Mitochondrial changes associated with viral infectious diseases in the paediatric population

Summary Infectious diseases occur worldwide with great frequency in both adults and children, causing 350,000 deaths in 2017, according to the latest World Health Organization reports. Both infections and their treatments trigger mitochondrial interactions at multiple levels: (i) incorporation of damaged or mutated proteins into the complexes of the electron transport chain; (ii) impact on mitochondrial genome (depletion, deletions and point mutations) and mitochondrial dynamics (fusion and fission); (iii) membrane potential impairment; (iv) apoptotic regulation; and (v) generation of reactive oxygen species, among others. Such alterations may result in serious adverse clinical events with considerable impact on the quality of life of the children and could even cause death. Herein, we use a systematic review to explore the association between mitochondrial alterations in paediatric infections including human immunodeficiency virus, cytomegalovirus, herpes viruses, various forms of hepatitis, adenovirus, T‐cell lymphotropic virus and influenza. We analyse how these paediatric viral infectious processes may cause mitochondrial deterioration in this especially vulnerable population, with consideration for the principal aspects of research and diagnosis leading to improved disease understanding, management and surveillance.


| INTRODUCTION
Our multidisciplinary team is composed of basic and translational researchers (mitochondriologists) as well as clinicians (experts in infectious diseases, paediatrics and internal medicine). Our expertise is therefore focused precisely on the three main fields carefully covered in this review: (i) mitochondrial metabolism, (ii) infectious diseases and (iii) paediatrics.
The fact that the activities of our team are mainly focused on the above-mentioned issues, has allowed us to coordinate on this common project over several months. There is an urgent need to depict mitochondrial alterations derived from infective processes in the paediatric population, as mitochondrial status has a great impact on the severity and progression of disease. As far as we are aware, this is the first time that mitochondrial impairment related to both viral infections and the anti-viral agents used for treatment in paediatrics has been reviewed. Also, special focus has been placed on children, as the most vulnerable population group.

| Mitochondria
Mitochondria are semi-autonomous, maternally inherited organelles present in the cytoplasm of virtually all eukaryotic cells. 1,2 They are essential for cell viability, due to their involvement in cellular respiration, apoptosis, catabolism and anabolism of metabolites, calcium homeostasis, thermogenesis, and, through the formation of adenosine triphosphate (ATP) molecules, energy production. 3 They are present in a variable number within cells depending on the energy requirements of each specific tissue. The greater the energy demand is, the greater the number of mitochondria, with the greatest numbers present in nervous and muscular tissues. 4 Mitochondria are not static structures within cells, but are dynamic, capable of merging and fissioning. They consist of (i) the outer mitochondrial membrane (OMM), permeable to ions, metabolites and polypeptides, due to porins and/or voltage- (iv) the mitochondrial matrix containing ions, oxidizable metabolites and the genetic material of the mitochondria, and the mitochondrial DNA (mtDNA).

| Mitochondrial physiology
Mitochondria respond to a series of genetic, metabolic and neuroendocrine signals through functional and morphological changes, and in turn generate signals that influence a large number of cellular functions that contribute to the complexity of physiology and pathology. This places the mitochondria in a privileged position, as a "portal" at the intersection of the cell and its environment. 5 Thus, mitochondria have been implicated in ageing, regulation of cell metabolism, control of the cell cycle, cell development, antiviral responses, signal transduction, among others. 6 The tricarboxylic acid (TCA) cycle, also called the Krebs cycle or the citric acid cycle, which takes place within the matrix of the mitochondria, is a series of eight enzymatic steps that consume, and then regenerate, citrate. It links the metabolism of carbohydrates, fats and proteins, since the catabolism of these compounds generates acetyl-CoA. This key molecule enters the TCA cycle, oxidizes, producing flavin and adenine dinucleotide hydrogen (FADH) and nicotinamide adenine dinucleotide (NADH), reducing molecules that will feed the mitochondrial respiratory chain (MRC) and OXPHOS. 7

| Mitochondrial pathology
The first patient with mitochondrial disease was described in 1962. 8 Human mitochondrial diseases are actually a very large collection of hundreds of very heterogeneous and rare diseases, since changes in any of literally thousands of genes can affect the mitochondrial function. 7 Hence, mitochondrial research is on the rise in the medical sciences. As evidence, the number of medical publications related to mitochondriopathies has surpassed those related to other alterations in other organelles, including the endoplasmic reticulum (ER), the Golgi apparatus and the nucleus. 5 Mitochondrial disorders represent a major challenge in medicine. 8 Similarly, the origin of pleiotropic and multisystemic symptoms in mitochondrial disorders is still poorly understood and often makes it difficult to diagnose this group of diseases. 5 Oxidative tissues, with high energy demand (including the brain, muscle, retina, cochlea, liver and kidney) are the most vulnerable to OXPHOS defects. 8 Clinical presentations in childhood include allergy, hypotonia, development of mental retardation, conduction failure, seizures, cardiomyopathy, hearing or visual impairment, movement disorders and lactic acidosis. 9

Anaerobiosis
In abnormal conditions, such as hypoxia or alterations in mitochondrial function, metabolic pathways are readjusted to continue contraction, chromatin degradation and by providing energy to the nucleus. Apoptosis processes are usually mediated by serine proteases called caspases, which are synthesized as zymogens (procaspases). Depending on the site of action, apoptotic caspases can be classified as initiators or effectors and three classic signalling pathways that lead to apoptosis are recognized: (i) the receptor or extrinsic pathway in which the stimulus is external and received by a cell surface receptor, (ii) the mitochondrial or intrinsic pathway in which the stimulus is internal and is regulated by the mitochondria, 14 and (iii) stress mediated in the ER.
Importantly, mitochondrial alterations can be classified as primary or genetic, when the origin is a genetic alteration, which affects a mitochondrial protein; or secondary or acquired, when the cause is external or environmental, due to the presence of a toxic agent, for example, cholesterol-lowering statin drugs, 1 or the presence of an infectious agent, such as HIV or human cytomegalovirus (HCMV), among others.
In this article, we will review those viral infections that involve the mitochondria, per se or by their treatment toxicity, and that can be considered of relevance in the paediatric population, taking into account the principal aspects of research and diagnosis leading to improved disease understanding, management and surveillance.

| Human immunodeficiency virus
HIV belongs to the genus Lentivirus in the family Retroviridae. 15 Two types of genetically and antigenically different viruses are known as HIV-1 and HIV-2. The vast majority of HIV infections in the global pandemic are caused by HIV-1. Most HIV-2 cases are confined to some West African countries with their epicentre in Guinea-Bissau. 16 HIV is present in body fluids as free virus particles and within infected immune cells and causes acquired immunodeficiency syndrome (AIDS). It primarily infects CD4 + T cells, macrophages and dendritic cells, in order to carry out its replication cycle. HIV infection is associated with a progressive decrease in CD4 + T-cell count and an increase in viral load. In the haematopoietic system, CD4 + T lymphocytes are the most visibly infected cell type since they express the CD4 molecule used by HIV as a receptor and can efficiently replicate the virus. Macrophages are also frequently found to be infected with HIV, but this infection may go unnoticed due to low viral production. 17 HIV kills CD4 + T cells by three mechanisms: (a) by direct viral destruction of infected cells, (b) by increasing apoptosis rates in indicated that the main routes of transmission of HIV were intimate sexual contact and contaminated blood. AIDS was initially described in homosexual and bisexual men and intravenous drug users, but its transmission as a result of heterosexual activity was also soon recognized. Furthermore, it became apparent that transfusion recipients and haemophiliacs could contract the disease by transfusion of blood or blood products and that mothers could transfer the causative agent to newborns as well. These three main means of transmission: parenteral, sexual and vertical (including during pregnancy at delivery and through breast milk) can be largely explained by the high concentrations of HIV in various body fluids. It is worth mentioning that the "optimal prevention scenario" would be the F I G U R E 2 Different stages of HIV infection over time. The stages are (a) acute infection (also known as primary infection), which lasts for several weeks and it can include symptoms like fever, lymphadenopathy, pharyngitis, myalgia, or mouth and esophageal sores. (b) The latency stage involves few or no symptoms and can last from 2 weeks to 20 years or more. (c) AIDS defined by low CD4 + T cell counts <200/μl, increased viral loads, various infections opportunists and cancers 1,2 pregnant woman adheres to treatment, under regular care and with a suppressed HIV viral load of <50 copies of RNA/ml throughout pregnancy and lactation. When these criteria are met, the theoretical risk of mother-to-child transmission is practically zero. 18,19 Vertical transmission of HIV occurs in 11%-60% of children born to HIVpositive mothers. 20 The latest findings suggest that the level of free infectious virus in maternal blood could predict the infection outcome of the newborn. 2,17 Vertical HIV transmission may decrease from 25% to 2% with the use of ARVs that include nucleoside analogues, during pregnancy. However, there is some evidence that exposure in the womb to nucleoside analogues can cause mitochondrial dysfunction symptoms in a small number of HIV-uninfected children. 21 More studies are needed to elucidate the mechanisms of the mitochondrial dysfunction, focusing on exposure in utero, and to identify the importance of mitochondrial variations in children without clinical signs of mitochondrial dysfunction. 21 2.1.1 | HIV structure HIV is a spherical particle 120 nm in diameter made up of three layers, including (i) a lipid envelope which is an external bilayer of phospholipids, coming from the infected host cell, containing class I and II histocompatibility antigens and some adhesion molecules that facilitate contact with target cells. It also contains 72 copies of a viral glycoprotein complex, called Env that protrudes through the surface to the outer environment. These glycoprotein complexes consist of a head of three gp120 glycoproteins and a body of three gp41 molecules, anchored to the molecules of the viral envelope. They allow the virus to bind and fuse cells to start the infectious cycle. Both surface proteins have been considered as possible targets for future development of treatments or vaccines. 22 23 When HIV enters the target cell, the viral RNA genome becomes double-stranded proviral DNA and is imported into the cell nucleus and integrated into the cellular DNA by a virally encoded integrase and host cofactors. 24 Once integrated, the virus can become dormant, allowing both the virus and its host cell to avoid detection by the immune system. Alternatively, the virus can be transcribed, producing new viral RNA and protein genomes that are packaged and released from the cell as new viral particles that begin the replication cycle. Importantly, most of the HIV proteins exert mitochondrial interactions, as depicted in Table 1. Mitochondrial changes derived from the viral interactions occur either in the host cells (mainly CD4 + T-cell lymphocytes and macrophages, but also other lymphocytes, neuronal and glial cells from the central nervous system (CNS), enterochromaffin cells from the gut and dendritic cells, including Langerhans cells, 33 or bystander cells. Apoptosis of uninfected bystander cells is a key element of HIV pathogenesis and represents a driving force to the important CD4 + loss which cannot be explained only by the direct infection. 34 While several viral proteins have been implicated in this process, the complex interaction between Env glycoprotein expressed on the surface of infected cells and the receptor and co-receptor expressing bystander cells has been proposed as a major mechanism. Laurent-Crawford et al. 35 were the first to demonstrate that the HIV Env glycoprotein alone expressed on the surface of cells is capable of inducing cell death in neighbouring T cells. Importantly, the effects of HIV proteins and/or ARV on mitochondria may differ depending not only on whether the target is a host or a bystander cell, but also on the cell type. As an example of the latter, HIV gp120 and Tat have been shown to alter autophagy and mitophagy in neurons and Tat also alters mitophagy in microglial cells. 36 Although this could certainly affect children, no data have been reported so far in paediatric population.
The viral cycle is divided into four stages: (i) fusion and entry of HIV: The gp120 glycoprotein binds CD4, undergoes a conformational change, and interacts with the cell co-receptor (CCR5 or CXCR4), prompting conformational changes in the viral gp41 glycoprotein. 37 Subsequently, once fused with the cell membrane, HIV releases its genetic material (viral RNA) into the cytoplasm of the cell, along with viral proteins. 2 (ii) Reverse transcription and integration of proviral DNA: single-stranded RNA is converted to double-stranded DNA through the activity of the viral reverse transcriptase enzyme. Viral proteins help double-stranded proviral DNA reach the nucleus and integrate into the cell genome, through the virus integrase enzyme. In the event of HIV entering a quiescent cell, the proviral DNA will accumulate in the cytoplasm without any integration, leading to latency. The latent provirus that exists as a reservoir within quiescent cells greatly hampers both the effective endogenous system and HIV treatment, as it avoids immune and exogenous control. 2  As mentioned previously, HIV replicates in CD4 + T cells and progressively destroys the immune system. In children, since the immune system is not fully developed, immune suppression as well as AIDS develops faster than in adults. Consequently, in the first years of life, viral loads remain very high in plasma in the absence of ARV.
The first symptoms of vertical HIV infection are usually nonspecific T A B L E 1 Viral proteins of HIV and mitochondrial interactions in the host cells

Type Protein Mechanism of action and mitochondrial interactions
Structural Env 17 i. Allows the virus to target and bind to specific cell types and infiltrate the cell membrane ii. Increases Bax (pro-apoptotic) iii. Decreases Bcl-2 (anti-apoptotic) iv. Activates mitochondrial apoptosis

Regulatory
Tat 17,[25][26][27][28] i. Reduces the expression of the mitochondrial superoxide dismutase 2 isoenzyme, (endogenous inhibitor of the permeability of the mitochondrial membrane) and triggers the loss of mitochondrial membrane potential ii. Increases Fas ligand expression in T cells, inducing apoptosis iii. Promotes Tat secretion by infected cells, promoting mitochondrial apoptosis in uninfected T cells iv. Induces apoptosis by a mechanism involving disruption of calcium homeostasis Rev 17 i. Ensures the replication of HIV in the infected cell ii. Targets the permeability transition pore, allowing the permeabilization of the mitochondrial membranes interstitial pneumonia is associated with slow progression. A third group of children (<5%) has also been described: very slow progressors, who remain with normal CD4 + T-cell counts and low viral loads for years, without any treatment.

| Mitochondrial changes in HIV infection
Mitochondrial impairment was first associated with HIV in the 1990s, 44 and in 2002, mtDNA depletion (a decrease in mtDNA copies) was described in mononuclear cells in the peripheral blood of HIV-infected patients who had never received ARV. 45 HIV causes mitochondrial impairment by triggering apoptosis; many viral proteins are known to have the ability to induce apoptosis, as already mentioned above. 46    to undetectable levels and increasing CD4 + T-cell counts. In addition, simplification of therapeutic administration has led to better adherence to therapy. In developed countries, due to ARV administration, HIV infection is considered a chronic disease rather than a lethal infection. 2 Importantly, several anti-HIV drugs may also lead to mitochondrial alterations at different levels, which has been summarized, including paediatric studies (

NRTI-derived clinical secondary events Monitoring biomarkers and altered clinical parameters Mitochondrial events
Paediatric studies in exposed and/or infected children i. Children exposed to nucleoside analogues during the perinatal period are at risk of a neurological syndrome associated with persistent mitochondrial dysfunction. 94 This is supported by findings observing the capacity of some ARV trespassing the blood-brain barrier and promoting mitochondrial damage in the brain 95 ii. Distal sensory polyneuropathy is a potential problem in children on d4T-based ARV 96 iii. HIV infection affects central nervous system structures mediating motor and spatial memory development, even in asymptomatic children. 97 This is also

NRTI-derived clinical secondary events Monitoring biomarkers and altered clinical parameters Mitochondrial events
Paediatric studies in exposed and/or infected children ii. Acute pancreatitis has never been reported as a presenting manifestation of acute HIV infection in children iii. Pancreatitis is uncommon in children and adolescents, and the causes are more varied than in adults 99 Lactic acidosis i. Increased lactate levels in serum ii. Seldom manifesting as acute lactic acidosis with evidence of hepatic steatosis probably the most worrisome toxicity (although this is not currently observed, since the most toxic ARVs are not being used) i. Overproduction of lactate derived from mitochondrial damage 100 ii. Insufficient oxidative phosphorylation i. In utero and perinatal exposure to NRTI trigger hyperlactatemia from mitochondrial toxicity 101,102 ii. The clinical presentation of lactic acidosis is unspecific in children and may include gastrointestinal symptoms (nausea and vomiting, abdominal pain) 91 iii. Chronic symptom-free hyperlactatemia has been reported in up to one-third of HIV-infected children v. However, mild tubular dysfunction is recognized in a substantial proportion of TDF-treated individuals and tends to increase with cumulative exposure 105 i. Severe renal damage associated with TDF use is uncommon and of multifactorial origin in children ii. The median blood urea nitrogen increases for every 6month increment in ARV duration in a cohort of children 106 12 of 34 -ROMERO-CORDERO ET AL.

NRTI-derived clinical secondary events Monitoring biomarkers and altered clinical parameters Mitochondrial events
Paediatric studies in exposed and/or infected children There is evidence that exposure to NRTI in utero and the neonatal period may cause lactic acidosis and a decrease in the number and function of mitochondria that may persist in the child, potentially affecting the growth and development of this otherwise healthy infant. Placental tissue of HIV-1-infected ARV-exposed pregnancies shows evidence of mtDNA depletion with secondary respiratory chain compromise. 112 Infants exposed to long-term ARV therapy are more likely than infants not exposed to ARVs to have fatty acid oxidation dysfunction as measured by acylcarnitine analysis. Thus, ARVs may adversely affect intermediate energy metabolism, particularly fatty acid oxidation, suggesting a mechanism of generalized mitochondrial dysfunction, likely due to OXPHOS disruption induced by NRTI. 113 The use of ARV drugs in human pregnancy is one of the most successful strategies to fight against HIV/AIDS, as it protects thousands of children worldwide from HIV infection. However, there are an increasing number of HIVuninfected children that were exposed in utero to HIV and ARVs.
Children exposed to HIV in utero generally function without problems, although some clinical studies and evidence from several biomarkers suggest that there may be progressive changes that will compromise important organs, such as the heart and brain, as ageing occurs. 113 As previously commented, in addition to infection and depletion of T cells, HIV rapidly enters the CNS where it productively infects macrophages, microglia and to some extent, astrocytes. 114 In fact, mitochondrial dysfunction has been claimed to potentially be a common pathway in HIV-associated neurological disorders and therefore a promising therapeutic target. 36 The presence of the virus leads to progressive cognitive disturbances in a large subset of infected individuals. Even with early ARV therapy, more than 50% of HIV patients in the United States develop HIV-associated neurocognitive disorders ranging from asymptomatic to severe dementia. From a behavioural point of view, HIV-associated neurocognitive disorder (TNAV) is characterized by executive dysfunction and memory problems, with significant problems in attention, multitasking and judgement, as well as memory encoding and retrieval. One of the distinctive neuropathological features that most correlates with these cognitive deficits in synaptodendritic damage, particularly decreased synaptic and dendritic density. 55,115 In neurocognitive disorders, neurons experience synaptodendritic abnormalities and damage that can lead to cell death.
A strong correlation factor for TNAV is believed to be oxidative stress.
Because mitochondria are the main source of ROS responsible for oxidative stress, mitochondrial abnormalities probably have a considerable contribution to the pathogenesis of these disorders. 39,116 Due to all these events, children may be more vulnerable than adults to the adverse effects of ARVs due to the potential negative impact on growth and development with their long-term exposure. However, information on the frequency and severity of long-term adverse effects in children is limited. 89  The replication of HCMV is associated with the sequential expression of three gene classes: the immediate early genes, early genes and late genes (  The impact of the congenital infection has led to the need to develop consensus regarding the prevention of infection in pregnant women, the diagnosis during pregnancy and its treatment. 126,139,140 Notably, the first months of life are a key stage in the child's development, in which the health problems will likely affect their future life.

Human herpes simplex virus (HSV) types 1 and 2 belong to the family
Herpesviridae. HSV is a ubiquitous viral pathogen capable of causing both productive and latent infections in its human host. 142 Infections are generally mild but can spread to the CNS, causing serious neurological damage. To enter its host, the virus must overcome a barrier of mucosal surfaces, skin or cornea. Keratinocytes are the main target during the initial entry to establish a primary infection in the epithelium, followed by a latent neuronal infection. 143  The HSV viral cycle can be divided into different phases including 146

| HSV in the paediatric population
Neonatal HSV infection causes high mortality and significant morbidity. Incidence estimates range from 1/3000 to 1/20,000 births. Type 2 predominates over type 1 HSV. 147,148 In the United States, paediatric HSV infections are common; as many as 36% of children <14 years of age have serologic evidence of HSV-1 infection. 149 Often around the age of 5, a child's first cold sore may appear. Cold sores (also called fever blisters or oral herpes) start as small blisters that form around the lips and mouth. After a few days, the blisters crust over and heal completely in a week. 150 ROMERO-CORDERO ET AL.

| Paediatric studies of mitochondrial interaction in HSV infection
Neonates are particularly susceptible to severe HSV disease upon primary infection. Infants infected with HSV rarely develop disseminated, multi-organ infections or encephalitis. 160,161 Life-threatening HSV-1 infections are also found in young children with inborn defects of innate immune signalling linked to type I IFN production.
HSV infection causes leakage of mtDNA, 161,162 and it has been found that aberrant mtDNA packaging promotes escape of mtDNA into the cytosol, where it engages the DNA sensor cGAS (also known as

| Hepatitis viruses
Viral hepatitis is classified as acute (<6 months) and chronic (>6 months). 163  presentation. For instance, HAV usually has a benign course and its evolution to chronicity has not been described. However, the fulminant form is the most serious complication with an estimated frequency of about 0.4% in children. 165 Parenteral hepatitis viruses replicate in the liver after entering the body and, rather than through a cytopathic mechanism, histological damage depends on the activation of the host immune system, which causes destruction of liver cells. The clinical manifestations will depend on the intensity and duration of the abovementioned response. 164

| Mitochondrial changes in viral hepatitis
When considering the relationship between mitochondria and hepatic impairment in paediatrics, a bilateral association must be acknowledged, as primary mitochondrial disorders may produce a variety of hepatological problems in childhood 189 and primary hepatitis is associated with mitochondrial dysfunction (Table 6).
HBV-specific T cells present the ability to switch to OXPHOS in the absence of glucose and subsequently lead to increases in mitochondrial size and a lower Δψm, indicating mitochondrial dysfunction. 190,191 The most pronounced mitochondrial abnormalities observed in hepatocyte-like degenerative cells in the course of chronic HBV infection are characterized by distinct inflammation, loss of mitochondrial ridges and the presence of myelin structures within the matrix. 192,193 On the other hand, HCV has been shown to induce mitophagy, although the precise underlying mechanism and the responsible effector protein remain unclear. The HCV 5A non-structural protein plays a key role in the regulation of cellular mitophagy.
Specifically, expression of HCV NS5A in hepatoma cells triggers distinctive features of mitophagy, including mitochondrial fragmentation, loss of mitochondrial membrane potential and Parkin translocation to mitochondria. Interestingly, NS5A expression concomitantly improves ROS production and treatment with an antioxidant attenuates the NS5A-induced mitophagy event. 194,195 To better clarify this, a detailed summary of mitochondrial damage associated with each type of virus has been summarized ( Table 6).  ii. It is estimated that only 10%-30% of cases present with symptoms: jaundice with pale stools and dark urine, stomach ache and fever 163,164 i. If the immune response is ineffective, the infection will become chronic, and asymptomatic for many years 163,164 ii. If the immune response is excessive, serious fulminant hepatitis and acute liver failure ii. Improvement of socio-sanitary and hygienic conditions 163 iii. HAV vaccine HBV vaccine 164,168 There is no specific vaccine or immunoglobulin 164   Most of the HDV-IgG-positive children show markedly elevated liver enzymes 187 In many developing countries, anti-HEV IgG seroprevalence studies show that most children under the age of 10 years have not been exposed to HEV.  163,164 In acute non-remitting HCV treatment with interferon may be indicated, 164 which has been associated with mitochondria due to its antioxidant capacity.
In HBV, the option of choice consists of two NRTIs: adefovir (ADV) and TDF, both of which cause mitochondrial dysfunction in renal tubular cells and reprogramming of glucose metabolism. 196 TDF has previously been discussed in the HIV section ( software for systematic reviews (http://rayyan.qcri.org), a free web and mobile app, that helps expedite the initial screening of abstracts and titles using a process of semi-automation while incorporating a high level of usability. 199 The studies were assessed for relevance and were blind selected by two independent investigators (SR and CM).
With respect to the inclusion criteria all randomized controlled studies in human models were included, as well as, case reports and review articles. Animal models were excluded for this review.

| RESULTS AND DISCUSSION
A summary of the viruses, related mitochondrial interactions and paediatric studies available is provided in ( Table 7).   Paediatric studies MtDNA levels are lower in HIV-positive patients exposed to HIV than in HIV-uninfected children. Peripheral blood mononuclear cell mtDNA levels are significantly altered in infants exposed to ARVs, not infected with HIV, and their infected mothers compared to infants and women not exposed to ARVs. At 5 years, peripheral blood mononuclear cell mtDNA levels increase to normal concentrations in children exposed to ARV but remain depressed in children not exposed to uptake Paediatric studies The regulatory non-structural proteins of HTLV-1, p13II, are associated with MIM, where it is proposed to function as a potassium channel. The entry of potassium through p13II into the matrix causes depolarization of the membrane and triggers processes that lead to T-cell activation or cell death through apoptosis. 213 impairment as an independent event from the observed depletion of mtDNA levels. 104,216 As observed in this review, not only the pathogens but also their treatments are frequently associated with mitochondrial changes. In this line, alterations in healthy infants exposed to ARVs have been reported. 40 Moreover, the reduction in activity of CI, CIII, and CIV and in general mitochondrial oxygen consumption rates in HIV infected paediatric patients either on or off treatment (in comparison to the healthy control population) is not attributable to a dysfunction of a single respiratory chain complex or a reduction of their protein synthesis rate. 216 In clinical practice, it is often difficult to differentiate whether mitochondrial abnormalities are exclusively related to the infection itself (e.g., HIV) or its treatment (e.g., NRTI). 2 Importantly, these abnormalities have been correlated with the onset of clinical symptoms in the paediatric population; that is, mitochondrial alterations are more evident in children presenting clinical manifestations (such as lipodystrophy under HAART) than in those who do not. 104 Coinfection with HIV/HCV is a main issue also in the paediatric population 218 and primary hepatitis is related to mitochondrial dysfunction, specifically OXPHOS and Δψm alterations. [189][190][191] In many viral infections, mitochondrial abnormalities can also lead to long-term metabolic complications, 110 135,136 or telaprevir and boceprevir use against HCV. 170 Another example of oxidative balance disruption and inhibition of mitochondrial ETC is HSV infection.
Specifically, HSV US3 protein inhibits electron transfer between CII and CIII. 151,152 Moreover, HSV types 1 and 2 induce changes in mitochondrial morphology and distribution in the early and late stages of productive infection in human keratinocytes, 143 evidencing the fact that mitochondrial affectations are not limited to molecular disruption but also to ultrastructural changes.
Sometimes the mitochondrial and cell changes triggered by the infective process are aimed at protecting the cell.
Importantly, in other cases, mitochondria of the infected cell turn out to be the main therapeutic target to treat the infection and pharmacological inhibition of a given mitochondrial performance may represent a key step to avoid pathogen replication.
Such is the case of pharmacological inhibition of complex III in HEV. Mitochondria-targeted pathogen products and the mitochondrial pathways affected by them provide potential novel targets for the rational design of drugs. Pathogen products may alter oxidative balance, mitochondrial PTP, ΔΨm, ETC and ATP production. 151 The finding that blocking of these functions inhibits pathogen growth in many systems suggests that drugs designed to affect viral mitochondrial products, or their targets will be effective in inhibiting the targeted pathogen. Understanding the mechanisms underlying the effects of viral mitochondrial products and their targeted pathways will enable rapid and efficacious drug design.
Mitochondrial performance is highly adaptive during a viral infectious process. For instance, during hepatitis viruses infections, switching to OXPHOS in the absence of glucose and the subsequent increase in mitochondrial size and a lower Δψm has been documented. 190,191 Figure 5 provides a summary of all mitochondrial changes derived from both viral and antiviral agents ( Figure 5). In line with these metabolic arrangements, immunometabolic mechanisms should also be taken into account. Although there is a lack of data in the children, interesting studies have found alterations in mitochondrial biogenesis in neurons and astroglia, which could ultimately modulate neuroinflammation processes together with immunometabolic imbalance in the brain derived from ARV have been reported. 219 Several studies indicate that upon activating glial cells, HIV proteins induce metabolic and inflammatory responses. 220 The metabolic responses include alterations in ROS, ATP production, lactate production, oxygen consumption and autophagic flux. These metabolic changes precede, or are concomitant with, induction of inflammatory gene expression. 221 In most cases, the number of children infected and receiving drug therapy against a given infection is increasing. Also, it is likely that if treatment ends up as indicated in pregnant women with acute infection, the number of treatment-exposed newborns will also increase. Since studies and information are limited, especially in children; it is essential to accurately assess the potential mitochondrial toxicity of such pharmacologic therapeutic agents in a population as susceptible as newborns and infants.
In the near future, the identification of pathways or metabolites that are common to multiple viruses and pathogens remains an important challenge. Additionally, metabolic alterations that are ROMERO-CORDERO ET AL.
-27 of 34 directly involved in pathogen replication and not just a consequence of the infection need to be identified. There are more data to come on the complex interaction between pathogens with mitochondrial metabolism. 216 It is known that disruption of mitochondrial integrity has been identified as a key virulence strategy of several viral pathogens. 222

| CONCLUSION
As a summary of the topics herein discussed: -Infants are a susceptible population group, which is especially vulnerable during specific infective processes.
-Despite the great number of studies in HIV and HCMV, there is a lack of mitochondrial studies in most infective processes in the paediatric population.
-Mitochondria are a main player in specific infections, due to (i) the reported molecular and ultrastructural alterations directly derived from the viral pathogen, (ii) the reported molecular and ultrastructural changes derived from the antiviral treatment, (iii) their role as a therapeutic target in the disease, (iv) their implication and correlation in further clinical manifestations, (v) their identification as a key virulence strategy of infective pathogens, (vi) their high adaptability during the infection process and (vii) their protective role during the infectious process.
-Once a given mitochondrial toxic agent (either the pathogen or its treatment) is withdrawn, the observed lesions are likely to be restored.
-There is an urgent need to carry out longitudinal studies monitoring long-term effects in the grown-up children.

ACKNOWLEDGMENTS
We wish to thank Richard Kirwan for final English supervision.

Constanza Morén
https://orcid.org/0000-0001-6848-7407 F I G U R E 5 General summary of the main mitochondrial changes associated to viral agents and antiviral drugs, described in the present review. To summarize, all viruses herein depicted are related to apoptosis and subsequent ROS production, often related to mitochondrial respiratory chain dysfunction. Specifically, HIV is able to promote metabolic changes and HIV-infected (and bystander) cells undergo apoptosis, present imbalance between oxidants and antioxidants, and Ca 2+ overload, as an HIV-derived toxic effect. HCMV, which presents both anti-and pro-apoptotic properties, also affects cell metabolism, and induces mitochondrial biogenesis and respiration, to facilitate its own replication, which otherwise triggers increased ROS. HSV is associated with inhibition of mitochondrial respiratory chain between CII and CIII, ROS/Ca 2+ overload and CytC release. HV affects mito-dynamics by promoting mitochondrial fragmentation and changes in mitochondrial morphology and mitophagy, in association with ROS generation. On the other hand, anti-HIV/anti-HCMV/anti-HV NRTIs are classically associated to mtDNA depletion, due to off-target inhibition of endogenous polymerases, whereas protease inhibitors are associated with mitochondrial network fragmentation (mitodynamics), apoptosis and ROS/calcium generation. Ca 2+ , calcium; CytC, cytochrome C; Mito-dynamics: mitochondrial fusion, mitochondrial fission and mitochondrial transport; ROS, reactive oxygen species; TCA, tricarboxylic acid