Hattori M. Antiviral activity of the marine alga Symphyocladia latiuscula against herpes simplex virus (HSV-1) in vitro and its therapeutic efficacy against HSV-1 infection in mice. Biol Pharm Bull. 2005;28(12):2258–62.

22. Sinha S, Astani A, Ghosh T, Schnitzler P, Ray B. Polysaccharides from Sargassum tenerrimum: structural features, chemical modification and anti-viral activity. Phytochemistry. 2010;71(2–3):235–42.

23. Sun QL, Li Y, Ni LQ, Li YX, Cui YS, Jiang SL, et al. Structural characterization and antiviral activity of two fucoidans from the brown algae Sargassum henslowianum. Carbohydr Polym. 2020;229:115487.

24. Vo TS, Ngo DH, Ta QV, Kim SK. Marine organisms as a therapeutic source against herpes simplex virus infection. Eur J Pharm Sci. 2011;44(1–2):11–20.

25. Wozniak M, Bell T, Denes A, Falshaw R, Itzhaki R. Anti-HSV1 activity of brown algal polysaccharides and possible relevance to the treatment of Alzheimer's disease. Int J Biol Macromol. 2015;74:530–40.

26. Guriec N, Bussy F, Gouin C, Mathiaud O, Quero B, Le Goff M, et al. Ulvan activates chicken Heterophils and monocytes through toll-like receptor 2 and toll-like receptor 4. Front Immunol. 2018;9:2725.

27. Denesvre C, Remy S, Trapp-Fragnet L, Smith LP, Georgeault S, Vautherot JF, et al. Marek's disease virus undergoes complete morphogenesis after reactivation in a T-lymphoblastoid cell line transformed by recombinant fluorescent marker virus. J Gen Virol. 2016;97(2):480–6.

28. Mallet C, Cochard J, Leclercq S, Trapp-Fragnet L, Chouteau P, Denesvre C. Hypoxia and HIF-1 trigger Marek's disease virus reactivation in lymphoma-derived latently infected T lymphocytes. J Virol. 2021;96(5):e0142721.

29. Sun Y, Niu L, Song M, Zhao X, Sun N, He J, et al. Screening compounds of Chinese medicinal herbs anti-Marek's disease virus. Pharm Biol. 2014;52(7):841–7.

30. Wild M, Kicuntod J, Seyler L, Wangen C, Bertzbach LD, Conradie AM, et al. Combinatorial drug treatments reveal promising Anticytomegaloviral profiles for clinically relevant pharmaceutical kinase inhibitors (PKIs). Int J Mol Sci. 2021;22(2):575.

31. Yang F, Feng C, Yao Y, Qin A, Shao H, Qian K. Antiviral effect of baicalin on Marek's disease virus in CEF cells. BMC Vet Res. 2020;16(1):371.

32. Bertzbach LD, Conradie AM, Hahn F, Wild M, Marschall M, Kaufer BB. Artesunate derivative TF27 inhibits replication and pathogenesis of an oncogenic avian alphaherpesvirus. Antivir Res. 2019;171:104606.

33. Duarte ME, Noseda DG, Noseda MD, Tulio S, Pujol CA, Damonte EB. Inhibitory effect of sulfated galactans from the marine alga Bostrychia montagnei on herpes simplex virus replication in vitro. Phytomedicine. 2001;8(1):53–8.

34. Aguilar-Briseno JA, Cruz-Suarez LE, Sassi JF, Ricque-Marie D, Zapata-Benavides P, Mendoza-Gamboa E, et al. Sulphated polysaccharides from Ulva clathrata and Cladosiphon okamuranus seaweeds both inhibit viral attachment/entry and cell-cell fusion, in NDV infection. Mar Drugs. 2015;13(2):697–712.

35. Damonte EB, Matulewicz MC, Cerezo AS. Sulfated seaweed polysaccharides as antiviral agents. Curr Med Chem. 2004;11(18):2399–419.

36. Sato Y, Morimoto K, Hirayama M, Hori K. High mannose-specific lectin (KAA-2) from the red alga Kappaphycus alvarezii potently inhibits influenza virus infection in a strain-independent manner. Biochem Biophys Res Commun. 2011;405(2):291–6.

37. Talarico LB, Zibetti RG, Faria PC, Scolaro LA, Duarte ME, Noseda MD, et al. Anti-herpes simplex virus activity of sulfated galactans from the red seaweeds Gymnogongrus griffithsiae and Cryptonemia crenulata. Int J Biol Macromol. 2004;34(1–2):63–71.

38. Cagno V, Donalisio M, Civra A, Volante M, Veccelli E, Oreste P, et al. Highly sulfated K5 Escherichia coli polysaccharide derivatives inhibit respiratory syncytial virus infectivity in cell lines and human tracheal-bronchial histocultures. Antimicrob Agents Chemother. 2014;58(8):4782–94.

39. Hardouin K, Bedoux G, Burlot A-S, Donnay-Moreno C, Bergé J-P, Nyvall-Collén P, et al. Enzyme-assisted extraction (EAE) for the production of antiviral and antioxidant extracts from the green seaweed Ulva armori-cana (Ulvales, Ulvophyceae). Algal Res. 2016;16:233–9.

40. Nyberg K, Ekblad M, Bergström T, Freeman C, Parish CR, Ferro V, et al. The low molecular weight heparan sulfate-mimetic, PI-88, inhibits cell-to-cell spread of herpes simplex virus. Antivir Res. 2004;63(1):15–24.

41. Levy AM, Heller ED, Leitner G, Davidson I. Effect of native chicken interferon on MDV replication. Acta Virol. 1999;43(2–3):121–7.

42. Thirumdas R, Kothakota A, Pandiselvam R, Bahrami A, Barba FJ. Role of food nutrients and supplementation in fighting against viral infections and boosting immunity: a review. Trends Food Sci Technol. 2021;110:66–77.

43. Weidner-Glunde M, Kruminis-Kaszkiel E, Savanagouder M. Herpesviral Latency-Common Themes. Pathogens. 2020;9(2):125.

44. Chang J, Renne R, Dittmer D, Ganem D. Inflammatory cytokines and the reactivation of Kaposi's sarcoma-associated herpesvirus lytic replication. Virology. 2000;266(1):17–25.

45. Hirsiger JR, Fuchs PS, Hausermann P, Muller-Durovic B, Daikeler T, Recher M, et al. Syphilis reactivates latent Epstein-Barr virus reservoir via toll-like receptor 2 and B-cell receptor activation. Open forum. Infect Dis. 2019;6(9):ofz317.

46. Bencherit D, Remy S, Le Vern Y, Vychodil T, Bertzbach LD, Kaufer BB, et al. Induction of DNA damages upon Marek's disease virus infection: implication in viral replication and pathogenesis. J Virol. 2017;91(24):e01658–17.

47. Hu J, Li H, Luo X, Li Y, Bode A, Cao Y. The role of oxidative stress in EBV lytic reactivation, radioresistance and the potential preventive and therapeutic implications. Int J Cancer. 2017;141(9):1722–9.

48. Li H, Liu S, Hu J, Luo X, Li N, MB A, et al. Epstein-Barr virus lytic reactivation regulation and its pathogenic role in carcinogenesis. Int J Biol Sci. 2016;12(11):1309–18.

49. Dorange F, El Mehdaoui S, Pichon C, Coursaget P, Vautherot JF. Marek's disease virus (MDV) homologues of herpes simplex virus type 1 UL49 (VP22) and UL48 (VP16) genes: high-level expression and characterization of MDV-1 VP22 and VP16. J Gen Virol. 2000;81(Pt 9):2219–30.

50. Blondeau C, Marc D, Courvoisier K, Vautherot JF, Denesvre C. Functional homologies between avian and human alphaherpesvirus VP22 proteins in cell-to-cell spreading as revealed by a new cis-complementation assay. J Virol. 2008;82(18):9278–82.

51. Brown AC, Nair V, Allday MJ. Epigenetic regulation of the latency-associated region of Marek's disease virus in tumor-derived T-cell lines and primary lymphoma. J Virol. 2012;86(3):1683–95.

52. Hicks JA, Trakooljul N, Liu HC. Alterations in cellular and viral microRNA and cellular gene expression in Marek's disease virus-transformed T-cell lines treated with sodium butyrate. Poult Sci. 2019;98(2):642–52.

53. Jarosinski KW, Margulis NG, Kamil JP, Spatz SJ, Nair VK, Osterrieder N. Horizontal transmission of Marek's disease virus requires US2, the UL13 protein kinase, and gC. J Virol. 2007;81(19):10575–87.

54. Remy S, Blondeau C, Le Vern Y, Lemesle M, Vautherot JF, Denesvre C. Fluorescent tagging of VP22 in N-terminus reveals that VP22 favors Marek's disease virus (MDV) virulence in chickens and allows morphogenesis study in MD tumor cells. Vet Res. 2013;44:125.

55. Jarosinski KW, Yunis R, O'Connell PH, Markowski-Grimsrud CJ, Schat KA. Influence of genetic resistance of the chicken and virulence of Marek's disease virus (MDV) on nitric oxide responses after MDV infection. Avian Dis. 2002;46(3):636–49.

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