Skip to main content
Download PDF

Canid alphaherpesvirus 1 infection alters the gene expression and secretome profile of canine adipose-derived mesenchymal stem cells in vitro

Virology Journal volume 21, Article number: 336 (2024) Cite this article

Abstract

Background

Canine adipose-derived mesenchymal stem cells (cAD-MSCs) demonstrate promising tissue repair and regeneration capabilities. However, the procurement and preservation of these cells or their secreted factors for therapeutic applications pose a risk of viral contamination, and the consequences for cAD-MSCs remain unexplored. Consequently, this research sought to assess the impact of canid alphaherpesvirus 1 (CHV) on the functional attributes of cAD-MSCs, including gene expression profiles and secretome composition.

Methods

To this end, abdominal adipose tissue from 12 healthy dogs was harvested to isolate cAD-MSCs. These samples were tested for CHV contamination before introducing a wild-type CHV strain via serial passages. Following CHV infection, real-time reverse transcription-polymerase chain reaction array and liquid chromatography with tandem mass spectrometry assessments enabled analyses of gene expression and secretome’s proteomic profile, respectively.

Results

This study showed that the initial cAD-MSC populations were devoid of CHV. cAD-MSCs showed susceptibility to infection with wild-type CHV, leading to notable modifications in gene expression and secretome profile. The observed genomic variations in gene expression indicate potential impacts on the stemness, migration, and other functional properties of cAD-MSCs, highlighting the need for further studies to evaluate their functional capacity post-infection. Moreover, gene expression and secretome analyses suggest a shift in stem cell differentiation toward an adipogenic phenotype.

Conclusion

To the best of our knowledge, this is the first study of the effects of virus infection on gene expression and secretome composition in cAD-MSCs. The outcomes of our study underscore the imperative of routine viral screening prior to the therapeutic use of cAD-MSCs. Moreover, these findings provide novel insights into the pathogenic mechanisms of CHV and pave the way for future canine stem cell and virus research.

Background

Mesenchymal stem cells (MSCs) have the capacity to regenerate tissue in various species, including canines, presenting significant potential for treating diseases with limited therapeutic options, such as osteoarthritis, spinal cord injuries, and chronic skin wounds [1,2,3]. One promising stem cell source in canines is adipose tissue, abundant with canine adipose-derived mesenchymal stem cells (cAD-MSCs), which are easy to obtain and proliferate rapidly compared to other types of MSCs [4,5,6]. The cAD-MSCs exhibit notable regenerative properties, particularly the ability to modulate immune responses by secreting relevant molecules, i.e., the secretome [5, 7, 8]. Moreover, the application of the cAD-MSC secretome for therapy, rather than the cells themselves, offers the potential for numerous lower-risk treatments [9,10,11,12].

Acquiring and storing cAD-MSCs and secretomes in therapeutic quantities necessitates in vitro cultivation, thereby introducing the risk of microbial contamination. Studies have indicated that stored MSC batches may be contaminated with bacteria, fungi, or viruses [13, 14]. Bacterial and fungal contamination can be effectively mitigated with antibiotics and antimycotics [13]; however, mitigating potential viral contamination remains challenging. Available data indicate that MSCs can be permissive to infection with RNA and DNA viruses, which can lead to cell death, persistent infection, or cellular transformation and may ultimately impair their functionality [15,16,17,18,19]. Viral infection of MSCs has been associated with the inhibition of differentiation [20], increased secretion of proinflammatory cytokines [21], and loss of immunomodulatory function [22]. In cAD-MSCs, one study reported the susceptibility to distemper virus [23], and recent findings confirmed the possibility of viral contamination of cryobanked cAD-MSC batches by detecting canine parvovirus, influenza, parainfluenza, and canid alphaherpesvirus 1 [24].

Canid alphaherpesvirus 1 (CHV) belongs to the species Varicellovirus canidalpha1 within the Orthoherpesviridae family, whose genome consists of double-stranded DNA [25]. The latent and subclinical persistence of CHV in dogs poses a significant challenge, potentially leading to infection oversight during routine clinical examination of cAD-MSC donors. Moreover, CHV is distributed globally, with seroprevalence ranging from 21.7% to 80% [26, 27], and nearly one-third of dogs are infected in Croatia [28]. This widespread prevalence makes CHV a considerable risk for contamination during sampling or culturing. Previous studies in human and equine MSCs have shown that herpesvirus infection can decrease the immunomodulatory effects of MSCs [22, 29, 30]. Since studies investigating this phenomenon in canines are currently lacking, this study aimed to assess the susceptibility and adaptability of cAD-MSCs to CHV through serial passages. Moreover, this study sought to explore the impact of CHV infection on the gene expression and secretome composition of cAD-MSCs.

Methods

Stem cell culture establishment and characterisation

Adipose tissue collection, cAD-MSCs extraction, and propagation

This study obtained adipose tissue samples from 12 clinically healthy dogs (Canis lupus familiaris), 11 females and one male who underwent elective surgery. The collection of adipose tissue, extraction of cAD-MSCs and propagation were performed according to previously established protocols [8, 31]. To accomplish the objectives of this investigation, we used cells from cAD-MSC donors 6/21, 9/21, 13/21, 14/21, 1/22, 2/22, 3/22, 6/22, and 7/22, which have been described in a prior publication [8]. In addition to these samples, cells from three novel donors (7/21, 8/21, and 8/22) were used following the same procedure. Nonetheless, this research provides a distinct objective, experimental framework, and conclusions by contrasting the baseline data from uninfected cAD-MSCs with new findings following CHV infection. Table 1 contains information on the age, breed, health status, adipose tissue collection site and mass of the donors. Sterility-tested cAD-MSCs for aerobic and anaerobic bacteria, fungi and mycoplasma were used for all experiments following a previously established protocol [31]. All donor cells were cryobanked in liquid nitrogen via the standard cryobanking procedure with 10% dimethyl sulfoxide (Sigma‒Aldrich, St. Louis, MO, USA, Cat. No. D2650-100ML) at passage 2 (P2) or P3 for future experiments.

Table 1 Canine adipose tissue donor information
Full size table

Immunophenotyping and multipotency testing of cAD-MSCs

As previously described [8], the immunophenotyping and multipotency testing of the cAD-MSCs were performed at P3. FACSVerse (BD, Franklin Lakes, NJ, USA) flow cytometry was used to confirm the immunophenotype, while adipogenic, osteogenic and chondrogenic in vitro differentiation was performed to verify multipotency, following the criteria of the International Society for Cellular Therapy [32].

Testing of established cAD-MSCs for CHV

All donors were tested for CHV to gain insight into the possible latent infection of cAD-MSCs extracted from adipose tissue. A cryobanked batch of cells per donor at P2/P3 was first transferred at -20 °C to induce lysis of the cell membranes. After 24 h, the cell lysate was thawed at room temperature for 30 min, vortexed and subjected to nucleic acid extraction using a MagMAX CORE nucleic acid purification kit (Thermo Fisher Scientific, Waltham, MA, USA, Cat. No. A32702) on a KingFisher Flex Purification System (Thermo Fisher Scientific) according to the manufacturer’s instructions. Quantitative real-time PCR (qPCR) was applied for the detection of the CHV glycoprotein B gene according to a previously published protocol [33] using a QuantiFast Pathogen PCR + IC Kit (Qiagen, Hilden, Germany, Cat. No. 211352) on a Rotor-Gene Q (Qiagen) instrument. Beta-actin served as an endogenous control, employing the same reagents, instrument, and a previously established protocol [34]. The reaction mixture setup and thermal cycling conditions were performed as recommended by the manufacturer. The reaction mixtures’ final primer and probe concentrations were adjusted to 1,000 nmol/L for CHV-For (5’-ACAGAGTTGATTGATAGAAGAGGTATG-3’) and CHV-Rev (5’-CTGGTGTATTAAACTTTGAAGGCTTTA-3’) and 500 nmol/L for CHV-Pb (5’-6-FAM-TCTCTGGGGTCTTCATCCTTATCAAATGCG-BHQ1-3’). For beta-actin, primers were adjusted to 83.3 nmol/L for ACT2-1030-F (5’-AGCGCAAGTACTCCGTGTG-3’) and ACT-1135-R (5’-CGGACTCATCGTACTCCTGCTT-3’) and 41.7 nmol/L for ACT-1081-HEX (5’-HEX-TCGCTGTCCACCTTCCAGCAGATGT-BHQ1-3’).

Isolation and characterisation of autochthonous wild-type CHV

CHV recovery from clinical specimen and virus stock production

The autochthonous wild-type CHV strain 29107 was obtained from the organs (liver, spleen, and lungs) of a 6-day-old golden retriever undergoing routine CHV diagnostics at the Croatian Veterinary Institute. The organ samples (1 × 1 cm each) were combined and homogenised with a cold mortar and pestle containing sterile sand and 10 mL of DMEM Low Glucose. The homogenate was freeze-thawed, centrifuged at 2,100 × g for 10 min, filtered using a Millex-HP syringe filter unit 0.45 µm (Merck, Darmstadt, Germany) and stored at − 80 °C. For in vitro propagation, the Madin-Darby Canine Kidney (MDCK) cell line (ATCC, Manassas, VA, USA; Cat. No. CCL-34), which is known to be susceptible to CHV [35], was used. Before inoculation, the MDCK cell line was confirmed to be CHV contamination-free. A 90% confluent MDCK (P34) monolayer in a T25 flask (Thermo Fisher Scientific) was infected with 1 mL of stock supernatant. Following two hours of adsorption at 37 °C (5% CO2, 80% humidity), 10 mL of basal medium (79% DMEM Low Glucose (Thermo Fisher Scientific, Cat. No. 31885049), 20% fetal bovine serum (FBS) (Thermo Fisher Scientific, Cat. No. 1027010), and 1% penicillin/streptomycin (Sigma‒Aldrich, Cat. No. P4333-100ML)) was added. Upon full CPE development (monitored using a Lux2 live imaging platform, Axion BioSystems, Atlanta, GA, USA) or 96 h postinfection (p.i.), the infected cell culture flask underwent a single freeze‒thaw-centrifugation cycle. The final CHV stock was generated after the third viral passage on MDCK cells in T75 flasks and stored at -80 °C.

CHV virus stock titration was conducted in triplicate using a confluent MDCK monolayer (P35) seeded in a 96-well microplate (Thermo Fisher Scientific). Eight separate tenfold dilutions of stock supernatant (100 µL per well) were added to the cells. After a two-hour adsorption period, 180 µL of the basal medium was added to the inoculum, and the plates were incubated at 37 °C with 5% CO2 and 80% humidity for 72 h. The virus titre (TCID50) was calculated using the Spearman–Kärber method.

Verification of the autochthonous wild-type CHV strain by NGS

To verify the autochthonous wild-type CHV strain 29107 and generate a whole-genome sequence, we performed next-generation sequencing (NGS). Specifically, viral DNA was extracted from 200 µL of CHV organ suspension homogenate using a DNA Blood and Tissue kit (Qiagen, Cat. No. 69506) according to the manufacturer’s instructions. Sequencing libraries were prepared using the Nextera XT DNA Library Preparation Kit (Illumina Inc., San Diego, USA, Cat. No. 15032354 and No. 15032355) with Nextera DNA UD Indexes (Illumina Inc., Cat. No. 20026934) and sequenced on a NextSeq 550 sequencer (Illumina Inc., Cat. No. SY-415-1002) loaded with a NextSeq 500/550 High Output Kit v 2.5 (300 cycles) (Illumina Inc., Cat. No. 20024908) following the manufacturer’s instructions. Library fragment size control and quantification were performed using a 2100 Bioanalyzer instrument with an Agilent High Sensitivity DNA Kit (Agilent Technologies, Santa Clara, CA, USA, Cat. No. 5067-4626) and a Qubit™ 4 Fluorometer with a Qubit dsDNA HS Assay Kit (Thermo Fisher Scientific, Cat. No. Q32854), respectively.

The sequence reads were assembled into contigs using the Spades software v3.15 [36]. The contigs were compared to known complete CHV genome sequences in NCBI GenBank. Sequence reads were mapped to the GenBank sequence MW353136 using Bwa mem v0.7.17 [37] and SAMtools v1.19 [38] and then used to generate a consensus assembly with Ivar v1.0 [39]. MW353136 was used as a reference because it was the most thoroughly covered by sequencing reads among the complete genome sequences of CHV (taxid:170325) in GenBank. The final novel sequence scaffold of the autochthonous wild-type CHV strain was scaffolded, curated and annotated manually. PROKKA software [40] was used to generate initial functional annotation. The autochthonous wild-type CHV strain 29107 genome sequence was deposited in GenBank under accession number PP349830.

Furthermore, phylogenetic analysis of the novel autochthonous wild-type CHV complete genome sequence was constructed from complete genome alignment of a total of 23 CHV sequences (22 reference sequences from the GenBank database) using IQTree2 software [41] and substitution model HKY + F + I. An optimal substitution model was found using ModelFinder [42]. Multiple sequence alignment was prepared using MAFFT software [43]. The phylogenetic tree was visualised with Python scripting with the help of the module Toytree with UFBoot node support [44] values shown. Calculation of the similarity plot was aided by the Python module Numpy, and visualisation was performed using the Toyplot module. Recombinations were analysed by RDP5 [45].

In vitro cAD-MSCs infection with wild-type CHV

CHV serial passages on cAD-MSCs

To demonstrate successful CHV infection in cultured cAD-MSCs, a cohort of six donors (9/21, 13/21, 14/21, 2/22, 3/22, and 7/22) was randomly chosen for five consecutive viral passage experiments. In contrast to prior experiments involving freshly utilized cAD-MSCs, cryopreserved cAD-MSCs at P2 or P3 were used for these specific infections. After thawing and expansion, cells from each donor were distributed as six replicates into 24-well plates (Thermo Fisher Scientific) at a density of 105 cells/well in 1 mL of basal medium and maintained at 37 °C and 5% CO2 (80% humidity) until they reached 90% confluence. Subsequently, the basal medium was removed, and three wells per donor were inoculated with CHV virus stock at a multiplicity of infection (MOI) of 0.5. Following a two-hour incubation period to allow virus adsorption, basal medium was added to the inoculum to a volume of 1 mL. The CHV infection experiments were conducted until the CPE reached 80% or for a maximum of 120 h if the CPE was minimal or absent. Upon meeting the criteria, the plates were frozen at − 80 °C. Thawed cell lysate suspensions from each donor triplicate were individually mixed, transferred to sterile 5 mL tubes (Eppendorf, Hamburg, Germany), and subjected to a second freezing cycle. After the second thaw, the cell lysate suspensions were centrifuged at 2665 × g for 10 min, and the supernatant was filtered through a 0.45 µm filter and stored in 2 mL cryovials (Cryoking, Newcastle, Australia). Subsequent passages of the CHV virus were initiated by preparing new 24-well plates as described previously, with 500 µL/well of the preceding viral passage used as inoculum for virus absorption. Five passages of the CHV virus were conducted for each cAD-MSC donor and control cell line (MDCK). The progression of CPE was documented using a microscopic camera (Axiocam ER/105/208/HD, Axio Observer D1, Zeiss, Jena, Germany) and a Lux 2 live imaging platform. Each passage included three wells of negative controls. Supernatants from each passage (CHV-infected and uninfected) were stored at -80 °C for further qPCR experiments.

Quantification of CHV DNA with qPCR

To confirm the success of the infection, i.e., the presence of viral DNA in the supernatant of infected cAD-MSCs, we quantified the number of CHV genome copies. Total DNA was extracted from 200 µL of filtered supernatant from each viral passage after two freeze–thaw cycles using a DNA Blood and Tissue Kit (Qiagen, Cat. No. 69506) according to the manufacturer’s cell extraction protocol. DNA was quantified using a Qubit 1X dsDNA High Sensitivity (HS) kit (Thermo Fisher Scientific, Cat. No. Q33230) on a Qubit 4 Fluorometer (Thermo Fisher Scientific). CHV detection was performed by qPCR as previously described.

For quantification, a triple 5-point standard curve was generated with quantitative genomic DNA from CHV strain D 004 (ATCC VR-552DQ, lot: 70054940). The following values of the standard curve were obtained: R2 = 0.99945, slope = − 3.57263, Y-intercept = 35.24627, and reaction efficiency = 91%. The limit of detection (LOD, ≥ 95% detection in 20 replicates) was 3.31 genomic copies (gc)/reaction. Theoretically, this assay provides an LOD of 6.44 × 102 gc/mL of cell lysate supernatant. The limit of quantification (LOQ, coefficient of variability ≤ 35% in 20 replicates) was set at 136.94 gc/reaction, theoretically providing a CHV LOQ of 2.66 × 104 gc/mL for the cell lysate supernatant. The results are presented as the mean ± SEM unless otherwise stated.

Gene expression profiling of CHV-infected cAD-MSCs

Gene expression analysis via RT‒qPCR array was conducted on P3 of CHV-infected and uninfected cAD-MSCs from twelve donors. Two T75 flasks (Thermo Fisher Scientific) were seeded with ≈106 cAD-MSCs per flask in basal medium and incubated until they reached 90% confluence (24–48 h). One flask was inoculated with CHV stock at an MOI of 0.5, allowing for a two-hour adsorption period, while the second flask served as a negative control. Twenty-four hours p.i., gene expression profiling was conducted following an established procedure [8]. In brief, RNA extraction was performed with an RNeasy Mini kit (Qiagen, Cat. No. 74106) following the manufacturer’s instructions. The quality of the extracted RNA was verified using an RNA QC kit (Qiagen, Cat. No. 50–727-743). Finally, gene expression profiling was conducted using the RT2 Profiler™ PCR Array for Dog Mesenchymal Stem Cells (PAFD-082ZR, Qiagen). The RNA QC and raw gene expression data from nine uninfected cAD-MSC donors (6/21, 9/21, 13/21, 14/21, 1/22, 2/22, 3/22, 6/22, and 7/22), which were previously published [8], were analysed together with new data from three additional uninfected cAD-MSC donors (7/21, 8/21, and 8/22). This way, a comparative analysis was performed with new results from all 12 CHV-infected cAD-MSC donors.

After data acquisition, the specialised RT2 Profiler PCR Array Data Analysis Software, accessible online at https://dataanalysis2.qiagen.com/pcr (accessed 19 March 2024), enabled normalisation and comprehensive analysis. The gene expression analysis results, researched gene names, symbols, and NCBI sequences are listed in Additional file 1. Statistical significance was determined via Student’s t-test applied to replicated 2^ (-Delta CT) values within both the control and treatment groups with p < 0.05. The software automatically established a fold change cut-off value of 2.0, corresponding to a log2fold change ± 1.0. Gene expression profile data were publicly deposited in the NCBI Gene Expression Omnibus database under accession number GSE267402. The data were visualised with GraphPad Prism 10.2.2.

Proteomic analysis of the CHV-infected cAD-MSCs secretome

The alterations in the proteomic composition of the secretome of cAD-MSCs were also analysed in P3 under two conditions, uninfected and CHV-infected cAD-MSCs, in six randomly selected donors (6/21, 9/21, 14/21, 1/22, 6/22, and 7/22). The cells were seeded in six replicates at 105 cells/mL density in 24-well plates (Thermo Fisher Scientific) and conditioned in the basal medium at 37 °C, 5% CO2 and 80% humidity until they reached 90% confluence. The culture medium was then aspirated, and three wells of uninfected cells were rinsed with 2 × 2 mL DMEM Low Glucose before being incubated in 2 mL of the same medium. On the other hand, three wells of CHV-infected cAD-MSCs were inoculated with a MOI 0.5 of CHV viral stock, the virus was allowed to absorb for 2 h, and then the CHV-infected cells were rinsed with 2 × 2 mL of DMEM Low Glucose and incubated in 2 mL of the same medium. Forty-eight hours later, the secretome of the cAD-MSCs was collected as previously described [8].

Following a previously published protocol [8], the samples were prepared for liquid chromatography with tandem mass spectrometry (LC–MS/MS) analysis. In brief, the secretome proteins were reduced and extracted from the culture medium. The protein concentrations were adjusted via the Bradford assay, and enzymatic digestion followed, with peptide separation conducted using the nanoLC EASY-nLC 1200 system (Thermo Fisher Scientific). The mass spectra were recorded using a Q Exactive Plus Hybrid Quadrupole-Orbitrap tandem mass spectrometer (Thermo Fisher Scientific).

Raw data analysis utilised Scaffold Quant Q + S 5.3.0, employing protein sequence data from the Canis lupus familiaris reference proteome (UniProt Proteome ID UP000805418, accessed on 30 October 2023, with a total of 20,991 entries). Scaffold Quant version 5.0.3 was utilised for subsequent analysis, employing untargeted label-free quantification and statistical analysis based on spectral counting. Statistical significance, verified via t-tests, was defined as p < 0.05, with proteins filtered to include only those with at least two identified peptide sequences. A cut-off value of 1.3 was applied, corresponding to a log2fold change ± 0.3785. The mass spectrometry proteomics data were deposited with the ProteomeXchange consortium via the PRIDE [46] partner repository with the dataset identifiers PXD052289 and https://doiorg.publicaciones.saludcastillayleon.es/10.6019/PXD052289. In this study, we incorporated previously published raw proteomic data from six uninfected cAD-MSC donors (6/21, 9/21, 14/21, 1/22, 6/22, and 7/22) [8] to facilitate a comparative analysis with new secretome proteome data from six cAD-MSCs following CHV infection.

Bioinformatics analysis of the detected proteins was performed with Gene Ontology (GO) Panther 18.0 to analyse cellular components, protein classes, molecular functions, and biological processes. GO enrichment analysis was used to determine affected protein pathways using Fisher’s exact test and false discovery rate (FDR) correction, with data presented as raw p values < 0.05 and FDR < 0.05. Additionally, a protein–protein interaction network analysis was conducted using STRING (v12.0) [47], employing a high confidence interaction score of 0.700, an FDR < 0.05, and a strength score > 0.75. To elucidate protein pathways and interactions lost due to CHV infection, the proteins secreted distinctively in uninfected samples were grouped with the biologically significant downregulated proteins (uninfected group). In contrast, distinct proteins secreted in CHV-infected samples were grouped with biologically significantly upregulated proteins (CHV-infected group) to elucidate the protein pathways activated after CHV infection. The data visualisation was performed with GraphPad Prism 10.2.2.

Results

Extracted cAD-MSCs showed stem cell properties and tested negative for CHV

The identity of the stem cells has already been published for nine cAD-MSC donors [8], except for donors 7/21, 8/21 and 8/22. All donors included in this study were positive for the mesenchymal markers CD90, CD105, CD44 and CD29 and negative for the hematopoietic markers CD34 and CD45 (Fig. 1). Furthermore, they differentiated into three mesodermal lineages: adipogenic, as confirmed by the presence of Oil Red O-positive fat droplets; osteogenic, as confirmed by purple staining indicating alkaline phosphatase activity; and chondrogenic, as confirmed by spheroids showing aggrecan (a proteoglycan in articular cartilage) when stained with Alcian blue (Fig. 1).

Fig. 1
figure 1

Canine adipose-derived mesenchymal stem cells (cAD-MSCs) multipotency and stem cell immunophenotype at passage 3 in vitro. A The morphology of undifferentiated cAD-MSCs: B osteogenic differentiation marked by the purple staining of alkaline phosphatase activity, C adipogenic differentiation evident by the presence of lipid droplets, stained red, D chondrogenic differentiation shown by Alcian Blue staining, with aggrecan appearing blue. A–D Microscopic images were captured using an Axiovert camera on an Axio Observer D1 microscope (Zeiss), with scale bars of 50 μm and 100 μm. E Whisker-box plot illustrates the Median Fluorescence Intensity (MFI) fold changes for surface markers CD90, CD105, CD44, and CD29 (upper plot), as well as CD73, CD271, CD45, and CD34 (lower plot) in cAD-MSCs at passage 3. The dotted line indicates the cut-off value of 1.5, and the legend shows individual MFI fold changes for each donor by colourful dots. F Representative flow cytometry histograms for each CD marker, with blue representing unstained cells, grey representing isotype controls, and black representing specific CD surface markers. Results of immunophenotype and differentiation analysis from 9 out of 12 canine-adipose derived mesenchymal stem cell donors (6/21, 9/21, 13/21, 14/21, 1/22, 2/22, 3/22, 6/22, 7/22) were previously published [8]

Full size image

qPCR analysis revealed that all 12 cAD-MSC donors were negative for CHV.

The autochthonous wild-type CHV strain was successfully isolated and verified by whole-genome sequencing

The autochthonous CHV strain was successfully recovered from the affected organs and further propagated on the MDCK cell line. The first signs of CPE were observed at ≈30 h p.i., characterised by cell rounding. The number of cells affected by viral activity increased progressively over time. Further progression of CHV infection induced clustering of rounded cells, leaving blank spaces between clusters. The characteristic CPE was fully developed at 96–120 h p.i. (Fig. 2A; Additional file 2). The established virus consistently exhibited CPEs at comparable intervals during the two subsequent passages on MDCK cells used to generate the CHV virus stock. The created CHV virus stock was titrated in triplicate, yielding an average titre of 105,04 TCID50/mL.

Fig. 2
figure 2

Development of cytopathogenic effects (CPE) following Canid alphaherpesvirus 1 (CHV) infection in vitro. A Progression of CPE after CHV virus stock infection in the Madin-Darby canine kidney cell line (MDCK) over time (marked in days, hours, minutes, and seconds). B Progression of CPE after CHV virus stock infection in canine adipose-derived mesenchymal stem cells (cAD-MSCs) over time (marked in days, hours, minutes, and seconds). C cAD-MSC negative control (left) and CPE development in six cAD-MSC donors 48 h after infection with the CHV stock (right). D CHV genome copies per mL of cell lysate supernatant for each virus passage in cAD-MSCs (boxplots) and MDCK cells (line graph). Microscopy images were acquired with the Lux 2 live imaging platform (Axion Biosystems) (A, B) or with the microscopic camera Axiocam ER/105/208/HD on Axio Observer D1 (Zeiss, magnification 50-100x, scale bar 200 µm) (C)

Full size image

The isolated autochthonous wild-type CHV strain 29,107 was verified by deep sequencing, and the complete genome was characterised. The consensus CHV genome sequence was generated by mapping reads to the GenBank sequence MW353136 and deposited to GenBank under the accession number PP349830. The CHV genome was 124,854 bases long and was covered by 234,338 sequencing reads (150 bp) with a mean base depth of 250 × and a mean base quality of 34.1 (PHRED). Phylogenetic analysis showed that the novel wild-type CHV was most closely related to the GenBank sequence MW353130 (Fig. 3A). MW353130 was most similar to PP349830 along the entire genome length, except in a region spanning positions 102,000 and 106,000, where it was most similar to MW353131 and MW353138 (Fig. 3B). This region encodes one set of copies of the virion proteins US10 and US1 and the regulatory protein V67. The abrupt drop in local similarity to the globally closest sequence suggests possible evolutionary forces, such as recombination, acting on virion proteins under selective pressure. RDP5 analysis detected weak recombination signals in this region of the CHV genome alignment. However, distinguishing recombination from other evolutionary processes is challenging given the similarity levels (global ~ 99%, local 92–93% to MW353131, and 86–87% to MW353130).

Fig. 3
figure 3

Phylogenetic analysis of the novel wild-type CHV complete genome sequence. A Phylogenetic placement of the wild-type autochthonous CHV (PP349830) among the 22 complete CHV sequences from the GenBank database. The wild-type CHV is highlighted in pink. Tips are labeled with GenBank accession numbers, collection locations, and dates. B Local similarity plot of PP349830 vs representative context sequences (KX828242, MW353130, MW353131, MW353136 and MW353138) along the length of the genome sequence. Context sequences shown were chosen to most representatively sample CHV genome diversity as represented by the phylogenetic tree (A). Local hamming similarities versus target sequences were computed in sliding windows (length = 1000 nt, step = 100)

Full size image

The CHV genome sequence contained all 75 alphaherpesvirus genes present in MW353130. The CHV genome contained 41 mutations (18 noncoding mutations, 11 synonymous mutations and 12 nonsynonymous mutations) with respect to MW353130, including seven insertions (four single nucleotides, one dinucleotide and two trinucleotide insertions), eight deletions (four single nucleotides, two dinucleotides, one trinucleotide and one heptanucleotide deletion) and 26 single nucleotide mutations. Coding mutations in CHV were localised to the CHV genes RS1 (n = 6), RS36 (n = 3), US1 (n = 2), and US10 (n = 2) and UL50, UL42, UL37, UL34, UL25, UL8, RL2, US7, US8 and US9 (n = 1 each). Mutations harboured by the wild type CHV are listed in Additional file 3.

The cAD-MSCs are susceptible to CHV

We demonstrated that CHV can infect cAD-MSCs from all 12 cAD-MSC donors, while six are represented in Fig. 2C. Similar to MDCK cells, all cAD-MSCs displayed focal cell rounding; however, the time to develop CPE was 24–48 h p.i. These rounded cells clustered together, creating empty spaces between them. At 72–96 h p.i., the typical CPE was observed throughout the culture (Fig. 2B; Additional file 4).

However, serial passages of CHV have shown that the time for cAD-MSCs to develop CPE has been prolonged. In CHV P3, CPE was observed in only 2/6 donors, and no observable CPE developed in any of the donor cAD-MSCs in later passages, CHV P4 and CHV P5. The qPCR results corroborated the in vitro observations. The CHV genome copy number decreased with each consecutive passage (Fig. 2D). However, it remained detectable at all five passages, detecting 7,550,146 ± 1,278,419 gc/mL, 3,757,414 ± 1,562,144 gc/mL, 1,338,312 ± 744,417 gc/mL, 301,236 ± 196, 130 gc/mL, and 79,131 ± 60,755 gc/mL in CHV P1, CHV P2, CHV P3, CHV P4, and CHV P5, respectively. Conversely, in the MDCK cell line, the CHV genome copy number increased from P1 to P2 and remained stable across all subsequent passages.

CHV infection significantly alters the gene expression of cAD-MSCs

The RNA QC results indicated high RNA quality across all uninfected and CHV-infected samples (Additional file 5). Gene expression analysis revealed significant alterations in 20.9% (18/85) of the total genes included in the array between uninfected and CHV-infected cAD-MSCs. Specifically, expression changes were observed for 16.7% (1/6) of the total stemness genes, 33.3% (6/18) of the total MSC-specific genes, 25.8% (8/31) of the total MSC-associated genes, and 9.7% (3/31) of the total MSC differentiation genes (Figs. 4A–D). Fold regulation values for all downregulated and upregulated genes are shown in Fig. 4E. The full report of the RT2 Profiler PCR Array Data Analysis Software is attached as Additional file 1.

Fig. 4
figure 4

Gene expression profile of canine adipose-derived mesenchymal stem cells following canine herpesvirus infection in vitro. Significantly downregulated and upregulated genes are labelled blue and orange, respectively. Volcano plots illustrate the expression profiles of genes related to stemness (A), mesenchymal stem cell (MSC)-specific genes (B), MSC-associated genes (C) and MSC differentiation genes (D) after CHV infection. The bar chart shows the fold-regulation of the significantly downregulated and upregulated genes (E). AGene expression was lower in the uninfected sample and more readily detectable in the CHV-infected sample, indicating that the fold change was at least as high as the calculated value

Full size image

CHV infection significantly alters protein secretion in the cAD-MSC secretome

The proteomic analysis of the cAD-MSC secretome identified 1,181 proteins. A comprehensive list of all detected proteins, including their accession numbers, gene names, molecular weights, t-test p values, and fold change data, is provided in Additional File 6. The 86.8% (1,025/1,181) of proteins were common between uninfected and CHV-infected samples. The commonly detected proteins were further compared by GO enrichment analysis, i.e., cellular components, protein classes, molecular functions, and biological processes (Fig. 5A–C). Similar involvement in all annotated functions was observed. Analysis of cellular components revealed that ≈ 60% (788/1181 and 754/1181 in uninfected and CHV-infected samples, respectively) of proteins belonged to cytoskeletal proteins, ≈ 19% (239 in uninfected and 235 in CHV-infected samples) belonged to protein-containing complexes, and the rest (154 in uninfected and 192 in CHV-infected) were not assignable to cellular component GO terms. More than 50% of the cAD-MSC secretome proteins were metabolite conversion enzymes, translational proteins, protein-modifying enzymes, and cytoskeletal proteins (Fig. 5A). Their molecular functions were mainly binding and catalytic activity (Fig. 5B), and they were predominantly involved in cellular and metabolic processes, biological regulation, response to stimuli and localisation (Fig. 5C). Among the commonly secreted proteins, 10 were significantly downregulated, whereas 66 were significantly upregulated (Fig. 5D; Additional file 6).

Fig. 5
figure 5

Proteomic analysis of the secretome from canine adipose-derived mesenchymal stem cells. A–C Complete functional protein classification of secretomes comparing uninfected and CHV-infected canine adipose-derived mesenchymal stem cells by protein class (A), by molecular function (B), and according to biological processes (C). Bars indicate the number of categorized secreted proteins. (D) Volcano plot showing commonly secreted proteins in the secretome of canine adipose-derived mesenchymal stem cells, comparing uninfected with CHV-infected cells. A comprehensive list of all detected proteins, including their accession numbers, gene names, molecular weights, t-test p values, and fold change data, is provided in Additional File 6. In the graphical representation and Additional file 6, the blue color represents significantly downregulated proteins (p < 0.05) with a fold change exceeding 1.3. Conversely, the orange color signifies proteins demonstrating statistical significance (p < 0.05) in upregulation, with a fold change greater than 1.3. (E) String protein interaction network representing significant protein interactions occurring due to CHV infection. The coloured nodes represent interactions with a high confidence score (0.700) and strength > 0.75. Red represents interactions of proteins involved in the aminoacyl-tRNA synthetase multienzyme complex; pink represents interactions involving the proteasome complex; yellow represents interactions involving pyruvate metabolism and carbon metabolism; green represents interactions involving glycolysis; and blue represents interactions involving the enolase, C-terminal TIM barrel domain and glycolysis

Full size image

Within the subset of proteins exhibiting distinctive secretion patterns (comprising 13.2% of the total proteins), 105 proteins were specific to uninfected samples, whereas 51 proteins were specific to CHV-infected samples. Subsequent bioinformatic analysis was subsequently conducted to gain insight into protein pathway involvement. The results of the GO enrichment analysis are presented in Table 2, while the outcomes of the STRING analysis are detailed in Table 3 and Fig. 5F.

Table 2 Protein pathway analysis of the uninfected and CHV-infected canine adipose MSC secretomes via Gene Ontology Panther
Full size table
Table 3 Protein pathway analysis of the secretome of uninfected and CHV-infected canine adipose MSCs via STRING
Full size table

Discussion

In vitro manipulations are unavoidable for acquiring and conserving therapeutic quantities of cAD-MSCs and their secretome. Nevertheless, these procedures involve an inherent risk of microbial contamination and the potential spread of pathogens, including viruses, originating from infected donor cells. Therefore, we aimed to investigate the interaction between globally distributed CHV and cAD-MSCs and to assess whether CHV infection affects the gene expression and secretome composition of cAD-MSCs. To the best of our knowledge, this study represents the first investigation of the effects of viral infection on the gene expression and secretome profile of cAD-MSCs. To achieve the aim, an autochthonous CHV strain was established. This strain should reflect the natural virus-host interplay more accurately than culture-adapted and extensively propagated CHV strains in vitro. The novel complete genome sequence of the autochthonous CHV strain from Croatia contributes to the knowledge of the complete genome diversity of CHV. Prior to this contribution, the GenBank database contained 22 complete CHV genome sequences, with only five sequences from Europe, three from the United Kingdom and two from Italy.

In vitro susceptibility to CHV, as indicated by the characteristic CPE of Orthoherpesviridae viruses [48], was likewise observed in cAD-MSCs and MDCK cells (Fig. 2A, B; Additional files 2 and 4). However, successive passages of CHV on cAD-MSCs exhibited a gradual reduction and disappearance of CPE, which was corroborated by diminishing yet persistent CHV genome copy numbers (Fig. 2C) in the supernatants of cell lysates, indicating abortive infection. The abortive infection has recently been documented in herpes simplex virus in vitro research [49], which proves that herpesviruses can infect nonneuronal cells, remain quiescent and be reactivated, challenging the current paradigm of herpesvirus latency. Our results seem to resemble the above scenario, but further experimental validation is needed to assign abortive infection status to this specific virus-host interaction.

The effects of CHV infection on cAD-MSCs were further explored at the gene expression level, and the results revealed that CHV infection significantly affected the expression of researched genes (Fig. 4). The upregulated genes were associated with proliferation [50], differentiation [51], and the immunosuppressive response [52,53,54]. Similar alterations in gene expression attributed to virus infections have been documented in previous studies on human stem cells [21, 55]. The most significantly upregulated gene in infected cAD-MSCs, TNF (Fig. 4C), encodes a protein responsible for various cellular processes, including proliferation, differentiation, and, interestingly, immune suppression in MSCs [54, 56]. It remains unclear whether canine MSCs can produce TNF; however, in human MSCs, there is clear evidence of their inability to produce TNF [57]. Nevertheless, cAD-MSCs are adipocyte progenitors, and adipocytes and their progenitors are well known for secreting TNF [58]. In this study, high TNF production combined with alterations in other genes primarily suggested increased adipocyte differentiation in CHV-infected cells. Moreover, the upregulation of ADIPOQ and NOTCH1 [59, 60] coupled with the downregulation of several genes related to stemness and regenerative capacity [61,62,63,64] further support the initiation of differentiation processes, predominantly adipogenesis, in CHV-infected cAD-MSCs. These findings indicate that CHV infection may drive cAD-MSC differentiation, affecting their regenerative potential and altering their typical stem cell properties.

The proteomic composition of the cAD-MSC secretomes further corroborated the initiation of adipogenesis in CHV-infected cells observed at the RNA level. GO enrichment analysis of the uninfected group revealed that proteins significantly involved in the WNT signalling pathway were absent in CHV-infected cells (Table 2). This finding suggests that CHV infection leads to the loss of the WNT signalling pathway, and its deactivation in MSCs is considered crucial for inducing adipogenesis [65, 66]. Furthermore, both GO enrichment and STRING analyses (Tables 2 and 3) revealed the presence of essential protein pathways involved in cell self-renewal, structure, survival, homing, and migration [67, 68] in the uninfected group, which were lost after CHV infection. These losses at the proteomic level align with the microscopically observed loss of cellular structure, survival, and migration following CPE development (Fig. 2; Additional files 2 and 4).

Additionally, the observed microscopic reduction in cell survival was further supported by findings from the CHV-infected group cAD-MSC secretomes. GO enrichment and STRING analyses revealed upregulated glycolysis and elevated levels of proteins associated with the enolase and pyruvate metabolism pathways (Tables 2 and 3). This virus’s takeover of host cell resources and metabolic machinery prioritises viral particle production over normal cellular functions, ultimately leading to cell damage and death. Similar alterations were previously documented in studies on viral-host interactions in other Orthoherpesviridae infections, such as human cytomegalovirus [69] and herpes simplex virus [70, 71] infections.

Although novel knowledge is unravelled in this study, future studies should deepen the understanding of cAD-MSC-CHV interactions by examining transcriptome and secretome alterations throughout serial passages and identifying any coinciding variants in the viral genome. Furthermore, as the AD-MSC secretome has recently been shown to have an antiviral effect [72] in felines, our research provides a direction for overcoming the currently limited knowledge on utilising stem cells to treat viral diseases in canines.

In conclusion, our study demonstrated the susceptibility of cAD-MSCs to CHV infection. The observed genomic variations in gene expression indicate potential impacts on the stemness, migration, and other functional properties of cAD-MSCs, highlighting the need for further studies to evaluate their functional capacity post-infection. Moreover, gene expression and secretome analyses suggest a shift in stem cell differentiation toward an adipogenic phenotype. These cumulative changes can negatively impact the regenerative properties of cAD-MSCs. These findings highlight the critical importance of screening cAD-MSC batches intended for therapeutic applications to ensure the absence of CHV prior to administration.

Availability of data and materials

The datasets generated and analysed during the current study are available in the NCBI GenBank [https://www.ncbi.nlm.nih.gov/nuccore/PP349830], Gene Expression Omnibus repository [https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE267402] and ProteomeXchange repository [https://www.ebi.ac.uk/pride/archive/projects/PXD052289]. This manuscript and additional files include other data generated or analysed during this study.

Abbreviations

cAD-MSCs:

Canine adipose-derived mesenchymal stem cells

CD:

Cluster of differentiation

CHV:

Canid alphaherpesvirus 1

CPE:

Cytopathogenic effect

DMEM Low Glucose:

Dulbecco’s modified eagle medium with low glucose

FBS:

Fetal bovine serum

FDR:

False discovery rate

GO:

Gene ontology

LOD:

Limit of detection

LOQ:

Limit of quantification

MDCK:

Madin-Darby canine kidney cell culture

MSC:

Mesenchymal stem cell

NGS:

Next-generation sequencing

P(number):

Passage (number)

p.i.:

Postinfection

qPCR:

Quantitative polymerase chain reaction

RT-qPCR:

Reverse transcription-quantitative polymerase chain reaction

TCID50:

50% tissue culture infectious dose.

References

  1. Voga M, Adamic N, Vengust M, Majdic G. Stem cells in veterinary medicine—current state and treatment options. Front Vet Sci. 2020;7:278. https://doiorg.publicaciones.saludcastillayleon.es/10.3389/fvets.2020.00278.

    Article  PubMed  PubMed Central  Google Scholar 

  2. Prišlin M, Vlahović D, Kostešić P, Ljolje I, Brnić D, Turk N, et al. An outstanding role of adipose tissue in canine stem cell therapy. Animals. 2022;12:1088. https://doiorg.publicaciones.saludcastillayleon.es/10.3390/ani12091088.

    Article  PubMed  PubMed Central  Google Scholar 

  3. El-Husseiny HM, Mady EA, Helal MAY, Tanaka R. The pivotal role of stem cells in veterinary regenerative medicine and tissue engineering. Vet Sci. 2022;9:648. https://doiorg.publicaciones.saludcastillayleon.es/10.3390/vetsci9110648.

    Article  PubMed  PubMed Central  Google Scholar 

  4. Russell KA, Chow NHC, Dukoff D, Gibson TWG, La Marre J, Betts DH, et al. Characterisation and immunomodulatory effects of canine adipose tissue- and bone marrow-derived mesenchymal stromal cells. PLoS ONE. 2016;11:e0167442. https://doiorg.publicaciones.saludcastillayleon.es/10.1371/journal.pone.0167442.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Villatoro AJ, Alcoholado C, Martín-Astorga MC, Fernández V, Cifuentes M, Becerra J. Comparative analysis and characterisation of soluble factors and exosomes from cultured adipose tissue and bone marrow mesenchymal stem cells in canine species. Vet Immunol Immunopathol. 2019;208:6–15. https://doiorg.publicaciones.saludcastillayleon.es/10.1016/j.vetimm.2018.12.003.

    Article  CAS  PubMed  Google Scholar 

  6. Humenik F, Maloveska M, Hudakova N, Petrouskova P, Hornakova L, Domaniza M, et al. A comparative study of canine mesenchymal stem cells isolated from different sources. Animals. 2022;12:1502. https://doiorg.publicaciones.saludcastillayleon.es/10.3390/ani12121502.

    Article  PubMed  PubMed Central  Google Scholar 

  7. Teshima T, Yuchi Y, Suzuki R, Matsumoto H, Koyama H. Immunomodulatory effects of canine adipose tissue mesenchymal stem cell-derived extracellular vesicles on stimulated cd4+t cells isolated from peripheral blood mononuclear cells. J Immunol Res. 2021;2021:2993043. https://doiorg.publicaciones.saludcastillayleon.es/10.1155/2021/2993043.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Prišlin M, Butorac A, Bertoša R, Kunić V, Ljolje I, Kostešić P, et al. In vitro aging alters the gene expression and secretome composition of canine adipose-derived mesenchymal stem cells. Front Vet Sci. 2024;11:1387174. https://doiorg.publicaciones.saludcastillayleon.es/10.3389/fvets.2024.1387174.

    Article  PubMed  PubMed Central  Google Scholar 

  9. Chandra V, Mankuzhy P, Sharma GT. Mesenchymal stem cells in veterinary regenerative therapy: basic physiology and clinical applications. Curr Stem Cell Res Ther. 2022;17:237–51. https://doiorg.publicaciones.saludcastillayleon.es/10.2174/1574888X16666210804112741.

    Article  CAS  PubMed  Google Scholar 

  10. Trzyna A, Banaś-Ząbczyk A. Adipose-derived stem cells secretome and its potential application in “stem cell-free therapy.” Biomolecules. 2021;11:878. https://doiorg.publicaciones.saludcastillayleon.es/10.3390/biom11060878.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Merlo B, Iacono E. Beyond canine adipose tissue-derived mesenchymal secretome characterization and applications. Animals. 2023;13(22):3571. https://doiorg.publicaciones.saludcastillayleon.es/10.3390/ani13223571.

    Article  PubMed  PubMed Central  Google Scholar 

  12. Vizoso FJ, Eiro N, Cid S, Schneider J, Perez-Fernandez R. Mesenchymal stem cell secretome: Toward cell-free therapeutic strategies in regenerative medicine. Int J Mol Sci. 2017;18:1852. https://doiorg.publicaciones.saludcastillayleon.es/10.3390/ijms18091852.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Szabłowska-Gadomska I, Humi M, Płaczkowska J. Microbiological aspects of pharmaceutical manufacturing of adipose-derived stem cell-based medicinal products. Cells. 2023;12(5):680. https://doiorg.publicaciones.saludcastillayleon.es/10.3390/cells12050680.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Martín PG, González MB, Martínez AR, Lara VG, Naveros BC. Isolation and characterisation of the environmental bacterial and fungi contamination in a pharmaceutical unit of mesenchymal stem cell for clinical use. Biologicals. 2012;40:330–7. https://doiorg.publicaciones.saludcastillayleon.es/10.1016/j.biologicals.2012.06.002.

    Article  PubMed  Google Scholar 

  15. Khatri M, O’Brien TD, Goyal SM, Sharma JM. Isolation and characterisation of chicken lung mesenchymal stromal cells and their susceptibility to avian influenza virus. Dev Comp Immunol. 2010;34:474–9. https://doiorg.publicaciones.saludcastillayleon.es/10.1016/j.dci.2009.12.008.

    Article  CAS  PubMed  Google Scholar 

  16. Khatri M, Saif YM. Influenza virus infects bone marrow mesenchymal stromal cells in vitro: implications for bone marrow transplantation. Cell Transpl. 2013;22:461–8. https://doiorg.publicaciones.saludcastillayleon.es/10.3727/096368912X656063.

    Article  Google Scholar 

  17. Nazari-Shafti TZ, Freisinger E, Roy U, Bulot CT, Senst C, Dupin CL, et al. Mesenchymal stem cell derived hematopoietic cells are permissive to HIV-1 infection. Retrovirology. 2011;8:3. https://doiorg.publicaciones.saludcastillayleon.es/10.1186/1742-4690-8-3.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Ma R, Xing Q, Shao L, Wang D, Hao Q, Li X, et al. Hepatitis B virus infection and replication in human bone marrow mesenchymal stem cells. Virol J. 2011;8:486. https://doiorg.publicaciones.saludcastillayleon.es/10.1186/1743-422X-8-486.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Sundin M, Örvell C, Rasmusson I, Sundberg B, Ringdén O, Le Blanc K. Mesenchymal stem cells are susceptible to human herpesviruses, but viral DNA cannot be detected in the healthy seropositive individual. Bone Marrow Transpl. 2006;37:1051–9. https://doiorg.publicaciones.saludcastillayleon.es/10.1038/sj.bmt.1705368.

    Article  CAS  Google Scholar 

  20. Roy E, Shi W, Duan B, Reida SP. Chikungunya virus infection impairs the function of osteogenic cells. mSphere. 2020;5:e00347-20. https://doiorg.publicaciones.saludcastillayleon.es/10.1128/mSphere.00347-20.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Behzadi Fard M, Kaviani S, Atashi A. Parvovirus B19 infection in human bone marrow mesenchymal stem cells affects gene expression of IL-6 and TNF-α and also affects hematopoietic stem cells differentiation. Indian J Hematol Blood Transfus. 2019;35:765–72. https://doiorg.publicaciones.saludcastillayleon.es/10.1007/s12288-019-01097-7.

    Article  PubMed  PubMed Central  Google Scholar 

  22. Meisel R, Heseler K, Nau J, Schmidt SK, Leineweber M, Pudelko S, et al. Cytomegalovirus infection impairs immunosuppressive and antimicrobial effector functions of human multipotent mesenchymal stromal cells. Mediators Inflamm. 2014;2014:898630. https://doiorg.publicaciones.saludcastillayleon.es/10.1155/2014/898630.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Altamirano-Samaniego F, Enciso-Benavides J, Rojas N, Iglesias-Pedraz JM, Enciso N, Fossatti M, et al. First report of canine morbillivirus infection of adipose tissue-derived stem cells from dogs with distemper. Vet World. 2022;15:1835–42. https://doiorg.publicaciones.saludcastillayleon.es/10.14202/vetworld.2022.1835-1842.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Pekker E, Priskin K, Szabó-Kriston É, Csányi B, Buzás-Bereczki O, Adorján L, et al. Development of a large-scale pathogen screening test for the biosafety evaluation of canine mesenchymal stem cells. Biol Proced Online. 2023;25:33. https://doiorg.publicaciones.saludcastillayleon.es/10.1186/s12575-023-00226-x.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. ICTV. Varicellovirus taxonomy [Internet]. 2024 [cited 2024 25 March]. Available from: https://ictv.global/report/chapter/orthoherpesviridae/orthoherpesviridae/varicellovirus

  26. Dahlbom M, Johnsson M, Myllys V, Taponen J, Andersson M. Seroprevalence of canine herpesvirus-1 and Brucella canis in Finnish breeding kennels with and without reproductive problems. Reprod Domest Anim. 2009;44:128–31. https://doiorg.publicaciones.saludcastillayleon.es/10.1111/j.1439-0531.2007.01008.x.

    Article  CAS  PubMed  Google Scholar 

  27. Krogenæs A, Rootwelt V, Larsen S, Sjøberg EK, Akselsen B, Skår TM, et al. A serologic study of canine herpes virus-1 infection in the Norwegian adult dog population. Theriogenology. 2012;78:153–8. https://doiorg.publicaciones.saludcastillayleon.es/10.1016/j.theriogenology.2012.01.031.

    Article  PubMed  Google Scholar 

  28. Gracin K. Dijagnostika herpesvirusne infekcije u pasa u Republici Hrvatskoj i njezin utjecaj na poremećaje reprodukcije. Ph.D. Thesis. University of Zagreb, Veterinary Faculty; 2020.

  29. de la Garza-Rodea AS, Verweij MC, Boersma H, van der Velde-van DI, de Vries AAF, Hoeben RC, et al. Exploitation of herpesvirus immune evasion strategies to modify the immunogenicity of human mesenchymal stem cell transplants. PLoS ONE. 2011;6:e14493. https://doiorg.publicaciones.saludcastillayleon.es/10.1371/journal.pone.0014493.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Claessen C, Favoreel H, Ma G, Osterrieder N, De Schauwer C, Piepers S, et al. Equid herpesvirus 1 (EHV1) infection of equine mesenchymal stem cells induces a pUL56-dependent downregulation of select cell surface markers. Vet Microbiol. 2015;176:32–9. https://doiorg.publicaciones.saludcastillayleon.es/10.1016/j.vetmic.2014.12.013.

    Article  CAS  PubMed  Google Scholar 

  31. Krešić N, Prišlin M, Vlahović D, Kostešić P, Ljolje I, Brnić D, et al. The expression pattern of surface markers in canine adipose-derived mesenchymal stem cells. Int J Mol Sci. 2021;22:7476. https://doiorg.publicaciones.saludcastillayleon.es/10.3390/ijms22147476.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Dominici M, Le Blanc K, Mueller I, Slaper-Cortenbach I, Marini FC, Krause DS, et al. Minimal criteria for defining multipotent mesenchymal stromal cells. The International Society for Cellular Therapy position statement. Cytotherapy. 2006;8:315–7. https://doiorg.publicaciones.saludcastillayleon.es/10.1080/14653240600855905.

    Article  CAS  PubMed  Google Scholar 

  33. Decaro N, Amorisco F, Desario C, Lorusso E, Camero M, Bellacicco AL, et al. Development and validation of a real-time PCR assay for specific and sensitive detection of canid herpesvirus 1. J Virol Methods. 2010;169:176–80. https://doiorg.publicaciones.saludcastillayleon.es/10.1016/j.jviromet.2010.07.021.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Wernike K, Hoffmann B, Kalthoff D, König P, Beer M. Development and validation of a triplex real-time PCR assay for the rapid detection and differentiation of wild-type and glycoprotein E-deleted vaccine strains of Bovine herpesvirus type 1. J Virol Methods. 2011;174:77–84. https://doiorg.publicaciones.saludcastillayleon.es/10.1016/j.jviromet.2011.03.028.

    Article  CAS  PubMed  Google Scholar 

  35. Eisa M, Loucif H, Grevenynghe J, Pearson A. Entry of the Varicellovirus Canid herpesvirus 1 into Madin-Darby canine kidney epithelial cells is pH -independent and occurs via a macropinocytosis-like mechanism but without increase in fluid uptake. Cell Microbiol. 2021;23:e13398. https://doiorg.publicaciones.saludcastillayleon.es/10.1111/cmi.13398.

    Article  CAS  PubMed  Google Scholar 

  36. Bankevich A, Nurk S, Antipov D, Gurevich AA, Dvorkin M, Kulikov AS, et al. SPAdes: a new genome assembly algorithm and its applications to single-cell sequencing. J Comput Biol. 2012;19:455–77. https://doiorg.publicaciones.saludcastillayleon.es/10.1089/cmb.2012.0021.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. Li H. Aligning sequence reads, clone sequences and assembly contigs with BWA-MEM. aRxiv [Internet]. 2013;1303:3997. Available from: http://arxiv.org/abs/1303.3997

  38. Danecek P, Bonfield JK, Liddle J, Marshall J, Ohan V, Pollard MO, et al. Twelve years of SAMtools and BCFtools. Gigascience. 2021;10:giab008. https://doiorg.publicaciones.saludcastillayleon.es/10.1093/gigascience/giab008/6137722.

    Article  PubMed  PubMed Central  Google Scholar 

  39. Grubaugh ND, Gangavarapu K, Quick J, Matteson NL, De Jesus JG, Main BJ, et al. An amplicon-based sequencing framework for accurately measuring intrahost virus diversity using PrimalSeq and iVar. Genome Biol. 2019;20:8. https://doiorg.publicaciones.saludcastillayleon.es/10.1186/s13059-018-1618-7.

    Article  PubMed  PubMed Central  Google Scholar 

  40. Seemann T. Prokka: rapid prokaryotic genome annotation. Bioinformatics. 2014;30:2068–9. https://doiorg.publicaciones.saludcastillayleon.es/10.1093/bioinformatics/btu153.

    Article  CAS  PubMed  Google Scholar 

  41. Nguyen L-T, Schmidt HA, von Haeseler A, Minh BQ. IQ-TREE: a fast and effective stochastic algorithm for estimating maximum-likelihood phylogenies. Mol Biol Evol. 2015;32:268–74. https://doiorg.publicaciones.saludcastillayleon.es/10.1093/molbev/msu300.

    Article  CAS  PubMed  Google Scholar 

  42. Kalyaanamoorthy S, Minh BQ, Wong TKF, von Haeseler A, Jermiin LS. ModelFinder: fast model selection for accurate phylogenetic estimates. Nat Methods. 2017;14:587–9. https://doiorg.publicaciones.saludcastillayleon.es/10.1038/nmeth.4285.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  43. Rozewicki J, Li S, Amada KM, Standley DM, Katoh K. MAFFT-DASH: integrated protein sequence and structural alignment. Nucleic Acids Res. 2019;47:W5–10. https://doiorg.publicaciones.saludcastillayleon.es/10.1093/nar/gkz342/5486273.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  44. Hoang DT, Chernomor O, von Haeseler A, Minh BQ, Vinh LS. UFBoot2: improving the ultrafast bootstrap approximation. Mol Biol Evol. 2018;35:518–22. https://doiorg.publicaciones.saludcastillayleon.es/10.1093/molbev/msx281.

    Article  CAS  PubMed  Google Scholar 

  45. Martin DP, Varsani A, Roumagnac P, Botha G, Maslamoney S, Schwab T, et al. RDP5: a computer program for analysing recombination in, and removing signals of recombination from, nucleotide sequence datasets. Virus Evol. 2021. https://doiorg.publicaciones.saludcastillayleon.es/10.1093/ve/veaa087/6020281.

    Article  PubMed  PubMed Central  Google Scholar 

  46. Perez-Riverol Y, Bai J, Bandla C, García-Seisdedos D, Hewapathirana S, Kamatchinathan S, et al. The PRIDE database resources in 2022: a hub for mass spectrometry-based proteomics evidences. Nucleic Acids Res. 2022;50:D543–52. https://doiorg.publicaciones.saludcastillayleon.es/10.1093/nar/gkab1038.

    Article  CAS  PubMed  Google Scholar 

  47. Szklarczyk D, Kirsch R, Koutrouli M, Nastou K, Mehryary F, Hachilif R, et al. The STRING database in 2023: protein-protein association networks and functional enrichment analyses for any sequenced genome of interest. Nucleic Acids Res (Oxford University Press). 2023;51:D638–46. https://doiorg.publicaciones.saludcastillayleon.es/10.1093/nar/gkac1000.

    Article  CAS  Google Scholar 

  48. Suchman E, Blair C. Cytopathic effects of viruses. In: ASM Conference Undergraduate Education 2007. Am Soc Microbiol [Internet]. 2007. p. 1–13. Available from: https://asm.org/Protocols/Cytopathic-Effects-of-Viruses-Protocols

  49. Cohen EM, Avital N, Shamay M, Kobiler O. Abortive herpes simplex virus infection of nonneuronal cells results in quiescent viral genomes that can reactivate. Proc Natl Acad Sci USA. 2020;117:635–40. https://doiorg.publicaciones.saludcastillayleon.es/10.1073/pnas.1910537117.

    Article  CAS  PubMed  Google Scholar 

  50. Han SH, Jang G, Bae BK, Han SM, Koh YR, Ahn JO, et al. Effect of ectopic OCT4 expression on canine adipose tissue-derived mesenchymal stem cell proliferation. Cell Biol Int. 2014;38:1163–73. https://doiorg.publicaciones.saludcastillayleon.es/10.1002/cbin.10295.

    Article  CAS  PubMed  Google Scholar 

  51. Park SR, Cho A, Kim JW, Lee HY, Hong IS. A novel endogenous damage signal, CSF-2, activates multiple beneficial functions of adipose tissue-derived mesenchymal stem cells. Mol Ther. 2019;27:1087–100. https://doiorg.publicaciones.saludcastillayleon.es/10.1016/j.ymthe.2019.03.010.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  52. Deng J, Li D, Huang X, Li W, Zhao F, Gu C, et al. Interferon-γ enhances the immunosuppressive ability of canine bone marrow-derived mesenchymal stem cells by activating the TLR3-dependent IDO/kynurenine pathway. Mol Biol Rep. 2022;49:8337–47. https://doiorg.publicaciones.saludcastillayleon.es/10.1007/s11033-022-07648-y.

    Article  CAS  PubMed  Google Scholar 

  53. Rubtsov Y, Goryunov К, Romanov A, Suzdaltseva Y, Sharonov G, Tkachuk V. Molecular mechanisms of immunomodulation properties of mesenchymal stromal cells: a new insight into the role of ICAM-1. Stem Cells Int. 2017;2017:6516854. https://doiorg.publicaciones.saludcastillayleon.es/10.1155/2017/6516854.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  54. Yang HM, Song WJ, Li Q, Kim SY, Kim HJ, Ryu MO, et al. Canine mesenchymal stem cells treated with TNF-α and IFN-γ enhance anti-inflammatory effects through the COX-2/PGE 2 pathway. Res Vet Sci. 2018;119:19–26. https://doiorg.publicaciones.saludcastillayleon.es/10.1016/j.rvsc.2018.05.011.

    Article  CAS  PubMed  Google Scholar 

  55. Li Q, Yu P, Wang W, Zhang P, Yang H, Li S, et al. Gene expression profiles of various cytokines in mesenchymal stem cells derived from umbilical cord tissue and bone marrow following infection with human cytomegalovirus. Cell Mol Biol Lett. 2014;19:140–57. https://doiorg.publicaciones.saludcastillayleon.es/10.2478/s11658-014-0187-3.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  56. Yan L, Zheng D, Xu R-H. Critical role of tumor necrosis factor signaling in mesenchymal stem cell-based therapy for autoimmune and inflammatory diseases. Front Immunol. 2018;9:1658. https://doiorg.publicaciones.saludcastillayleon.es/10.3389/fimmu.2018.01658/full.

    Article  PubMed  PubMed Central  Google Scholar 

  57. van den Berk LCJ, Jansen BJH, Siebers-Vermeulen KGC, Roelofs H, Figdor CG, Adema GJ, et al. Mesenchymal stem cells respond to TNF but do not produce TNF. J Leukoc Biol. 2010;87:283–9. https://doiorg.publicaciones.saludcastillayleon.es/10.1189/jlb.0709467.

    Article  CAS  PubMed  Google Scholar 

  58. Cawthorn WP, Sethi JK. TNF-α and adipocyte biology. FEBS Lett. 2008;582:117–31. https://doiorg.publicaciones.saludcastillayleon.es/10.1016/j.febslet.2007.11.051.

    Article  CAS  PubMed  Google Scholar 

  59. Nedvídková J, Smitka K, Kopský V, Hainer V. Adiponectin, an adipocyte-derived protein. Physiol Res. 2005;54:133–40.

    Article  PubMed  Google Scholar 

  60. Shan T, Liu J, Wu W, Xu Z, Wang Y. Roles of notch signaling in adipocyte progenitor cells and mature adipocytes. J Cell Physiol. 2017;232:1258–61. https://doiorg.publicaciones.saludcastillayleon.es/10.1002/jcp.25697.

    Article  CAS  PubMed  Google Scholar 

  61. Tanabe S, Kawabata T, Aoyagi K, Yokozaki H, Sasaki H. Gene expression and pathway analysis of CTNNB1 in cancer and stem cells. World J Stem Cells. 2016;8:384–95. https://doiorg.publicaciones.saludcastillayleon.es/10.4252/wjsc.v8.i11.384.

    Article  PubMed  PubMed Central  Google Scholar 

  62. Enciso N, Ostronoff LLK, Mejías G, León LG, Fermín ML, Merino E, et al. Stem cell factor supports migration in canine mesenchymal stem cells. Vet Res Commun. 2018;42:29–38. https://doiorg.publicaciones.saludcastillayleon.es/10.1007/s11259-017-9705-x.

    Article  PubMed  Google Scholar 

  63. Cao Z, Xie Y, Yu L, Li Y, Wang Y. Hepatocyte growth factor (HGF) and stem cell factor (SCF) maintained the stemness of human bone marrow mesenchymal stem cells (hBMSCs) during long-term expansion by preserving mitochondrial function via the PI3K/AKT, ERK1/2, and STAT3 signaling pathways. Stem Cell Res Ther. 2020;11:329. https://doiorg.publicaciones.saludcastillayleon.es/10.1186/s13287-020-01830-4.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  64. Wang S, Mo M, Wang J, Sadia S, Shi B, Fu X, et al. Platelet-derived growth factor receptor beta identifies mesenchymal stem cells with enhanced engraftment to tissue injury and pro-angiogenic property. Cell Mol Life Sci. 2018;75:547–61. https://doiorg.publicaciones.saludcastillayleon.es/10.1007/s00018-017-2641-7.

    Article  CAS  PubMed  Google Scholar 

  65. Visweswaran M, Pohl S, Arfuso F, Newsholme P, Dilley R, Pervaiz S, et al. Multi-lineage differentiation of mesenchymal stem cells—to Wnt, or not Wnt. Int J Biochem Cell Biol. 2015;68:139–47. https://doiorg.publicaciones.saludcastillayleon.es/10.1016/j.biocel.2015.09.008.

    Article  CAS  PubMed  Google Scholar 

  66. Ling L, Nurcombe V, Cool SM. Wnt signaling controls the fate of mesenchymal stem cells. Gene. 2009;433:1–7. https://doiorg.publicaciones.saludcastillayleon.es/10.1016/j.gene.2008.12.008.

    Article  CAS  PubMed  Google Scholar 

  67. Zhang Z, Liu M, Zheng Y. Role of Rho GTPases in stem cell regulation. Biochem Soc Trans. 2021;49:2941–55. https://doiorg.publicaciones.saludcastillayleon.es/10.1042/BST20211071.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  68. Alqahtani S, Butcher MC, Ramage G, Dalby MJ, McLean W, Nile CJ. Acetylcholine receptors in mesenchymal stem cells. Stem Cells Dev. 2023;32:47–59. https://doiorg.publicaciones.saludcastillayleon.es/10.1089/scd.2022.0201.

    Article  CAS  PubMed  Google Scholar 

  69. Moreno I, Rodríguez-Sánchez I, Schafer X, Munger J. Human cytomegalovirus induces neuronal enolase to support virally mediated metabolic remodeling. Proc Natl Acad Sci. 2022;119:e2205789119. https://doiorg.publicaciones.saludcastillayleon.es/10.1073/pnas.2216830120.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  70. Kun-Varga A, Gubán B, Miklós V, Parvaneh S, Guba M, Szűcs D, et al. Herpes simplex virus infection alters the immunological properties of adipose-tissue-derived mesenchymal-stem cells. Int J Mol Sci. 2023;24:11989. https://doiorg.publicaciones.saludcastillayleon.es/10.3390/ijms241511989.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  71. Zhuo C, Zheng D, He Z, Jin J, Ren Z, Jin F, et al. HSV-1 enhances the energy metabolism of human umbilical cord mesenchymal stem cells to promote virus infection. Future Virol. 2017;12:349–60. https://doiorg.publicaciones.saludcastillayleon.es/10.2217/fvl-2017-0038.

    Article  CAS  Google Scholar 

  72. Teshima T, Yasumura Y, Suzuki R, Matsumoto H. Antiviral effects of adipose tissue-derived mesenchymal stem cells secretome against feline calicivirus and feline herpesvirus type 1. Viruses. 2022;14:1687. https://doiorg.publicaciones.saludcastillayleon.es/10.3390/v14081687.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgements

We thank Ivana Ljolje, Petar Kostešić, and all canine donors and their owners for providing the canine adipose tissue. Furthermore, we thank Manuela Zadravec, Mihaela Stuparić Komušar, Dunja Vlahović, and Marko Močibob for their generous help during sterility testing, chondrodifferentiation preparation, chondrodifferentiation interpretation, and proteomic analysis, respectively.

Funding

This research was funded by the Croatian Science Foundation within the Installation Research Project (UIP-2019–04-2178) “Revealing transcriptome and secretome of mesenchymal stem cells” (SECRET). The next-generation sequencing analysis was funded by the Slovenian Research and Innovation Agency (grants P3-0083 and J3-3062). Proteomic analysis was partially funded by the European Regional Development Fund (infrastructural project CIuK, grant number KK.01.1.1.02.0016). The funding agencies did not contribute to the study’s conceptualisation, design, data collection, analysis, decision to publish, or manuscript preparation.

Author information

Authors and Affiliations

  1. Virology Department, Croatian Veterinary Institute, Zagreb, Croatia

    Marina Prišlin Šimac, Vjekoslava Kostanić, Valentina Kunić & Dragan Brnić

  2. Department for Pathological Morphology, Croatian Veterinary Institute, Zagreb, Croatia

    Šimun Naletilić

  3. Institute of Microbiology and Immunology, Faculty of Medicine, University of Ljubljana, Ljubljana, Slovenia

    Tomaž Mark Zorec, Mario Poljak, Doroteja Vlaj & Rok Kogoj

  4. Department of Microbiology and Infectious Diseases With Clinic, Faculty of Veterinary Medicine, University of Zagreb, Zagreb, Croatia

    Nenad Turk

Authors
  1. Marina Prišlin Šimac
    View author publications

    You can also search for this author inPubMed Google Scholar

  2. Šimun Naletilić
    View author publications

    You can also search for this author inPubMed Google Scholar

  3. Vjekoslava Kostanić
    View author publications

    You can also search for this author inPubMed Google Scholar

  4. Valentina Kunić
    View author publications

    You can also search for this author inPubMed Google Scholar

  5. Tomaž Mark Zorec
    View author publications

    You can also search for this author inPubMed Google Scholar

  6. Mario Poljak
    View author publications

    You can also search for this author inPubMed Google Scholar

  7. Doroteja Vlaj
    View author publications

    You can also search for this author inPubMed Google Scholar

  8. Rok Kogoj
    View author publications

    You can also search for this author inPubMed Google Scholar

  9. Nenad Turk
    View author publications

    You can also search for this author inPubMed Google Scholar

  10. Dragan Brnić
    View author publications

    You can also search for this author inPubMed Google Scholar

Contributions

MPŠ, DB, and NT developed the study concept. MPŠ, TMZ, DV, RK, and DB developed the methodology. MPŠ, ŠN, MPo, RK, and DB provided resources. MPŠ, ŠN, VKu, TMZ, DV, and DB performed the investigation. MPŠ, VKo, and TMZ collected the data and performed the formal analysis. TMZ performed the software analysis. MPŠ, VKo, TMZ and DB validated the results. MPŠ visualised the results. DB acquired funding; MP and RK acquired funding for NGS. DB provided project administration. DB, MP, RK, and NT supervised the study. MPŠ wrote the original draft, while TMZ and DB wrote sections of the manuscript. All authors reviewed the manuscript drafts and approved the final version of the manuscript.

Corresponding authors

Correspondence to Marina Prišlin Šimac or Dragan Brnić.

Ethics declarations

Ethics approval and consent to participate

The animal research was evaluated and approved by the Ethics Board of the Croatian Veterinary Institute (approval code Z-IV-4–2022/19) and the Veterinary Ethics Committee at the Faculty of Veterinary Medicine, University of Zagreb, Croatia (approval codes 640–01/20–17/10, 640–01/20–17/55, and 640–01/22–02/07). Canine donor owners provided written informed consent before sampling.

Consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

Additional information

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary Information

12985_2024_2603_MOESM1_ESM.pdf

Additional file 1 This additional report was generated through the analysis of gene expression data employing the RT² Profiler PCR Array Data Analysis Software. In addition to the analysis results, the investigated gene names, symbols, and corresponding NCBI sequences were comprehensively described. (PDF 321 KB)

Additional file 2 This movie additionally illustrates the progression of cytopathogenic effects in the Madin-Darby canine kidney cell line after infection with canid alphaherpesvirus 1. A scale bar of 200 µm is provided, along with a time bar delineated in days, hours, minutes, and seconds. (MP4 18067 KB)

12985_2024_2603_MOESM3_ESM.xlsx

Additional file 3 The table lists all mutations found in the wild-type CHV (GenBank accession number PP349830) relative to the closest relative, MW353130. Mutation positions, effects, gene genes affected, and protein-level consequences are also listed. (XLSX 13 KB)

Additional file 4 This movie additionally illustrates the progression of cytopathogenic effects in canine adipose-derived mesenchymal stem cells after infection with canid alphaherpesvirus 1. A scale bar of 200 µm is provided, along with a time bar delineated in days, hours, minutes, and seconds. (MP4 20489 KB)

12985_2024_2603_MOESM5_ESM.xlsx

Additional file 5 This additional table displays the RNA quality control (QC) analysis results, including the RNA integrity score and 28:18S ratio for all assessed total RNA samples. (XLSX 10 KB)

12985_2024_2603_MOESM6_ESM.xlsx

Additional file 6 This table provides each identified protein’s accession number, gene name, and molecular weight. Furthermore, t-tests were used to determine the significance and fold changes in secretome protein secretion between CHV-infected and uninfected samples. The list is organised in ascending order based on the t-test p values. The blue color represents significantly downregulated proteins (p < 0.05) with a fold change exceeding 1.3. Conversely, the orange color signifies proteins demonstrating statistical significance (p < 0.05) in upregulation, with a fold change greater than 1.3. A “missing value” indicates that the protein is secreted exclusively in uninfected samples, while a “reference missing” indicates that the protein is secreted solely in CHV-infected samples. (XLSX 108 KB)

Rights and permissions

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Prišlin Šimac, M., Naletilić, Š., Kostanić, V. et al. Canid alphaherpesvirus 1 infection alters the gene expression and secretome profile of canine adipose-derived mesenchymal stem cells in vitro. Virol J 21, 336 (2024). https://doiorg.publicaciones.saludcastillayleon.es/10.1186/s12985-024-02603-8

Download citation

  • Received:

  • Accepted:

  • Published:

  • DOI: https://doiorg.publicaciones.saludcastillayleon.es/10.1186/s12985-024-02603-8

Keywords

Virology Journal

ISSN: 1743-422X