Zika virus (ZIKV) was discovered in Uganda in 1947 and was originally isolated only in Africa and Asia. After a spike of microcephaly cases in Brazil, research has closely focused on different aspects of congenital ZIKV infection. In this review, we evaluated many aspects of the disease in order to build its natural history, with a focus on the long-term clinical and neuro-radiological outcomes in children. The authors have conducted a wide-ranging search spanning the 2012–2021 period from databases PubMed, PubMed Central, Web of Science, Medline, Scopus. Different sections reflect different points of congenital ZIKV infection syndrome: pathogenesis, prenatal diagnosis, clinical signs, neuroimaging and long-term developmental outcomes. It emerged that pathogenesis has not been fully clarified and that the clinical signs are not only limited to microcephaly. Given the current absence of treatments, we proposed schemes to optimize diagnostic protocols in endemic countries. It is essential to know the key aspects of this disease to guarantee early diagnosis, even in less severe cases, and an adequate management of the main chronic problems. Considering the relatively recent discovery of this congenital infectious syndrome, further studies and updated long-term follow-up are needed to further improve management strategies for this disease. ZIKV is a single-stranded ribonucleic acid (RNA) flavivirus transmitted by arthropod vectors, in particular, Aedes aegypti  and Aedes albopictus . The virus replicates in the insect’s epithelial cells and, eventually, in the salivary glands. ZIKV infection used to be considered a tropical disease, but global interest has increased since 2015 due to an epidemic of neonatal microcephaly in Brazil. The virus has been found capable of crossing the placenta in all gestational periods, but mainly in the first trimester. The infection has also been shown to be transmissible sexually and through transfusions . AlthougThe authors have conducted a wide-ranging search spanning the 2012–2021 period from databases PubMed, Web of Science, Medline, and Scopus using search strings consisting of keywords such as Zika, pathogenesis, epigenetics, and microcephaly. Among these papers, we found review/meta-analysis, cohort/retrospective cohort studies  and case-report/case series (reporting the autopsy findings of fetuses that died in utero or shortly after birth). The results and evidence found in the various studies were schematized and both points of agreement and divergence were discussed. The various sections are designed to comprehensively describe the ZIKV congenital infection syndrome. This schematization was used to discuss and propose possible management strategies.ZIKV has a unique ability among flaviviruses to cross the placental barrier  . HypotheTechnological advances are increasingly contributing to clarifying the key factors that affect and shape the dynamics of emerging infections, including ZIKV. Techniques such as genome-wide association studies , high-thThe complex ways in which the pathology of ZIKV infection is affected by genetic traits will be instrumental in laying out therapeutic pathways and enhancing surveillance and prevention capabilities to counter the spread of ZIKV . ReverseThe gold standard for prenatal diagnosis of ZIKV infection is the polymerase chain reaction essay (PCR) on amniotic fluid. Virus-specific IgM antibodies may be detected 3 days after the onset of symptoms. The serology findings can determine false positives due to cross-reaction with other flaviviruses . After birth, the viral genome is detectable in saliva, breast milk, urine, and serum within several days after delivery; these data support the possible perinatal transmission of the infection .Another limitation for prenatal diagnosis of the infection is persistent viremia and viruria in pregnancy, even weeks and months after primary exposure . In the The distinctive features of the congenital ZIKV syndrome in the new-born are the following: severe microcephaly with partially collapsed skull, redundant scalp skin, early closure of the fontanelles, macular scarring and focal pigmentary retinal mottling, congenital contractures of one or more joints  and extrapyramidal motor symptoms with marked hypertonia. According to some authors, microcephaly may develop in new-borns with a normal head circumference at birth ,47. In sNeuroimaging with computed tomography (CT) or magnetic resonance (MRI) has found calcifications of the cortico-medullary junction with band distribution, more frequently in the basal ganglia and less commonly in the thalamus. Other alterations include cysts, mainly in the occipital area, delayed myelination and ventriculomegaly . The latIn congenital ZIKV infection, the whole development of the brain is compromised, with a reduction in cortical gyrification, cerebellar hypoplasia, and hypo/dysmyelination of the white matter in almost all affected subjects. These pathologic alterations are visible both with CT performed after birth and with fetal MRI .Vasco Aragao et al. and Pires et al. have reported that the calcifications of the junction between the cortical area and the white matter are the most frequent finding, followed by alterations of the gyrification, such as pachygyria and micropolygyria, especially in the frontal lobes; hypoplasia of the corpus callosum, ventriculomegaly, hypoplasia of the cerebellum and brainstem (mainly of the pons) or global delay of myelination are less frequent ,51. CalcComparing MRI pathological changes of ZIKV infection with those of CMV infection, , the speThe impact of congenital Zika virus infection on the brain of children has been investigated by several authors through autopsy findings in fetuses (stillbirths or interrupted pregnancy) and children who died shortly after birth. Mlakar et al., in a fetal autopsy, observed complete agyria, calcifications of the junction between the cortex and white matter, hydrocephalus and focal inflammation loci. These alterations are consistent with the neuroimaging described in the previous paragraph. Molecular biology investigations with RT-PCR on these tissues led to isolation of the viral genome .Brasil Martines et al. analyzed brain tissue of children who died after birth with microcephaly and severe arthrogryposis and placental tissues of first trimester miscarriages. In both cases, it was possible to detect viral antigens and viral genome with RT-PCR technique .In children with minor morphological damage, the main neurological problems are infantile cerebral palsy, epilepsy  and neuThis review set out to analyze the main features of congenital ZIKV syndrome, including the pathogenesis of placental and neuronal damage, the methods of prenatal diagnosis, the clinical diagnosis of affected children, neuroimaging and long-term developmental outcomes. Given the large percentage of asymptomatic infections in pregnant women, screening seems to be the only method that really guarantees an early diagnosis and serology, with IgM detection. It should, therefore, be proposed to all pregnant women in endemic areas. Since this test is easily burdened by false positives in the endemic areas, it should be associated with obstetric ultrasound to detect specific markers already in the first trimester of pregnancy. Everyone should be aware that microcephaly can only be seen in the second trimester ultrasound and that some of the affected children will not develop microcephaly. In the case of strong clinical suspicion, PCR on amniotic fluid ought to be carried out .Long-term outcomes of affected children are another very important point of interest. The majority of children who develop microcephaly have an overall developmental delay and, often, severe cerebral palsy. Conversely, among children with normal head circumference, most have a regular pattern of development. In these cases, with established prenatal exposure, even in the absence of microcephaly, it is important to monitor regular development with accurate tests such as GMA. Obviously, GMA, in addition to suggestive clinical signs, can also help to assess cases that have eluded prenatal diagnosis (or doubtful cases due to isolated laboratory positivity). Knowledge of the neuroradiological markers of infection is certainly helpful in cases of clinical suspicion. Children diagnosed with neurodevelopmental issues should start specific rehabilitation protocols as soon as possible. Furthermore, it is essential to investigate epilepsy, possibly with serial electroencephalographic insights and monitoring .In conclusion, shedding light on key aspects of this disease will allow for early diagnosis, even in less severe cases, and adequate management of chronic issues. Given the relatively recent discovery of this congenital infection syndrome, further studies, with updated long-term follow-up of affected children are needed to further improve management strategies.


Cell death by apoptosis is a major cellular response in the control of tissue homeostasis and as a defense mechanism in the case of cellular aggression such as an infection. Cell self-destruction is part of antiviral responses, aimed at limiting the spread of a virus. Although it may contribute to the deleterious effects in infectious pathology, apoptosis remains a key mechanism for viral clearance and the resolution of infection. The control mechanisms of cell death processes by viruses have been extensively studied. Apoptosis can be triggered by different viral determinants through different pathways as a result of virally induced cell stresses and innate immune responses. Zika virus (ZIKV) induces Zika disease in humans, which has caused severe neurological forms, birth defects, and microcephaly in newborns during the last epidemics. ZIKV also surprised by revealing an ability to persist in the genital tract and in semen, thus being sexually transmitted. Mechanisms of diverting antiviral responses such as the interferon response, the role of cytopathic effects and apoptosis in the etiology of the disease have been widely studied and debated. In this review, we examined the interplay between ZIKV infection of different cell types and apoptosis and how the virus deals with this cellular response. We illustrate a duality in the effects of ZIKV-controlled apoptosis, depending on whether it occurs too early or too late, respectively, in neuropathogenesis, or in long-term viral persistence. We further discuss a prospective role for apoptosis in ZIKV-related therapies, and the use of ZIKV as an oncolytic agent. Flaviviridae family, was in the spotlight. [The recent COVID pandemic reminds us of how vulnerable the world’s population is to zoonotic RNA viruses of medical concern. Between 2007 and 2016, the emergence of epidemic strains of mosquito-borne Zika virus (ZIKV), a member of the flavivirus genus of otlight. .The pathogenicity of an infectious agent depends largely on the cytopathic effects it will produce in its target cells. In contrast, programmed cell death (PCD) is an urgent and necessary defense response, implemented by the infected host to disrupt virus multiplication and propagation, thereby preserving other cells and surrounding tissue . The deaDespite the tissue damage and the subsequent critical loss of cell populations, PCD by apoptosis is thus considered a ‘necessary evil’ of the body’s response in its efforts to get rid of the infectious agent.In the case of ZIKV infection and pathogenesis, massive apoptosis of neuronal progenitor cells (NPCs), early in fetal development, has been proposed as an explanation for the specific induced central nervous system (CNS) disorders in newborns and infants ,9. At thIn this review, we propose examining the available data on ZIKV-induced apoptosis and the state of knowledge regarding the virus’s ability to manipulate, delay, or inhibit the cell death response. We also discuss antiviral therapies based on the remediation of apoptosis, and given its unique dual relationship with cell death, the possibility of using ZIKV as an oncolytic agent.Apoptosis is one of the genetically programmed cell deaths that regulates tissue homeostasis and aims to eliminate non-viable, stressed, injured, or infected cells . Cell deSeveral typical situations are capable of initiating apoptosis . ReceiviEach stage of a viral infection cycle is likely to drive pro-apoptotic signaling. First, some viruses can trigger an extrinsic pathway, just upon attachment and entry. In the cases reported in the literature ,) functional proximity between the viral cell surface binding site and death receptors seems to be involved . Second,In addition to metabolic reprogramming, the increasing amounts of viral compounds following virus replication are likely to generate stresses that lead to apoptosis initiation. The considerable input of proteins to be processed by the endoplasmic reticulum leads to ER stress and UPR. This adaptive response is implemented by cells for its antiviral effects at the end. UPR is a way to reduce viral protein synthesis and ultimately results in the self-destruction of the cell, thus bypassing the virus. Many viruses have replication cycles that lead to UPR-dependent apoptosis. This kind of viral induced cellular stress, followed by cell death has been extensively documented, for example, in Japanese encephalitis virus (JEV) and herpes simplex virus (HSV) infections ,28,29. VThe literature on virus-induced apoptosis frequently reports that viruses can change the dominant relationship between pro- and anti-apoptotic factors. It has been shown that Dengue virus (DENV2) induces apoptosis via the expression of XIAP associated factor 1 (XAF1), an inhibitor of the anti-caspase activity of the XIAP protein . AdditioFurthermore, it is increasingly suggested that pattern recognition receptor (PRR) activation by viral compounds, in particular viral genomes in all their forms  and the subsequent innate immunity signaling, can result in apoptosis . The mitAedes (Ae.) [Flaviviridae family and the Flavivirus genus such as DENV, WNV, and many others. It was initially isolated in 1947 from a sentinel rhesus monkey during a study on the circulation of the Yellow Fever virus (YFV) in Uganda and found later in Ae. Africanus mosquitoes [Zika virus (ZIKV) is a pathogenic arbovirus, vectorized by mosquitoes of the genus es (Ae.) . It belosquitoes . The firsquitoes , but litsquitoes , then thsquitoes , and affsquitoes . Today, squitoes . Furthersquitoes ,51.Ae. Aegypti, but also Ae. Albopictus, Ae. Polynesiensis, Ae. Vittatus, Ae. Unilineatus, and Ae. Hensilli [Associated with the virus’s emergence, new modes of transmission have been discovered. Several hypotheses have been put forward to explain the extent and rapid expansions of the last outbreaks. Among them was the switch from historical African and sylvatic species of mosquitoes to more urban vectors, mainly Hensilli . More unHensilli ,54. MateHensilli .Clinical forms are in most cases common to many arbovirosis and include fever, rash, arthralgia, and sometimes conjunctivitis due to direct eye infection . The febHowever, serious complications have occurred after infection with ZIKV. The most outstanding ones have been neurodevelopmental injuries during mother-to-fetus transmission, but peripheral nervous system disorders such as Guillain-Barré syndrome in adults have also alerted the international scientific community. High levels of anti-ganglioside antibodies in affected patients with this syndrome may be linked to this pathology ,58.Numerous cases of congenital Zika syndrome (CZS) including microcephaly due to arrested development of the cerebral cortex , brain cAnother surprising feature of Zika infection was the discovery that viral RNA could be found until 40 days in serum and until 120 days in semen after the onset of symptoms ,63. MoreThe ZIKV genome is composed of a positive 11 kb single-stranded RNA that encodes 10 proteins. Among these proteins, three are structural  and seven are non-structural  as shown in Although not classified as an encephalitic flavivirus, ZIKV has been responsible for debilitating CNS disorders in neonates in the severe forms of infection described from 2013 in Polynesia and 2015 mainly in Brazil . Most of−/− mice) had reproduced the main features of the human disease including infection of the placenta and neuro-invasiveness, leading to reduced brain development and fetus size. Many studies have shown that in these models, the virus could accumulate in various organs such as the brain with associated damages, in the spinal cord, in eye associated tissues, male and female reproductive tracts, gonads, and kidneys. Neurotropic pathogenesis has been attributed to the depletion of neural progenitor cells; however, their infection does not appear to be the only cause of their loss [Multiple strains of ZIKV of African or Asian-American origin have been isolated and used for the study of ZIKV tropism and pathogenesis. Some studies have used recent epidemic clinical isolates and other infectious clones for the analysis of (i) viral tropism and (ii) molecular determinants of pathogenesis . Some teeir loss ,75,76. IIn the field of in-vitro models to investigate the interaction of the virus with its human host, Marazzo and colleagues recently reviewed how the development of neurosphere organoid cultures has provided insights into the specific neuropathological mechanisms of ZIKV . The difFinally, ZIKV was shown able, in-vitro and in-vivo, to infect trophoblasts, fetal endothelial cells of the placenta, placental macrophages (Hofbauer cells), and perivascular cells ,80. It hAs the cytopathic activity of the virus may explain the pathogenic processes and some clinical outcomes, many studies have evaluated the viral-induced forms of PCD in in-cellulo, ex-vivo, in-vivo, and in animal models of ZIKV infection. The onset timing of cytopathic effects and the search for markers specifying the virally mediated cell death were conducted and compared to the course of infection and completion of the viral cycle. Various apoptosis data obtained for different ZIKV strains and infection models have been compiled in Most of the studies carried out suggest that ZIKV elicits apoptosis in vitro, around 48- or 72-h post-infection in cultured cell models. We and others have thus evidenced the mitochondrial events typical of PCD by apoptosis and the implication of BAX and caspases in fibroblast HT1080, in epithelial cell lines such as A549, HEK 293 ,94, in gCells that may be key players in the unique pathogenic process of ZIKV, which depends on maternal–fetal transmission, blood–brain barrier crossing, or important points in the neurodevelopment, were specifically studied.One of the main issues raised by the CZS observed during the last outbreaks was how ZIKV spread into the fetal compartment early in gestation and whether apoptosis could be involved in the process. The mechanisms of crossing the uterine–placental interface (UPI) by ZIKV have been extensively studied and reviewed. Many cell types of the UPI support ZIKV replication. Placental infection, associated cytopathic effects and injury were characterized ex vivo, in villus explants from the first-trimester of pregnancy, in primary cells isolated from placental tissues, and in several relevant cell-lines ,99,100.Santara et al. found that in the first-trimester placenta, infected trophoblasts under severe ER stress were being destroyed, particularly through direct killing by maternal decidual natural killer cells (dNK) . Thus, MTo complete the available data on the mechanisms of maternal–fetal transmission, it is also worth mentioning that a pro-inflammatory context, likely to alter the physical placental barrier function, a cell-to-cell infection process, and a Trojan horse role for infected Hofbauer cells would be the major causes of viral dissemination through the placenta and then to the fetus. It is therefore interesting to note that primary Hofbauer cells have been shown to be resistant to virus-induced cell death . FurtherTo gain an insight into the pathophysiology of ZIKV in neurodevelopment, infections have been conducted in neuronal models. Apoptosis was then shown to be induced in human neural progenitors (hNPC) and associated with P53 and cell cycle impairments . In addiAmong the studies conducted ex vivo, sliced human fetal brain tissues from 14 to 21 weeks of gestation were infected with ZIKV. Activated caspase 3 and DNA fragmentation could be found in numerous infected and non-infected cells. Targeted cells were intermediate progenitor cells (IPCs) and post-mitotic neurons, confirming that ZIKV-induced neuropathogenesis could be the consequence of the disappearance of these cell populations .Several in vivo study models support this assertion. In a mouse model of intracranial infection at different stages of embryonic development, co-immunoreactivity throughout the brain of cleaved caspase-3 and ZIKV are indicators of deleterious cell death . In a reIt should be noted that the study of ZIKV-induced patho-neuro-physiology has greatly benefited from the development of complex neurosphere and brain organoid models. Organoids are 3D cell cultures of self-organized progenitor cells that mimic the tissue architecture and development . SeveralTranscriptome analyses performed on infected hNPCs, SH-SY5Y neuroblastoma cells or brain organoids revealed that ZIKV modified the expression of several factors related to apoptosis, neuronal development, and differentiation ,117,118.As a first look at all the data linking cell death and post-infection pathophysiology, it seems that a consensus has emerged on the capacity of ZIKV to induce an excess of apoptosis too soon during the particular situation of gestation and fetal development. This cell death would occur at the level of placental cells, which would facilitate the dissemination of the virus to the fetus. Apoptosis would then concern the neuronal progenitors and astrocytes. It would be responsible for a depletion of neuronal populations and for definitive damage during the development of the fetal brain.A delayed apoptosis controlled by ZIKV was supported by mathematical models  and expeA body of evidence points to the ability of ZIKV to manipulate cell death in order to limit the antiviral action produced by the cell auto-destruction. Thus, several studies have shown that apoptosis has an antiviral effect on ZIKV. Addition of the pan-caspase inhibitor, zVAD-FMK during infection leads to an intracellular increase in viral RNA . The useIn the case of ZIKV infection and its unique pathogenesis with neurological complications, the role of anti-apoptotic factors and the control of their expression or activity by the virus have been examined. A clinical study of cases of fatal microcephaly found that the parenchyma of infected newborns had a two-fold increase in the anti-apoptotic Bcl-2 protein. This feature appears to be specific to CZS as microcephalies of other origin do not have variation in Bcl-2 expression . Based oWe investigated which protagonists of apoptosis ZIKV was able to act upon and also observed that the amount of anti-apoptotic protein Bcl-2 was increased during in vitro infection. A privileged role for the Bcl-2-family of anti-apoptotics was confirmed by the use of the inhibitor ABT-737, which abrogated ZIKV-mediated protection, led to restoration of apoptosis, and reduced viral infection . As prevThis viral ability to inhibit one of the main cellular defense responses to infection legitimately raises the question of its effect on the outcome of infection and, if unsuccessful, the possibility that the virus will not be properly eliminated. There is an extensive literature on the strategies developed by infectious agents to manipulate apoptosis and the link between impaired apoptosis, deficiencies in viral clearance, and viral persistence in privileged niches ,127,128.Initially puzzling to the scientific community, a first case of sexual transmission of ZIKV was reported in 2011 . The conHigh-level and persistent viruria could also be observed for up to 15 days after the onset of infection symptoms. Several cell types of the infected renal tissue such as glomerular cells or renal proximal tubular epithelial cells (hRPTEpiCs) may be susceptible as reservoirs for long-term excretion of ZIKV in urine ,131.The maintenance of viral replication in these different cells, whether or not associated with their survival and incomplete apoptosis, deserves further investigation.Then, the first clinical indications that the virus might also persist in the CNS were given when worrying cases of neurological complications were reported in infants a long time after their birth and their in-utero exposure to ZIKV ,133.In conclusion, a body of evidence indicates that ZIKV manipulates apoptosis, which is an adverse response of the cell to virus multiplication. This ability could account for the persistence of the virus in immunoprivileged niches. Inhibited or delayed apoptosis and viral persistence could influence pathogenesis and could explain the unique features of ZIKV infection. These main singularities are the non-vectorial routes of transmission and the ability to induce neuropathogenesis long after infection.In mosquito cells, arbovirus infections rarely cause cytopathic effects, and the limited data currently available assessing the role of apoptosis in vector competence suggests that apoptosis is detrimental to the virus. Virus/vector interactions appear to have co-evolved by limiting apoptosis, probably to an initial regulation by non-retroviral integration RNA virus sequences (NIRVS) and the piRNA antiviral pathway . In inveInfection with ZIKV leads, in particular, to a reprogramming of cellular glucose metabolism in a human cell model by enhancing ATP production via the use of the tricarboxylic acid cycle . In contAll of these different apoptosis control mechanisms are important for vector competence, and thus virus spread between mosquitoes and humans.In addition to preventive measures, which mainly involve personal protection against mosquito bites, it is essential to respond to the Zika threat by developing treatments and vaccines. Currently, we have neither one nor the other, although a vaccine candidate using mRNA technology has recently shown promising results in mice . The devOne approach to containing the infection is to fight the virus early by acting on the virus particle itself or at a particular stage of the viral cycle . Unfortunately, although many studies have been carried out and have proposed molecules with promising effects in vitro, no validated treatment has yet been improved ,140,141.Alternative strategies focus on enhancing the natural antiviral responses of host cells by repurposing already known active therapeutic molecules. Trials using the proteasome inhibitor bortezomib have demonstrated antiviral activity in in vivo models and have provided some hope ,143. TheBased on the observation that many viruses, and this is the case of ZIKV, manipulate the cell death response and that this capacity contributes to infectious processes , therapeutic options using apoptosis remedial action have been explored. The underlying idea is that restoring apoptosis can improve or accelerate the resolution of infection and free the body from potential virus persistence . ApoptosInterestingly, ZIKV has been considered as an oncolytic candidate, notably for therapy of brain cancer . The modAs aforementioned, ZIKV has a privileged tropism for neuronal cells in which it produces cytopathic effects ,111. ThiConsistent with these results, intracerebral injection of a live attenuated Zika virus (ZIKV-LAV) in a mouse model of human GBM significantly reduced intracerebral tumor growth and prolonged animal survival by selectively killing SOX2+ GSCs within the tumor . RecentlIt has also been shown that ZIKV oncolytic activity requires CD8+ T cell recruitment to the tumor microenvironment, as the survival benefits are lost if CD8+ T cells are depleted . ZIKV inHowever, as discussed above, ZIKV can persist in the body and has a broad tissue tropism. There is therefore concern that it may affect several organs other than the target tumor. These issues could be circumvented by improving ZIKV specificity to tumor cells. Indeed, receptors essential for ZIKV entry such as AXL and CD24 that are overexpressed in glioblastoma and neuroblastoma cancer cells, respectively, are also present on many other cell types ,159. ThuFinally, and regarding the ambiguity revealed by our literature review on the ability of ZIKV to promote apoptosis too early or too late, it would appear that delayed or attenuated cell death is not a pitfall, but may be an advantage in oncolytic therapy. Thus, cytolytic effects on infected cells, by being delayed, will promote immunogenicity with a progressive and sustained recruitment of immune cells ,162. ThiIn the last decade, a series of epidemics has put global human health at risk. This pressure is due to the threat of mainly viral pathogens that do not spare any region, regardless of the economic levels or medical capacities of countries. In this context, better knowledge of infectious agents, their mechanisms of dissemination/transmission in the environment, and their interactions with their hosts, particularly humans, is essential. This is necessary to face the risks of viral emergence or re-emergence as well as current and future epidemics and pandemics. Understanding the mechanics of host–pathogen interactions at the cellular and molecular levels is also essential for the development of preventive and/or therapeutic strategies. Among the cellular and molecular responses that are crucial for eliminating the pathogen, but that may also be involved in pathological processes is cell death by apoptosis. Just as humans evaluate the benefits and risks of an action, the cell must make a choice between survival and self-killing to resolve an infection. The decision to commit suicide is based on a precise control of pro-apoptotic and anti-apoptotic factors. The process of ZIKV infection seems to be a wonderful example of manipulation of this pro-death or survival balance of the host cell.ZIKV, similar to other flaviviruses , is capable of inducing apoptosis in various types of infected cells. Therefore, an excess of placental cell apoptosis, early in gestation, seems to be one of the main causes of horizontal maternal–fetal transmission. Dissemination of the virus into fetal tissues, infection of the brain, and early apoptosis of neuronal progenitors seem to be involved in Zika pathology and SCZ development .Conversely, cell death by apoptosis has been shown to occur late in many cell types. This delay in the onset of cell death involves anti-apoptotic proteins such as Bcl-2, whose stability and half-life are increased by a mechanism that remains to be defined. It also involves a defect in the induction of the expression of pro-apoptotic factors  that are cruelly lacking to tip the balance. This defect is in part due to the ability of ZIKV to ‘hack’ the communication pathway between ER stress, UPR, and apoptosis by inhibiting the CHOP factor. Other studies have described that paraptosis is sometimes induced by ZIKV, with hyper vacuolation of cells without caspase activation. This could be in place of apoptotic death and apparently allows the virus to replicate more abundantly in the cell . This co


Orchids form mycorrhizal symbioses with fungi in natural habitats that affect their seed germination, protocorm growth, and adult nutrition. An increasing number of studies indicates how orchids gain mineral nutrients and sometime even organic compounds from interactions with orchid mycorrhizal fungi (OMF). Thus, OMF exhibit a high diversity and play a key role in the life cycle of orchids. In recent years, the high-throughput molecular identification of fungi has broadly extended our understanding of OMF diversity, revealing it to be a dynamic outcome co-regulated by environmental filtering, dispersal restrictions, spatiotemporal scales, biogeographic history, as well as the distribution, selection, and phylogenetic spectrum width of host orchids. Most of the results show congruent emerging patterns. Although it is still difficult to extend them to all orchid species or geographical areas, to a certain extent they follow the “everything is everywhere, but the environment selects” rule. This review provides an extensive understanding of the diversity and ecological dynamics of orchid-fungal association. Moreover, it promotes the conservation of resources and the regeneration of rare or endangered orchids. We provide a comprehensive overview, systematically describing six fields of research on orchid-fungal diversity: the research methods of orchid-fungal interactions, the primer selection in high-throughput sequencing, the fungal diversity and specificity in orchids, the difference and adaptability of OMF in different habitats, the comparison of OMF in orchid roots and soil, and the spatiotemporal variation patterns of OMF. Further, we highlight certain shortcomings of current research methodologies and propose perspectives for future studies. This review emphasizes the need for more information on the four main ecological processes: dispersal, selection, ecological drift, and diversification, as well as their interactions, in the study of orchid-fungal interactions and OMF community structure. Biological interactions are key for the construction and maintenance of communities . Fungi iOrchids are the second largest flowering plant family after Asteraceae. In addition to being an “ideal model” group for research on the biodiversity and evolution of interactions , this fa13C, 15N, 18O, and 2H abundances suggests that they acquire some fungal biomass  many species are uncultivable and (ii) taxonomic discrimination between morphologically similar fungal species is difficult, as exemplified by case of ermifera . Howeverermifera . Moreoveermifera . Fungal ermifera  such as To facilitate an extensive understanding of the diversity and ecological dynamics of orchid-fungal associates and to promote the resource conservation and regeneration of rare or endangered orchids with high research or commercial potential, this article summarizes six research modules: the research methods of orchid-fungal diversity, the primer selection in HTS, the fungal diversity and specificity in orchids, the difference and adaptability of orchid mycorrhizal fungi (OMF) in different habitats, the comparison of OMF in orchid roots and soil, and the spatiotemporal variation patterns of OMF. Thus, this review provides avenues for in-depth insight into this field.in vitro fungal isolation in the understanding and experimenting on OM. Moreover, the identifications of some relatively easy-to-isolate strains and biological effect detection have been reported in recent years  is considered as the most reliable and accurate method  and molecular identification  locus, mainly the internal transcribed spacer (ITS), were used in the initial OM diversity studies . HoweverTulasnella species. This set has been widely used for the identification of OM via cloning and sequencing  are the key mycorrhizal symbionts of orchids, mainly its clades A and B. Compared to clade A, clade B is well differentiated and hardly amplified by general primers or even by Tulasnellaceae-specific primers . AccordiOne may be aware that no primer set is perfect and consider using several sets available at the time of designing the study. Based on this and a series of works from our research team (unpublished data), it is highly recommended to sue multiple pairs of primers with low amplification overlap. They can be amalgamated and combined with a nested PCR amplification method to identify the maximum number of orchid mycorrhizal partners . The useOrchids are often associated with phylogenetically and ecologically diverse fungi. Basidiomycota and Ascomycota, with very few Chytridiomycota, Glomeromycota, Zygomycota, or Mucoromycota, are widely distributed in the aerial roots of epiphytic orchids and in the underground roots or rhizomes of terrestrial and lithophytic orchids . Fungi iSerendipita (= Piriformospora) indica is orchid mycorrhizal in Brazilian orchids  as well as Serendipitaceae . Most of these three fungal taxa have ecological niches ranging (and mixing in many cases) saprotrophy, i.e., exploiting decaying matter, and endophytism in non-orchid plants . This cl orchids . In addiloeselii . Furtherleborine , and in leborine .Peziza, and Inocybe , OMF diversity tends to decline from the seed stage to the seedling stage and to often increase again in the adult stage in an hourglass-like pattern. In terrestrial orchids, OMF from protocorms or seedlings are conventionally subsets of those found in early germinating seedlings and adult plants . This ha species . In addi species . To summ species , 2018.Xylaria spp. and Hypoxylon spp.) and Helotiales  are the main ONF associated with tropical and temperate orchids, respectively; Chaetothyriales, Hypocreales, Helotiales, and Capnodiales are also frequent in tropical epiphytic orchids  vermifera  on limestone vary significantly ; hence, the co-occurring orchid species with different OMF compositions may also result from a strong OMF selection by the host orchids , tree richness, altitude, mean annual precipitation, and mean annual temperature . ElucidaIn terms of biotic factors, changes in habitat vegetation influence the OMF communities associated with orchid roots , by chanWith respect to abiotic factors, several studies suggest that many soil characteristics  explain the distribution of OMF in habitat patches . DependiConsidering the effect of latitude on the species and functional group richness of OMF in orchids, diversity decreases with increasing latitude; however, whether the orchid diversity follows similar trends remains unclear . The divAt the same latitude, orchids in areas with higher temperatures and fewer seasonal changes exhibit higher OMF diversity. Environmental conditions, such as high temperature or high humidity, may increase the activity of some saprotrophic fungi, enabling them to obtain more carbon so as to support the growth of MH orchids . RecentlIn conclusion, climatic changes may strengthen the filtering effect of environmental factors on OMF associated with orchids by perturbing biological interactions. Furthermore, in addition to ecological factors, the phylogeny, ploidy levels, and genome composition of host orchids as well as the sampling effort itself influences the OMF communities . TherefoDespite extensive research on the diversity of OMF colonizing orchid roots, their spatial distribution and abundance in soil has received more limited attention. Several reports suggest that OMF either exist in saprophytic form in the soil or form ECM or endophytic colonization on adjacent plants ; howeverEarly seed germination experiments and a few pieces of molecular evidence revealed that most OMF distributed in orchid roots are also widely present in the soil. Moreover, with increase in radial distance between the soil sampling point and the adult orchid, the corresponding OMF richness and abundance often decreased . InteresGastrodia flavilabella were rarely found in the surrounding soil  in 21 semi-natural calcareous grasslands suggested that the available evidence for spatial clustering of OMF on a local scale was limited , while ONF displayed a more distinct spatial distribution pattern  the different nutritional requirement of host orchids at different spatiotemporal points; (2) the differences in the ability of OMF to grow in soil and provide nutrients; and (3) the different adaptability of OMF to environmental conditions . Notably, fungal dormancy may play a key role in the spatiotemporal turnover of fungal communities, since dormancy can bypass environmentally imposed choices and allow the consistent presence of genetic variations in the environment, thereby increasing the genetic diversity of fungal communities .Fungi are a major pillar of biodiversity in ecosystems, occupying a wide range of niches. Owing to the relatively easy growth of some fungi, fungal diversity and strain collection have always been hot spots in the field of fungal research . OM symbThe emergence of culture methods that differ from traditional technologies, such as genomic information methods based on membrane protein expression and microfluidic technology , brings The roles played by the deterministic processes based on niche theory and the stochastic processes based on neutral theory in the construction of communities have been well-established in the field of microbial ecology . HoweverThus, future research on OMF diversity should primarily focus on how the four main ecological processes interact in the assembly and function of OMF communities. In addition to improve certain shortcomings mentioned in the various sections of this review, considering the overall framework required for further research on the fungal diversity of orchids, we have added several keys or difficult issues that are noteworthy for further explorations in the future:in situ seed baiting, the transplantation of seedlings, and the identification of OMF contained in barks or substrates of phorophytes at different sites, are needed to elucidate the interactions among orchids, OMF, and phorophytes.Compared to terrestrial orchids, little is known about the OMF in tropical epiphytic orchids and their distribution on phorophytes. The phorophytes of orchids reportedly cover at least 46 plant families . HoweverAlternaria, Clonostachys, Aspergillus, Penicillium, Phomopsis, etc.) and OMF in orchid tissues is strongly recommended.The selection method for OMF-specific primers, in addition to the method combining multiple pairs of complementary primers mentioned in this review, can be further improved by employing epicPCR technology, which can focus on two genes in a genome at the same time . MoreoveSeveral recent studies have revealed that the difference in relative abundance generated by amplicon sequencing technology is unable to reflect the difference in true absolute abundance of microorganisms in samples, as it ignores the influences of changes in the overall microbial abundance on hosts . Althougvia HTS are truly rare species remains controversial. However, an in-depth analysis of the fungal metabarcoding data obtained from 16 HTS analyses of representative ribosomal RNA gene regions indicates that less than half of these sequences are artifacts . To improve the efficiency and accuracy of such predictions, it is recommended to use platforms or software, such as MetaCoMET, COREMIC, BURRITO, and PhyloCore, that consider more critical ecological information  to predict and analyze key species in a given microbial community. It is also necessary to standardize their application for orchid-fungal communities based on the effect of various methods, thus facilitating better comparability of core fungal taxa of different or the same orchid species from different case studies.Clarifying the architecture of the OMF networks formed by co-existing orchids can help to comprehend how these hyper-diverse interacting guilds are maintained and co-evolve in their habitats. This can further reveal the species, lineages, or functional taxa that are significant to ecosystem services.Research on orchid-fungal diversity often involves cryopreservation, especially in case of large-scale sampling to study the mycorrhizal networks. This may result in a decline in the vitality of orchid tissues, affecting the OMF. Therefore, it is highly critical to explore the influence of liquid nitrogen or cryopreservation at −80°C on the composition and structure of OMF communities – a prerequisite for truly reflecting the symbiotic patterns of orchids with OMF in natural habitats.Mycena, Thelephoraceae, and Russulaceae) demonstrate a relatively wide distribution in orchids. Further investigations are needed to identify (i) their exact ecology, between pure saprotrophy and pure endophytism in non-orchids and (ii) the geographical hot spots of these OMF. This is crucial for the conservation and regeneration of rare and endangered orchids.Although it is known that OMFs occur in various habitats, not only the rhizoctonia taxa  but also some frequent non-rhizoctonia taxa  have increasingly become a hot issue in the study of ecology and evolution of plant microbiomes . The recWith the joint efforts of the growing number of orchid experts from all over the world, the exploration and understanding of elusive and complex mechanisms underlying orchid-fungal interactions are expected to gradually gain momentum and reveal the much sought-after answers.JG and M-AS designed the outline of the manuscript. TL, JG, M-AS, WY, and SW collected the data and wrote the manuscript. M-AS and JG polished the article. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Until recently, pain was viewed as an unpleasant symptom of disease or injury that neededto be either alleviated or tolerated, but that did not inflict harm in and of itself. We nowknow that pain is much more than a symptom and, when chronic, can be viewed as a diseaseitself. The opioid crisis, along with an aging population and the novel threat of painrelated to long COVID, has drawn new light to the consequences of pain and led to the searchfor a better understanding of pain mechanisms and possible non-opioid treatments.The Oxford Handbook of the Neurobiology of Pain is a timely offering,providing a comprehensive compendium of scholarly articles about pain, ranging from thehistory of pain research, through non-human models, to detailed chapters about mechanisms atperipheral and central nervous system levels, and finally pain conditions and treatmentmodalities. Individual chapters are dedicated to some common chronic pain syndromes, such asheadache, visceral pain, neuropathic pain and cancer pain. Other chapters delve intomolecular mechanisms, pain genetics, neuroimmune interactions and pain, and developmentalaspects of pain.The handbook is not organized into sections, and there is little coordination betweenchapters, leading to a fair amount of redundancy, particularly in terms of primary afferentand spinal cord mechanisms. This is not necessarily bad, since it allows readers to selectindividual chapters to read, without committing to a thorough perusal of the more than 800pages of text. Each chapter has enough autonomy to be a stand-alone analysis of the topicand serve as a fundamental resource for scientists and trainees working in the field.A stellar array of more than 50 experts in the field have contributed to the handbook, eachpresenting state-of-the art analyses of their topic. It is not possible to address eachchapter individually, but some highlights include an engrossing historical overview ofpurported pain mechanisms and analgesic treatments throughout the ages (Chapter 1) and athought-provoking analysis of the translation from rodent models to humans that clearlyshows the problems but presents an optimistic view of the future (Chapter 2).A number of chapters offer an excellent reference for the serious pain scientist, withdetailed analyses of most aspects of nociception. Chapters are dedicated to the dorsal rootganglia (5), heat and cold pain (7), noxious mechanoreception (8), signaling channels in theperiphery and dorsal horn , and dorsal horn mechanisms (16).The chapters on basic pain mechanisms may be too detailed for clinicians or neuroscientistsnot specializing in pain, but other chapters could be extremely useful to theresearch-minded clinician by providing a link between pre-clinical data and clinicalsyndromes. Such chapters include a discussion of sympathetic nervous system and pain (6);neuroimmune interactions and pain (13), developmental and sex-related aspects of pain (14),mechanisms of migraine pain (19), relationship between sleep and pain (20), the diversity ofneuropathic pain conditions (22), the transition from acute to chronic (23), cancer pain(24) and opioid mechanisms (25).Although the handbook is quite comprehensive, there are some important gaps in the topics.In terms of pre-clinical models, it was nice to see the inclusion of a chapter oninvertebrate models of nociception, but a discussion of human and non-human primate studiesof nociception is conspicuously absent. Additionally, although several chapters discussdescending pain modulation systems and their role in pharmacological pain modulation, thereis little discussion of non-pharmacological pain treatments and their mechanisms. Given theproblems with opioids and the uneven effectiveness of other pharmacological treatments forchronic pain, a deep dive into non-pharmacological pain treatments would have beenwelcome.The Oxford Handbook of the Neurobiology of Pain is anoutstanding collection of chapters addressing a wide range of topics in the field. With afew gaps, it is inclusive of both basic and clinical aspects of pain. The volume shouldappeal most to the dedicated pain scientist, but it could also be a useful tool forclinicians and students. At a price-tag of more than $200 US, and with free reviews of manytopics available within scientific journals, the appeal of this book to trainees may,unfortunately, be diminished. Nevertheless, it is a definitive volume that brings togetherthe important issues in the field of pain, discussed by the most preeminent experts.In conclusion,


Some say that small is beautiful, and if beauty could be measured by levels of diversity and complexity, we could definitely say that viruses are beautiful. This fascinating particle of life has developed complex mechanisms to infect a large variety of eukaryote and prokaryote hosts. Although some mechanisms such as the exchange and duplication of genes can occur, genetic diversity amongst viruses is mainly generated through inter- and intra-species genetic recombination and mutation. Nucleotide substitution is the most common mechanism of genetic variation and results from errors during the replication of the virus by viral polymerases. The lack of proofreading and repair activities by RNA virus polymerases explains why RNA viruses have higher mutation frequencies than DNA viruses. Nevertheless, some DNA viruses can avoid high-fidelity replication mechanisms and have high mutation frequencies ,2,3. TheViruses, we collected peer-reviewed research articles focusing on different aspects of viral genetic diversity.In this Special Issue of Interestingly, Armero et al. suggest human-to-human transmission of nonsynonymous intra-host variants of SARS-CoV-2 . They prAnother respiratory virus, the respiratory syncytial virus (RSV), poses a public health problem in terms of morbidity and mortality. The annual burden of the disease associated with RSV infection is high, particularly in countries with low resources and where access to care is limited, resulting in elevated mortality among children under five years of age . To reduAfrica and South America encompass hotspots of biodiversity, not only among their fauna and flora, but also all along their living landscapes. Somehow, the diversity of species impacts the diversity of viruses. We discovered, in this Special Issue, that viral diversity in the neotropical rodents of the Amazonian basin is influenced by their habitat types . In partAnother example of the high degree of viral genetic diversity is in Picobirnaviruses (PBVs). These small, segmented RNA viruses have been detected in a wide variety of hosts, both vertebrate and invertebrate, as well as in the environment. Perez et al. have published their work in this issue, which shows that the optimization of phylogenetic analysis is essential to better understand the phylogenetic diversity of PBVs . FollowiIn this Special Issue, we sought to highlight recent advances in understanding the genetic diversity of viruses. We are grateful for the tremendous work conducted by the authors of the articles published. The diversity of topics presented in these papers reflects the diversity of virology, and we are convinced that our issue will provide interesting new knowledge and insight to the virology community.


Background: The aim of the study was to identify, interpret, and compare the current perspectives of regulatory agencies in six member countries of BRICS-TM  on the different criteria used for biosimilar development and marketing authorisation process.Methods: A semi-quantitative questionnaire was developed covering the organisation of agency, biosimilar development criteria and marketing authorisation process and sent to seven regulatory agencies covering the BRICS-TM countries. All data was kept anonymous and confidential. Data processing and analysis was carried out; descriptive statistics were used for quantitative data and content analysis was employed to generate themes for qualitative data.Results: Out of the seven regulatory agencies included in the study, six representatives provided the responses. The perspectives of these six regulatory agencies varied on a number of aspects relating to the review criteria for biosimilar development and licencing process. The most prevalent model for data assessment is the “full review” of a marketing authorisation application. There is lack of a standard approach across the agencies on sourcing of the reference biological product, in vivo toxicity studies and confirmatory clinical studies. Most agencies restrict interaction with biosimilar developers and any scientific advice is non-binding. The marketing authorisation approval depends on scientific assessment of the dossier, sample analysis and GMP certification. The agencies do not issue any public assessment report specifying the summary basis of biosimilar approval.Conclusion: Regulatory agencies across the six emerging economies are steadily improving the regulatory mechanism in the area of biosimilars. However, there remains scope for increasing the effectiveness and efficiency of the processes by encouraging open and transparent interaction with developers, adopting a flexible approach toward accepting advanced analytical data in lieu of clinical studies and enhancing regulatory reliance amongst agencies. This will help to simplify the new biosimilar development programmes and make them more cost-effective. Emerging economies represent 70% of the world population accounting for a 31% share of global GDP and more than 30% of pharmaceutical spending . BiosimiIn the last 2 decades, the mature regulatory agencies, in particular in ICH jurisdictions, have made significant progress toward establishing, revising, and updating biosimilar guidelines to match the dynamic innovation in biotechnology. However, there remains scope for improvement in establishing regional standardisation for regulatory requirements of biosimilar development and approval process.The main objectives of the study were to:evaluate and compare technical capabilities of the six regulatory agencies of the emerging economies in the area of biosimilars,identify similarities and differences in regulatory requirements of biosimilar development criteria i.e., biosimilarity principles, comparative studies including physicochemical characterisation, non-clinical, and clinical studies,evaluate and compare “must submit documents” as part of biosimilar application for marketing authorisation in these six emerging economies,map the biosimilar marketing authorisation approval pathway specifically for key milestones, scientific advice meetings, clinical trial mandates, and backlogs.The regulatory authorities included in this study were those which are part of the BRICS-TM grouping. This refers to the countries of Brazil, Russia, India, China, South Africa, Turkey, and Mexico deemed to be developing countries at a similar stage of newly advanced economic development, on their way to becoming developed countries and also known for their significant influence on regional affairs. It was initially developed as BRICS and since 2009, their governments have met annually at formal summits. Russia hosted the most recent, 12th BRICS-TM summit on 17 November 2020, virtually due to the COVID-19 pandemic –12. TherThe potential study participants were identified via each respective authority's general email addresses obtained from agency websites, LinkedIn, the research team's personal contacts, ex-employee, and local leading regulatory consultants for each authority. They were selected based on their work experience in the biologic or biosimilar division of the authority, having held a position as a general manager or above or a leading regulatory consultant with a close working relationship with the relevant authority in the biosimilar space. They were sent an electronic mail with brief information about project and the questionnaire, the objective of the study, the number of authorities to be included and requesting their agreement to participate in the study.Responses and conditions of acceptance were different across all seven authorities. It took ~18 months to receive agreement from two agencies i.e., Agência Nacional de Vigilância Sanitária (ANVISA), the Brazilian Health Regulatory Agency, Brazil, and the South African Health Products Regulatory Agency (SAHPRA). The respondents from the Central Drug Standards Control Organisation (CDSCO), India agreed to participate on anonymity and the Türkiye Ilaç ve Tibbi Cihaz Kurumu (TITCK), Turkish Medicines and Medical Device Agency data was gathered from public sources of the Agency such as Activity report, official website, and Agency's publicity manual. Two agencies including the Russian MoH and Comisión Federal para la Protección contra Riesgos Sanitarios (COFEPRIS), the Federal Commission for the Protection Against Sanitary Risks, Mexico did not respond to the letter of invitation. Consequently, local senior regulatory consultants were engaged as proxy for Russia and Mexico. Despite tremendous efforts to establish direct contact with the National Medical Products Administration (NMPA) of China or via local Chinese regulatory consultants, the outcome was unsuccessful.On receipt of agreement to participate from the recruited six countries, the self-administered Biosimilar Development, Evaluation, and Authorisation (BDEA) questionnaire was sent via email for completion by the respective authorities. This was followed up by a face-to-face or virtual meetings after receipt of the completed questionnaire. Such meetings were arranged to understand and further interpret the respective agency's view and to verify the validity of the responses to the questionnaire. In addition, copies of the relevant guidelines were requested as part of the questionnaire to verify the responses and to correlate the actual regulatory requirements. This phase of data collection period took place between March and October 2020.A semi-quantitative questionnaire, Biosimilar Development, Evaluation, and Authorisation (BDEA) was developed (in English) . This waData for the comparator authorities was collected in 2019–2020. The BDEA questionnaire which standardises the review process allowing key milestones, activities and practises of the seven regulatory authorities to be identified was completed by a senior member of the biosimilar licencing division and validated by the head of the division/authority. The final version of the BDEA questionnaire dated March 2020, consists of 35 pages and the questions are grouped under 22 categories and grouped into three major sections as follows:Part I - Organisation of the agency - This part of the BDEA questionnaire consist of current agency structure, resources in the biosimilar domain, and types of review models i.e., review models employed for scientific assessment ].For the purpose of clarity, the results will be presented in three parts:Part I – Organisation of agency;Part II – Biosimilar development criteria; andPart III – Marketing authorisation process.Out of the seven regulatory agencies invited to take part in the study, four agencies, ANVISA (Brazil), SAHPRA (South Africa), CDSCO (India), and TITCK (Turkey) agreed to take part and completed the questionnaire. Leading regulatory consultants working closely with the agencies for biosimilar medicines from Russia and Mexico also participated in the study. However, multiple efforts to reach NMPA (China) either directly or via regulatory experts were unsuccessful. The individuals who completed the questionnaire held senior positions  within the biologic divisions of their regulatory authority. The regulatory consultants were Chief Executive Officers (CEO) of their respective consulting firms.This provided information on agency size and the strength of the biological division including the number of internal assessors with their minimum qualifications and details on support obtained from external assessors or committees .ANVISA - The capacity of the biological department is around 1.6% in comparison with the total size of the agency. The agency does not engage with external assessors, and the applications are reviewed by qualified internal assessors all of whom hold a PhD as their qualification. The agency relies on type III data assessment (full review of the marketing authorisation application) for most of the applications.Russian MoH - There is no distinction between internal assessors for the review of biological or non-biological marketing authorisation applications, resulting in the same assessors reviewing both types of applications. Product approval is based solely on self-assessment by internal assessors applying type III review model.CDSCO - The capacity of the biological division within CDSCO is 2% representing common internal assessors for the review of all new biological and biosimilar applications. The agency mandates a master's degree in pharmacy as the minimum qualification for internal assessors and takes expert advice from external assessors for the review of both non-clinical and clinical parts of the dossier. The agency has several bodies with different responsibility including: Subject Expert Committee (SEC) for clinical review which comprises external physicians and regulators; the Review Committee for Genetic Manipulation (RCGM) for non-clinical data; and the Department of Biotechnology (DBT) for developing and defining regulatory guidelines. The agency follows the type II (abridged) review if the biosimilar has been approved by at least one recognised reference agency and waives the non-clinical studies if the product is already approved by more than one agency, including China and South Korea subject to positive review outcomes. The reference agencies defined for type II review model are EMA, MHRA, USFDA, TGA, and BRDD. In addition, the agency also carries out type III (full review) review but does not mention verification review model.SAHPRA - The overall size of the agency is around 200 personnel. There is a total of five reviewers for biological applications with minimum qualification as shown in TITCK – The agency follows the type III review model and takes advice from external assessors also for CMC, non-clinical and clinical review.COFEPRIS - The biological division represents 1% of the overall size of the agency with a bachelor's degree as the minimum qualification for internal assessors. The agency relies more on external experts under both the committees, the SEPB  and the NMC (New Molecule Committee) headed by COFEPRIS. The COFEPRIS is the only agency among these regulatory agencies of the emerging economies following the type I data assessment model relying on other reference agencies' evaluation including the EMA, USFDA, and TGA. In addition, the agency also conducts type III full dossier evaluation for biosimilars.It is evident from in vitro analytical testing and quality characterisation, non-clinical comparative pharmacology testing to toxicology, PK/PD studies, and clinical trials   . Althougfficacy) , there iin vitro non-clinical PK/PD studies and literature based clinical performance evaluation, additional in vivo safety data plus confirmatory clinical safety and efficacy trial. However, expectations for local or global clinical studies vary among the agencies. The ANVISA, SAHPRA, TITCK accept clinical studies performed in any country globally, while CDSCO and COFEPRIS mandate a local study. The Russian MoH accepts global studies as long as a trial includes Russian patients. In addition, extrapolation of indications is allowed subject to fulfilment of conditions defined by each agency.All the six regulatory agencies of these emerging economies expect the sponsor to demonstrate biosimilarity of the proposed biosimilar product with the reference product. This includes proving satisfactory physicochemical and biological characterisation with Furthermore, these regulatory agencies of the emerging economies (except Russian MoH and TITCK) do not regulate interchangeability by law and allow a prescriber to decide based on a patient's need. However, in Russia, biosimilar products can be interchangeable with the reference product by law whereas in Turkey, the reimbursement institution authorises interchangeability.Selection Criteria. In response to questions on the RBP selection, the agencies mostly indicated mandatory requirements for locally authorised reference product  for comparability studies , non-clinical studies (in vivo), and development studies such as “quality target product profile” (QTPP) which is a summary of the quality characteristics of the respective biosimilar. These quality characteristics are essential to ensure that the finished product meets the required standard of quality.Use of RBP Authorised in Emerging Countries. None of the agencies accept authorised reference products from other emerging countries, except CDSCO which may then only consider this in emergency situations such as the COVID-19 pandemic.Criteria of RBP Batches. Unlike the Russian MoH and COFEPRIS, the regulatory agencies of Brazil, India, South Africa, and Turkey also mandate the use of multiple batches of RBP with varied expiry dates. However, ANVISA, has provisions for the changeover of RBP during development and comparability studies.Bridging Study Requirement. All the six regulatory agencies of BRICS-TM has not specified the bridging study requirements.Data Sharing Arrangements. ANVISA established data sharing arrangements with advanced regulatory agencies such as EMA, USFDA, PMDA, and MHRA. CDSCO also holds a data sharing agreement with EMA and USFDA. In contrast, COFEPRIS does not have a data sharing arrangement with other advanced regulatory agencies and expects a full dossier submission for products approved by a foreign agency  indicated the need for comparative accelerated and stress stability studies, along with real time, real condition stability studies conducted in their respective climatic zone to support the shelf-life. CDSCO (India) does not require comparative studies and TITCK (Turkey) considers it only as supportive data for biosimilar development.Comparative stability data is essential for “totality-of-evidence” to determine biosimilarity  and is ain vitro comparative functional assays such as biological assays, binding assays, and enzyme kinetics; in vivo pharmacokinetics, pharmacodynamics and immunogenicity studies; and in vivo comparative repeat dose toxicity studies are requisite for non-clinical studies. In addition, local tolerance studies and other toxicological studies are expected by CDSCO, TITCK, and COFEPRIS. Safety pharmacology studies are required by SAHPRA. In TITCK, the evaluation and acceptability are on case-by-case basis.The six regulatory agencies of the emerging economies state that in vivo studies, the Russian MoH advises the use of transgenic animal/ transplant models in a GLP setting while CDSCO suggests toxicity studies in rodent and non-rodent animals for proving statistical difference and advises to submit scientific justification for the choice of animal model. If a relevant non-rodent model is not available in India, then non-rodent studies can be waived by the Review Committee on Genetic manipulation (RCGM). The TITCK reported that the evaluation and acceptability of non-clinical studies is solely on a case-by-case basis in alignment with EU and ICH guidelines  approval process; and dossier review process including validation of application, queuing, scientific assessment, sample analysis, GMP certification, and product approval .in vitro non-clinical data; in vivo clinical data and justification of differences and clinical safety and efficacy trial protocol design and approval; and overall dossier content. The advanced agencies such as the USFDA  offer pre-submission advice for the biosimilar developers. The advice from ANVISA can be obtained through face-to-face meetings, electronic mails, or written correspondence whereas CDSCO and SAHPRA prefer face-to-face meetings. The expert advice received through such meetings is not legally binding on both parties, however, agencies do expect compliance to their comments during development of the biosimilar. The Russian MoH, TITCK, and COFEPRIS are yet to establish any formal meeting procedures.The absence of scientific advisory meetings in TITCK has also been highlighted in an earlier study  where thThe CTA is evaluated and approved by specific committees designated by the agencies such as: Coordenação de Pesquisa Clínica (ANVISA); Subject Expert Committee (CDSCO); and Clinical Trials Committee (SAHPRA). The Russian MoH and TITCK assign internal assessors to review the application. No clarity on this topic was received from COFEPRIS. An integral part of the CTA is the Ethics Committee (EC) approval letter, which is to be obtained from the Institutional Review Board of hospitals or institutions where the clinical trial is intended to be performed. The Russian MoH and COFEPRIS require an EC letter as part of the initial application, whereas the rest of the agencies are flexible and will accept such letter during the review process or post approval of the CTA.All the regulatory agencies have varied timelines for CTA approval as shown in The six regulatory agencies of the emerging economies accept electronic CTD dossiers as the format for marketing authorisation applications (MAA) for biosimilar products. The Certificate of Pharmaceutical Product (CPP) is a mandatory document as part of the initial dossier by the Russian MoH and CDSCO, for acceptance of the application by the agency. The ANVISA, COFEPRIS, and TITCK provide relaxation for the CPP submission before granting a marketing authorisation. In addition, TITCK also accepts any marketing authorisation certificate and published approvals from the relevant agencies' official websites.Post submission by the sponsor, the product dossier passes through different stages such as screening against a checklist, acceptance for further review, queuing for review and scientific assessment resulting in approval or non-approval of the application by the agency.As part of the screening or validation process, all the agencies verify applications against a standard checklist and request additional data (except CDSCO) if some documents are missing. In case of CDSCO, submission will not be uploaded on the SUGAM online portal if the dossier is inadequate. Further, all the information pertaining to “milestone” dates are recorded during the review process into an electronic tracking/recording system maintained by the agencies, i.e., DATAVISA (ANVISA), GRLS (Russian MoH), SUGAM (CDSCO). In case of SAHPRA and COFEPRIS, there is no specific system in place, whereas no information available from TITCK on the same.The queue time for dossiers awaiting review ranges from 4 weeks to 1 year as displayed in Scientific assessment of biosimilar applications depends on the outcomes of the dossier review, sample analysis, and GMP certification.For dossier review, CDSCO and SAHPRA use external assessors, however, there is no contractual agreement defining the timelines for review of the technical data. ANVISA and TITCK issue an emergency letter to sponsors in the case of a sudden unforeseen crisis as and when they review different sections of the dossier, while the rest of the agencies collate quality, safety, and efficacy deficiencies in one batch and send it to the applicant. The obligatory time for developers to respond to queries varies between 3 and 6 months and referred to as “clock stop.” Failure to meet the stipulated time, leads to rejection of the application with forfeiting of the fees with ANVISA, the Russia MoH, CDSCO, and COFEPRIS. The TITCK sends official letters for rejection to the sponsor, but the company can object to the same; however, there is no predetermined deadline in this aspect, while SAHPRA allows for extensions. Further, in case of a negative opinion from the scientific committee, CDSCO has provisions for sponsors to approach the technical committee and apex committee for their intervention and decision. The Russian MoH and ANVISA have no such additional provision and also there was no clarity received in this regard from COFEPRIS. The defined target timeline for scientific review by each of the regulatory agencies also varies as detailed in Sample Analysis. Most of the regulatory agencies of these emerging economies expressed a requirement of sample analysis at specified approved quality control laboratories as part of the dossier approval process. The ANVISA and SAHPRA rely only on technical documentation for biosimilar products and do not require sample analysis. The Russian MoH, TITCK, and CDSCO expect sponsors to submit samples along with analytical specifications and methods, reference/working standard and analytical columns. The CDSCO additionally require an analytical validation package. The maximum time to analyse samples is 110 calendar days as defined by FGBU (Russia) while no such deadlines are specified by other agencies.GMP Inspection. These six regulatory agencies of emerging economies also mandate on-site GMP inspections for biological substances and biosimilar product manufacturing sites. Generally, each agency (except CDSCO) performs inspection during the dossier evaluation process, whereas CDSCO inspects site/s after completion of the dossier assessment. For TITCK, separate site GMP application is required, and the agency conducts inspection before scientific assessment of dossier, unless there are priority products. Also, CDSCO and SAHPRA accept GMP certification from reference agencies i.e., EMA (EU), BRDD (Canada), MHRA (UK), USFDA (USA) instead of on-site inspections. In addition, CDSCO accepts TGA  certification whereas COFEPRIS accepts EMA (EU), TGA , and USFDA (USA) certification. The TITCK does not accept foreign agencies' GMP inspections.Across these emerging economies, the final decision maker on the marketing authorisation is the head of the agency.Except for ANVISA, the regulatory agencies of these emerging economies are yet to establish procedures for the issuance of a public assessment report or clarifying the basis for approval for the product. In such scenarios, measuring real approval timelines for biosimilars becomes arduous. The biosimilar approval metrics for the duration of 2017–2019 for ANVISA is presented in Biosimilar products are complex molecules produced using highly complex manufacturing processes. Due to the complexity of the biosimilar products, regulatory requirements for analytical comparability, non-clinical, and clinical studies vary with the geographies , particuRegarding the type of dossier assessment and allocation of resources for the dossier evaluation by the regulatory agencies, external evaluators are involved for review of applications by SAHPRA, TITCK, CDSCO, and COFEPRIS, while Russian MoH has common assessors for biologic and non-biologic applications. All the six emerging agencies follow “Type III - Full review of the marketing authorisation application” data assessment model. In addition, CDSCO follows “Type II - Abridged review” and COFEPRIS follows “Type I – Verification of marketing authorisation application”, with a clear indication of less prevalence of Type I and Type II review models among these countries. It has been commonly cited that building capacity and expertise in a national regulatory authority is a long-term process and quick resolutions lie in relying on information from other regulatory authorities or a shared or abridged review models . The stuin vitro non-clinical studies, additional in vivo safety data along with confirmatory clinical safety and efficacy data. While ANVISA, SAHPRA, TITCK accept global clinical studies, Russia MoH, CDSCO, and COFEPRIS mandates conduct of clinical efficacy trials in the local population. The non-acceptance of global clinical data and repetition of clinical studies mandatorily in local population by these regulatory agencies adds to unnecessary development costs  for comparability studies. While few agencies like CDSCO, TITCK, and COFEPRIS provide flexibilities for sourcing RBP from ICH/ reference countries, ANVISA, Russian MoH, and SAHPRA have stringent regulation on using only the locally licenced RBP. In Russia, a comparator product cannot be sourced from another regulatory jurisdiction since it is only allowed to use a reference comparator drug that has Russian marketing authorisation . Furtherin vitro comparative functional assays are required by all the six emerging agencies along with in vivo pharmacokinetics, pharmacodynamics and immunogenicity studies. There is a mandatory requirement for in vivo repeat dose toxicity studies from all the countries; with TITCK evaluation in accordance with EU and ICH guidelines, on a case-by-case basis. Further, CDSCO expects non-clinical studies in rodent and non-rodent animal and Russian MoH in transgenic animal/transplant models to be conducted. However, there is no clarity on the type of study or species or other requirements from other agencies. The study clearly shows the lack of consistency in the regulations on non-clinical aspects from these countries. Also, such mandatory requirements for non-clinical studies demonstrates a lack of scientific approach toward the assessment of data indicating lack of full implementation of a “step-wise approach” for proving biosimilarity , clinical safety and efficacy studies along with comparative immunogenicity data as part of a biosimilar application. However, the Russian MoH does not provide clarity on expectations on immunogenicity studies nor for extrapolation of immunogenicity data to other indications in most of the agencies. Further, as discussed earlier, the acceptance of clinical efficacy data from foreign patient data is not supported by all the agencies. Such a mandate on confirmatory clinical efficacy studies shows the agencies lack in science-based approach for review of dossier. It is apparent that the understanding by these agencies on the importance of comprehensive analytical comparability studies and the evaluation of comparability data for any structural and functional differences is inadequate. Further, non-recognition and non-acceptance of global data leading to duplication of studies, impacts on development costs and delays in approval of the biosimilar product . EmphasiScientific advice helps to ensure that developers perform the appropriate tests and studies, so that no major objections regarding the design of the tests are likely to be raised during the evaluation of the marketing authorisation application. This also helps avoid patients taking part in studies that will not produce useful evidence. Such pre-submission advice for biosimilar developers to get agencies' opinions on the biosimilar development process is only offered by a few of these emerging agencies like ANVISA, CDSCO, and SAHPRA. These advisory meetings are through face-to-face meetings, electronic mail or written responses. However, there are no set procedures for any formal meeting in the rest of these emerging agencies. The absence of a communication channel between the biosimilar developer and the national health authority greatly impacts the overall development process. With an increasing number of biosimilar developers across the globe, the scientific advice requests to developed agencies like EMA is expanding. EMA has also launched a pilot project in 2017  for “taiThe dossier content requirements for biosimilar Marketing Authorisation Application (MAA) are similar within these regulatory agencies of emerging economies. All these agencies accept electronic CTD and mandate CPP as part of the dossier, however the flexibility over time of the submission of such administrative documents  varies. Relaxation in terms of provision of other published approvals and authorisation documents in lieu of CPP by few agencies, exists. The dossier screening and validation process against a standard checklist is uniform across all the six emerging agencies, however the acceptance of the MAA with insufficient data differs with the agencies. The queueing time for dossier review varies from 4 weeks to 1 year, with almost all the agencies discounting the priority products (except the Russian MoH and COFEPRIS). The biosimilar application is considered for scientific assessment based on the outcome of the dossier review, sample analysis, and GMP certification. Although most of the agencies evaluate the dossier internally, a few opt for external evaluators for dossier evaluation. This is partly due to the full review of dossier (Type III data assessment model) by the agencies. Joint or shared review of the dossiers will ease the resource constraint or the dossier review process among these agencies . FurtherDespite the technical dossier, the requirement for samples by all these emerging agencies (except ANVISA and SAHPRA) along with reference standard/working standards for testing at qualified laboratories for the biosimilar approval process extends the overall biosimilar approval timelines. Additionally, each of these agencies mandate on-site GMP inspections for biological substances and biosimilar product manufacturing sites. Though inspection of the manufacturing site is essential for ensuring compliance to global manufacturing standards and assuring the quality of the product, individual or separate inspections by each of the emerging agencies leads to duplication. Instead, acceptance of reference agency GMP certification , as permitted by CDSCO, SAHPRA, and COFEPRIS will improve the process efficiency of the agencies . Also, cBased on the evaluation of the study results, following key outcomes from this study can be considered for an effective biosimilar development and approval process among these emerging agencies -This study emphasises the need to foster effective collaboration between regulators and developers in six emerging agencies in order to streamline the development strategies and approval pathways for biosimilar products.in vivo non-clinical studies. This may also help to shorten the overall review and approval timeline.A formal approach to regular, appropriate, and tailored scientific advice from regulatory agencies to developers will help to align expectations on both sides and support step-by-step development, thereby reducing the need for certain studies i.e., Significant challenges in sourcing RBP for comparative studies necessitates regulatory flexibility in norms for sourcing the comparator. Allowing RBP from other emerging countries will also facilitate the use of common biosimilar development programs.While appropriate resource allocation and upskilling of regulators needs to be considered, adoption of an alternative regulatory framework such as abridged review models might help in optimising the use of resources within the biosimilar departments of these six emerging agencies.The biosimilar therapy in emerging economies is still in the infancy stage with little or no presence but expected to show strong growth  remains Although there were no remarkable changes in biosimilar guidelines in the six emerging economies between 2018 and 2020, there have been progress toward relaxing few guidelines with regards to conduct of clinical trials and GMP inspections. For instance, ANVISA, Brazil, has introduced certain relaxation of clinical trial procedure and allowed sponsors to modify or amend protocol without ANVISA's authorisation. In addition, if clinical study is related to COVID-19 then clinical trial consent can be obtained immediately upon formal submission of the protocol. As per the resolution of the Collegiate Board of ANVISA, RDC no 346/2020 of March 13th, ANVISA has adopted an alternative route for GMP certification of Active Pharmaceutical Ingredient (API), drugs and health products, based on remote inspection or reliance from other health authorities. If the manufacturer is accredited by PIC/s GMP certification, then ANVISA can process a faster GMP certification . RDC no The CDSCO has upgraded regulatory standards for clinical trials via the New Drug Clinical Trials Rule 2019 . In IndiFurthermore, The TITCK, Turkey, announced some flexibility due to COVID-19 such as postponing marketing authorisation certificate's annotation process, online stakeholder meetings regarding marketing authorisation activities, accepting CPP or similar certificate without apostille, readability test waiving until end of 2021, extension of GMP validity period to end of 2021 .The study presented covers evaluation of responses obtained from 6 out of 7 (85.7%) regulatory agencies. While ANVISA, Russia MoH, CDSCO, SAHPRA, TITCK, and COFEPRIS responses were obtained, the multiple efforts to reach NMPA (China) either directly or via regulatory experts were unsuccessful. Though the non-participation of China in this study could be considered as a limitation, however the survey (even without China) encompasses a large, diverse and important segment of the world population and pharmaceutical market, so it should provide strategic information to pharma companies, as well as national regulatory authorities and international bodies. The response pertaining to biosimilar approval metrics i.e., applications received, applications screened and accepted for further review, biosimilars approved, biosimilars refused and average approval times was received only from ANVISA. The response from other agencies would have helped understand the process efficiency and provided benchmark in terms of basis of approval to biopharmaceutical companies.To conclude, many medical treatments and medicines now lay in Biotechnology, where understanding of the patient's physiology and cell make up is the key to treatment. Biological drugs bring that value in the treatment of many disabling and life-threatening chronic diseases, including inflammatory arthritis, certain types of cancer, diabetes, inflammatory bowel disease, Crohn's disease, psoriasis, and COVID-19. Biosimilars can help the public gain access to health through affordability, and that is where the need for the regulatory guidelines of biosimilars can contribute through harmonisation and simplification. The research undertaken presents an effort in that direction. This study has, for the first time, evaluated the regulatory requirement for approval and development of biosimilars in these six emerging economies and has identified a lack of alignment in certain areas that would benefit from standardisation. There remains scope for improving transparency in the national regulatory frameworks and aligning regulatory standards among these emerging economies. This would impact the overall review and approval process as well as enable a common development programme across these countries. Further, a future study could focus on developing proposals for an improved regulatory model for approval and development of biosimilars in these emerging economies. Integration of regulatory standards across emerging economies would also enable streamlined biosimilar development programmes and expedited licencing processes, thereby facilitating improvements in patient care and access to these life-saving medicines.The original contributions presented in the study are included in the article/HR: designing of research methodology, questionnaire development, the study sample selection, online interviews, data collection, interpretation, and writing of the first draft of the manuscript. AS: review of the questionnaire, support in identifying appropriate participants for the study, and review of the first draft of the manuscript. GS: support in providing data from ANVISA, Brazil and review of the first draft of the manuscript and comments. CD: support in providing data for Russia and review of the first draft of the manuscript and comments. SP-T and CL-M: support in providing data for Mexico and review of the first draft of the manuscript and comments. SS: development of the concept, supervision of the study, review of the questionnaire, research coordination, interpretation of the results, and review of the first and final draft of the manuscript. All authors contributed to the article and approved the submitted version.HR was employed by the company Metina PharmConsulting Pvt. Ltd. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.