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Down-Regulation of MicroRNA-210 Confers Sensitivity towards 1’S-1’-Acetoxychavicol Acetate (ACA) in Cervical Cancer Cells by Targeting SMAD4

MicroRNAs (miRNAs) are short non-coding RNAs that regulate genes posttranscriptionally. Past studies have reported that miR-210 is up-regulated in many cancers including cervical cancer, and plays a pleiotropic role in carcinogenesis. However, its role in regulating response towards anti-cancer agents has not been fully elucidated. We have previously reported that the natural compound 1’S-1’-acetoxychavicol acetate (ACA) is able to induce cytotoxicity in various cancer cells including cervical cancer cells. Hence, this study aims to investigate the mechanistic role of miR-210 in regulating response towards ACA in cervical cancer cells. In the present study, we found that ACA down-regulated miR-210 expression in cervical cancer cells, and suppression of miR-210 expression enhanced sensitivity towards ACA by inhibiting cell proliferation and promoting apoptosis. Western blot analysis showed increased expression of mothers against decapentaplegic homolog 4 (SMAD4), which was predicted as a target of miR-210 by target prediction programs, following treatment with ACA. Luciferase reporter assay confirmed that miR-210 binds to sequences in 3′UTR of SMAD4. Furthermore, decreased in SMAD4 protein expression was observed when miR-210 was overexpressed. Conversely, SMAD4 protein expression increased when miR-210 expression was suppressed. Lastly, we demonstrated that overexpression of SMAD4 augmented the anti-proliferative and apoptosis-inducing effects of ACA. Taken together, our results demonstrated that down-regulation of miR-210 conferred sensitivity towards ACA in cervical cancer cells by targeting SMAD4. These findings suggest that combination of miRNAs and natural compounds could provide new strategies in treating cervical cancer.

INTRODUCTION
Cervical cancer is one of the leading malignancies affecting women worldwide. In 2012, an estimated 528,000 new cases were diagnosed and 266,000 deaths were reported (Torre et al., 2015). Chemotherapy is often used alongside surgery and radiotherapy to improve the overall response and survival in cancer patients. Despite this, clinical outcomes remained dismal, especially in patients with recurrent or advanced cancer due to drug resistance and toxicities (Crafton and Salani, 2016;Gottesman, 2002). Hence, there is an on-going effort to develop effective targeted therapies for cervical cancer.
The use of naturally occurring compounds is promising because they are able to target multiple signaling pathways, besides having lower toxicities compared to some conventional chemotherapy agents (Millimouno et al., 2014). Some examples of the plant-derived natural compounds currently used in chemotherapy are paclitaxel, topotecan, vinblastine and vincristine (Cragg and Newman, 2005). The 1'S-1'acetoxychavicol acetate (ACA) is a natural compound isolated from the wild ginger, Alpinia conchigera. We have previously reported that ACA is able to induce comparable level of cytotoxicity to cisplatin when used as stand-alone, and potentiates the effects of cisplatin when used in combination on various cancer cell lines including cervical cancer (Awang et al., 2010;Phuah et al., 2013).
MicroRNAs (miRNAs) are highly conserved short non-coding RNAs which regulate gene expression by binding to its target sequences in messenger RNAs (mRNAs) to induce mRNA degradation or suppress translation (Lagos-Quintana et al., 2001). Since one miRNA can target many genes and one gene can be targeted by different miRNAs (Wu et al., 2010), they are able to regulate various cellular processes such as cell death (Othman et al., 2013), metastasis (Ho et al., 2014), proliferation (Liang et al., 2015) and chemosensitivity (Kim et al., 2015). Studies have shown that natural compounds such as curcumin and resveratrol can modify miRNA expression, and changes in the expression of these miRNAs can subsequently affect their anti-cancer activities (Phuah and Nagoor, 2014).
The up-regulation of miR-210, which is associated with poorer prognosis, have been reported in various cancers such as acute myeloid leukemia (Tang et al., 2015), breast (Camps et al., 2008), pancreatic (Greither et al., 2010) and cervical cancer (Rao et al., 2012). Over the years, a growing body of evidence has revealed the pleiotropic roles of miR-210 in cancer initiation and progression by targeting genes involved in regulating cell proliferation, apoptosis, cell cycle, angiogenesis, metastasis and DNA repair (Dang and Myers, 2015;Qin et al., 2014). However, the mechanistic role of miR-210 in regulating sensitivity towards anti-cancer agents has not been fully elucidated.
In this study, we found that miR-210 is down-regulated following treatment with ACA in both cervical cancer cell lines. Functional studies carried out indicated that suppression of miR-210 enhanced sensitivity towards ACA by decreasing cell proliferation and promoting apoptosis. The protein expression of SMAD4, which was identified as a putative target of miR-210 by miRNA target prediction programs, was found to be up-regulated following treatment with ACA. The direct binding of miR-210 to SMAD4 was subsequently confirmed using luciferase reporter assay. We also demonstrated that overexpression of miR-210 reduced SMAD4 protein expression while inhibition of miR-210 increased SMAD4 protein expression. Lastly, we showed that ectopic expression of SMAD4 augmented the antiproliferative and apoptosis-inducing effects of ACA.

Cell culture and ACA
The human cervical cancer cells Ca Ski and SiHa obtained from American Type Cell Culture (ATCC) were cultured in RPMI-1640 (Hylcone, USA) and DMEM (Hyclone, USA) containing 10.0% (v/v) fetal bovine serum (FBS) (Hyclone, USA), and maintained at 37.0℃ and 5.0% CO 2 in humidified atmosphere. The ACA was provided by the Centre for Natural Product Research and Drug Discovery (CENAR), Department of Chemistry, University of Malaya, Malaysia. Transient transfection miRNA transfection: Cells were plated at density of 3.0  10 5 cells and allowed to attach overnight. Cells were then transfected with 100 nM of miRIDIAN microRNA human hsa-miR-210 mimic or miRIDIAN microRNA human hsa-miR-210 hairpin inhibitor (Thermo Fisher Scientific, USA), complexed with DharmaFECT 1 Transfection Reagent (Thermo Fisher Scientific, USA) for 24 h according to the manufacturer's protocol. miRIDIAN microRNA Mimic Negative Control #1 and miRIDIAN microRNA Hairpin Inhibitor Negative Control #1 (Thermo Fisher Scientific, USA) were used as negative controls.
Plasmid transfection: Cells were plated at density of 1.5  10 5 cells and allowed to attach overnight. Cells were then transfected with 50 ng of pCMV6-SMAD4 (Origene Technologies, USA) complexed with DharmaFECT 1 Transfection Reagent (Thermo Fisher Scientific, USA) for 12 h according to manufacturer's protocol. The empty vector pCMV6 (Origene Technologies, USA) was used as negative control.

Quantitative real-time RT-PCR (RT-qPCR)
The total RNA was extracted using miRNeasy Mini Kit (Qiagen, Germany) and cDNA was synthesized with TaqMan  MicroRNA Reverse Transcription Kit (Applied Biosystems, USA) using Veriti 96-Well Thermal Cycler (Applied Biosystems, USA) according to the manufacturer's protocol. The miRNA expression level was determined with TaqMan  Mi-croRNA Assays (Applied Biosystems, USA) according to the manufacturer's protocol using Bio-Rad CFX96 ™ Real-Time PCR Detection System (Bio-Rad Laboratories, USA) and analyzed with Bio-Rad CFX Manager v2.1 (Bio-Rad Laboratories, USA). The U6 small nuclear RNA was used as an internal control to normalize RNA input. Fold changes were calculated using the 2 -ΔΔCt method (Livak and Schmittgen, 2001), and presented as normalized fold expression.

MTT cell viability assay
Transfected cells were treated with ACA for 12 h (plasmid transfection) or 48 h (miRNA transfection). Following incubation, 30.0 l of 3-(4,5-dimethylthiazol-2-l)-2,5-diphenyltetrazoliumbromide (MTT) reagent (5.0 mg/ml) was added into each well and incubated in the dark at 37℃ for 1 h. Media containing excess MTT reagent was then removed and formazan crystals were dissolved with 200.0 l dimethyl sulfoxide. Results were obtained by measuring the absorbance at 570 nm using microtiter plate reader (Tecan, Switzerland).
Annexin V/PI assay Transfected cells were treated with ACA (20 M for Ca Ski and 30 M for SiHa) for 12 h (plasmid transfection) or 48 h (miRNA transfection). Apoptotic cells were detected using BD Pharmingen Annexin V-FITC Apoptosis Detection Kit (BD Biosciences, USA) according to the manufacturer's protocol. Results were obtained using BD FACSCanto II flow cytometer (BD Biosciences, USA) and analyzed with BD FACSDiva software (BD Biosciences, USA).

Vector construction
The total RNA extracted was first converted into cDNA using RevertAid First Strand cDNA (Thermo Fisher Scientific, USA). The 3UTR regions of SMAD4 containing the predicted binding site were then amplified from the cDNA and inserted into the pmirGLO Dual-Luciferase miRNA Target Expression Vector (Promega, USA) to construct the luciferase reporter constructs. Site-directed mutations in the predicted binding site for SMAD4 were perfomed using the QuikChange Multi Site-Directed Mutagenesis Kit (Stratagene, USA) according to the manufacturer's protocol. The sequences of the constructs were verified by sequencing.

Dual-luciferase assay
Cells were co-transfected with 40 ng of 3UTR reporter constructs containing wild-type or mutated binding sites and 100 nM of miR-210 mimic or mimic negative control using DharmaFECT 1 Transfection Reagent (Thermo Fisher Scientific, USA). The firefly and Renilla luciferase activities were measured 48 h post-transfection with Dual-Glo  Luciferase Assay System (Promega, USA) according to the manufacturer's protocol using GLOMAX  -Multi Jr (Promega, USA). The firefly luciferase activity was normalized to Renilla activity, which was used as an internal control.

Western blot
Proteins were extracted using RIPA buffer containing 1 Halt Protease and Phosphatase Inhibitor Cocktail (Thermo Fisher Scientific, USA) and quantified using BCA Protein Assay Kit (Pierce, USA) according to manufacturer's protocol. Total cell lysates were fractionated by 12% sodium dodecyl sulfatepolyacrylamide (SDS-PAGE) gel electrophoresis and transferred onto nitrocellulose membranes. Membranes were blocked for 1 h with 5% (w/v) non-fat milk followed by overnight incubation at 4℃ with primary antibodies against SMAD4 (1:1000; Cell Signaling Technology, USA) and GAPDH (1:1000; Cell Signaling Technology, USA). Detection was carried out using secondary IgG HRP-linked antibody and anti-biotin HRP-linked antibody (1:1000; Cell Signaling Technology, USA). Bands were visualized using Western-  Bright Quantum (Advansta, USA) on Fusion FX7 system (Vilber Lourmat, France) and quantified using the ImageJ Analyst software (NIH, USA), with band intensities normalized to GAPDH, which was used as loading control.

Statistical analysis
All experiments were carried out in triplicates and presented as mean values ± standard error mean. Student's t-test was used to determine the statistical significance of results, whereby value of P < 0.05 was considered as statistically significant.

ACA down-regulates mir-210 expression and suppression of miR-210 confers sensitivity towards ACA
To study the effects of ACA on miR-210 expression, the expression of miR-210 in cells treated with ACA was determined using RT-qPCR. Figure 1A indicated that miR-210 expression is down-regulated following treatment with ACA in both Ca Ski and SiHa cells. To study the function of miR-210, we transfected miR-210 hairpin inhibitor and hairpin inhibitor negative control into both cervical cancer cell lines. Results from RT-qPCR showed that transfection with miR-210 hairpin inhibitor successfully suppressed miR-210 expression level when compared to hairpin inhibitor negative control (Fig. 1B). Next, the effects of ACA on the transfected cells were analyzed using MTT cell viability assay. As shown in Fig. 1C, cells transfected with miR-210 hairpin inhibitor were more sensitive towards ACA, indicating that downregulation of miR-210 conferred sensitivity towards ACA. However, no significant changes in sensitivity towards ACA were observed when miR-210 was overexpressed (data not shown).

Suppression of miR-210 increases ACA-induced apoptosis
To determine if the effects from combinatorial treatment with miR-210 hairpin inhibitor and ACA were modulated by apoptosis, Annexin V/PI and Caspase 3/7 assays were utilized. Transfection with miR-210 hairpin inhibitor markedly increased the apoptotic cells following exposure to ACA ( Fig.  2A). Correspondingly, Fig. 2B showed that suppression of miR-210 induced higher Caspase 3/7 activity in ACA-treated cells. Taken together, these results showed that suppression of miR-210 promoted ACA-induced apoptosis. No significant differences were observed when miR-210 was overexpressed in the cells (data not shown).

miR-210 directly targets SMAD4
To identify the potential targets of miR-210, miRNA target prediction programs were used. Both TargetScan v7.1 and miRanda predicted SMAD4 as a putative target of miR-210.
Western blot was carried out to study the effects of ACA on SMAD4 protein expression, and results showed that SMAD4 is up-regulated following treatment with ACA (Fig. 3A). To confirm that miR-210 directly targets the 3UTR of SMAD4, luciferase reporter vector containing wild-type or mutated binding site for SMAD4 (Fig. 3B) were constructed. Figure  3C showed that miR-210 overexpression significantly reduced luciferase activity when co-transfected with vector containing wild-type binding site but not in vector containing mutated binding site, confirming that SMAD4 is direct target of miR-210. To assess if miR-210 can regulate SMAD4 protein expression, western blots were performed. Results showed that overexpression of miR-210 reduced SMAD4 protein level, while inhibition of miR-210 increased SMAD4 protein level (Fig. 3D).
Overexpression of SMAD4 augments anti-proliferative and apoptosis-inducing effects of ACA To assess the role of SMAD4 in regulating response towards ACA, SMAD4 was transiently overexpressed using pCMV6-XL5 vector containing SMAD4 sequence (pCMV6-SMAD4) while empty vector lacking SMAD4 sequence (pCMV6) was used as negative control. To evaluate the anti-proliferative effects of ACA on the transfected cells, MTT cell viability assay was carried out. Results showed a significant reduction in the cell viability when SMAD4 is overexpressed (Fig. 4A).
To determine if SMAD4 overexpression augments ACAinduced apoptosis, transfected cells treated with ACA were analyzed using Annexin/PI assay. As seen in Fig. 4B, a significant increase in the number of apoptotic cells was observed when SMAD4 is overexpressed. To verify this, the activation of Caspase 3/7 was assayed. Similarly, a marked increase in Caspase 3/7 activity was observed in SMAD4-overexpressing cells treated with ACA (Fig. 4C). These results demonstrated that overexpression of SMAD4 augmented the antiproliferative and apoptosis-inducing effects of ACA.

DISCUSSION
The use of cisplatin-based regimens has long been reported to be most active in treating cervical cancer despite median overall survival being only around 1 year (Monk et al., 2009). However, the addition of targeted therapy such as bevacizumab, a humanized monoclonal antibody against vascular endothelial growth factor A (VEGF-A), has greatly improved the overall survival in advanced or recurrent cervical cancer compared to the use of chemotherapy alone. Subsequently, US Food and Drug Administration approved the use of bevacizumab in persistent, recurrent or metastatic cervical cancer in combination with chemotherapy in 2014 (Crafton and Salani, 2016). This advancement has spurred interests to identify new combinations to improve efficacies in chemotherapy.
The miR-210 is frequently up-regulated in many cancer types, including cervical cancer (Rao et al., 2012). In cervical cancer patients, miR-210 was also found to be expressed at higher levels in HPV-positive patients than HPV-negative patients (Lou et al., 2016). Since miR-210 is overexpressed in HPV-positive Ca Ski and SiHa cells compared to normal cervical tissues and HPV-negative cervical cancer cell line (C-33a) (Martinez et al., 2008), these two cell lines were selected to be used for this study.
Previous studies have reported that down-regulation of miR-210 in combination with radiotherapy enhanced antitumor effects on human hepatoma cell lines and xenograft (Yang et al., 2012;. Beside this, an association between miR-210 plasma levels and trastuzumab resistance in patients with HER2-positive breast cancer was also observed (Jung et al., 2012). These studies revealed the potential role for miR-210 in cancer therapies. In the present study, we demonstrated that down-regulation of miR-210 enhanced sensitivity towards ACA by augmenting ACA-induced apoptosis, indicating that miR-210 plays a role in regulating response towards anti-cancer agents.
In addition, we have also identified SMAD4 as a direct target of miR-210. Loss or reduction in SMAD4 function has been reported in many cancers such as breast (Liu et al., 2015), colorectal (Miyaki et al., 1999) and cervical cancer (Baldus et al., 2005;Kloth et al., 2008). Additionally, reduced SMAD4 expression was also observed in HPV-positive tumors compared to HPV-negative tumors (Baez et al., 2005;Kloth et al., 2008). Contrastingly, SMAD4 was reported to be oncogenic in other cancers such as hepatocellular carcinoma (Hernanda et al., 2015), suggesting that its role might be cell-context-dependent. Past studies have shown that ectopic expression of SMAD4 induced apoptosis on SiHa cells (Lee et al., 2001), and reduced cervical cancer growth in nude mice (Klein-Scory et al., 2007), indicating a tumor suppressive role for SMAD4 in cervical cancer. Different groups have reported a positive correlation between SMAD4 expression and sensitivity towards cetuximab in head and neck squamous cell carcinoma (Cheng et al., 2015) as well as 5-fluorouracil in breast (Yu et al., 2013) and colorectal cancer (Papageorgis et al., 2011;Zhang et al., 2014;. In this study, we showed that overexpression of SMAD4 in cervical cancer cells augmented antiproliferative and apoptosis-inducing effects of ACA, indicating that SMAD4 plays a role in regulating response towards anti-cancer agents.
In summary, our results demonstrated that down-regulation of miR-210 enhanced sensitivity towards ACA in cervical cancer cells by regulating SMAD4. These findings suggest that combination of miRNAs and natural compounds could provide new strategies in treating cervical cancer.


extfieldsofstudy: Biology Medicine

s2fieldsofstudy: Biology Medicine

text:
α7 nicotinic acetylcholine receptor agonist attenuates the cerebral injury in a rat model of cardiopulmonary bypass by activating the Akt/GSK3β pathway

α7 nicotinic acetylcholine receptor (α7nAchR) agonist treatment may provide a promising therapeutic effect for cerebral injuries. However, it is unclear whether the activation of α7nAchR agonist may reduce cerebral injuries induced by cardiopulmonary bypass (CPB). A total of 96 male Sprague-Dawley rats were randomly divided into four groups (n=24/group): i) Sham operation group; ii) CPB group; iii) CPB + α7nAchR agonist group; and iv) CPB + α7nAchR agonist + α7nAchR antagonist group. Following treatment, 24 rats from each group were sacrificed and the serum and hippocampal tissues were collected. The serum expression levels of S100β, interleukin 6 and tumor necrosis factor α were evaluated by ELISA, hippocampal tissues were analyzed by histopathological examination using hematoxylin & eosin and terminal deoxynucleotidyl-transferase-mediated dUTP nick-end labeling (TUNEL) staining and Caspase 3 expression in the hippocampal tissues was evaluated by immunohistochemistry. In addition, Caspase 3, Akt and glycogen synthase kinase 3β (GSK3β), as well as phosphorylated (p)-Akt and (p)-GSK3β were examined by western blot assay. The present study demonstrated that α7nAchR agonist treatment was able to alleviate pathological damage and inhibit hippocampal cell apoptosis and inflammatory response. α7nAchR agonist treatment also increased the expression levels of p-Akt and p-GSK3β, which indicated an upregulation in Akt/GSK3β signaling. These data suggested that α7nAchR agonist may provide a promising new therapeutic approach for cerebral injury caused by CPB.

Introduction
Cardiopulmonary bypass (CPB) is considered indispensable during heart operations, but the potential adverse effects on sensitive organs, such as the brain or the kidneys, cannot be ignored (1). In particular, many of the patients who undergo CPB surgery suffer from adverse cerebral outcomes, which may include stroke, postoperative cognitive dysfunction and transient ischemic attacks (2). The underlying molecular mechanism of cerebral injuries induced by CPB is unknown; however, the pathological changes may in part be due to microemboli and impaired cerebral perfusion, as well as cerebral ischemia and inflammatory damage (3,4).
It has been previously reported that CPB may initiate systemic inflammatory reaction syndrome (SIRS) owing to the blood comprehensive contact with non-biological materials (5); CPB may also activate cerebral inflammation in the presence of blood-brain barrier injury or disruption (3,6). Therefore, inflammatory responses serve important roles in the progression of cerebral injures induced by CPB, and reducing inflammation would be of great benefit for CPB surgery of (5,7). For example, ulinastatin treatment exhibited neuroprotective effects on an animal model of CPB, possibly through beneficial effects on anti-inflammatory systems (8).
The cholinergic anti-inflammatory pathway (CAP) is an endogenous neural feedback regulation mechanism and can regulate peripheral inflammatory responses (9). Therefore, the physiological regulation of CAP has been used to treat infectious or inflammatory animal models (9)(10)(11)(12)(13)(14). Stimulation of the efferent vagus nerve releases the important neurotransmitter acetylcholine, which acts through the α7 nicotinic acetylcholine receptor (α7nAchR) expressed in the macrophages and the brain. Notably, it has been revealed that activation of α7nAchR may effectively decrease the expression of proinflammatory cytokines and inhibit the inflammation process (10,(15)(16)(17)(18). In addition, the α7nAchR agonist PHA568487 has been used to α7 nicotinic acetylcholine receptor agonist attenuates the cerebral injury in a rat model of cardiopulmonary bypass by activating the Akt/GSK3β pathway treat neuroinflammation following tibia fracture and endotoxemia in mice (15), as well as ischemic stroke injury (16) and brain injury in a subarachnoid hemorrhage model rats (19). Therefore, the α7nAchR agonist may provide promising therapeutic effects for cerebral injuries. However, it is still unclear whether activation of the α7nAchR agonist is able to reduce cerebral injuries induced by CPB. The present study evaluated the therapeutic effects and the molecular mechanisms of the α7nAchR agonist on CPB-induced brain injury in a rat model. The results indicated that the α7nAchR agonist may effectively inhibit the inflammatory response and reduce apoptosis by activating the Akt/GSK3β signaling pathway. CPB animal model establishment. CPB surgery was performed as previously reported (7), with minor modifications. Briefly, rats received an intraperitoneal (i.p.) injection of 10% chloral hydrate (300 mg/kg; Shanghai Ziyuan Pharmaceutical Co., Ltd., Shanghai, China) for anesthesia. Photopic oral intubation was performed using a 16 G intravenous (i.v.) catheter, and animals were mechanically ventilated with a small animal ventilator (settings: Frequency, 60 beats/min; tidal volume, 3 ml/kg; inspiratory to expiratory ratio, 1:1.5) connected to a monitor to observe the heart rate, oxygen saturation and rectal temperature of the rats. During surgery, anesthesia was maintained with i.v. injection of pipecuronium bromide (0.1 mg/kg; Hangzhou Minsheng Pharmaceutical Co., Ltd., Hangzhou, China).

Materials and methods
The puncture site was sterilized with iodophor (Shandong Lierkang Disinfection Technology Co., Ltd., Dezhou, China), followed by exposure and puncture of the vein. Right femoral vein catheterization (24G) was performed to open the fluid path, which was transfused with 6% hydroxyethyl starch (Guangdong Jiabao Pharmaceutical Co., Ltd., Qingyuan, China) and connected to a microinfusion pump. The left femoral artery was catheterized (22G) and used to monitor blood pressure. Coccygeal artery catheterization (22G) and right internal jugular vein catheterization (18G) were performed to drain blood for CPB. The drainage tube, a homemade blood storage device, a constant peristaltic pump (Baoding Longer Precision Pump Co., Ltd., Baoding, China), silicone tubing (internal diameter, 4 mm) and a rat membrane oxygenator (Guangdong Kewei Medical Instrument Co. Ltd., Dongguan, China) were installed between the two puncture sites to establish the CPB circuit. Heparin sodium (300 IU/kg; Shenyang Haitong Pharmaceutical Co., Ltd., Shenyang, China) was injected into the left femoral vein once the activated clotting time reached 480 sec.
CPB was performed with the membrane oxygenator to supply oxygen. The low-flow CPB velocity was 35 ml/kg/min, which was later increased to 100-120 ml/kg/min at full-flow bypass. To prevent air embolism, 1-2 ml of blood was retained in the blood storage device. Mean arterial pressure was maintained at >60 mmHg, partial CO2 pressure at 35-45 mmHg, base excess at -3-3 mmol/l mmHg, pH at 7.35-7.45 and hematocrit at >0.25. Rats were treated with 2-20 µg/100 g epinephrine hydrochloride (Wuhan Grand Pharmaceutical Group Co., Ltd., Wuhan, China) and fluids during surgery to maintain a stable circulation.
Groups and treatments. Rats were randomly divided into four groups (n=24/group): i) The Sham group (S group), in which intubation and mechanical ventilation were performed in the right femoral artery only and the right internal jugular vein was catheterized without bypass; ii) the CPB surgery group (C group), which received the CPB surgery aforementioned; iii) the α7nAchR agonist group (P group), which received an i.p. injection of the α7nAchR agonist PHA568487 (0.8 mg/kg; Tocris Bioscience; Bio-Techne, Minneapolis, MN, USA) 30 min prior to CPB establishment; and iv) the PHA568487 + α7nAchR antagonist group (M group), which were also pretreated with PHA568487 (0.8 mg/kg) for 30 min, followed by i.p. injection of the α7nAchR antagonist methyllycaconitine (MLA; 6 mg/kg; Sigma-Aldrich; Merck KGaA, Darmstadt, Germany). CPB surgery was performed 60 min after MLA injection.
Specimen collection and processing. Arterial and venous blood samples were collected prior to CPB (T0), upon completion of CPB surgery (T1), 2 h post-CPB (T2) and 6 h post-CPB (T3); subsequently, rats were sacrificed with 2% pentobarbital sodium (40 mg/kg by i.p. injection; Merck Sharp & Dohme, Shanghai, China). The systemic circulation system of the rats was infused with saline (250-400 ml), and the whole brain was collected on the ice and divided into two halves along the median sagittal line. The hippocampus was isolated from each of the two halves, the right half was fixed in 4% paraformaldehyde (PFA) at room temperature for 24 h, and the left side was stored at -80˚C for western blot analysis. Sera were separated by centrifugation at 1,000 x g for 10 min at 4˚C, and stored at -80˚C.
Histopathological assessment. Fixed hippocampal tissues were gradually dehydrated with ethanol and embedded in paraffin. Paraffin blocks were subsequently sectioned (5 µm) and stained with a Hematoxylin & Eosin (H&E) staining kit (Nanjing Jiancheng Bioengineering Institute, Nanjing, China). Double-blind evaluation of hippocampal injury was performed by two expert pathologists. Images of the histopathological examination were captured by a light microscope (Olympus Corporation, Tokyo, Japan) at x400 magnification.
Tissue apoptosis assay. A terminal deoxynucleotidyl-transferase-mediated dUTP nick-end labeling (TUNEL) Assay kit (Shanghai Fusheng Industrial Co., Ltd., Shanghai, China) was used, according to the manufacturer's protocol, to determine the effects of α7nAchR agonist treatment on apoptosis in the fixed and mounted hippocampal sections. DAPI was used as a nuclear stain, with sections stained with 100 ng/ml DAPI for 5 min. Apoptotic rates were examined and images captured using a light microscope (Olympus Corporation, Japan) at a magnification of x400, and the densitometric scanning was finally analyzed by using the MetaMorph BX41 Image Analysis System (Olympus Corporation, Japan). A total of 5 images were captured randomly for each section at x400 magnification and integral optical density was calculated using Microscopic Image Analyzer (MetaMorph BX41 Image Analysis System). Percentages of TUNEL-positive cells above untreated controls were calculated as follows: %apoptosis = (number of TUNEL-positive cells / number of total cells) x 100.
Immunohistochemistry. To further determine the effects of α7nAchR on apoptosis in the hippocampus, expression levels of the cellular apoptosis maker Caspase 3 was examined by immunohistochemical analysis. Briefly, dimethylbenzene was used to remove the paraffin from the hippocampal sections, followed by immersion in distilled water. Subsequently, antigen retrieval was conducted by placing the slides in a microwave in 10 mmol/l citrate buffer, pH 6.0, for 15 min. The slides were washed with 0.01 mmol/l PBS (HyClone; GE Healthcare Life Sciences, Logan, UT, USA) every 5 min for 3 times, followed by incubating in TBS + 0.3% H 2 O 2 + 0.1% saponin at room temperature for 15 min to block the endogenous peroxidase. The slides were blocked with goat serum (Sigma-Aldrich; Merck KGaA) in TBS + 0.1% saponin for 20 min at room temperature, followed by incubating with polyclonal rabbit anti-Caspase 3 (1:300; ab13847; Abcam, Cambridge, UK) overnight at 4˚C. The slides were incubated with biotin-conjugated secondary antibody (1:2,000; ab6720; Abcam) for 30 min, and 3,3'-diaminobenzidine stain (8 min at room temperature) was used to visualize Caspase 3 expression in the hippocampus. Images of Caspase 3 expression were captured with a light microscope (Olympus, Japan) at a magnification of 400x. A total of 5 images were captured randomly for each section at x400 magnification and integral optical density was calculated using Microscopic Image Analyzer (MetaMorph BX41 Image Analysis System).
Statistical analysis. Quantitative data were expressed as the mean ± standard deviation. Statistical analyses were performed with GraphPad Prism software, (version 6.00; GraphPad Software, Inc., La Jolla, CA, USA). Multiple comparisons were analyzed with one-way analysis of variance, followed by an appropriate multiple comparison test (Tukey's procedure). P<0.05 was considered to indicate a statistically significant difference.

α7nAchR agonist alleviates pathological injury caused by CPB.
To determine the protective effects of α7nAchR agonist on the morphological alterations of the hippocampus, sections were evaluated at 6 h post-CPB, the T3 time point, by H&E staining. There was no detectable morphological damage to the hippocampal tissues in the S group (Fig. 1A), whereas clear cellular degeneration and abnormal cell arrangements were observed in the samples of CPB-injured rats (Fig. 1B), which indicated that the rat model of cerebral injury caused by CPB was successfully established. Following pretreatment with the α7nAchR agonist, only a slight morphological change was observed in the P group as compared to those in the C group (Fig. 1C), which suggested that the α7nAchR agonist may have alleviated the pathological injury of the CPB-injured rats; however, the typical vacuolated degenerations in hippocampal neurons were observed in those co-treated with α7nAchR antagonist (Fig. 1D), indicating the protective effects of α7nAchR agonist may be inhibited by MLA treatment. These results suggested that activation of α7nAchR may alleviate CPB-induced pathological injury. α7nAchR agonist inhibits CPB-induced apoptosis of hippocampal neurons. To determine the effects of α7nAchR agonist treatment on apoptosis in hippocampal neurons, the T3 sections were also evaluated by TUNEL staining. Compared with the control neurons in the S group, the neurons in the C group exhibited typical signs of apoptosis ( Fig. 2A); neuronal apoptosis appeared to be lower in the P and M groups when compared with the C group ( Fig. 2A). To further determine the effects of the α7nAchR agonist on hippocampal neuron apoptosis, the integrated OD average of apoptosis positive area was quantified in captured images from all experimental groups. Compared with the S group, apoptosis was significantly increased in CPB-injured rats in groups C, P and M, which suggested that hippocampal cell apoptosis may be induced following CPB surgery. Notably, a lower rate of neuronal apoptosis was observed in rats pretreated with the α7nAchR agonist compared with the C group (P<0.05; Fig. 2B); however, apoptosis was significantly increased in rats co-treated with the α7nAchR antagonist compared with the P group (Fig. 2B). These results indicated that CPB-induced apoptosis of hippocampal neurons may be effectively reduced by pretreatment with the α7nAchR agonist.
In the light of the inhibition of α7nAchR agonist on apoptosis of hippocampal neurons, the protein expression levels of Caspase 3, a key downstream inducer of apoptosis (20), was evaluated by western blot assay. In the T0 and T1 tissue specimen, no significant differences were detected in Caspase 3 expression between any of the groups, which suggested that apoptosis was not induced at this period in time. Conversely, tissues at T2 and T3 exhibited increased Caspase 3 expression in the CPB-injured rats in groups C, P and M compared with expression in the S group rats (P<0.05; Fig. 3A and B), which implied that apoptosis was activated 3-6 h post-CPB surgery. Caspase 3 expression was significantly decreased in P group rats following pretreatment with α7nAchR agonist compared with the C group (P<0.05), whereas this effect was reversed in M group rats co-treated with the α7nAchR antagonist (P<0.05; Fig. 3A and B).
To confirm the location of Caspase 3 expression in the hippocampus, immunohistochemical analysis was used to determine the expression at T2, as the Caspase 3 expression reached a peak in the CPB-injured rats at T2 according to the western blotting data aforementioned. Caspase 3 expression was detected in the neurons of hippocampus (Fig. 3C), and Caspase 3 expression was significantly inhibited in the P group compared with the C group (P<0.05; Fig. 3D), which was consistent with western blotting results. Therefore, these results indicated that the α7nAchR agonist may effectively inhibit apoptosis in hippocampal neurons, which may partly be accomplished by suppressing the expression of Caspase 3. α7nAchR agonist pretreatment reduces serum levels of S100β, TNF-α and IL-6 in CPB-injured rats. Serum expression levels of S100β, TNF-α and IL-6 were measured to evaluate the inflammatory response in rats with CPB injury. Compared with the control S group, rats in the CPB groups C, P and M exhibited significantly increased levels of S100β, TNFα and IL6 at experimental time points T1-T3 (P<0.05; Fig. 4A-C, respectively), which was considered as an indicator of serious cerebral injury. The levels of S100β, TNFα and IL6 were significantly decreased rats in the P group following pretreatment with 7nAchR agonist compared with the expression levels in CPB model rats in the C group at the T1-T3 experimental time points (P<0.05); however, rats in the M group exhibited an increase in serum expression levels compared with the P group (P<0.05; Fig. 4A-C). The apparent improvement of  were observed in rats in the P group following pretreatment of α7nAchR agonist compared with those in the C group at T2 and T3. (C) Immunohistochemistry for Caspase 3 in hippocampus at T2. Magnification, x400; scale bar, 20 µm; red arrows indicate positive expressions. (D) Integrated OD average analysis indicated the decreased expression levels of Caspase 3 in P group rats pretreated with the α7nAchR agonist compared with expression in the C group model rats. Data are presented as the mean ± standard deviation; n=24/group; * P<0.05 vs. S group; # P<0.05 vs. C group; ∆ P<0.05 vs. P group. α7nAchR, α7 nicotinic acetylcholine receptor; C group, CPB surgery only; CPB, cardiopulmonary bypass; M group, CBP + α7nAchR agonist PHA568487 + α7nAchR antagonist methyllycaconitine; P group, CBP + α7nAchR agonist PHA568487; S group, Sham operation; T0, prior to CPB; T1, upon completion of CPB; T2, 3 h post-CPB; T3, 6 h post-CPB. inflammation suggested that α7nAchR agonist pretreatment may have a beneficial effect on anti-inflammatory systems of the rat model of CPB. α7nAchR agonist pretreatment promotes phosphorylation of Akt and GSK3β. To further explore the underlying mechanisms by which α7nAchR agonist alleviated the cerebral injuries caused by CPB, Akt/GSK3β pathway activation was examined to determine the protective effects of α7nAchR agonist on the CPB-injured rats, as the Akt/GSK3β pathway was previously identified as a significant cell survival pathway (21). CPB rats in the C group exhibited a significant increase in the expression of p-Akt and p-GSK3β compared with expression levels in the S group (P<0.05; Fig. 5); whereas the expression levels of p-Akt and p-GSK3β were significantly increased in the P group following pretreatment with the α7nAchR agonist, Figure 4. α7nAchR agonist pretreatment reduces the serum expression levels of S100β, TNF-α and IL-6 in CPB-injured rats. The serum levels of (A) S100β, (B) TNF-α and (C) IL-6 were measured to evaluate the anti-inflammation effects of α7nAchR agonist on CPB-injured rats. Serum levels of S100β, TNFα and IL6 were significantly decreased in P group rats following pretreatment with the α7nAchR agonist compared with levels in the C group model rats. Data are presented as the mean ± standard deviation; n=24/group; * P<0.05 vs. S group; # P<0.05 vs. C group; ∆ P<0.05 vs. P group. α7nAchR, α7 nicotinic acetylcholine receptor; C group, CPB surgery only; CPB, cardiopulmonary bypass; M group, CBP + α7nAchR agonist PHA568487 + α7nAchR antagonist methyllycaconitine; IL, interleukin; P group, CBP + α7nAchR agonist PHA568487; S group, Sham operation; T0, prior to CPB; T1, upon completion of CPB; T2, 3 h post-CPB; T3, 6 h post-CPB; TNF-α, tumor necrosis factor α. Figure 5. α7nAchR agonist promotes expression of p-Akt and p-GSK3β. (A) Western blot analysis for Akt and p-Akt in the hippocampus at T3. (B) p-Akt expression levels were significantly increased in the P group following pretreatment with the α7nAchR agonist compared with expression in the C group model rats. (C) Western blot analysis for GSK3β and p-GSK3β in the hippocampus at T3. (D) p-GSK3β expression levels were significantly increased in the P group following pretreatment with the α7nAchR agonist compared in the C group model rats. Data are presented as the mean ± standard deviation; n=24/group; * P<0.05 vs. S group; # P<0.05 vs. C group; ∆ P<0.05 vs. P group. α7nAchR, α7 nicotinic acetylcholine receptor; C group, CPB surgery only; CPB, cardiopulmonary bypass; GSK3β, glycogen synthase kinase 3β; M group, CBP + α7nAchR agonist PHA568487 + α7nAchR antagonist methyllycaconitine; p, phosphorylated; P group, CBP + α7nAchR agonist PHA568487; S group, Sham operation; T3, 6 h post-CPB. compared with the S group and C group, which suggested that the α7nAchR agonist may promote the phosphorylation of Akt and GSK3β. Rats in the M group that were co-treated with the α7nAchR antagonist exhibited a significant decrease in p-Akt and p-GSK3β expression levels compared with the P group (Fig. 5). By contrast, no significant differences in the expression levels of total Akt and total GSK3β were identified between the groups (Fig. 5), which implied that the α7nAchR agonist did not affect the expressions of Akt and GSK3β. Therefore, these results indicated that the α7nAchR agonist may effectively upregulate the activation of the Akt/GSK3β signaling pathway in the CPB-injured rats.

Discussion
Cerebral injury is a serious complication following the use of CPB in the cardiac surgery (2,22,23). This pathological lesion may be due to several aspects, including impaired cerebral perfusion and oxygenation, cerebral microemboli and SIRS (3,4). Among these factors, SIRS is one of great significance for CPB; therefore, minimizing SIRS is widely considered as a prerequisite strategy for inhibiting the inflammatory response (5). It is generally accepted that proinflammatory cytokines such as TNF-α may further increase the permeability of the blood-brain barrier and subsequently promote the invasion of inflammatory cytokines and immune cells (24). Results from the present study demonstrated that expression levels of the proinflammatory cytokines, including the TNF-α and IL-6, were significantly increased in CPB-injured rats, which was consistent with previous reports (7,25). Therefore, reducing proinflammatory cytokines levels may alleviate neuronal injury and improve functional recovery.
As a physiological regulation of the innate immune system, CAP has been widely used to inhibit the expression of proinflammatory cytokines for treating infectious and inflammatory diseases (16). According to that report, activation of the main regulatory target, α7nAchR, may aid in the reduction of proinflammatory cytokines. Therefore, the present study hypothesized that the α7nAchR agonist may effectively inhibit the serum levels of TNF-α and IL-6 in the CPB-injured rats, which suggested that the α7nAchR agonist may provide a promising strategy for reducing SIRS post-CPB. S100β is regarded as a reliable serum maker of cerebral injury following the breakdown of the blood-brain barrier (26)(27)(28). In the present study, an increased serum level of S100β was observed in the CPB model rats compared with normal rats, whereas the α7nAchR agonist was able to decrease the serum level of S100β, which demonstrated that the CPB model was successfully established and that the neuroprotective effects may be achieved by pretreatment with the α7nAchR agonist.
Several previous reports suggested that the hippocampus is sensitive to ischemia and reperfusion injury caused by CPB (1,29). In the present study, clear pathological damage and an increase in cell apoptosis and Caspase 3 expression levels in the hippocampus were observed in the CPB-injured rats, which confirmed that pathological changes occur in the hippocampus following CPB surgery. Notably, these pathological injuries were effectively inhibited in rats pretreated with the α7nAchR agonist, which demonstrated the protective effects of the α7nAchR agonist on CPB rats.
Additional studies have demonstrated that the Akt/GSK3β pathway serves a central role in cell survival in a number of neurological diseases (30)(31)(32). In particular, activation of the Akt/GSK3β pathway may attenuate apoptosis, which is closely related to the regulation of Caspase 3 expression (33)(34)(35). Based on the present results that demonstrated the inhibitory effects of the α7nAchR agonist on apoptosis and Caspase 3 expression, activation of the Akt/GSK3β pathway was further examined for the protective effects of α7nAchR agonist on CPB. The results indicated that p-Akt and p-GSK3β expressions were upregulated following α7nAchR agonist pretreatment, which suggested that the α7nAchR agonist may be able to inhibit hippocampal cell apoptosis by activating the Akt/GSK3β pathway.
To further determine the protective effects of the α7nAchR agonist on CPB, the α7nAchR antagonist was concurrently administered in the present study. By contrast to pretreatment with the α7nAchR agonist alone, co-treatment with the α7nAchR antagonist resulted in significant increases in the serum levels of S100β, TNF-α and IL-6, as well as the pathological damage, increased apoptosis and increased Caspase 3 expression, and a significant decrease in the expression levels of p-Akt and p-GSK3β. These results further demonstrated the neuroprotective effects of α7nAchR agonist on CPB-injured rats.
In conclusion, the present study demonstrated that the α7nAchR agonist may reduce pathological damage and apoptosis in the hippocampus by upregulating Akt/GSK3β signaling. The α7nAchR agonist may provide a promising therapeutic approach for cerebral injury caused by CPB.


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Viral component of the human genome

Relationships between viruses and their human host are traditionally described from the point of view taking into consideration hosts as victims of viral aggression, which results in infectious diseases. However, these relations are in fact two-sided and involve modifications of both the virus and host genomes. Mutations that accumulate in the populations of viruses and hosts may provide them advantages such as the ability to overcome defense barriers of host cells or to create more efficient barriers to deal with the attack of the viral agent. One of the most common ways of reinforcing anti-viral barriers is the horizontal transfer of viral genes into the host genome. Within the host genome, these genes may be modified and extensively expressed to compete with viral copies and inhibit the synthesis of their products or modulate their functions in other ways. This review summarizes the available data on the horizontal gene transfer between viral and human genomes and discusses related problems.

INTRODUCTION
The relationships between viruses and their hosts are in fact more complex and diverse than is generally perceived. If their interactions are transient and limited to the single virus host paradigm, it largely determines the diagnosis of an infection, and the virus is considered an absolute parasite; this is the approach currently practiced by clinicians. However, on a larger time scale, transient interactions of this kind represent only a specific case of a more general role of viruses, since they are the basis of the evolutionary progression of the whole biological system where viruses and their hosts are constantly adapting to each other, either gaining certain advantages or suffering considerable losses. This is a continuous, ongoing process with a varying rate. Both sides act on the population level and exhibit different extents of aggression and plasticity. For this reason, it can be extremely difficult to determine the causes and the nature of a pathology (especially chronic) in a given individual because they are often modified by virological events that occurred many generations ago. Therefore, it seems fairly reasonable to consider a potential viral origin (in a broad sense) for almost any human disease with unclear etiology, even though it appears noninfectious, especially taking into account that, in particular, this origin implies the possibility of horizontal gene transfer, a phenomenon that is most efficiently mediated by viruses and largely determines the evolutionary progress as the consequence of the total sum of numerous elementary interspecies interactions [1].
The initiation of a viral infection depends on the presence of specific receptors on host cells, i.e., on the host sensitivity to a particular virus. The host's ability to resist infection or to develop a mutual relationship with the virus determines a favorable outcome, while weaker elements can be eliminated from the host population by the lethal course of infection. The distribution of genes that encode these receptors in the human genome is shown in Fig. 1 [2].
The parallel evolution of viruses and their host's sensitivity on the immune response level can lead to a decrease in the virus's pathogenicity [3]. For instance, this process underlaid the spreading of the low-virulence poxvirus variant, alastrim, in the years that preceded the eradication of smallpox [4][5][6].
Viruses recognize specific receptors on the surface of host cells; otherwise, if the expression of these receptors is inhibited, they become noninfectious. For instance, this happens if the human gene that encodes the CCR5 receptor to HIV is damaged by a 32-bp deletion [7]. In some cases, a virus receptor can be blocked by a protein, the gene of which was previously acquired by the host from the same virus via horizontal transfer. On the molecular level, a mutualistic solution has the form of a latent infection; a viral agent can per-REVIEWS UDC 578. 264.9 sist for a long time in a host's cells, which protect it from external factors, while the host organism can make use of the reactivation of the virus, the expression of certain viral genes, or the production of latent RNAs for its own benefit; at the same time, sporadic reactivation and release from the host body enable the virus to maintain the level of genomic variation sufficient for its evolutionary promotion.
Horizontal transfer involves fragments of genetic information that vary strongly in size, in particular depending on the buffer genome capacity of each participant. In the human genome, this capacity is determined by the portion of chromosomal DNA, which does not contain species-specific protein-encoding sequences and, thus, can basically make a place for novel information that will be modified to reach a new balance. If we consider full-size genes, the essential sequences occupy ~10% of the human genome, while only 2.5% of the genome are gene exons.
The reverse process, i.e., the acquisition of host genes or shorter sequences by viruses, is also possible, although viral genomes obviously have a lower abso-lute capacity for storing the acquired material. However, there still are certain provisions; for instance, it was shown that up to 80% of genes in the herpesvirus [8][9][10][11] and adenovirus [12][13][14][15] genomes can be removed and substituted with foreign DNA without losing virus viability.
In some cases, it is difficult to identify the direction of the initial horizontal transfer (i.e., to determine whether a gene was transferred from the virus to the host or the other way around), because these genes start to perform important functions in both the virus and the host.
Considerable interest is drawn to viral tRNA-like (clover leaf, or L-form) structures present in the human genome and in some viral genomes, such as alphavirues and endogenous retroviruses. These structures can participate in the stabilization of viral RNA, as well as in viral replication and translation; all of these functions are determined by the folding of these structures [16,17].
In human DNA, viral insertions can be present as full-size genome sequences, but also as smaller    I  II  III  IV  V  VI  VII  VIII  IX  X   (b) genome segments, individual viral genes or their clusters, and short sequence fragments. The genetic material of all known virus types using all possible replication strategies can reach animal germ cells and be transferred to subsequent generations, which determines the evolutionary role of the gene flow from viruses to animals [1].

RNA viruses
Retroviruses have certainly left the most extensive and frequent evolutionary ancient viral traces in host DNA: in the human genome, sequences of human endogenous retroviruses (HERV) amount to ~8% of its total size [18][19][20] and are derived from at least 504 phylogenetically different sources [20]. This fact is related to specific characteristics of the retrovirus replication machinery, which transcribes the genetic information carried by the viral DNA into DNA that is subsequently incorporated into the host genome. These incorporated viral sequences can be maintained in the host genome for a long time, either in the initial form, or with some modifications, and can be inherited. Modifications are determined by the activity of a number of different factors, including mobile elements of the host genome and transposons, which makes it usually very difficult to identify the source of a given insertion. Further on, viral genes integrated into host chromosomes can act as alleles that modify the host phenotype and sometimes provide a considerable selective advantage. For example, it is believed that this phenomenon contributed to the evolution of viviparous placental mammals; their genomes carry retroviral insertions that encode syncytins, proteins that serve to form the syncytiotrophoblast layer of the placenta and to ensure the immunological tolerance of the mother towards the embryo. Importantly, not all of the mammals (and not only mammals) possess a well-developed placenta, but those who do also have syncytins derived from surface glycoproteins of different retroviruses, the insertion of which occurred at different moments of mammalian evolution. In retroviruses, these surface glycoproteins contain immunosuppressing domains [2,[21][22][23][24][25][26], and it is these domains that are used by the novel hosts. Relic retroviral sequences (HERV-K) can be found in the genomes of human ancestors, Old World primates, nearly to the moment of their separation from the New World primates [27]. Human chromosomal DNA contains 30-50 HERV-K copies, some of which contain genes that exhibit low levels of expression in normal testicular and placental tissue [28]. At the same time, endogenous retroviruses and retrotransposons can induce carcinogenesis in somatic cells [29,30]. MOLECULAR  Other RNA viruses. In fact, the scope of the described phenomena is not limited to retroviruses as such, since the ubiquity of retroviral elements in animal genomes, their activity in germline cells [31], along with the fact that viral replication depends significantly on RNA expression, allow retroviruses to contribute in different ways to the insertion of nonretroviral genes into animal germline cells.
The genomic integration of nonretroviral genes can be mediated by nonhomologous recombination with chromosomal DNA [32] or by interaction with retroelements of the host cells [33,34]. It has been shown that retrotransposons can help the host borrow sequences from different RNA and DNA viruses. For example, recombination between the RNA of the lymphocytic choriomeningitis virus and a murine IAP retrotransposon results in reverse transcription of the RNA and in its integration into the host genome [35]. In some species, such insertions were shown to provide advantages; for example, bees that have acquired the gene that encode structural proteins of dicistrovirus become resistant to this agent, which causes acute paralysis in wild-type individuals [36]. This phenomenon is very common in the kingdoms of plants and fungi [37,38].
In human, genomic incorporation of nonretroviral sequences has been described for such RNA viruses as Ebola [21,22,39,40] and Marburg viruses (family Filoviridae), agents of the Born disease (family Bornaviridae) [23,37,38,41], and polioviruses (order Picornavirales) [32]. The first two families belong to the order Mononegavirales, which also includes paramyxoviruses and rabdoviruses. These viruses have different virion structures, but share a common trait: their genome is composed of a single-stranded negative-sense RNA molecule up to 19 kb long. Filoviruses cause extremely severe acute infections in humans (with 60-90% lethality), and bornaviruses causes an equally severe disease in horses (with up to 90% lethality). Bornaviruses have also been detected in humans; in patients with severe mental disorders, such as schizophrenia [42], as well as in individuals without any pronounced clinical presentation in the cells of which these viruses persisted over long periods of time [43].
Fragments of filo-and bornavirus genomes are inserted into the host genome via interaction with LINEs [44], the most common mobile elements in higher eukaryotes, which bear a reverse transcriptase gene. The most frequent findings in the host genomes are inserted fragments containing bornavirus genes N and L, which encode nucleocapsid protein N and RNA-dependent RNA polymerase (p190), respectively. It is difficult to say when these fragments were inserted into the host genomes; presumably, this event occurred about 40 MA ago. Its initiation and rate varied among different hosts and for different bornaviruses species. At present, in many animal species, bornavirus-derived genes have evolved into homologous own genes, EBLN1 and EBLN2 [44]. These acquisitions provided an important selective advantage, enabling the host to resist devastating bornavius epizootics. It was shown that animals that possess EBLN genes are resistant to species-specific bornavirus infections, or the course of the disease is less severe in them [37]. The molecular basis of this resistance is the excessive synthesis of a protein N analog, which inhibits p190 polymerase and, thus, decreases the virus yield. The initial functions of inserted genes can undergo gradual modification with time; this phenomenon is referred to as exaptation.
Filovirus genes appropriated by vertebrates are those that encode NP nucleoprotein and structural VP35 protein, which inhibits interferon production in the host. Cellular analogs of these genes have been found in bats, bandicoot, wallaby, and other animals of the area. The borrowed viral genes can be partially transcribed, and the resulting truncated N-terminal NP fragments (the full-size sequence of the host gene apparently is not expressed [44]) compete with the corresponding viral component, inhibiting the replication of the virus [45].
Retroviruses are not the only group for which nucleotide sequences can be fixed in eukaryotic genomes. In the 1970s, V.M. Zhdanov, an outstanding Soviet virologist, hypothesized that it should be possible for other RNA viruses [46]. In a later work, L.Yu. Frolova et al. showed that tRNA-encoding sequences in the human genome homologous to tRNA-like elements encoded by LTRs of endogenous retroviruses, can act as targets for alphaviruses [47].
Presumably, virus-specific revertase is not at all necessary for a viral nucleic acid to insert into a host genome [22]. Some features of this process are similar in very different viruses and are worth a detailed analysis. For example, the stable incorporation of viral genes into the host genome typical for retroviruses was also described in filoviruses (although it is less frequent), while in many other viruses, this feature is less prominent or unknown at all.
The Ebola virus genome has an interesting feature, which probably does not explain its ability to integrate into the host genome (most probably occurring by homologous recombination), but is nevertheless worth mentioning. It is a short (25-27 amino acids) immunosuppressing fragment p15E, which exhibits a high level of homology in Ebola and retroviruses [22]. Importantly, the function of this domain is activated as a result of the incorporation of an additional adenine, which results in a reading frame shift. This adenine insertion in p15E serves as a marker of pathogenicity of both retroviruses and the Ebola virus when they infect a new host.
The human genome contains a number of immunosuppressing fragments: they are expressed within syncytin genes in plancetal cells [23]. Insertions of nonretrovirus genes can basically have occurred as result of interactions between the gene source and a retrovirus (most commonly, a LINE retrotransposon).
Among the proteins, the genes of which were borrowed from a virus, those that perform a primarily protective function are the most likely to be fixed in their new environment. For example, Fv1 is a protein similar to CA protein of the murine leukemia virus and competing with it; it binds the viral capsid and blocks reverse transcription, providing insensitivity to infection. In human and other animals, there are also protective proteins encoded by genetic elements of viral origin incorporated in the host genome. In particular, TRIM5 (and its analogs present in some primates) can inhibit the proliferation of some retroviruses in largely the same way as Fv1 and, at the same time, affects the proinflammatory transcription factors NF-κB and AP-1, which control the expression of genes involved in immune response, apoptosis, and cell cycle [48].

DNA Viruses
DNA retroviruses. Elements derived from genomes of DNA retroviruses (e.g., the duck hepatitis B virus) are found significantly less frequently in host DNA than those originating from RNA retroviruses [1,44,49]), even though they possess revertase, which could be expected to enable efficient incorporation of viral sequences into the host genome.
Other DNA viruses. Host genomes also bear traces of encounters with DNA viruses. Most commonly, these are members of the large parvovirus family (Parvoviridae): dependoparvoviruses (adenoassociated agents that can replicate only in the presence of a helper adenovirus or herpesvirus), nonpathogenic in humans. Dependoparvovirus genes have been found in the DNA of pigs, cattle, rats, mice, and other animals [1,50]. Probably (although it has not been proven), these insertions serve to protect against parvovirus infections. Papillomaviruses were also shown to integrate into human genome [51].
Apart from the above agents, it was also found that the pig genome contains relic copies of the circovirus genome (noninfectious for humans). Genes of nanoand geminiviruses were found in plant genomes [1,50]. Human DNA was also found to contain genes and larger genome elements of herpesviruses, including the Epstein-Barr virus (human herpesvirus 4, HHV4), human herpesvirus 6 (HHV6), and other members of this superfamily [52].
Finally, at least 34% of the human genome is composed of fairly large virus-like sequences: the so-called selfish DNA, the origin of which is unclear, while the only observed type of activity is autoreplication. The most active group are transposons of the LINE class (long interspersed elements); in human, they harbor approximately one in thousand genetic mutations [53][54][55]. On the whole, virus-like components of the human genome account for nearly a half of the chromosomal DNA, and some of them play an important role in the host organism, but hardly anything is known about the origin and functions of the others. Antiviral host response can transform an acute infection into the chronic or even the latent form (as it happens with herpesviruses), and a reservoir of viruses is thus conserved in their natural host, who will remain their target in case of reactivation.
It should be underlined that, for many important reasons, the body of relevant data available from the existing publications is far from being complete [1]. First of all, not all of the known viruses have been studied as potential sequence donors for the host genome, and not all the potential host species have their genomes sequenced. Secondly, an insertion of a viral sequence may represent a temporary outcome of a single infection event and will not be maintained in subsequent host generations. Moreover, the host species may also be eliminated from the evolution (as a result of extinction), which means that the acquired insertions will only be conserved if they are no longer limited to the extinct species and have become specific for a more general taxonomic branch, such as genus, order, or higher. Next, the relatively recent insertions may be insufficiently widespread to be identified and thus evade observation. Finally, the ability to incorporate parts of the viral genome into the chromosomal DNA of host germline cells can vary strongly among different taxonomic groups of viruses, i.e., orders, families, genera, and even species If insertions of viral sequences remain functionally active in the host cell genome, they can give rise to either proteins that function in a new environment or untranslated RNAs of different sizes. If these insertions are inactive, they can merely witness a history of close and evolutionary long-term interactions between the virus and the host.
The characteristic trait of human herpesviruses is that, in their typical latent state, they can persist and replicate in the form of an episome in the direct vicinity of the host genomic DNA. These viruses have long coexisted with their hosts and the hosts' phylogenetic ancestors, and their genomes carry full-size genes that in turn were captured at some moment from the host and can often be expressed in their new environment [52,56]. The nucleotide sequences of these genes, the encoded amino acids, and even the functions of the resulting proteins do not correspond strictly to their cell counterparts, but the range of their functions in the virus certainly suggests a relationship between the viral and the host genes. Some herpesviruses (such as HHV8 associated with Kaposi's sarcoma) have acquired genes of serpentines (G-protein-coupled receptors; GPCR), which they employ at the early stage of lytic infection of lymphoid cells and of sarcoma itself; moreover, the inhibition of viral DNA synthesis does not affect the functioning of these MOLECULAR BIOLOGY Vol. 51 No. 2 2017 genes. Their function is not quite clear, since they act mainly at the early stages of infection: GPCR transcripts, which have mainly bicistronic structure, protect the 5'-region of the coding sequence of another HHV8 gene, К14; while monocistronic transcripts analyzed in model experiments did not exhibit such properties. This probably indicates that the translation of GPCR transcripts may be reinitiated and suggests the need for further analysis of all the functions of GPCR itself in the pathogenesis of Kaposi's sarcoma. The product of the GPCR-encoding gene BILF1 captured by HHV4 acts as a specific inhibitor of class I major histocompatibility complex. The reverse process can also be imagined easily, as well as subsequent GPCR modifications as a result of the repeated gene capture with the acquisition of new functions. In fact, no such data are currently available, but the research has only begun very recently.
A recent study detected the first endogenous herpesvirus (genus Roseolovirus) in the genome of the Philippine tarsier (order Primates), while insertions of nearly full-size HHV8-and HHV4-like genomes were found in the DNA of other primates: aye-aye, lemur, and chimpanzee [57].
In this context, it is also worth mentioning that more data exist on the integration of herpesviruses into host genomes [58], as well as on the incorporation of herpesvirus 6 DNA (HHV6; also of the genus Roseolovirus) into the telomeric zones of human chromosomes. The significance of these insertions (which can be transmitted vertically if occur in germline cells) for human diseases or for the functioning of the immune system is currently absolutely unclear [56,59,60].
Similar data were obtained for viruses of other taxonomic groups. A systematic analysis showed that sequences derived from a wide range of animal viruses other than retroviruses are present as endogenous elements in mammalian, avian, and insect genomes. These elements of animal genomes represent the full spectrum of viral replication strategies [1]; moreover, the larger the sample of animal genomes, the wider the diversity of endogenous viral elements. Obviously, the more ancient these elements are, the smaller the number of host species needed to detect them. To identify more recent viral insertions, a much wider sample on the order and genus level is required. However, as we have pointed out above, the currently available data are far from complete.
The diversity of the known virus isolated as represented in virus gene/protein banks is but a small portion of the total virus diversity. In view of their likely ancient origin, members of the many virus families may be much more widely distributed among their mammalian hosts than we currently imagine, both as separate entities and as genome fragments in the host DNA. This is also reflected in the virus phylogeny, which was constructed using endogenous virus insertions along with exogenous viruses; close exogenous relations frequently are either not identified or were only described in the past decade [61][62][63]. The recently discovered relationship between filoviruses and marsupials has suddenly specified this infraclass of mammals as a potential filovirus reservoir. The presence of viral insertions may become an important factors for evaluation of findings obtained using metagenomic approaches [48,64,65].
All of these data have been discussed in several comprehensive reviews [1,2,66]. The capture of certain fragments of a viral genome by the host may be a random event; however, should the captured genes reach germline cells and prove useful to the host, they can become fixed in subsequent generations.
It may prove interesting to consider information that concerns shorter insertions of viral origin. They can be expected to exist in much higher numbers, since the probabilities of their incorporation, as well as of transposition or multiplication, seem higher than for larger insertions. Fragments of 20-30 nt long would have the same size as the biologically active RNAs, such as those involved in RNA interference.
However, it is currently hardly possible to identify the targets of microRNAs based on their sequences only, since they do not need to be strictly complementary. In addition to the CLIP-seq and PAR-CLIP techniques that employ immunoprecipitation of RNA-protein complexes with subsequent sequencing [81], it may prove helpful to analyze the data on the coexpression of miRNAs and their putative gene tar-gets [82]. Two other groups of molecules participating in RNA interference are siRNAs and so-called piwiRNAs (or piRNAs). The latter are 24-29 nt long; their activity is mediated by a specific mechanism involving enzymes of the PIWI family, and their targets are usually transposons, retrotransposons, and endogenous retroviruses [83]. The 20-30-nt long fragments that form siRNAs and piRNAs must be almost fully complementary to their target sequences (with mismatches of no more than 2 nt). These RNAs are mainly targeted at exons, which facilitates the in silico search for putative small insertions in the recipient DNA. The functional activity of siRNAs and piRNAs should be determined experimentally. The detection of nucleotide fragments of the specific size in the Escherichia coli genome invited the hypothesis that bacteria possess an antiviral immune defense system [84], which later became the basis for the development of the revolutionary gene editing tools, CRISPR Cas9/Cpf1 [85,86].
We attempted to identify 20-to 30-nt-long homology stretches (which we provisionally refer to as hits) in the human genome and in the genomes of human adenoviruses and herpesviruses and showed that their number was significantly higher than in extended samples that included either viruses that do not directly infect human, such as bacteriophages, or artificially generated sequences of the same as the herpesvirus genome [87].
Later, we showed that, even in a given group of viruses (HHVs), the portion of the viral exome that corresponds to hits, i.e., short 20-28-nt long sequences homologous between the virus and the host, is specific to each virus type and, as the first approximation, can be related to the destructive effect of the viral infection [88].
We have also proposed a hypothesis that reactivated HHV1 could exhibit oncolytic properties in vivo [88], because the body of RL1 (which encodes ICP34.5 protein) contains a large number of virus/human homologous sequences (hits); in artificial HHV1-based oncolytic constructs, this gene is switched off first. Obviously, our hypothesis requires experimental verification.

Homo sapiens AS A RECIPIENT OF VIRAL INSERTIONS
The Fig. 2 schematically shows the load of human DNA with viral relics. We define the known viral insertions and their derivatives, including transposons, as relics, since we are currently able to identify only those that were incorporated into the host genome long time ago.
Human translated genes constitute approximately 2% of the total information volume of genomic DNA, whereas as virus relics and virus-like structures (with some reservations, in addition to endogenous retrovi-ruses, these may be assumed to include retrotransposons and DNA transposons) amount to nearly half of the human genome.
This information suggests that there might be a need to reconsider the notion of a virus. From the traditional medical point of view, a virus is a parasite that infects a sensitive cell and ultimately causes its destruction. The virus (its genome) can also infiltrate the host DNA, occupying the free space or adding to its size. By replicating in the host genome, the virus ensures its genetic diversity and at the same time acquires a safe depot protecting it from external factors. This viral activity can be reasonably classified as parasitic.
However, from this viewpoint, we overlook the potential evolutionary advantages that the host population may gain as result of a viral infection: viruses are the most efficient vectors, which might transfer novel genetic information. For this reason, we propose to define a virus as an information carrier indistinguishable from a specific one and possessing an autonomous autoreplication program that employs the addressee's reading, synthesizing, and metabolic machinery for its own realization. This definition underlines the major evolutionary function of viruses as carriers of genetic information and is free from moral judgment, which is foreign to nature. Moreover, this definition has a more general character than the one specifying viruses a parasites.
Certainly, these considerations do not imply that Homo sapiens does not need to struggle with destructive viral infections. Moreover, the fact that, in nature,
As for the short regions of homology to herpesvirus or adenovirus DNA (which can basically be viral insertions), their distribution in the human genome is specific for every virus species, and on the whole, as we showed previously [87], it can hardly be random.

CONCLUSIONS
It was not the main objective of our brief review to list all of the available data on the question. The body of these data is rapidly growing, which, however, does not seem to change the existing consensus view on the problem. Obviously, mutual insertions of genomic fragments in virus-host pairs occur regularly, if not very frequently. Their exact frequency remains to be determined both on the population level and on the level of single individuals. The objective is not as much to determine the number of already incorporated foreign genes but rather to find out how this frequency depends on the type of the virus and its host. It is also important to determine the consequences of each insertion, either transient or fixed, can have for the human host within the lifetime of the hosts or of their ancestors. It should be evaluated how often these insertions reach the host's germline cells and what key factors govern these events. There are currently no answers to these questions, but these issues are addressed in different areas of study, which sometimes produces unexpected results. For instance, the Haldane and Waddington problem about the number of generations were required to obtain recombinant inbred strains was initially only solved by the authors for the cases of two and three genes [92]. Recently, for the general case Samal and Martin proposed an approach based on a statistical formalism rarely used in areas other than physics [93]; surprisingly, it provided an exact solution to the problem involving any given number of genes. Although the above example lies in the area of population genetics, while the number of studies on our topic of interest is insufficient to allow population-level generalizations, it nevertheless confirms the need to continue the efforts and emphasizes the productivity of breaking traditional thinking patterns, even in such conservative fields as medical diagnostics and therapy. The exchange of genetic information between living organisms is a complex albeit an infrequent phenomenon and generates a considerable uncertainty when we attempt to provide a comprehensive explanation of the causes and nature of a given pathological condition. The dynamic equilibrium between the human genome sensu stricto and the integrated viral sequences that perform protective and regulatory functions in the host organism represents a much deeper relationship than the organism's interaction with the internal microbiota. Currently, these relationships constitute the subject of separate omicstype research. They are the consequence of a multisided (not just a two-sided) encounter, where one side is represented by a viral population, which is heterogeneous in each individual case and consists of different proportions that are both fully featured and defective, as well as mutant particles, while the other side is a multicellular organism that developed as a result of macro-and microevolution. Viruses have enriched the host genome in functional virus relics, the amounts and diversity of which greatly exceed the total of the host's own genes. On the other hand, the multicellular organism has developed a system of antiviral defense and continues to develop it while employing the full range of available molecular mechanisms. Predicting the consequences of this encounter and the results of their targeted modification for the benefit of the infected macroorganism is a problem that can only be solved after considerable advances in the techniques of analyzing and modeling the underlying mechanisms.


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Genetic characterization of Rhipicephalus sanguineus (sensu lato) ticks from dogs in Portugal

Background The taxonomic status of the brown dog tick Rhipicephalus sanguineus (sensu stricto) is a subject of on-going debate; there is a consensus that populations of this tick species should be referred to as R. sanguineus (sensu lato) until its taxonomic status is resolved. Recent genetic studies revealed the existence of more than one lineage of R. sanguineus (s.l.) in temperate countries. In this study, we assessed the genetic identity of ticks collected from rural dogs living in several areas located in all major geographical regions of Portugal. Methods A total of 347 ticks were collected from rural dogs living in different regions of Portugal. These ticks were morphologically identified and partial mitochondrial 16S rRNA gene sequences (~300 bp) were obtained from representative specimens. Results The ticks were morphologically identified as Ixodes ricinus (seven males and 27 females), Rhipicephalus bursa (one male), Rhipicephalus pusillus (one female) and R. sanguineus (s.l.) (two larvae, 101 nymphs, 108 males and 100 females). Partial mitochondrial 16S rRNA gene sequences were obtained from 58 R. sanguineus (s.l.) specimens, and all of them were genetically identified as belonging to the so-called temperate lineage of R. sanguineus (s.l.) Conclusions These results strongly suggest that the temperate species of R. sanguineus (s.l.) is the only representative of this tick group found on dogs in Portugal. It also adds weight to the hypothesis that Rhipicephalus turanicus is not present in this country, although further investigations are necessary to confirm this. Electronic supplementary material The online version of this article (doi:10.1186/s13071-017-2072-1) contains supplementary material, which is available to authorized users.

Background
Ticks are important vectors of pathogens to companion animals, livestock and humans. Current global changes (e.g. climate changes, deforestation, changes in land use, urbanization, increased trade and travel) are affecting animal host populations worldwide [1], favouring the establishment of ticks and their associated pathogens into previously free areas.
The brown dog tick Rhipicephalus sanguineus (sensu stricto) is a species of major medical and veterinary significance [2]. The taxonomy of this tick species is a subject of an ongoing debate, mainly because there is no typematerial and no bona fide morphological description [3,4]. For this reason, there is a consensus that populations of this tick species should be referred to as R. sanguineus (sensu lato) (s.l.) until its taxonomic status is resolved.
Genetic studies have consistently reported the existence of two well-defined lineages within "R. sanguineus": the southern lineage (also referred to as temperate species/lineage) and the northern lineage (tropical species/ lineage) [5][6][7][8][9][10][11]. Available scientific data indicate that the situation regarding the taxonomic status of R. sanguineus (s.l.) in Europe is even more complicated. In particular, a higher diversity of cryptic species seems to occur in the Mediterranean region [8] as compared with Latin America [5][6][7]. Furthermore, the diversity of Rhipicephalus spp. appears to increase from West to East in the Eurasia [8]. Indeed, the so-called temperate species/lineage (also referred to as "Rhipicephalus sp. II" by Dantas-Torres et al. [8]) of R. sanguineus (s.l.) appears to be the only representative of this species group in western European countries such as Portugal and Spain [8]. On the other hand, additional operational taxonomic units (OTUs) (e.g. Rhipicephalus sp. I) and/or species (e.g. Rhipicephalus turanicus) are apparently present in countries such as Italy and Greece.
A considerable amount of genetic data have been generated from R. sanguineus (s.l.) ticks in recent years. However, only few studies have assessed the genetic variability of ticks from different regions within the same country (e.g. Brazil; [5]), in order to define which tick species/lineage is present. In this study, we assessed the genetic identity of R. sanguineus (s.l.) ticks collected from dogs from all major geographical regions of Portugal to assess two hypotheses: (i) R. turanicus is not found on dogs from Portugal; and (ii) the temperate species/lineage is the only representative of the R. sanguineus (s.l.) group infesting dogs in this country.

Methods
From June 2013 to April 2014, ticks (n = 347) were collected from rural dogs living in different regions of Portugal ( Table 1). The owners physically restrained dogs and ticks were collected manually and placed in labelled vials containing 70% ethanol. Identification was carried out under a stereomicroscope using morphological keys [12][13][14]. In particular, we considered all the morphological characters detailed in Dantas-Torres et al. [8], such as idiosoma, dorsal scutum, basis capituli, hypostomal dentition, female porose areas, female genital opening, spiracular plates, dorsal tail of spiracular plates, lateral and postmediam grooves, cervical pits, cervical fields, internal and external cervical grooves, marginal lines, male adanal and accessory plates, male caudal process, spur on trochanter I, and body colour. Ticks morphologically compatible with the description of Walker et al. [13] for "R. sanguineus" were referred to as "R. sanguineus (s.l.)".
A total of 61 ticks, including at least one tick from each geographical site, were selected for genetic analysis. DNA extraction was performed using a commercial kit (DNeasy Blood & Tissue Kit, Qiagen GmbH, Hilden, Germany), in accordance with the manufacturer's instructions. Partial mitochondrial 16S rRNA (~300 bp) gene sequences were generated and analysed. Primers and PCR conditions have been described elsewhere [5]. Each reaction consisted of 4 μl of tick genomic DNA and 46 μl of PCR mix containing 2.5 mM MgCl 2 , 10 mM Tris-HCl (pH 8.3), and 50 mM KCl, 250 μM of each dNTP, 50 pmol of each primer and 1.25 U of AmpliTaq Gold (Applied Biosystems, California, USA). Approximately 100 ng of genomic DNA (with the exception of the no-template control) were added to each PCR. Amplified products were examined on 2% agarose gels stained with GelRed (VWR International PBI, Milan, Italy) and visualized on a GelLogic 100 gel documentation system (Kodak, New York, USA).
Amplified products were purified and sequenced, in both directions using the same primers as for PCR, employing the Big Dye Terminator v.3.1 chemistry in a 3130 genetic analyzer (Applied Biosystems, California, USA). The 16S rRNA gene sequences were aligned using ClustalW program [15] and compared with those available in GenBank using the BLASTn tool (http:// blast.ncbi.nlm.nih.gov/Blast.cgi). The percentage of nucleotide variation (pairwise comparison -Pwc) amongst all haplotypes identified was calculated using the Kimura 2-parameter substitution model with gamma distributed rates among sites [16], implemented in the MEGA 6 software [17].
For phylogenetic analyses, we included sequences from each haplotype obtained herein as well as individual or consensus sequences (GenBank accession numbers: KC243835-KC243838, KC243843-KC243847, KC 243851-KC243854, KC243855, KC243856-KC243867 and KC243871) for the other Rhipicephalus spp. (for more details, see Additional file 1: Table S1) available from a previous study [8]. In particular, consensus sequences from selected tick species were generated after alignment with ClustalW program [15] and using the BioEdit software [18]. A homologous gene sequence from Ixodes ricinus (GenBank JF928527) was used as the outgroup. Phylogenetic relationships were inferred by maximum likelihood analysis [16] with the general time reversible model in MEGA 6 [17]; bootstrap values are based on 8,000 replicates.

Results
Morphologically, ticks were identified as I. ricinus (seven males and 27 females), Rhipicephalus bursa (one male), Rhipicephalus pusillus (one female), and R. sanguineus (s.l.) (two larvae, 101 nymphs, 108 males and 100 females). Most (all but 12) R. sanguineus (s.l.) ticks resembled (e.g. large, dark-coloured ticks, with males presenting elongated spiracular plates with narrow dorsal tails and females presenting typical U-shaped genital opening) the so-called temperate species (data not shown). Morphological variations in ticks collected from different regions and even within the same region were noticeable. In particular, some male ticks (ten from Bragança and two from Lourinhã) presented spiracular plates with short and large dorsal tails, which could resemble those of R. turanicus.
With regard to genetic data, a total of 59 partial 16S rRNA gene sequences were obtained; for two ticks no amplification was obtained. One of these sequences shared 98% identity with a sequence of R. pusillus available in GenBank (KC243855). The remaining 58 sequences were from ticks identified as R. sanguineus (s.l.) and were all genetically assigned to the temperate species/lineage, including specimens resembling morphologically R. turanicus (see Additional file 1: Table S1). BLAST analysis revealed that the eight haplotypes identified shared high nucleotide identity (99-100%) with those of Rhipicephalus sp. II (= temperate species/ lineage) available in GenBank (KC243843-KC243846). Indeed, one haplotype was identical to haplotype II of Rhipicephalus sp. II previously identified in Portugal and in northern Italy (GenBank KC243844).
The new representative sequence types were named as haplotypes VI-XII. The haplotype VI was the most prevalent haplotype (n = 30; 51.7%) identified and found in all surveyed areas (Table 2), followed by haplotype II (n = 10; 17.2%). The nucleotide sequence variation, upon pairwise comparison, ranged from zero to 1.1% (mean 0.7%), and a high nucleotide difference (1.1%) was recorded between haplotypes II and X, which were obtained from specimens collected in the same area (Alijó). All representative new haplotypes obtained are available in the GenBank database under accession numbers: KY216135-KY216141.
Phylogenetic analysis was concordant in clustering all haplotypes identified in the same clade with the consensus sequence of the temperate species/lineage, supported by a high bootstrap value, to the exclusion of other Rhipicephalus spp. (Fig. 1).

Discussion
In this study, all R. sanguineus (s.l.) ticks collected from rural dogs living in different regions and areas of Portugal were morphologically compatible with the so-called temperate species/lineage. Twelve male ticks presented spiracular plates with large and short dorsal tails, resembling those of R. turanicus (data not shown), but all of them were genetically confirmed as indistinct from the temperate species/lineage. Our data further indicate that the "classic" morphological identification of R. turanicus based on only spiracular plates does not correlate with molecular findings. This also indicates that ticks previously identified as R. turanicus in Portugal (e.g. [19]) were actually R. sanguineus (s.l.) (temperate species/lineage), as already suggested by Santos-Silva et al. [20]. Indeed, our study indicates that the temperate species/lineage is the only representative of R. sanguineus (s.l.) infesting dogs in Portugal. The existence of a single species referred to as "R. sanguineus" with a high level of morphological polymorphism has been discussed elsewhere [20]. However, the existence of R. turanicus and even other species/lineages of R. sanguineus (s.l.) parasitizing other animal species in Portugal, particularly wildlife, cannot be ruled out and deserves further investigation.
Studies conducted in Latin America revealed the presence of two main lineages of R. sanguineus (s.l.) in the Neotropical region: tropical species (occurring from northern Mexico, Central America, and tropical areas of South America) and temperate species (found temperate and cold localities from the southern cone of South America) [5][6][7]. This notion is supported by more recent studies [8][9][10], indicating that ticks currently identified as "R. sanguineus" actually belong to at least two distinct species.
Because dogs are travelling with their owners (and with their ticks) around the world, the existence of two different lineages is intriguing. According to a recent study, the separation of these species seems to be driven by climate variables, particularly the annual mean temperature. Data indicate that the tropical species/lineage is present in areas with annual mean temperature > 20°C, whereas the temperate species/ lineage occurs in areas with annual mean temperature < 20°C [11]. Whether ongoing climate changes will favour the establishment of populations of the tropical/lineage species in areas currently occupied exclusively by the temperate species/lineage and vice versa has yet to be determined. The oldest record of a tick on a dog comes from ancient Egypt [21]. Ticks collected from a dog mummy found in a tomb surrounding a Roman fortress in El Dei were morphologically identified as belonging to the temperate species/lineage. Actually, the separation of the two main lineages of R. sanguineus (s.l.) might have occurred millions of years ago, as these species are reproductively isolated [22]. Moreover, differences in their complete mitochondrial genomes are estimated to be in the order of~10% [23].
Finally, the known circulation of several pathogens (e.g. Babesia vogeli, Cercopithifilaria sp. II, Ehrlichia canis, Hepatozoon canis, Rickettsia conorii and Rickettsia massiliae) among R. sanguineus (s.l.) ticks, dogs, cats and foxes in Portugal [24][25][26][27][28] indicates that the temperate species/lineage is playing a role in the transmission of these agents in this country. Interestingly, a study conducted by Moraes-Filho et al. [29] suggested that the absence or scarcity of cases of canine monocytic ehrlichiosis due to E. canis in the southern cone of South America might be a result of vector incompetence of the R. sanguineus (s.l.) ticks that are found on dogs in this part of South America. Whether the different haplotypes of the temperate lineage of R. sanguineus (s.l.) found in Portugal and other European countries present different vector capacities for each of the abovementioned pathogens has yet to be determined.

Conclusions
In conclusion, the data presented herein add weight to the hypotheses that the temperate species/lineage is the only representative of R. sanguineus (s.l.) in Portugal and that R. turanicus does not occur in this country.

Additional file
Additional file 1: