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Transcriptomic analysis of differential gene expression reveals an increase in COX2 levels during in vitro canine herpesvirus infection/Analise transcriptomica da expressao genica diferencial revela um aumento nos niveis de COX2 durante a infeccao in vitro por herpesvirus canino.

INTRODUCTION

The canine herpesvirus causes a contagious viral infection among canids in many countries (ROBINSON et al., 2005; RONSSE et al., 2005). Etiological agent of canine herpesvirus belongs to the species Canine alphaherpesvirus 1 (CaHV-1), genus Varicellovirus, subfamily Alphaherpesvirinae, and family Herpesviridae (ICTV 2017). It is a virus in which double-stranded DNA is contained in a nucleocapsid, immersed in the tegument, and enclosed into a lipoprotein envelope (DAVISON et al., 2009). Naturally infected dogs with CaHV-1 represent an economic loss to breeding kennels due to reproductive, respiratory, and neurologic problems, and death by systemic disease in immunosuppressed adults and neonates (MALONE et al., 2010; EVERMANN et al., 2011; LARSEN et al., 2015). However, the pathogenesis of canine herpesvirus infection is not well known. The inactivated subunit vaccine against CaHV-1 is available in Europe, where it is used on breeding females, conferring short-duration passive immunity (CARMICHAEL & GREENE, 2012).

Herpesviruses are ubiquitous pathogens, well adapted to their hosts through many regulation mechanisms related to viral replication and cellular process domains (HOULDCROFT & BREUER, 2015; WEI et al., 2016). During productive infection, herpesviruses establish a complex and robust standard for viral genic expression (JURAK et al., 2011). Additionally, host response to viral infections involves intricate sets of regulatory processes (LAN et al., 2018). Therefore, the herpesviruses develop a mechanism to ensure the efficient translation of both viral and cell mRNA (GALE JR et al., 2000). The expression of the gene encoding prostaglandin-endoperoxide synthase 2 (PTGS2), better known as cyclooxygenase 2 (COX2), is induced by inflammatory response and infections, including those caused by viruses, in which COX products and derivatives may play a role in host response (KIRKBY et al., 2011; KUNZMANN et al., 2013). COX2 is involved in the production of prostanoids, which contribute to vasodilation, leukocyte chemotaxis, fever, and potential nociception (KIRKBY et al., 2011). However, during herpesvirus infection, the transcription of COX2 is essential for viral replication and virus-host interaction (RAY & ENQUIST, 2004). Under resting conditions, COX2 mRNA is not expressed by most cells (PABLOS et al., 1999).

Transcriptomic analysis is essential to study functional genome elements, subjacent mechanisms and disease triggers (YANG & KIM, 2015). RNA-sequencing (RNA-seq) using the next generation sequencing (NGS) platform has enabled the analysis of millions of transcripts from biological samples (MUTZ et al., 2014). Furthermore, RNA-seq is considered the gold standard for whole transcriptome gene expression quantification (EVERAERT et al., 2017). Thus, through transcriptional profile analysis, information on the biology of cells during certain conditions, as well as mechanisms and cell response to viral infection, can be obtained (WANG et al., 2009). Therefore, the purpose of this study was to analyze differential cellular gene expression during CaHV-1 infection in vitro. Additionally, the levels of COX2 expression in response to viral replication were evaluated. To our knowledge, this is the first transcriptomic analysis of CaHV-1 infection in vitro using RNA-seq.

MATERIALS AND METHODS

Cell culture and viral infection

For viral infection, a viral suspension of CaHV-1 strain BTU-1 (1061 TCID50) was obtained from the kidney of an infected neonatal puppy that died of a systemic infection. The genomic sequence of CaHV-1 strain BTU-1 was deposited in GenBank (KT828242). Subsequently, MDCK cells were subcultured in a 24-well plate in Dulbecco's modified Eagle medium (DMEM) and 10% fetal bovine serum (FBS), and maintained at 37[degrees]C in the presence of 5% CO2 and 80% humidity. After incubation for 24h, when 90% monolayer confluence was achieved, containing approximately 9.24x[10.sup.5] cells/well. Cells were infected with CaHV-1 at a multiplicity of infection (MOI) of 3. Next, the cell culture plate and viral suspension were maintained at 4[degrees]C for 1h to allow the adsorption and synchronization of the infection, as described by FLORES & KREUTZ, 2012. Next, the cell monolayer was washed three consecutive times with sterile phosphate-buffered saline (PBS) solution (0.01M P[O.sub.2.sup.-4], 0.14M NaCl, pH 7.4). Finally, DMEM containing 1% FBS was added, and the cells were incubated at 37[degrees]C, under conditions of 5% C[O.sub.2] and 80% humidity atmosphere. Nucleic acid extraction from cells was performed at different time periods after infection in duplicate.

Isolation of nucleic acids

For RNA extraction, the Total RNA Purification Kit (Norgen Biotek Corp.) was used according to the manufacturer's instructions. RNA samples were evaluated for purity by spectrophotometry using a Nanodrop ND1000 (Thermo Scientific), and the concentration was measured by fluorimetry using a Qubit 2.0 Fluorometer (Invitrogen Corp.). The integrity and quality of the RNA were analyzed using a Bioanalyzer 2100 (Agilent Technologies); a RNA integrity number (RIN) above 8.0 was considered acceptable. DNA extraction was performed using a Thermo Scientific GeneJET Genomic DNA Purification Kit (Thermo Fischer Scientific Inc.), following the manufacturer's instructions.

Sequencing libraries

Samples for sequencing libraries were prepared using the Sure Select Strand-specific RNA Library Prep reagents of the Illumina[R] Multiplexed Sequencing mRNA library kit (Agilent Technologies), following the manufacturer's instructions. Specifically, duplicate samples collected at pre-inoculation, 0h (post-adsorption), 1h post-inoculation (pi), 4h pi, 16h pi, 20h pi, 24h pi, 28h pi, and 32h pi were used. Quantification of the sample libraries was performed based on the absolute standard curve of the quantitative polymerase chain reaction (qPCR), using the Kapa Library Quantification Kit Illumina[R] platform (Kapa Biosystem Inc.), following the manufacturer's instructions. Amplification reaction of the library fragments was performed using an AB 7500 Fast thermal cycler (Applied Biosystem). After quantification, each sample library was normalized to 0.5nM to prepare a library pool. Capillary electrophoresis was performed using a Qiaxel Advanced System (Qiagen Company) to evaluate the presence of dimers and estimate the fragment length of the libraries. Additionally, the qPCR products were loaded onto an agarose gel (1.5%), stained with Neotaq Brilliant Plus DNA Stain (NeoBio) at 0.5[micro]L/mL, and electrophoresed. Gel bands were observed under ultraviolet light using a Fluor Chem FC2 Imaging system (Alpha Innotech Corp.) and analyzed using the Alpha View Program version 3.2.2 (Cell Biosciences Inc.).

Clustering and sequencing

For sequencing of mRNA libraries, the High Output Kit for up to 400M cluster-V2 75 cycles (Illumina) was used; sequencing was performed on the Illumina Next seq 500/550 platform (Illumina) for single-end and 75-bp sequences.

Differential expression analysis

The sequences obtained by RNA-seq were analyzed using the CLC Genomics Workbench 7.0.4 software (CLC Bio, Qiagen Company). Sequences were cleaned with a quality index limit of 0.08 and maximum limit of 2 ambiguous nucleotides and fragments above 40bp. In addition, the analysis of the components of differentially expressed genes (DEGs) reported in each library was performed. Sequence reads were analyzed with the RNA-seq software on the CLC platform, and mapped onto the mRNAs of the Canis lupus familiaris genome. Expression levels were assessed using the RPKM (reads per kilobase million mapped reads) as a method of analysis, and submitted to quantile normalization (ROBINSON & SMYTH, 2008). Next, transformed into log2 expression values for subsequent statistical analysis. The Kal test (Z-Test) was employed to identify differentially expressed transcripts (KAL et al., 1999). In DEG analysis, data filtering at least two fold-change (FC) values (>2 and <-2), P-values <0.05, a false positive rate (FPR) <0.05, and RPKM values [greater than or equal to] 2 were used.

Functional enrichment analysis based on gene ontology

Enrichment analysis was performed for differentially expressed transcripts using an FC value greater than 2 across the Gene Ontology Database released by the Gene Ontology Consortium (GOC; <http://www.geneontology.org>.

Reverse transcription (RT)-qPCR for COX2 gene expression validation

Total RNAs were isolated from cells at various times after viral infection (pre-inoculation, 0h, 1 h, 4 h, 16h, 20h, 24h, 28h, and 32h) using the Total RNA Purification Kit (NorgenBiotek Corp.). The samples were incubated with deoxyribonuclease I (Invitrogen Corp.) to remove residual DNA.RTqPCR experiments were carried out on a 7500 Fast Real-Time PCR System (Applied Biosystem) using Go Script Reverse Transcriptase reagents (Promega), 0.2 [micro]L of each primer (10 [micro]M), and nuclease-free water q.s.p.to achieve a final reaction volume of 20 [micro]L. Reverse transcription was performed at 45[degrees]C for 10min, followed by reverse transcriptase inactivation and Taq polymerase activation at 95[degrees]C for 2min, 40 cycles at 95 [degrees]C for 10s, and 60[degrees]C for 30s. Primers used for COX2 target amplification were based on CLEMENTE et al. (2013). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as the reference gene, and the sequence primers were based on those used in a study by PARK et al. (2013). Normalization of the COX2 expression data was performed to canine GAPDH, according to a previously published method (LARIONOV et al., 2005). The expression FC value was calculated using the 2-[DELTA][DELTA]Ct method, according to LIVAK & SCHIMITTGEN (2001).

Construction of the plasmid standards for the qPCR assay

Afragment of the TK gene (494bp) was used as the CaHV-1 standard DNA, with an external primer set described by SCHULZE and BAUMGARTNER (1998). This fragment sequence was cloned into the pGEM-T Easy Vector plasmid (Promega), according to the manufacturer's instructions. Escherichia coli JM109 was used as the cloning strain. The recombinant plasmids were purified using E.Z.N.A Plasmid Mini Kit (Omega Bio-tek), and quantified by fluorimetry using the Qubit[R] Quantification System (Invitrogen Corp.). For preparing the absolute standard curve, the recombinant plasmid DNA containing CaHV1TK region was serially diluted 10-fold, i.e. dilutions ranging from 10-1 to 10-10 were prepared in nuclease-free water. Aliquots (2[micro]L) of the diluted plasmid were used in duplicate in the qPCR for the amplification of TK using the internal primer set, as described by SCHULZE & BAUMGARTNER (1998).

Estimation of the viral load during in vitro infection by qPCR

Samples of the infected cells were collected in duplicate to perform DNA extraction for the determination of the viral load at the following infection times: 0h, 1h pi, 4h pi, 16h pi, 20h pi, 24h pi, 28h pi, and 32h pi. The mixture for the qPCR was prepared using the master mix qPCR Gotaq Sybr Green (Promega), 0.4[micro]L of each primer (0.01 [micro]M), 2 [micro]L of sample DNA, and nuclease-free water q.s.p, to achieve a final reaction volume of 20[micro]L. Amplification of TK was conducted using the thermocycler AB 7500 Fast (Applied Biosystem) under the following conditions: 95[degrees]C for 2min, 40 cycles at 95[degrees]C for 15s, 57[degrees]C for 10s, and 72[degrees]C for 30s. The amplification run contained control reactions. The negative control contained DNA extracted from uninfected MDCK cells, whereas the positive control contained the DNA extracted from CaHV-1 stocks. Nuclease-free water was used instead of DNA as the reaction control.

RESULTS AND DISCUSSION

Analysis of differentially expressed genes

After filtering and treatment of the sequences for the analysis of DEGs, RNA-seq generated 164.81 million reads, which were mapped to the canine genome to yield 124.02 million reads, which were analyzed by CLC Genomics Workbench 7.0.4 software. Of the 2,999 DEGs identified 1,662 were positively regulated and 1,335 were negatively regulated.

The collection times were initially selected to evaluate the cycle of infection of the virus in the cell, being 1h and 4h pi to evaluate the virus entry into the cell. Viral replication was evaluated at 16h pi. At 20h pi, it was evaluated the onset of cytopathic effects in infected cells and then monitored until 32h to evaluate the possible effects of cytolysis and apoptosis.

Cell genes expressed most up-regulated verified during in vitro CaHV-1 infection are shown in table 1. Among them, were verified genes associated with apoptosis or cell cycle arrest, cell proliferation and immune response. The genes with higher expression levels throughout the course of the infection were EGR-1 (1h pi, FC=35.30 [+ or -] 5.52), FOS (1h pi, FC=33.38 [+ or -] 0.89), and COX2 (32h pi; FC=26.41 [+ or -] 0.74). Thus, EGR-1 and FOS well as well DUSP-1, are important in response to the immune system to infection, including virus (BAHRAMI & DRABL0S, 2016). However, EGR-1 can increase viral replication by activating promoters of viral productive cycle genes and suppressing the promoter of the latent gene (CHEN et al., 2009). In a previous transcriptomic analysis, PAULUS et al. (2016) verified that the expression of FOS and DUSP-1 was activated by immediate stress response in cells infected with Suid alphaherpesvirus 1 (more commonly known as pseudorabies virus or Aujeszky's disease virus). Therefore, in the early stages of the viral infection, the highest expression of cellular immediate early genes (IEGs) was verified. IEGs are genes that encoding the first phase of mRNAs expressed from the viral genome of pre-existing host cell proteins (TINDALL and CLERK, 2014). Additionally, IEGs are expressed rapidly and transiently, induced in cells by mitogenic factors due to changes in the physiological state, stress and pathogens (RAHAUS & WOLFF, 2003; PRADO & DEL BEL, 1998).The ATF3 (activating transcription factor) is an IEG and designated as a stress-inducible gene (LU et al., 2006). The induction transcriptional mechanism of ATF3 by DNA damage response is rapid and transient (FAN et al., 2002). Thus, at 1h pi the ATF3 was up-regulated (FC=8.86 [+ or -] 0.81), but at 32h pi it expression was noted as being 2.3 times higher (FC=20.37 [+ or -] 2.24) than at 1h pi. Thus, ATF3 late expression may play a role as proapoptotic gene being induced by pro inflammatory cytokines and hypoxia (ZHANGA et al., 2002; LU et al., 2006). Table 2 contains the 20 major inhibited cell genes during CaHV-1 infection in MDCK cell. Most of the genes were associated with inhibition of maintenance or structure cell while the remainder were associated with cell regulation cycle or apoptosis and immune. Thus, the virus interact with cell cycle regulatory and rapidly shuts off cellular protein synthesis, dampening host responses to infection and allowing viral mRNAs to dominate translational machinery (FLEMINGTON, 2001; SMILEY, 2004). The most inhibited gene, ENSCAFG00000004590 (PTHR11143) which encodes the 60S ribosomal protein L26 (RPL26) family, showed the maximum inhibition at time 24h pi (FC=-236.25). In cells subjected to DNA damage or stress, RPL26 binds to the mRNA responsible for inducing the translation of the p53 protein, thus leading to cellular halt response or apoptosis (KASTAN & BARTEK, 2004). However, in cells with DNA damage and RPL26 inhibition, p53 protein levels do not increase (TAKAGI et al., 2005; CHEN & KASTAN, 2010). During infection, alphaherpesviruses use anti-apoptotic strategies to maintain their replication and propagation (YOU et al., 2017). Therefore, it is possible that CaHV-1 uses the mechanism of RPL26 expression inhibition to complete its life cycle. Considering that, alpha herpes virus infections occurs lytic replication in epithelial cells before becoming latent in the trigeminal ganglion (COHRS& GILDEN, 2016; LUECKE & PALUDAN, 2015). Therefore, in MDCK cell, inhibitory process of RPL26 genic expression could ensure viral multiplication and complete viral cycle.

The ATP5G2 and MRPS17 genes down-regulation can be related to compromising mitochondrial function. BRUGGEMANN et al. (2017), verified that ATP5G2 down-regulation reduced activity of the mitochondrial electron chain and this leads to reduced oxidative phosphorylation. The MRPS17 is required for the maintenance of respiratory-proficient mitochondria (HANLON et al., 2004). Some studies related the damage in this organelle by herpesvirus that induce shutoff of host functions, and contribute to the cell death and tissue damage (SAFFRAN et al., 2010; MURATA et al., 2000).

COX2 expression validation by RT-qPCR

Figure 1 shows the temporal evolution of PTGS2 (COX2) expression levels during infection, based on the RPKM values and relative quantification by RT-qPCR. Interestingly, both showed similar performance and a peak elevation at 32h pi. Additionally, expression of COX2 during CaHV-1 infection has been analyzed by comparing the FC values obtained with RNA-seq and RT-qPCR (Figure 2). The comparison performed between RNA-seq and RT-qPCR reveals a correlation between both techniques used for the quantification of COX2 expression level. Thus, it was verified that the RNA-seq technique can be reliable for the quantification of gene expression, and it was possible to confirm the increase in COX2 expression level.

In cells infected with PRV, the highest level of COX2 transcription occurred at 8h pi, showing a 11-fold elevation (RAY et al., 2004). In HSV-1-infected cells, the transcription of COX2 mRNA reached a fold-change peak of 14.5 at 8h pi (LIU et al., 2014). However, in CaHV-1 infection, the onset of COX2 elevation occurred 16h pi with FC = 2.54 [+ or -] 0.16 (RT-qPCR) and 1.51 [+ or -] 0.32 (RNA-seq). The peak of COX2 expression in CaHV-1 infection was observed later in relation to PRV and HSV-1 infection, reaching maximum levels 32h pi with FC values of 25.40 [+ or -] 2.37 (RT-qPCR) and 26.41 [+ or -] 3.27 (RNA-seq). These different expression patterns might occur due to infection cycle of CaHV-1 be longer than those exhibited by PRV and HSV-1. During in vitro PRV infection, the cytopathic effects occurs at 16h pi (PASDELOUP et al., 2013). In this study, virus isolate presents onset of cytopathic effect at 20h post inoculation and evidenced at 24h pi (data not shown). Additionally, LARA and colleagues (2016) observed late cytopathic effects during CaHV-1 infection after 48h pi, while DE PALMA et al. (2010) at 72h pi.

Hypoxia Response (GO: 0001666) was among the biological processes related to COX2 expression. Hypoxia inducible factors (HIFs) modulate the gene expression of DNA viruses, such as herpesviruses, due to the presence of elements responsive to hypoxia in their promoters, which increase viral replication (TOMASKOVA et al., 2011). High COX2 expression levels occur during inflammation, and oxygen levels decrease, thus leading to the activation of adaptive responses related to the expression of HIFs via nitric oxide (CARIA et al., 2014). In addition, hypoxia promotes vessel growth through the positive regulation of several pro-angiogenic pathways (KROCK et al., 2013). Interestingly, angiogenesis has been linked to elevated COX2 levels due to the increase of the levels of associated pro-angiogenic growth factors (YAO et al., 2011). Our gene ontology analysis confirmed the presence of the biological process Regulation of angiogenesis (GO: 0045765), among the up-regulated genes. Therefore, hypoxia, DNA damage response, and apoptosis may indicate the occurrence of viral replication and consequent increase in COX2 levels. Gene expression levels associated with hypoxia-related genes, with the exception of COX2, remained constant after their detection (24h pi). However, levels of other genes related to COX2 increased 32h pi, indicating that COX2 possibly interacts or activates other genes that promote other cellular biological processes. Thus, gene ontology analysis of the positively regulated genes revealed that when the FC value of COX2 increased, the number of biological processes that were involved with the gene increased as well (Figure 3).

Absolute viral load quantification and COX2 gene expression

Viral load during infection was measured by qPCR and correlated to COX2 expression by RT-qPCR (relative quantification) and RNA-seq (RPKM). Interestingly, both viral load and COX2 expression increased during the evolution of the infection, with the onset occurring 20h pi. Considering pre-inoculation COX2 levels as basal values, it may be suggested that viral replication is involved in the induction of COX2 expression. Moreover, the highest viral load (~1.62x[10.sup.6]copies/[micro]L) was reached 32h pi, which is when the expression level of COX2 also reached its maximum (Figure 1).The increase in both COX2 expression and viral load throughout the viral infection time can indicate a correlation between the two parameters.

COX2 transcripts are important for viral replication and virus-host interaction (RAY et al., 2004; REINOLDS & ENQUIST, 2006). Thus, herpesviruses exploit the induction of the COX2 cellular pathway for viral replication (CHENG et al., 2015). Therefore, the increase in COX2 transcription reported in this study was verified in infections by other herpesviruses, including bovine alphaherpesvirus 4 (BoHV-4), human herpesvirus 4 [HHV-4, also known as Epstein

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Barr virus (EBV)], human herpesvirus 6 (HHV-6), HSV-1, and Kaposi's sarcoma-associated herpesvirus (KSHV) (JANELLE et al., 2002; DONOFRIO et al., 2007; SHARMA-WALIA et al., 2010; AL-SALAM et al.2013; LIU et al., 2014). The role of COX2 in the viral replication cycle is unclear (REINOLDS & ENQUIST, 2006). However, in HHV-6, viral gene transcription is required for COX2 promoter activation (JANELLE et al., 2002). In cytomegalovirus infections, blockage of COX2 inhibited the viral mRNA by interrupting protein and DNA synthesis (ZHU et al., 2002).The increase in COX2 expression can lead to the enhanced production of prostaglandin (PG), which is an important factor in the inflammatory cascade reaction (LIU et al., 2014). The rise in prostaglandin levels was associated to latent herpesvirus activation (BAKER et al., 1985). In contrast, previous studies indicated that the PGs act as inhibitors of replication in some negative-strand RNA viruses such as the Sendai (SeV), Mayaro (MAYV) and polivirus (SANTORO et al., 1983; CONTI et al., 1996; BARBOSA & RABELLO, 1998). In these viruses, the PGs can affect an early and a late phase of viral production, including the synthesis and/or maturation of viral proteins (SANTORO et al., 1994).

COX2 inhibitors have been tested for the treatment of the infection and, interestingly, have proven efficient in reducing the frequency and severity of the lesions (CHENG et al., 2015). Moreover, use of selective COX2 inhibitors may favor interferon responses during infections, and increase antiviral immunity (KIRKBY et al., 2011). Considering the availability of drugs that selectively inhibit the COX2 pathway, further studies to assess their therapeutic potential for use against CaHV-1 infections are needed. However, these results may serve for as a basis for future studies on the use of selective COX2 inhibitors for the suppression of CaHV-1 replication in active infection states.

CONCLUSION

In summary, RNA-seq allowed for the transcriptomic analysis of differentially expressed genes in host cell in vitro, during CaHV-1 infection. COX2 (i.e., PTGS2) expression from infected cells was among the most induced. High expression levels of this gene were related to replication and viral load enhancement during CaHV-1 infection. In contrast, RPL26 was the most inhibited gene, possibly because of apoptosis inhibition, which contributed to viral infection in the host cells.

Therefore, the findings verified in this study can contribute to determining important targets for inhibiting or preventing canine herpesvirus infection either by cellular or viral mechanisms and lessening clinical signs and mortality during disease outbreaks.

http://dx.doi.org/ 10.1590/0103-8478cr20170945

ACKNOWLEDGMENTS

This study was financed in part by Coordenacao de Aperfeicoamento de Pessoal de Nivel Superior (CAPES) - Finance Code 001 and Fundacao do Instituto de Biociencias (FUNDIBIO) of Universidade Estadual Paulista (UNESP) Campus Botucatu.

BIOETHICS AND BIOSSECURITY COMMITTEE APPROVAL

The experimental design was approved by the local Ethics Committee for the Use of Animals (CEUA) of the UNESP, under the protocol number 141/2016, in accordance with the standards of the National Council for the Control of Animal Experimentation (CONCEA).

DECLARATION OF CONFLICTING INTERESTS

The authors declare no conflict of interest. The founding sponsors had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, and in the decision to publish the results.

AUTHORS' CONTRIBUTIONS

The authors contributed equally to manuscript.

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Jacqueline Kazue Kurissio (1) * Joao Pessoa Araujo Junior (1)

(1) Departamento de Microbiologia e Imunologia, Instituto de Biociencias, Instituto de Biotecnologia, Faculdade de Medicina Veterinaria e Zootecnia (FMVZ), Universidade Estadual Paulista (UNESP), Botucatu, 18618-691, SP, Brasil. E-mail: jkkurissio@yahoo.com.br.

* Corresponding author.

Caption: Figure 1-Correlation between COX2 expression and CaHV-1 viral load during infection. Comparison between COX2 expression levels obtained by RNA-sequencing (RNA-seq; RPKM), quantitative reverse transcription PCR (RT-qPCR; relative quantification, x1000), and CaHV1 viral load obtained by qPCR (absolute quantification x [10.sup.-3]). The CaHV-1 viral load at 0h was average of 5,190.18copies/[micro]L while at 32h pi was 1,215,253.76 copies/[micro]L increasing to a 234 fold number of viral genome.

Caption: Figure 2-Correlation of the fold-change values of COX2 expression. The values were obtained from RNA- sequencing (RNA-seq) and quantitative reverse transcription PCR (RT-qPCR).

Caption: Figure 3-Gene ontology of the genes regulated positively related to COX2 expression. The values are represented as fold-change (cutoff >2) involving biological process of cellular genes. RNA-seq analyses did not show the differential expression of PTGS2 at the time periods before 16h pi.
Table 1--List of 20 most up-regulated genes during in vitro CaHV-1
infection as a function of time (H) and quantity (FC).

Gene Symbol           Description             Gene product function

1. EGR-1         Early growth response      Transcriptional regulator
                       protein 1

2. FOS            Proto-oncogene c-Fos          Essential for cell
                                                proliferation and
                                                 differentiation

3. COX2        Prostaglandin g/h synthase    Increase cell adhesion,
                           2                   phenotypic changes,
                                             resistance to apoptosis
                                              and tumor angiogenesis

4. CHAC1       Cha C glutathione specific    Pro-apoptotic component
                         gamma-
               glutamylcyclotransferase 1

5. DDIT4          DNA damage-inducible        Responses to cellular
                  transcript 4 protein      energy levels and cellular
                                                stress, including
                                             responses to hypoxia and
                                                   DNA damage.

6. ATF3           Cyclic amp-dependent        This protein binds the
               transcription factor ATF-      camp response element
                           3                  (CRE), present in many
                                                viral and cellular
                                             promoters, repressor of
                                              inflammatory response

7. ISG20         Interferon stimulated          Interferon-induced
                  exonuclease gene 20       antiviral exoribonuclease

8. BTG2          BTG anti-proliferation     Anti-proliferative protein
                        factor2

9. RPL4         RPL4 ribosomalprotein L4         Compose a large
                                            ribonucleoprotein complex
                                               responsible for the
                                             synthesis of proteins in
                                                     the cell

10. DUSP1       Dual specificity protein       Cellular response to
                     phosphatase 1             environmental stress

11. MARCKSL1          MARCKS like1          Controls cell movement by
                                                 regulating actin
                                             cytoskeleton homeostasis

12. TNFAIP3    TNF alpha inducedprotein3         Involved in the
                                             cytokine-mediated immune
                                                 and inflammatory
                                                    responses.

13. SON               Protein SON           DNA-binding, tumorigenesis
                                                and apoptosis, but
                                            functional significance of
                                            this protein had not been
                                                   recognized.

14. MRPL33_1    Mitochondrial ribosomal        Large mitoribosomal
                      protein L33           subunit protein, possible
                                                  involvement in
                                            mitochondrial translation.

15. YPEL3            Yippee like 3            Inhibitory effects in
                                              normal and tumor cell
                                                      lines

16. SMIM10L1    Small integral membrane      The function SMIM10L1 is
                   protein 10 like 1                not clear

17. MYH14        Myosin heavy chain 14      Regulate the cytokinesis,
                                            motility and polarity cell

18. EIF4A1       Eukaryotic translation      Proliferation and growth
                 initiation factor 4A1                 cell

19. RPL37        Ribosomal protein L37         Component of the 60S
                                                     subunit

20. UBALD1     UBA like domain containing        No characterized
                           1

Gene Symbol    Time    FC

1. EGR-1        1h    35.30

2. FOS          1h    33.38

3. COX2        32h    26.41

4. CHAC1       32h    24.74

5. DDIT4       32h    20.83

6. ATF3
               32h    20.37

7. ISG20       32h    12.75

8. BTG2        32h    11.44

9. RPL4         1h    11.13

10. DUSP1       1h    10.79

11. MARCKSL1   32h    9.79

12. TNFAIP3    32h    9.52

13. SON
               16h    9.42

14. MRPL33_1   16h    9.14

15. YPEL3      32h    9.08

16. SMIM10L1   16h    8.13

17. MYH14      28h    7.76

18. EIF4A1     16h    7.38

19. RPL37      16h    6.61

20. UBALD1      0h     6.6

Table 2--List of 20 most down-regulated genes during in vitro CaHV-1
infection as a function of time (H) and quantity (FC).

Gene Symbol          Description            Encodes function protein

1. RPL26        Ribosomal protein L26          Cell proliferation

2. ATP5G2          ATP synthase, H+            Mitochondrial ATP
                    transporting,           synthesis coupled proton
              mitochondrial F0 complex,            transport
                      subunitc-2

3. ATP5MC2      ATP synthase membrane       Lipid binding and proton
                  subunit c locus 2        transmembrane transporter
                                                    activity

4. TMED9          Trans membrane p24       Transport proteins located
                trafficking protein 9          in the endoplasmic
                                              reticulum and Golgi
                                                    complex

5. RPL23A       Ribosomal protein L23a     Cell growth and regulation

               Ribosomal protein S27 or    Extra-ribosomal functions,
6. RPS27        metallopan-stimulin 1         regulate growth and
                       (MPS-1)              survival cell as well as
                                                 DNA repair and
                                                 transcription

7. ARPC3      Actin related protein 2/3         Control of actin
                  complex subunit 3         polymerization in cells

8. RPL29       RPL29 ribosomal protein         Cell proliferation

                         L29

9. S100A2        S100 calcium binding           Cell regulation
                      protein A2

10. CRYAB         Crystallin alpha B          Apoptosis inhibition

11. EEF1A1      Eukaryotice longation       Instead to upregulation,
                    factor 1 alpha           the down regulation of
                                            eEF1A1, is required for
                                                 the apoptosis

12. TMEM97     Transmembrane protein 97        Cell proliferation

13. DLADRA      MHC class II DR alpha          Cellular immunity
                        chain

14. MRPS17     Mitochondrial ribosomal       Mitochondrial proteins
                     protein S17                   synthesis

15. DHRS2      Dehydrogenase/reductase2    Cycle cell regulation and
                                                   apoptosis

16. MMP13     Matrix metallopeptidase 13       Cell proliferation

17. KRT14             Keratin 14              Structural molecule
                                              activity and keratin
                                                filament binding

18. SMIM7      Small integral membrane         Integral component
                      protein 7                     membrane

19. TMEM179B  Transmembrane protein 179B        Membrane protein
                                                  transporter

20. CCL17     C-C motif chemokine ligand    Mediating by chemotaxis
                          17                the infiltration of Th2
                                             cells in inflammatory
                                                    reaction

Gene Symbol    Time      FC

1. RPL26       24h     -236.25

2. ATP5G2      16h     -99.11

3. ATP5MC2      4h     -46.36

4. TMED9       16h     -38.05

5. RPL23A       0h     -35.08

6. RPS27        0h     -26.26

7. ARPC3        4h     -24.84

8. RPL29       16h     -24.54

9. S100A2      16h     -19.79

10. CRYAB       0h     -15.40

11. EEF1A1      4h     -14.12

12. TMEM97     16h     -12.94

13. DLADRA     28h     -12.26

14. MRPS17      4h     -11.17

15. DHRS2      28h     -10.95

16. MMP13       4h     -10.68

17. KRT14       0h     -10.65

18. SMIM7       4h     -10.39

19. TMEM179B    0h      -9.71

20. CCL17       4h      -9.69
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Title Annotation:MICROBIOLOGY
Author:Kurissio, Jacqueline Kazue; Araujo, Joao Pessoa, Jr.
Publication:Ciencia Rural
Date:Oct 1, 2018
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