**Selenium and Health: An Update on the Situation in the Middle East and North Africa**

#### **Sohayla A. Z. Ibrahim 1, Abdelhamid Kerkadi <sup>2</sup> and Abdelali Agouni 1,\***


Received: 29 May 2019; Accepted: 19 June 2019; Published: 27 June 2019

**Abstract:** Selenium (Se) is an important trace element that should be present in the diet of all age groups to provide an adequate intake. Se is incorporated in 25 known selenoproteins, which mediate the biological effects of Se including, immune response regulation, maintenance of thyroid function, antioxidant defense, and anti-inflammatory actions. A balanced intake of Se is critical to achieve health benefits because depending on its status, Se has been found to play physiological roles or contribute to the pathophysiology of various diseases including, neurodegenerative diseases, diabetes, cancer, and cardiovascular disorders. Se status and intake are very important to be known for a specific population as the levels of Se are highly variable among different populations and regions. In the Middle East and North African (MENA) region, very little is known about the status of Se. Studies available show that Se status is widely variable with some countries being deficient, some over sufficient, and some sufficient. This variability was apparent even within the same country between regions. In this review, we summarized the key roles of Se in health and disease and discussed the available data on Se status and intake among countries of the MENA region.

**Keywords:** selenium; chronic diseases; Middle East; North Africa

#### **1. Introduction**

Selenium (Se) is a semi-solid metal that was discovered in 1817 as a byproduct of sulphuric acid synthesis. It belongs to group 6 of the periodic table with an atomic number of 34. It is a red colored powder; however, in vitreous form it is observed as black, and in crystalline form it is observed as metallic gray. Se exists in many different oxidation states including 2+, 4+, 6+, and 2- [1]. Se is present in small amounts in food mostly in organic forms (selenomethionine and selenocysteine) and rarely in inorganic forms (selenate and selenite) [2]. Selenomethionine is usually derived from animal sources in addition to cereal products grown in areas where the soil is rich in Se whereas selenocysteine is only obtained from animal sources [3]. Inorganic forms are the major sources of Se incorporated into dietary supplements [2]. Se concentrations in the soil where plants are grown or where animals are raised are important indicators of Se intake in a country leading to tremendous variations among different countries in Se status [2,4]. The most common sources of Se are Brazil nuts, cereals, offal, fish, eggs, poultry, and vegetables [3–5].

Se is a necessary trace element that should be present in the diet of all age groups. The Recommended Dietary Allowance (RDA) of Se is generally based on the optimum amount that can maximize the activity of selenoprotein glutathione peroxidase [2–4]. This value was estimated to be 55 μg/day for both males and females [2–4,6]. The Estimated Average Requirement (EAR) was found to be around 45 μg/day for 19–50-year-old men and women and the tolerable upper intake level (UL) is around 400 μg/day [3,4]. However, the optimal amount required to achieve maximum health benefits is unknown [3]. The World Health Organization (WHO) recommends that the optimum serum

concentration of Se for healthy adults is 39.5–194.5 ng/ml and that the concentration that maximizes glutathione peroxidase activity is between 70 and 90 ng/ml [7,8].

Very limited data exist on Se toxicity in humans; however, animal studies are available on chronic Se poisoning [4]. It was reported that an intake of around 800 μg/day of Se would cause no observed adverse effect level (NOAEL), 1,540 to 1,600 μg/day of Se would result in low observed adverse effect level (LOAEL) and 5,000 μg/day of Se is the toxic level where selenosis is expected to happen [3]. LOAEL is associated with symptoms of hair and nail brittleness and loss, garlic like breath odor, gastrointestinal disturbances, and fatigue [3,4]. Selenosis can be associated with serious respiratory, renal, and cardiac complications [3].

Se status can be assessed through blood, urine, nails, and hair levels. Blood specimens can be either whole blood, erythrocytes, serum, or plasma. Erythrocytes, hair, and nails reflect long-term status opposing to plasma or serum which reflect short-term levels. It is challenging to compare Se levels across different countries due to the wide variations in methodologies of Se detection among different laboratories [9,10].

#### **2. Selenium: A Focus on Biology**

#### *2.1. Selenoproteins as Mediators of Se Actions*

Se is an essential nutrient to human biology. The beneficial roles of Se to human health and its requirement for life have been known for several decades. Se exists in 25 known selenoproteins, some of which have important known biological functions [11]. The critical role of Se in human health is particularly underscored by being the sole trace element specified in the genetic code as selenocysteine, today recognized as the 21st amino acid, which has its proper genetic code, specific biosynthesis pathway, and insertion mechanism. This amino acid exists in the active site of many selenium-dependent enzymes that are responsible for multiple essential functions in the human body [1,12,13]. In this case, Se acts as a redox center. For example, thioredoxin reductase, a selenoenzyme, participates in DNA synthesis by reducing nucleotides which then helps in controlling the redox state intracellularly [3]. Another example is iodothyronine deiodinases which activates thyroid hormone [1]. Other selenoproteins have different functions including anti-inflammatory and antioxidant effects; however, some of those functions are yet to be identified. The biological effects of Se also include its participation in immune responses and thyroid function whereas its serum level can also be linked with risk of diabetes, cardiovascular disease (CVD), and cancer [5]. Table 1 summarizes the main functions and significance of the different known selenoproteins.




**Table 1.** *Cont.*

#### *2.2. Synthesis and Co-Translational Incorporation of Selenocysteine in Humans*

The cycle of selenocysteine synthesis and incorporation in humans begins with the attachment of L-serine to non-cognate tRNASec by seryl-tRNA synthetase, an error that is not edited. This seryl group gets phosphorylated by O-phosphoseryl-tRNASec kinase (PSTK) resulting in phosphoseryl (Sep)-tRNASec intermediate. Finally, this Sep-tRNASec is converted into selenocysteinyl (Sec)-tRNASec through O-phosphoseryl-tRNASec:selenocysteinyl-tRNASec synthase (SepSecS) in the presence of selenophosphate and pyridoxal phosphate. Selenophosphate is the main Se donor in humans synthesized from adenosine triphosphate (ATP) and selenide, the products of selenocysteine degradation, by selenophosphate synthetase (SPS2). A specialized elongation factor (EFsec) then delivers Sec-tRNASec to the ribosome. SElenoCysteine Insertion Sequence (SECIS), located in the 3 -UTR, helps localize EFsec and Sec-tRNASec near the translation site and forms a stem loop structure which is necessary for decoding of selenocysteine UGA codon and its insertion into the nascent protein [14–17]. The cycle of selenocysteine synthesis and incorporation into selenoproteins in humans is summarized in Figure 1.

**Figure 1.** Synthesis and co-translational incorporation of selenocysteine into selenoproteins in humans.

#### **3. Selenium in Health and Disease: The Importance of a Balanced Intake**

Se depending on its status has been reported to play crucial roles in normal physiology or contribute the pathophysiology of various diseases. Se has antioxidant and anti-inflammatory actions and therefore various studies have assessed the impact of Se status on conditions characterized by inflammation and oxidative stress including, neurodegenerative diseases, diabetes, cancer, and cardiovascular disorders [18].

#### *3.1. Pregnancy*

During pregnancy, the demand for multiple nutrients increase owing to many physiological changes, resulting in deficiencies of many vitamins and minerals [9,19]. Those deficiencies might sometimes lead to complications to the fetus or to the mother. Se levels were found to be significantly reduced during pregnancy [9,19]. During the first trimester, GPx activity was found to be reduced significantly and this low level was maintained throughout the second and third trimesters with a slight increase during delivery [20]. Subsequently, in the second and third trimesters, Se levels were noted to be significantly reduced with further reduction during delivery [20]. Due to this increased demand, the RDA for Se during pregnancy was increased from 55 μg/day to 60 μg/day [19,21]. Se deficiencies in pregnant women might have adverse effects on the developing fetus, particularly the nervous system. A case-control study of pregnant women found a positive correlation between low Se levels and the occurrence of neural tube defects [22]. However, it is important to note that defects in the nervous system development are affected by multiple other factors and not solely by Se deficiencies. Se deficiencies during pregnancy were also found to cause oxidative stress partly resulting in miscarriages, pre-term deliveries, intrauterine growth retardations, preeclampsia, thyroid dysfunctions, gestational diabetes, and cholestasis [9,23]. Therefore, it is important to maintain optimal Se levels during pregnancy by increasing Se intake to meet the increased demand.

#### *3.2. Diabetes Mellitus*

The relationship between Se and diabetes was investigated in multiple studies with contradicting results [18]. Those studies were conducted initially following the assumption that Se is likely to exert protective effects against diabetes due to its antioxidant actions. In support of this assumption, Se was found to delay the onset and progression of diabetes. Se was also found to act as an insulin mimetic when in the form of selenite. Moreover, several in vitro and in vivo studies have suggested that Se plays an important role in the regulation of glucose homeostasis [18,24]. Human studies have also investigated the impact of Se on diabetes yet with conflicting conclusions. Both low and high levels of Se have been shown to be associated with a risk of diabetes [2]. The role of Se in diabetes was assessed in few randomized control trials (RCT). The first large RCT was the Nutritional Prevention of Cancer (NPC) trial which aimed at assessing the effect of 200 μg of daily Se supplementation on reducing the risk of skin cancer compared to placebo [25]. A secondary analysis of the data after a follow-up of 7 years suggested a statistically significant increase in the incidence of type 2 diabetes among those who received Se supplementation compared to placebo [26]. In contrast, the Se and Vitamin E Cancer Prevention Trial (SELECT) found no statistically significant increase in the incidence of type 2 diabetes after Se supplementation [27]. The third trial is the Se and Celecoxib (Sel/Cel) Trial. The intervention was a randomized, placebo controlled trial of Se (200 μg/day) and cyclooxygenase 2 selective inhibitor, celecoxib (400 mg, once per day), alone or in combination, for the prevention of colorectal adenoma in men and women. After exclusion of participants who had type 2 diabetes prior to the trial, 1640 participants were assessed for the presence of type 2 diabetes during the follow-up period. Authors found that 31 participants randomized to Se and 25 randomly assigned placebo were diagnosed with type 2 diabetes; however, despite a higher number of cases in the Se arm, the difference was not statistically significant. Interestingly, the study showed a statistically significant increase in the risk of type 2 diabetes in elderly individuals, which suggests that with old age supplementation with Se may increase the risk of type 2 diabetes [28]. These findings mirror the controversy on whether higher Se concentrations could increase the risk of type 2 diabetes or not.

Observational studies have also shown a non-linear association between Se and type 2 diabetes. A recent, comprehensive meta-analysis that was conducted in 2019 among 20 observational studies concluded that high Se levels are significantly associated with type 2 diabetes (pooled odds ratios, 1.88; 95% confidence interval, 1.44 to 2.45). However, significant heterogeneity was observed within different studies and the analysis of the funnel plot revealed significant publication bias. Subgroup analysis was also conducted according to the method of Se measurement in each study. Significant association was shown among studies that used blood, diet, and urine as specimens for Se level detection but not in studies that used nails as a sample for Se measurement. Sensitivity analysis was conducted in addition to the trim and fill analysis to account for the heterogeneity and the publication bias, yet the results were consistent upon all adjustments [18]. Another non-linear dose–response analysis confirmed that both levels below and above the physiological range are potential risk factors for diabetes which is consistent with other review articles that suggested a U-shaped relationship between Se and diabetes. Positive associations were detected among patients with low Se levels (<100 μg/l) and patients with high Se levels (>130 μg/l) [29].

#### *3.3. Cardiovascular Disease (CVD)*

Se as an antioxidant was assumed to play an essential role in protecting against atherosclerotic events and CVD [30]. Se deficiency was directly linked to Keshan disease, an endemic cardiomyopathy that is found in regions where Se intake is very low [31]. Multiple studies were carried out to assess the impact of Se on CVD risk. Both low and high levels of Se were found to adversely affect heart function [2]. In Eastern USA, a post hoc analysis of the NPC trial was conducted with a follow-up of 7.6 years to study the effect of Se supplementation on the prevention of CVD. The incidence of myocardial infarction, total cerebrovascular accidents and CVD were assessed, and the results revealed that there is no overall benefit of supplementing 200 μg/day of Se to prevent CVD [26]. The SELECT trial did not also find any significant effect of Se on overall cardiovascular events [27]. A meta-analysis of RCTs showed that oral Se did not have an overall effect on cardiovascular events regardless of Se formulation or dose [32]. Observational studies have shown conflicting conclusions. A meta-analysis of observational studies concluded a nonlinear, U-shaped relationship, between Se levels and CVD risk. The suggested range of Se concentrations that results in significant beneficial effects against CVD is from 55 to 145 μg/l [32].

#### *3.4. Cancer*

The role of Se in cancer prevention was extensively studied in various cancer types. Se exerts its action through the antioxidant activity of selenoproteins in addition to several other mechanisms [33]. However, some laboratory studies suggested that Se can promote malignant cell transformation and progression [34]. Therefore, whether Se promotes or prevents cancer is not fully clear. Various RCTs investigated Se supplementation for cancer prevention. The first RCT is the NPC trial that aimed at assessing the influence of Se on the development of non-melanoma skin cancer. The NPC trial concluded that Se does not exert a chemoprotective effect against the recurrence of non-melanoma skin cancer, yet it significantly reduced the risk for all cancers, and for esophageal, prostate, colorectal,

and lung cancers [25]. Another smaller trial among patients who received organ transplants found an unexpected increase in the incidence of non-melanoma skin cancer as a secondary outcome in Se-supplemented group [35]. The SELECT trial was a pivotal study that was carried out in male general population without high risk of prostate cancer to assess the incidence of prostate cancer among Se-supplemented participants compared to placebo. The study found no significant difference in prostate cancer incidence between Se-supplemented and placebo groups. It also detected no difference in overall cancer risk or any other cancer types [27]. Moreover, three recent RCTs evaluated Se impact on participants with high risk for prostate cancer and all showed no beneficial effect of Se on the incidence of cancer [36–38]. Additionally, a trial among women with high risk of breast cancer due to mutations to breast cancer type 1 (BRCA1) susceptibility gene showed increased risk of all cancers and primary breast cancer in Se-supplemented arm compared to placebo [39]. Observational studies have also assessed Se effect on cancer risk with strongly conflicting results. A recent meta-analysis of observational cohort studies showed an overall lower risk of cancer among participants with the highest baseline exposure levels, yet the included studies are subject to a substantial risk of bias [40].

#### **4. Regional Variability of Se Status: A Focus on the Situation in the Middle Eastern and North Africa (MENA) Region**

The status of Se across the MENA region is widely variable with some countries deficient, some over sufficient and some sufficient in addition to several countries with unknown Se status. This variability was apparent even between provinces from the same country. These tremendous variations may be linked to the varying concentration of Se in the soil were food consumed is gown. We summarized in this review the available evidence on Se status and intake among countries of the MENA region. It is important to note that most of the studies that assessed Se concentrations in biological samples were case-control studies with small sample sizes that cannot be generalized to the whole population. Few studies addressed Se status in a small number of countries with total paucity of information in many other countries of the MENA region. Available evidence on Se status among countries of the MENA region are summarized in Figure 2.

**Figure 2.** Available data on Se status in children and adults among countries of the MENA region.

#### *4.1. Turkey*

A study that analyzed Se concentrations in colostrum, transitional and mature breast milk among healthy lactating women concluded that Se content is below the international reference range set at 18.5 μg/l among all different samples throughout the lactation period [41]. Moreover, analysis was extended to dairy milk products in Turkey collected from different cities and its findings revealed that goat milk had the highest content followed by sheep milk and finally cow milk had the lowest Se content [41]. A more recent study included all dairy products in the analysis and

found that concentrations varied based on the type of food with butter and cheese having higher concentrations; however, other products such as ice cream, milk, and yogurt had almost no Se content [42]. The estimated daily intake of Se among children was reported as 30-40 μg/day [43]. However, with regards to Se concentrations in blood, multiple case-control studies were conducted among different healthy and patient populations including infants, children, adults, pregnant women, and postmenopausal women. Other studies also used samples from erythrocytes and semen to assess Se concentrations [44,45]. Overall, values were widely variable ranging from 50–138 μg/l in healthy populations [43–55].

#### *4.2. Jordan*

A survey of groundwater was conducted to assess the levels of Se in multiple aquifers in Amman Zarqa Basin. Se levels were various among different aquifers ranging from 0.09 to 742 μg/l with an average of 24 μg/l, which exceeds WHO recommended threshold for drinking water [56]. A later case-control study of colorectal cancer patients assessed the dietary intake of Se which was found to be 59.26 ± 8.91 μg/day for controls. This value is higher than the RDA (55–70 μg/day) [57]. On the other hand, Se concentrations were analyzed in two studies using blood and hair samples [58,59]. Blood concentrations in non-smokers were found to be around 187 μg/l which is relatively high [58].

#### *4.3. Iran*

Multiple studies were conducted in Iran among various populations, children, adults, and elderly [60–62]. Se intake was found to be adequate in children and adults [60,61], whereas in postmenopausal women, Se intake was significantly lower than the RDA (55 μg/day) [62]. Se levels in rice samples from Iran were assessed and they were found to be relatively high [63]. Se status in Iran was assessed in various studies in healthy and patient populations. Blood concentrations ranged from 58–123 μg/l in children, adults and pregnant women [64–77].

#### *4.4. Kingdom of Saudi Arabia (KSA)*

A study was conducted to assess the content of Se in wheat grains grown in KSA. This study reported highly variable concentrations ranging from 8 to 293 μg/kg [78]. Other recent studies that quantified the intake of Se estimated the intake to be 75–121.65 μg/day and 93 μg/day in the regions of Jeddah and Riyadh, respectively [79,80]. Moreover, surveys of infant milk formulas, breast milk and cow's milk were conducted to determine Se intake in infant/children populations. The findings of those surveys showed that infant milk formulas contained adequate amounts of Se whereas some breast-fed infants might have lower than recommended Se intakes [81,82]. On the other hand, Se status was assessed in multiple case-control studies in KSA. Those studies were done using various samples; serum, urine, toenails, whole blood, umbilical cord blood, and placental tissue [5,80,83–91]. Blood concentrations varied widely from as low as 32 μg/l to 195 μg/l [5,80,85–88,90,91].

#### *4.5. Egypt*

Se intake among healthy children in Egypt was found to be 8.3 ± 2.3 mg/day [92]. Case-control studies in Egypt were mainly done on children, and reported Se concentrations ranging from 65–83 μg/l [93–95]. Studies have also assessed Se concentrations in neonates and their mothers and the concentrations were found to be 86 μg/l and 118 μg/l, respectively [96].

#### *4.6. Qatar*

In Qatar, no direct studies were conducted to assess the intake of Se among Qatari population. However, according to Qatar General Electricity & Water Corporation (Kahramaa) drinking water quality requirements report, Se is not expected to be present in Qatar's water system [97]. This indicates that the Qatari population is not expected to have any Se intake from drinking water sources. Additionally, a market basket survey of Se in rice imports in Qatar, the primary staple dish, concluded that for Qatari citizens, rice compensates for more than 100% of RNI Se (30 μg/day) regardless of the gender or the type of rice consumed. However, for non-Qatari expatriates (over 80% of the population), with a much lower rice consumption, the percentages were variable according to gender and type of rice consumed, yet they were all below 100% of RNI Se [98]. Further analysis included rice-based infant cereals in Qatar which was found to provide around 63% of RNI Se based on the recommended daily serving [98]. This can conclude that rice consumption in Qatar is a significant contributor to the daily intake of Se. However, Se status in Qatar was not assessed in any of the few studies available.

#### **5. Conclusions**

Se plays a very important role in health and hence Se status and intake are very important to be known for a specific population as the levels of Se are highly variable among different populations and regions. Studies conducted in the MENA region to assess Se status and intake are very limited and most of those available were designed as case-control protocols with small sample sizes. For those countries with data available such as Iran, Turkey, and KSA, more powered, rigorous, and well-designed studies should be conducted to assess Se status among different populations in that specific country or regions that share more or less similar lifestyle and climate such as the six countries of the Gulf Cooperation Council (GCC). Se intake should also be assessed using food surveys and through surveying Se content in staple food (e.g., rice) especially that most of food products consumed in a large number of countries of the MENA region are imported from various parts of the world which may have variable Se content in the soil. Moreover, for many countries within the MENA region, including Qatar and most North-African countries, data about Se status is totally absent. This warrants the need for rigorous and well-constructed studies to determine Se intake and status among the general population and specific populations, such as children and pregnant women, in those countries. Furthermore, studying the relationship between Se status and the incidence of chronic diseases, such as diabetes and CVD in the MENA region would help in devising novel preventive approaches for these disorders particularly prevalent in many countries of this region.

**Author Contributions:** A.A. conceptualized the idea. A.A. and S.A.Z.I. wrote the manuscript. A.K. contributed to selected sections and edited the manuscript. A.A. coordinated the writing up and the submission process. All authors approved the final version for submission.

**Funding:** Our work is supported by award NPRP-8-1750-3-360 from Qatar National Research Fund (a member of Qatar Foundation) to A.A. The statements made herein are solely the responsibility of the authors.

**Acknowledgments:** The figures in this article were created with BioRender.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**


© 2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).

### *Review* **Selenium, Selenoproteins and Viral Infection**

**Olivia M. Guillin 1,2,3,4,5, Caroline Vindry 1,2,3,4,5, Théophile Ohlmann 1,2,3,4,5 and Laurent Chavatte 1,2,3,4,5,\***


Received: 30 July 2019; Accepted: 27 August 2019; Published: 4 September 2019

**Abstract:** Reactive oxygen species (ROS) are frequently produced during viral infections. Generation of these ROS can be both beneficial and detrimental for many cellular functions. When overwhelming the antioxidant defense system, the excess of ROS induces oxidative stress. Viral infections lead to diseases characterized by a broad spectrum of clinical symptoms, with oxidative stress being one of their hallmarks. In many cases, ROS can, in turn, enhance viral replication leading to an amplification loop. Another important parameter for viral replication and pathogenicity is the nutritional status of the host. Viral infection simultaneously increases the demand for micronutrients and causes their loss, which leads to a deficiency that can be compensated by micronutrient supplementation. Among the nutrients implicated in viral infection, selenium (Se) has an important role in antioxidant defense, redox signaling and redox homeostasis. Most of biological activities of selenium is performed through its incorporation as a rare amino acid selenocysteine in the essential family of selenoproteins. Selenium deficiency, which is the main regulator of selenoprotein expression, has been associated with the pathogenicity of several viruses. In addition, several selenoprotein members, including glutathione peroxidases (GPX), thioredoxin reductases (TXNRD) seemed important in different models of viral replication. Finally, the formal identification of viral selenoproteins in the genome of molluscum contagiosum and fowlpox viruses demonstrated the importance of selenoproteins in viral cycle.

**Keywords:** reactive oxygen species; glutathione peroxidases; thioredoxin reductases; influenza virus; hepatitis C virus; coxsackie virus; human immunodeficiency virus; molluscum contagiosum virus; viral selenoproteins; immunity

#### **1. Introduction**

Selenium is an essential trace element for mammalian redox biology. Numerous epidemiological studies have revealed an association between selenium deficiencies and the increased risks of developing several pathologies, including cancers, neurogenerative diseases, cardiovascular disorders and infectious diseases [1–13]. The ability of selenium supplementation to reverse or reduce these risks has been reported in many human or animal models although it remains controversial [14]. Unlike other trace elements that act as cofactors, selenium is covalently bound to organic molecules. Most of the beneficial effects of selenium is due to its incorporation in the form of selenocysteine into an essential group of proteins that are called selenoproteins. Selenocysteine is the 21st proteinogenic amino acid and is encoded by an UGA codon which is normally the signal for termination of protein synthesis [15–23]. Selenocysteine is a structural and functional analog of cysteine in which a selenium

atom replaces sulfur to confer an enhanced catalytic activity. Amongst the twenty-five selenoprotein genes identified to date, several have important cellular functions in antioxidant defense, cell signaling and redox homeostasis [24]. Within the well characterized selenoproteins we find the following sub-families: Glutathione peroxidase (GPX1–GPX4 and GPX6) that reduce hydrogen and lipid peroxides [25], thioredoxin reductases (TXNRD1–TXNRD3) which are essentials in the homeostasis of thiol systems [26–29], methionine sulfoxide reductase (MSRB1) [30] and selenoproteins located in the endoplasmic reticulum (DIO2, SELENOF, SELENOK, SELENOM, SELENON, SELENOS and SELENOT) exhibit important functions in protein folding and in the endoplasmic reticulum stress response [31–33]. The other half of the selenoproteome remains without a, yet, defined function. Selenoproteins are present in many organelles or cellular compartments, with a specific tissue distribution and sensitivity to selenium level changes. Selenoproteins are therefore important components of antioxidant defense systems maintaining redox homeostasis, which also include catalase (CAT), superoxide dismutase (SOD), glutathione (GSH), vitamin E, carotenoids, and ascorbic acid.

Reactive oxygen species (ROS) are produced during viral infections with both beneficial and deleterious consequences for the cell (Figure 1). The viruses associated with ROS production are human immunodeficiency virus (HIV), hepatitis B virus (HBV), hepatitis C virus (HCV), Epstein-Barr virus (EBV), herpes simplex virus type 1 (HSV-1), vesicular stomatitis virus (VSV), respiratory syncytial virus (RSV), human T cell leukaemia virus type 1 (HTLV-1) and influenza viruses [34]. The mechanisms of ROS generation by the various viruses are diverse, but in several cases the host antioxidant defense enzymes, and especially members of the selenoproteome, are targeted.

#### **2. Reactive Oxygen Species (ROS) in Immunity and Viral Infection**

#### *2.1. ROS and Oxidative Stress*

The term "reactive oxygen species" (ROS) refers to series of side-products derived from molecular oxygen (O2) generated during mitochondrial oxidative phosphorylation in every respiring cells (Figure 1). ROS can also arise from exogenous sources including drugs, xenobiotics, metals, radiation, smocking and infection [35]. ROS consist of radical and non-radical oxygen species formed by the partial reduction of molecular oxygen. They include superoxide anion radical (O2 •−), hydrogen peroxide (H2O2), and hydroxyl radical (HO•). At low concentration, ROS are also essential molecules in physiological processes such as cell signaling, proliferation, tumor suppression, and maintenance of the immune system. Oxidative stress arises when an imbalance between ROS and the cellular antioxidant defense system occurs (Figure 1). This could be due to an increase in ROS levels or a decrease in the cellular antioxidant capacity. Oxidative stress leads to direct or indirect ROS-mediated damage of nucleic acids, proteins, and lipids, and this phenomenon has been implicated in many pathological conditions including carcinogenesis [36], neurodegeneration [37,38], atherosclerosis, diabetes [39], and aging [40].

The production of ROS can be assessed indirectly either by using redox-sensitive dyes that are oxidized by ROS into quantifiable fluorescent products, such as 20,70-dichlorodihydrofluorescein diacetate (DCFHDA) or by quantification of cellular oxidation products such as oxidized DNA (8-hydroxydeoxyguanosine), lipids (malondialdehyde, F2-isoprostane, 7-ketocholesterol, and 7-hydroxycholesterol), proteins (carbonyl, 4-hydroxynonenal or glycated oxidation products). Many enzymatic assays are also available to evaluate the antioxidant function of the organisms [41].

**Figure 1.** Balance between the generation of reactive oxygen species (ROS) and their scavenging systems in human. This equilibrium can be unbalanced during viral infections, resulting in oxidative stress. The main ROS producing systems include the mitochondrial oxidative phosphorylation, the phagocytic cell NAPDH oxidases (PHOX), the NADPH oxidases/dual oxidases (NOX/DUOX) and the xanthine oxidase (XO). The main ROS scavenging systems include the catalase, the superoxide dismutases (SODs), the peroxiredoxins (PRXs), the glutathione peroxidases (GPXs), the thioredoxins (TRXs) and the balance between reduced and oxidized glutathione (GSH/GSSG). The viruses for which an oxidative stress has been reported are herpes simplex virus type 1 (HSV-1), influenza viruses, vesicular stomatitis virus (VSV), Epstein-Barr virus (EBV), human immunodeficiency virus (HIV), human T cell leukaemia virus type 1 (HTLV-1), hepatitis B virus (HBV), respiratory syncytial virus (RSV) and hepatitis C virus (HCV) [34].

#### *2.2. ROS Function in Immunity and Cell Signaling*

ROS have an important role in host defense and immunity [42]. The most characterized example is the mechanism by which phagocytic cells produce large amounts of ROS to eliminate a wide variety of pathogens without altering the host cell viability. The nicotinamide adenine dinucleotide phosphate (NADPH) oxidase enzyme complex of phagocytic cells (PHOX) produces superoxide anion radical in the phagocytic vacuole via the transfer of one electron from NADPH to molecular oxygen [41]. The conventional idea is that this (O2 •−) molecule dismutates to form H2O2 and other ROS by further chemical or enzymatic reactions [43,44]. Indeed, the myeloperoxidase (MPO) that is an abundant protein released from the granules into the vacuole can further process H2O2 into HOCl. While the mechanism by which ROS can neutralize the invading micro-organisms in the phagosome is still a matter of debate, the production of HOCl by MPO seems to have a predominant role [43,44].

In addition to microbicidal activity, ROS also act as signaling mediators during cell death/apoptosis but also in processes that control cellular proliferation and differentiation. The family of NADPH oxidases (NOX) and Dual Oxidases (DUOX), referred to as NOX/DUOX, are homologs to PHOX and expressed in a variety of tissues, including colon, kidney, thyroid gland, testis, salivary glands, airways and lymphoid organs. A clear role for cytoplasmic ROS generated by NOX2 as well as DUOX1 has been shown in T cell receptor signaling as well as downstream activation and differentiation of T cells [45–48]. ROS production by mitochondrial complex III is required for antigen-induced T cell activation and production of interleukin-2 which is the cytokine essential for T cell proliferation [49].

#### *2.3. ROS and Viral Infection*

Viral infection is often accompanied by alteration of intracellular redox state of the host cell [34,41,50–60] (Figure 1). Viruses are known to induce ROS-generating enzymes, including NOX/ DUOX and xanthine oxidase (XO) and to disturb antioxidant defenses. XO is implicated in the catabolism of purine nucleic bases by producing H2O2. Increased of NOX/DUOX and XO activities were observed both in vitro and in vivo during viral infection [41]. Infection by the HIV is associated with decreased levels of GSH and an increased production of ROS [61–63]. The latter can be caused directly by virus and/or by the inflammatory response of the host. The viral TAT protein increases intracellular ROS levels by inhibiting the antioxidant enzyme manganese superoxide dismutase MnSOD [64]. In chronic hepatitis C, direct interaction of core protein with mitochondria is an important cause of the oxidative stress [57]. The increase of ROS production has been well documented during HIV, HBV, HCV, EBV, HSV-1, VSV, RSV, HTLV-1 and Influenza viral infections [34]. With HIV-1, ROS were found to stimulate viral replication with the nuclear transcription factor NF-kB, which is necessary for viral replication, being activated by oxidative stress in vitro [54,57,65].

#### **3. Selenium, Selenoproteins and ROS**

#### *3.1. Selenium Insertion in Selenoproteins*

Food is the primary source of selenium intake for mammals, but only five molecules (selenocysteine, selenomethionine, selenoneine, selenite, and selenate) constitute the bioavailable selenium in food intake [9,11]. The recommended daily intake of selenium in adults is comprised between 50 and 70 μg per day. A repeated daily intake above 400 μg leads to selenosis and eventually death. However, in certain regions of China, continual intakes of ~1000 μg Se/day are not associated with adverse effects other than fragile hair and fingernails due to keratin disruptions. Importantly, the concentration of selenium measured in the soil and water determines its levels in the living organisms and crops growing in these territories, and notably the components of human food chain, including microorganisms, plants, cereals, vegetables, fruits, farm animals, etc. The importance of selenium as a trace element in human health has been evidenced in a selenium-deprived area of China named Keshan, which then provided the name to the disease, as described in Section 4.1 and in [66]. Strikingly, Keshan disease has been fully eradicated by selenium supplementation [66]. Other regions around the globe are particularly deprived from selenium (<0.1 mg·kg<sup>−</sup>1), and are located in China, New Zealand, Finland, South-East of USA, and in the UK [9].

It is now well admitted that the biological activity of selenium comes from its insertion into selenoproteins as a rare amino acid, selenocysteine [15–23]. The human selenoproteome is encoded by 25 selenoprotein genes and is highly regulated by selenium bioavailability [9,10,15,22,67–70]. Many reports have evidenced a prioritized regulation of the selenoproteome in response to selenium depletion that maintains the expression of essential selenoenzymes at the expense of others. Upon a normal diet, tissue concentration of selenium in the human body ranges from highest levels to lowest: Kidney, liver, spleen, pancreas, heart, brain, lung, bone and skeletal muscle [11]. Interestingly, in animals fed with a low selenium diet, selenium levels are drastically reduced in most tissues, including the normally selenium-rich ones kidneys and liver, but are maintained in very few tissues such as the brain and neuroencrine glands [71]. This phenomenon, described at the scale of organism, tissue, or cell lines, is referred to as selenium or selenoprotein hierarchy [15,22].

The process of selenocysteine insertion relies on a translational mechanism that is unique in many aspects. Selenocysteine was the first addition to the genetic code and is therefore referred to as the 21st amino acid. This amino acid is encoded by the UGA codon, which is normally a stop codon [15,17]. Thus, the cell has evolved a dedicated machinery to recode UGA as selenocysteine in selenoprotein mRNAs while maintaining its use as a stop codon in other cellular mRNAs [15,17]. The selenocysteine insertion sequence (SECIS) located in the 3 UTR of the mRNA [72] and the selenocysteine-tRNA (Sec-tRNA[Ser]Sec) [16], together with their interacting protein partners allow the co-translational incorporation of a selenocysteine amino acid in selenoproteins. This mechanism is rather inefficient (between 1 and 5%), and mostly results in a truncated protein, the UGA codon being read as a stop codon [73–78]. Interestingly, more and more reports support the idea that the

UGA-selenocysteine recoding event by the ribosome is a limiting stage, and its efficiency dictates selenoprotein expression [15,17,22,67–70,74].

#### *3.2. Role of Glutathione Peroxidases in Antioxidant Defense*

Twenty five selenoprotein genes are present in the human genome [79–81], and most of them are involved in a redox reaction [24]. Among the selenoproteome, the GPXs are major components of the mammalian antioxidant defense. In humans, eight GPXs paralogs have been identified, five of them contain a selenocysteine residue in the catalytic site (GPX1–GPX4, GPX6), and three have a cysteine instead (GPX5, GPX7 and GPX8). Among the selenoenzymes, GPX1 and GPX4 are ubiquitously expressed and represent two of the most abundant selenoproteins in mammals. GPX1 is only cytoplasmic while GPX4 is localized in cytoplasmic, mitochondrial, and nuclear cellular compartments. GPX3 is a glycosylated protein secreted in the plasma mostly by the kidney, and its enzymatic activity is commonly used to evaluate the selenium status of the organism as its level fluctuates with selenium intake. GPX2 has initially been described as a gastrointestinal-specific enzyme but is present in other epithelial tissues (lung, skin, liver). Finally, the recently characterized GPX6, is only found in the olfactory epithelium and embryonic tissues. The role of the GPXs is to reduce hydrogen peroxides and organic hydroperoxides before they cause oxidative damage by reacting on cellular components. GPXs use GSH as a cofactor which is subsequently recycled by glutathione reductases [25], Figure 2A. In vitro, GPXs are able to reduce a wide variety of substrates that include H2O2, tert-butyl hydroperoxide, cumene hydroperoxide, ethyl hydroperoxide, linoleic acid hydroperoxide, paramenthane hydroperoxide, phosphatidylcholine hydroperoxide and cholesterol hydroperoxide (Figure 2A). As reported in [82,83] the different GPXs have overlapping enzymatic activities but they exhibit strong substrate specificities. For example, GPX4 is thought to be specialized in the reduction of lipid hydroperoxides while GPX1 is involved in the regulation of H2O2 metabolism.

The individual role of GPX members has been revealed by gene inactivation in mice. Interestingly, while the inactivation of *Gpx4* gene is embryonically lethal [84], mice deficient in *Gpx1* or *Gpx2* genes are perfectly healthy, fertile and show no increased oxidative stress as compared with wild-type (WT) animals in normal growth conditions [85–87]. However, when *Gpx1*−/<sup>−</sup> and WT mice are exposed to lethal doses of pro-oxidant, such as paraquat or H2O2, an eight-fold decrease in survival is observed for *Gpx1*−/<sup>−</sup> knockout (KO) mice [88,89]. This data suggests that GPX1 has a major role in protecting cells against strong oxidative stress but plays a limited role during normal development and under physiologic conditions. GPX2 appears to have a dual role in cancer, behaving either as a protector of carcinogenesis or a promoter of tumor growth, as revealed by various models [90].

#### *3.3. Role of Thioredoxin Reductase in Antioxidant Defense, Redox Homeostasis and Redox Signaling*

The two major reductive systems in mammalian cells are the thioredoxin (Txn) and GSH pathways. The Txn system is completely dependent on selenium as the three thioredoxin reductases (TXNRD1–TXNRD3) are selenoproteins with the selenocysteine residue at the penultimate position of the C-terminal end of the protein [26,28]. TXNRD1 and TXNRD2 are ubiquitously present in the cytoplasm and mitochondria, respectively, while TXNRD3 expression is restricted to specific tissues. The primary substrates of TXNRD1 and TXNRD2 are Txn1 and Txn2, respectively, Txn2 being localized in the mitochondria. TXNRDs catalyze the NADPH-dependent reduction of oxidized thioredoxin (Figure 2B). The Txns catalyze the reduction of protein disulfides such as in ribonucleotide reductase, peroxiredoxins (PRX), MSRB1, protein disulfide-isomerase (PDI), and are therefore critical for DNA synthesis, the defense against oxidative stress and disulfide formation within the endoplasmic reticulum [20]. Peroxiredoxins are able to reduce H2O2, organic hydroperoxides and peroxynitrite in order to protect cellular components from oxidative damage. However, the existence of multiple peroxide-removing enzymes such as catalase, GPX and PRX indicates that these peroxidases are not simply used in oxidant defense [91]. During inflammation, high levels of peroxides are produced by phagocytes to kill microorganisms. It has been well established that PRXs play cytoprotective

antioxidant role in inflammation. Recently, it has been proposed that PRXs may play key roles in innate immunity and inflammation [91]. It becomes clear that, in addition to fighting oxidative stress, PRXs are important modulators of peroxide signaling. In addition to Txn, TXNRDs can reduce other small molecules containing sulfur, selenium, or oxidized semiquinone and therefore participate in many other cellular processes [20,26,28,92], Figure 2B.

**Figure 2.** Enzymatic activities of the two most important families of selenoproteins involved in antioxidant defense in mammalian cells: the glutathione peroxidase (GPX) and the thioredoxin reductase (TXNRD). (**A**) GPXs use two molecules of reduced glutathione (GSH) to reduce hydrogen peroxides and organic hydroperoxides (ROOH) in their respective alcohols (ROH). The various peroxide substrates of mammalian GPXs are listed next to the bracket; (**B**) TXNRDs use NADPH to catalyze the reduction of thioredoxins and therefore participate in many cellular functions but can also reduce other sulfur or selenium containing compounds. Ox, oxidized molecule; Red, reduced molecule.

#### **4. Selenium, Selenoproteins and Viral Replication**

It is now recognized that the nutritional status of the host plays a leading role in the defense against infectious diseases [93–97]. Many studies show that nutritionally deficient humans or animals are more susceptible to a wide variety of infections. For a long time, researchers have believed that this was only the result of an impaired host immune response due to the deficiency of a particular nutritional element. However, as described below, the mechanism is more complex in that nutritional deficiency impacts not only the immune response of the host but also the viral pathogen itself. Thus, dietary selenium deficiency that causes oxidative stress in the host can alter a viral genome, so that a normally benign or mildly pathogenic virus becomes highly virulent in the deficient host under oxidative stress. This phenomenon has been reported in animal models for influenza and coxsackie viruses [95,98,99], but the molecular mechanism remains unclear. Once the viral mutations occur, even hosts with normal diet would be sensitive to the newly pathogenic strain. The link between selenium levels and viral infection has been reported for many viral groups, see Table 1.

#### *4.1. Coxsackie Virus*

The coxsackie virus is a nonenveloped, linear, positive-sense single-stranded RNA virus that belongs to the family of *Picornaviridae* (Group IV), genus *Enterovirus*. These enteroviruses, which also include poliovirus and echovirus, are among the most common and important human pathogens [100,101]. Coxsackie viruses are divided into group A (23 serotypes) and group B (six serotypes) viruses. In general, coxsackie viruses from group A infect the skin and mucous membranes, while viruses from group B infect the heart, pleura, pancreas, and liver [100].

In the early 1930s an endemic cardiomyopathy termed Keshan disease was first described in Heilongjiang province, Northeast China. This disease mainly affects infants, children and women in childbearing age [66]. It is characterized by cardiac enlargement, congestive heart failure, pulmonary edema and death. Keshan disease spread in another 12 provinces across China between the 1940s and 1960s. Approximately eight million people lived in the affected areas during that period of time, and thousands of people died of Keshan disease every year from this pathology. It is only in the 1970s and even the early 1980s, that the selenium contents in soil, water, food, and human body fluids were found extremely deficient in the areas affected by Keshan disease as compared with adjacent provinces [102]. Selenium fertilizer was applied to the soil in order to increase its content in the food [66]. In addition, selenium supplementation of the diet was also given to the people of these areas. The result was the complete eradication of this disease in these provinces of China [103]. However, several features of the Keshan disease, especially the annual or seasonal fluctuation in the incidence of the disease, did not wholly fit with a selenium deficiency. It appears that this disease has a dual etiology, i.e., selenium deficiency and an infectious cofactor, namely the coxsackie virus B [103–106].

Animal models were used to understand the relationship between host selenium nutritional status and coxsackie virus infection [53,93,94,96–99,107–112]. Coxsackie virus B3 (CVB3) infection of mice can cause myocarditis, similar to that found in human populations afflicted with Keshan disease. Interestingly, as illustrated in Figure 3 the work from Beck and co-authors showed that a non-virulent stain of CVB3 (designated CVB3/0) that do not lead to myocarditis, although replicating, is able to evolve in a virulent strain when inoculated in selenium deficient mice [98,99,109–111]. Remarkably, this is also true when *Gpx1* knockout mice were infected with the benign strain CVB3/0. The sequencing of the viral genomic RNA isolated from selenium deficient and *Gpx1*−/<sup>−</sup> mice demonstrated that a viral genome change had occurred during the infection and replication of the virus as compared to the viral genome replicated in selenium adequate animals, resulting in a highly pathogenic virus [108]. Out of the ten-nucleotide positions that were reported to co-vary with cardio-virulence in CVB3 strains, six reverted to the virulent genotype in virus isolated in Se-deficient mice, and seven in *Gpx1*−/<sup>−</sup> mice. Interestingly, a similar finding was also reported with the deficiency of another essential antioxidant, namely Vitamin E [93,94].

These experiments performed in animal models demonstrate that the host nutritional status, and particularly its antioxidant defense system is an important virulence factor, which can greatly contribute to the evolution of benign viral genomes into more virulent viruses. However, the molecular mechanism involved in this process remains to be elucidated.

#### *4.2. Influenza Virus (Orthomyxoviridae)*

Influenza viruses are enveloped, linear, negative-sense single-stranded RNA viruses belonging to the *Orthomyxoviridae* family (Group IV). There are four genus of this family: A, B, C and Thogotovirus, but only three influenza viruses are infectious for humans (A, B and C) [113]. The viral genome consists of eight segmented single-stranded RNA segments (seven for influenza C virus) encoding from 9 to 12 proteins, including hemagglutinin (HA) and neuraminidase (NA) surface glycoproteins, three ribonucleic acid (vRNA) polymerase subunits (vRNP: PA, PB1, PB2), non-structural protein (NS1), and matrix proteins M1 and M2 [113].

Various subtypes of the most common influenza A viruses are classified based on the diversity in the structure of HA and NA proteins. Influenza viruses can be divided into 16 different HA and

NA combinations. Influenza A and B viruses cause epidemics, whereas influenza C virus tends to cause infections with less severe symptoms [113]. According to the World Health Organization (WHO), the seasonal epidemics result every year in 3 to 5 million cases of severe illness and in 250 to 500 thousands deaths worldwide (https://www.who.int/influenza/en/). People at highest risk for mortality are the elderly and individuals with chronic diseases of the lung and heart. However, safe and effective vaccines are available but often do not perfectly match the circulating subtypes or become ineffective due to viral antigenic drift [113]. It is therefore necessary to engineer new vaccines and revaccinate people at risk every year.

**Figure 3.** Evolution of the pathogenicity of Coxsackie virus as a function of selenium intake or selenoprotein knockout [53,93,94,96–99,107–111]. Coxsackie virus B3 (CVB3) infection of mice can cause myocarditis, similarly to that found in human disease. A non-virulent stain of CVB3 (referred to as CVB3/0, and shown in blue) does not lead to myocarditis in this animal model, although replicating in the mice heart fed with adequate selenium diet (left column). In case of selenium deficient mice, a group of animals was fed with a selenium-deficient diet for four weeks before infection with the benign strain CVB3/0 (second column from the left). A control group of animals was fed with an adequate-selenium diet and infected in parallel [98]. In case of selenium-deficient mice, they developed severe myocarditis. The sequencing of the CVB3 viral genome isolated from the heart of selenium-deficient mice showed mutations at nucleotide positions known to co-vary with cardio-virulence of CVB3 strains (shown in yellow). In comparison, the sequence of CVB3 isolated from selenium adequate mice showed no genetic variation (first column). To determine the consequences of the genetic alterations of the virus, CVB3 isolated from selenium deficient mice was inoculated in animals fed with a selenium-adequate diet (third column from the left) [98]. This experiment confirmed that the mutations of the viral genome increased the cardio-virulence of the virus, which can now induce severe myocarditis even in selenium adequate mice. To investigate whether the most abundant selenoprotein, GPX1, which expression correlates with selenium intake, is involved in the virulence of CVB3, a similar study was performed with *Gpx1*−/<sup>−</sup> mice (right column) [108]. These mice, infected with the benign strain CVB3/0, developed myocarditis and nucleotide mutations of the viral genome isolated from their heart, similarly to selenium deficient mice.

The patients infected with influenza virus display a marked increase in DNA, lipid and protein oxidation products in blood plasma and urine [41,114–116]. Models of mice and cell lines infected with influenza viruses also show an enhanced production of ROS together with an imbalance of antioxidant defense [117–120]. These models are relevant to study the changes in redox homeostasis induced by the influenza virus.

The work from Beck's laboratory extended this novel concept that host nutritional status (especially selenium deficiency) is an important virulence factor in a viral family other than enteroviruses, as shown in Figure 4 [95,121–124]. Indeed, a rapid change in the pathogenicity of the virus in selenium deficient host has been also reported for influenza virus similarly to what was found for coxsackie virus. As shown in Figure 4, mice were fed with a diet either deficient or adequate in selenium for 4 weeks. Then, influenza A/Bangkok/1/79 (H3N2), a strain that induces mild pneumonitis in normal mice, was inoculated to both groups of mice. Interestingly, at all-time points post-infection a clear difference in pathology was observed between the two groups of mice [95,121–124]. The virus was much more virulent in selenium deficient mice, although with a similar virus titer than the selenium adequate mice. In addition, the sequencing of the HA, NA and M genes of viruses isolated from selenium-adequate and selenium-deficient mice demonstrated a strong impact of selenium status on virus mutation.

It appears that the selenium deficiency of the host promotes rapid genomic evolution of the virus in HA and NA genes as compared with selenium adequate animals [53,95,122–125]. Strikingly, these mutations are not stochastic as they were identical in three independent mice fed in selenium deficient diet. In comparison, very few mutations were detected in animals fed with adequate selenium diet. These data further confirm the impact of selenium status of the host in viral genome evolution.

#### *4.3. Human Immunodeficiency Virus (HIV)*

The human immunodeficiency virus (HIV) is an enveloped, linear, positive-sense single-stranded RNA virus that belongs to the family of *Retroviridae* (Group VI), genus *Lentivirus*. Two types of HIV have been characterized: HIV-1 and HIV-2 [126]. Given that HIV-1 is more virulent and more infective than HIV-2, HIV-1 has spread worldwide while HIV-2 is mostly confined to West Africa [127]. HIV is the etiologic agent of acquired immunodeficiency syndrome (AIDS) and is responsible for a weakened immune system as it infects immune cells [126]. HIV affects more than 35 million people worldwide and causes the death of about 1.5 million patients per year (http://www.who.int/hiv/en/). HIV infection is now considered as a chronic disease that requires intensive treatment and can present a variable clinical course. No vaccine is available until now, but an effective medication in decreasing the viral load and increasing the number of CD4 T-lymphocytes has been developed and is referred to highly active antiretroviral therapy (HAART) [128]. This treatment consists in the combination of three or more drugs that target different aspects of HIV replication [129].

HIV genome is highly compact and contains three genes encoding viral structural proteins (*gag*, *pol* and *env*), two genes for essential regulatory elements (*tat* and *rev*) and at least four genes encoding accessory regulatory proteins (*nef*, *vpr*, *vpu* and *vif*). As in any retrovirus, the RNA viral genome is reverse-transcribed in dsDNA that is then integrated in the host genome by the viral integrase. HIV-1 infects immune cells that harbor the CD4 receptor and a co-receptor belonging to the chemokine receptor family (CCR5 and CXCR4) [126]. Therefore, cells infected by HIV-1 are CD4 T-lymphocytes, monocytes, macrophages and dendritic cells. The replication but also the latency of the virus is extremely variable from one cell type to another.


**Figure 4.** Evolution of the pathogenicity of influenza virus as a function of dietary selenium intake in mice. Influenza A/Bangkok/1/79 (H3N2) virus was inoculated in mice that were previously fed with selenium adequate or deficient diet for four weeks. This virus induces mild pneumonitis in selenium-adequate mice but a severe lung pathology in selenium deficient mice [95,121–124]. Various parameters, including the time of lung inflammation, the number of immune cells, the nucleotide mutations of the isolated influenza viruses, the oxidation status of glutathione (reduced/oxidized), the GPX and SOD enzymatic activities in the lung, were evaluated and compared between selenium adequate (left column) and deficient (middle column) mice. The low and high virulent H3N2 viruses are represented in blue and yellow respectively. The virus recovered from selenium-deficient mice was inoculated in selenium-adequate mice to evaluate its pathogenicity. Consistent with the observations made with coxsackie virus, the mutations of the influenza viral genome increased the pathogenicity of the virus, which can now induce severe lung pathology even in selenium adequate mice [95,121–124].

Lentiviruses are characterized by a long incubation period after the primo infection that is highly variable from one patient to another. During this time, humans infected with HIV are under chronic oxidative stress. The redox status of the patient is strongly disturbed in HIV infected patients as revealed by the decrease of antioxidant defense (selenium, ascorbic acid, alpha-tocopherol, carotenoids, superoxide dismutase, glutathione, and glutathione peroxidase) and the increase in ROS production (hydroperoxides, malondialdehyde, and clastogenic factors) [130]. The altered redox status seems to contribute to HIV pathogenesis in several ways. In vitro, increasing oxidative stress enhances the replication of HIV through the activation of NF-kB. Several mechanisms have been reported to explain the cellular enhancement of ROS production in HIV infection. Most of them imply the following viral proteins: Gp120, Tat, Nef, Vpr, and Retrotranscriptase (RT), as reviewed in [50]. A dramatic consequence of this chronic oxidative stress is the fatal decrease in the number of CD4 T-cells by apoptosis, and ultimately a failure of the immune system leading to death.

The nutritional deficiencies of the HIV-infected patient can affect the responsive capacity of the immune system and the progression to AIDS. Selenium is nowadays understood as an essential micronutrient for antioxidant defense and also immune function [131,132]. HIV infection simultaneously increases the demand for micronutrients and causes their loss which leads to a deficiency that can be compensated by micronutrient supplementation [133–135]. Low selenium levels are associated with a lower number of CD4 T-cells, faster progression of AIDS, and 20% increase in the risk of death [133,136]. However, little has been done in term of intervention studies by selenium supplementation or at the cellular and molecular levels to establish the link between selenium, selenoprotein and HIV infection. For example, selenium supplementation is only effective in slowing HIV progression for a subgroup of patients, for which serum selenium levels, CD4 count and viral load were improving in contrast to selenium non-responders or placebo group [137–139]. However, the cellular and molecular mechanism for this unequal response remains elusive. Although efficient at controlling viral load and restoring immune function, HIV antiretroviral therapies, especially the protease and reverse transcriptase inhibitors, have been shown to induce oxidative stress [50,140]. Interestingly, a long time treatment (more than 2 years) with antiretroviral therapy improves selenium levels as compared with HIV-infected patients not receiving the treatment [141].

The field awaits further investigations to understand the role of selenium and selenoproteins during HIV infection at the molecular level. The only in vitro data available reported a modification of the pattern of selenoprotein expression in response to HIV infection in lymphocytes [142] but these experiments were performed before the complete characterization of the selenoproteome. The impact of selenium status on viral genome mutations and in particular the shift to more virulent viruses has not yet been tested for HIV as it has been done for coxsackie and influenza.

#### *4.4. Hepatitis C Virus (HCV)*

The hepatitis C virus (HCV) is an enveloped, linear, positive-sense single-stranded RNA virus that belongs to the family of *Flaviviridae* (Groupe IV), genus *Hepacivirus*. Nowadays, about 3% of the world's population is infected with HCV, which represents approximately 170 million people. Although HCV replication occurs in hepatocytes, the virus also propagates in immune cells. In 80% of the patients with acute hepatitis C, the disease evolves to chronic hepatitis, with 2% developing liver cirrhosis and 1–5% developing liver cancer [143,144]. Many characteristics of oxidative stress have been reported during chronic hepatitis C, including a decrease in GSH, increase in MDA, HNE and caspase activity [145,146]. Zinc and selenium deficiencies increase the risk of chronicity and malignancy [147]. In addition, there is a high prevalence of HCV coinfection in HIV infected patients. The genome of around 9600 nucleotides encodes a unique polyprotein which is co- and post-translationally cleaved into 10 structural and non-structural proteins.

The infection by HCV is another well-documented example of virus-induced generation of ROS. The nucleocapsid protein of HCV, and to a lesser extent NS3, NS5A, E1, E2 and NS4B, are involved in generating oxidative stress in the liver [51,148–151]. In parallel, the plasma levels of selenium

together with erythrocyte GPX activities were significantly lower in HCV-infected patients than in healthy controls. An inverse correlation of selenium levels with viral load was also observed [152]. Interestingly, in HCV and HIV co-infected patients, an even lower serum selenium concentration was measured than in HIV-infected patients [153]. Endoplasmic reticulum stress and unfolded protein response are induced by HCV gene expression [154]. A selenoprotein involved in these mechanisms, SELENOM, has been reported to be upregulated in human hepatocellular carcinoma (HCC) cell lines and liver biopsies of patients with HCV-related cirrhosis [155]. Whether this is true for other endoplasmic reticulum located selenoproteins remains to be investigated.

#### *4.5. Other Viruses*

The Hepatitis B virus is an enveloped virus with a circular and partially double-stranded DNA that belongs to the *Hepadnaviridae* family (Group VII). HBV includes several viruses that infect liver cells and cause hepatitis in humans and animals. In the viral genome, the large negative stranded DNA encodes the envelope, core and non-structural proteins, the DNA polymerase and an oncogenic transactivator [156,157]. The synthesis of the short strand is completed by cellular DNA polymerases after infection. There are 8 HBV strains, from A to H that differ from their geographic repartition [156,157]. Worldwide, between 2 and 8% of the population is infected by HBV but in most of the cases being asymptomatic. An acute HBV infection is however characterized by yellow eyes and skin, severe fatigue, vomiting and abdominal pain. In less than 5% of the cases, the infected people could develop a chronic infection which can further lead to a cirrhosis (in 20% of the cases) [156,157]. Several studies showed an association link between plasma selenium levels and progression of HBV infection [158–160]. For example, the selenium level is not correlated with the responsiveness to interferon treatment [161] but an elevated plasma selenium concentration is associated with a low level of transaminases [161]. Theses hepatic enzymes are implicated in amino acid catabolism, and their release in the plasma is linked to hepatocellular damage. In intervention studies, selenium supplementation decreased cancer incidence in HBV infected patients [162], but when the supplementation was stopped, the incidence became similar to control patients. Finally, in vitro, when hepatic cell lines were grown with different selenium concentration, lower viral proteins, viral transcripts and viral genomic DNA were detected with high selenium culture conditions [161].

The Porcine Circovirus 2 (PCV2) is a non-enveloped virus with a circular single-stranded DNA genome which belong to the *Circoviridae* family (Group II) [163]. Two strains exist, type 1 and type 2, but only type 2 causes a disease in swine, namely the Postweaning Multisystemic Wasting Syndrome (PMWS), a dramatic disease for pig-production industry. The severity of this syndrome is thought to highly dependent on intrinsic factor such as the status of the immune system. It is one of the smallest virus characterized so far, encoding only a capsid protein and two necessary proteins for viral replication [163]. It has been shown that selenomethionine supplementation in cell culture inhibits viral replication [164–168]. Furthermore, addition of H2O2 or ochratoxin A that induced oxidative stress enhanced viral replication. This effect was prevented by selenium supplementation [164–168] or by selenoproteins SELENOS and GPX1 [164–168]. It appears that this mechanism involved the autophagy pathway [164–168]. Finally, in infected mice, selenium supplementation was able to decrease histological lesions by reducing inflammation [164–168].


*Nutrients* **2019**, *11*, 2101


*Nutrients* **2019**

, *11*, 2101


**Table1.***Cont.*

131

#### *Nutrients* **2019**, *11*, 2101


**Table 1.** *Cont.*

132

#### *Nutrients* **2019**, *11*, 2101


**Table 1.** *Cont.*

#### **5. Selenoproteins in Viral Genomes**

#### *5.1. 1998: First Example of a Viral Selenoprotein Encoded in Molluscum Contagiosum Virus Genome*

Molluscum contagiosum is a viral infection that affects the skin and is caused by the dermatotropic poxvirusmolluscum contagiosum virus (MCV) [237,238]. Unlike smallpox and humanmonkeypox diseases, MCV is nonlethal, mostly common in children and young adults and present worldwide [237,238]. However, MCV causes severe skin infections in immunosuppressed adults [237,238]. A typical feature is the apparition of single or multiple papules on the skin, which may persist for a few years. Most cases resolve in six to nine months without specific treatments [237,238]. Such a prolonged infection implies that MCV successfully manipulates the host environment. In 1998, the analysis of the MCV genome sequence predicted the presence of a candidate selenoprotein, homologous to mammalian GPX, with 75% amino acid sequence identity with human GPX1 [175], see Figure 5A,B. This viral GPX protein is encoded by *MC066L* gene that presents every features of a selenoprotein gene, i.e., an in-frame UGA codon, a stop codon different from an UGA (in this case UAG), and a SECIS element in the 3'UTR of the mRNA (Figure 5A). The absence of homologs of this gene in vaccinia and variola viruses suggests that the GPX-like gene was acquired by the MCV after the divergence of the *Molluscipoxvirus* and *Orthopoxvirus* genera. The expression of this predicted selenoprotein was tested experimentally in mammalian cells. Indeed, when a plasmid containing the *MC066L* gene was transfected in human skin cell lines, many evidences supported the insertion of a selenocysteine residue at the UGA codon in the full-length protein, the functionality of the SECIS elements and the cellular antioxidant activity of the MC066L protein [175,239]. Remarkably, this viral selenoprotein has been shown to be protective for human keratinocytes against cytotoxic effects of UV-irradiation and hydrogen peroxides [175,239], suggesting an important function for the virus in defending itself against environmental stress and inflammation. How and when this selenoprotein is expressed in the context of viral infection remains poorly investigated. The first transcription map of the MCV genome was provided by the transcriptome sequencing (RNA-seq) of the RNAs synthesized in abortively infected cultured cells and human skin lesions [240]. These next generation sequencing experiments showed that MC066L mRNA was only detected in cutaneous lesions, but not in MRC-5, Huh7.5.1 and Vero cells infected in vitro by the MCV virus isolated from these same skin lesions.

#### *5.2. 2007: A Second Example of an Encoded Viral Selenoprotein in Fowlpox Virus Genome*

Almost ten years later, another example of an encoded viral selenoprotein was reported in fowlpox viral genome [176], see Figure 5C,D. This was due to the increasing number of viral genome sequenced but also to the development of novel bioinformatic tools dedicated to the discovery of selenoprotein genes in newly sequenced genomes [79–81]. In this viral genome, a coding region homolog to the mammalian GPX4 gene was found, with an in-frame UGA codon, and a predicted SECIS element downstream of the UGA codon but this time within the open reading frame instead of being in the 3'UTR. This finding represented a great opportunity to investigate whether this putative viral SECIS or a canonical SECIS could function within the open reading frame. The authors demonstrated that mammalian cell lines supported the expression of selenoproteins with in-frame SECIS element from both viral and mammalian origin. This fowlpox SECIS element was the second example of a functional viral SECIS element with a structure being identical to the mammalian SECIS. Interestingly, in an evolutionary related virus, the canarypox virus (CPV), this gene has evolved in a Cys-containing GPX4 with a fossil SECIS element still present in the coding region (Figure 5C,D). The potential of this fossil SECIS to trigger recoding of an UGA codon in selenocysteine has not been investigated. It appears that there was a recent mutation of the selenocysteine into cysteine codon in canarypox virus, as it has happened multiple times during evolution of the selenoproteomes in *Eukarya*, *Bacteria* and *Archaea* [79,80]. Note that cysteine is encoded by UGC and UGU codons, and that a single mutation is able to change a selenocysteine to cysteine codon and vice-versa. The presence of a fossil SECIS

element indicates that the GPX4 selenoprotein gene was first acquired from the host and recently converted to the Cys form.

**Figure 5.** Gene structures and amino acid sequences of the selenoproteins present in the viral genomes of molluscum contagiosum virus subtype 1 (MCV1) (**A**,**B**) and of fowlpox virus (FPV) (**C**,**D**) in comparison with their respective human orthologs, GPX1 and GPX4 [175,176]. (**A**) Location of the typical features of a selenoprotein gene (coding sequence, TGA codon and SECIS element) in human *GPX1* gene in comparison with those of MCV1 *GPX* gene. For clarity reasons, the introns of human gene have been removed, but the position of splice sites is indicated by a dashed bar. (**B**) Amino acid sequence alignment of human GPX1 (P07203) with MCV1 GPX (Q98234). Identical and similar amino acids in both sequences are highlighted in black and grey, respectively. The selenocysteine amino acid (U, in one-letter code) is highlighted in yellow. (**C**) Comparison of the location of selenoprotein gene features in human *GPX4* gene with those of FPV *GPX* and Canarypox virus (CPV) *GPX* genes. The replacement of the TGA (selenocysteine) codon by a TGC (Cysteine) one in CPV *GPX* gene is indicated in green. (**D**) Amino acid sequence alignment of human GPX4 (P36969) with FPV GPX (Q70H87) and CPV GPX (Q6VZR0). In-frame SECIS elements in the C-terminal region of FPV GPX are highlighted in red.

The fact that at least two selenoproteins are encoded by viral genomes suggests that these proteins provide a substantial advantage for viruses. Similar to molluscum congatiosum GPX1, the fowlpox GPX4 may provide survival benefits for the virus. These two proteins are, so far, the only proven examples of genetically encoded viral selenoproteins.

#### *5.3. Putative Selenoproteins in Other Viral Genomes*

These two examples of selenoprotein gene snatching from eukaryotic genomes in the viral genomes of fowlpox and molluscum contagiosum viruses lead to the careful investigation for further examples of selenoprotein genes sequences with viral genomes. The first analysis searched for GPX modules within viral genomes where an in-frame UGA codon would be in an amino acid environment close to the catalytic site sequence of eukaryotic GPXs. Several candidates with sequence identities greater than 25% were found in the genomes of HIV-1, HIV-2, HCV, coxsackie virus B3 and measles viruses [183]. Despite these in silico data that GPX-related features are present in a number of RNA viruses, no RNA structure similar to the SECIS element can be evidenced. Additionally, no biochemical data demonstrated the expression of viral selenoproteins in any of these cases. It is possible that viruses have developed somewhat unique mechanisms for Sec insertion, as suggested in [195], but this remains purely hypothetical in the absence of further experimental proofs.

Perhaps, the most advanced study concerns a putative GPX protein coded in the third reading frame of the envelope (Env) gene of HIV-1 [241]. Indeed, it contains the typical catalytic triad selenocysteine (U), Glutamine (Q) and Tryptophan (W) and this putative HIV-GPX protein has been predicted to adopt the overall GPX fold, as deduced from computerized calculations [233]. In addition, it appears that the HIV-GPX gene is conserved in laboratory strains of HIV-1, as well as in long-term non-progressor isolates, but most of HIV isolates from patients with progressive disease presented deleterious mutations (mostly premature stop codons). In order to grasp the cellular function of this putative HIV-GPX in mammalian cells, the corresponding coding sequence has been fused to a mammalian SECIS element and transfected in mammalian cells [234]. The expression of the HIV-GPX seems to have an anti-apoptotic activity, by conferring cytoprotection against exogenous or endogenous ROS. Indeed, several viral proteins are known to induce apoptosis via redox-sensitive effects during HIV-1 viral cycle. Therefore, the presence of a HIV-GPX could be pertinent in the long-term non-progressor patients. Note that these experiments were performed before the emergence of the HAART.

Another putative viral selenoprotein gene has been reported in the -1 reading frame of the NS4 region of Japanese encephalitis virus (JEV). JEV belongs to the *Flaviviridae* family, which also includes dengue fever virus (DENV), yellow fever virus (YFV) and West Nile virus (WNV). The gene named NS4-fs encodes a potential 104 amino acid sequence with three predicted selenocysteine residues, i.e., three in frame UGA codons [191]. This putative selenoprotein displays 30.3% identity and 45.8% similarity with an aligned family of ferredoxin with cysteine instead of selenocysteine. Noteworthy, these three UGA codons are highly conserved, as they are present in all of the 15 full genomic JEV sequences analyzed. A 3D structure of the protein has been modeled [191] where the selenocysteine residues are proposed to maintain the conformation of the [Fe2S2] cluster center. Interestingly, ferredoxin usually acts as an electron transfer agent in biological redox reactions, and this may somehow be important for JEV infection or replication. Again, in this example, neither SECIS elements were found nor any biochemical evidence of selenoprotein expression was provided.

#### **6. Conclusions**

During viral infections, there are many ways that the host metabolism could be affected, leading to a dysregulation of redox homeostasis. The viral pathogens induce oxidative stress via the increase generation of ROS and the alteration of cellular ROS scavenging systems. As part of antioxidant defense, selenoproteins, such as GPXs, TXNRDs and those located in the ER, play an important role in controlling oxidative stress. Selenium deficiency creates a weakening of the defense against infectious diseases

by reducing selenoprotein expression. However, nutritional status of the host can also lead to viral genome mutations from a benign or mildly pathogenic virus to a highly virulent one under oxidative stress that could further spread in hosts with adequate selenium intake. The molecular mechanism leading to the site-specific genome evolution of the virus toward more pathogenic strains awaits further experiments, especially to understand the implication of the selenoproteins.

**Funding:** This work was supported by ANRS, INSERM, CNRS and ENS de Lyon 'Emerging Project' (LC). OMG was a recipient of a Master fellowship from Labex Ecofect (ANR-11-LABX-0048) of the Université de Lyon, within the program Investissements d'Avenir (ANR-11-IDEX-0007) operated by the French National Research Agency (ANR). OMG is a recipient of a Ph.D fellowship from the Université Claude Bernard Lyon 1. CV is a recipient of a Post-doctoral fellowship from ANRS.

**Acknowledgments:** In several figures and in the graphical abstract, we used illustrations from (https://smart. servier.com/) and (https://biorender.com/) websites.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**


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