High-throughput sequencing (HTS) and its use in recovering and assembling novel virus sequences from environmental, human clinical, veterinary and plant samples has unearthed a vast new catalogue of viruses. Their classification, known by their sequences alone, sets a major challenge to traditional virus taxonomy, especially at the family and species levels, which have been historically based largely on descriptive taxon definitions. These typically entail some knowledge of their phenotypic properties, including replication strategies, virion structure and clinical and epidemiological features, such as host range, geographical distribution and disease outcomes. Little to no information on these attributes is available, however, for viruses identified in metagenomic datasets. If such viruses are to be included in virus taxonomy, their assignments will have to be guided largely or entirely by metrics of genetic relatedness. The immediate problem here is that the International Committee on Taxonomy of Viruses (ICTV), an organisation that authorises the taxonomic classification of viruses, provides little or no guidance on how similar or how divergent viruses must be in order to be considered members of new species or new families. We have recently developed a method for scoring genomic (dis)similarity between viruses (Genome Relationships Applied to Virus Taxonomy – GRAViTy) among the eukaryotic and prokaryotic viruses currently classified by the ICTV. At the family and genus levels, we found large-scale consistency between genetic relationships and their taxonomic assignments for eukaryotic viruses of all genome configurations and genome sizes. Family assignments of prokaryotic viruses have, however, been made at a quite different genetic level, and groupings currently classified as sub-families are a much better match to the eukaryotic virus family level. These findings support the ongoing reorganisation of bacteriophage taxonomy by the ICTV Phage Study Group. A rapid and objective means to explore metagenomic viral diversity and make evidence-based assignments for such viruses at each taxonomic layer is essential. Analysis of sequences by GRAViTy provides evidence that family (and genus) assignments of currently classified viruses are largely underpinned by genomic relatedness, and these features could serve as a guide towards an evidence-based classification of metagenomic viruses in the future. Viral diversity is, however, far greater than that of other organisms, with major differences in their genetic material (RNA or DNA) and configurations (double or single stranded), as well as the orientation of their encoded genes. Viral genomes may, furthermore, be distributed across several segments, sometimes packaged together in a virion, or often in separate virus particles, all of which are needed to infect a cell for replication to occur. Viral genomes come in various sizes, reflecting their diverse replication mechanisms and cellular interactions, as well as the varying structural complexity of their virions. The smallest virus genomes range from less than 2,000 bases, containing two genes, to 2.5 million base pairs, containing over 2,500 genes [Virus taxonomy is an essential element in the description of viruses and acts as a unified catalogue of their vast diversity and genetic interrelationships. Viruses are assigned in a hierarchy of taxonomic levels by the International Committee on Taxonomy of Viruses  are vital resources underpinning the whole virus classification field.The current taxonomy of viruses has itself gradually evolved since the formation of what is now the ICTV in 1966. The vastly and rapidly expanding knowledge of virus diversity since that time and the advent of molecular methods for virus discovery and genetic characterisation have greatly increased the number of taxa assigned, with the current totals being 9 orders, 131 families, 46 subfamilies, 803 genera and 4,853 species, following the ratification vote after the 49 meeting . IncorpoHerpesvirales is based on their morphology, and is independent of genomic relationships, which do not place their members into a coherent genetic group. Contrastingly, membership of the order Tymovirales is based upon possession of genetically related RNA polymerase genes, irrespective of the highly variable virion structures of their members.Nevertheless, you might discover on closer inspection that the unified virus taxonomy is a rather ramshackle construction, with taxonomic assignment rules often being based on quite different and inconsistent criteria between virus groups. For example, the assignment of viruses to the order Yellow fever virus, Carnation mottle virus and Salmon pancreas disease virus are typical examples of literally hundreds of such disease-focussed designations. As nucleotide sequence information on classified species gradually accrued in the 1980s-1990s, it became clear that species were typically genetically distinct from each other, but there was no fixed sequence divergence threshold that defined members of the same and different species. As an example of what typifies large numbers of other species assignments throughout the ICTV taxonomy, members of different flavivirus species show pairwise distances in the RNA-dependent RNA polymerase (RdRp) gene of only 1.5% , while members of San Perlita virus and Ilheus virus differ by 44%. As an example of even greater discrepancy, members of the species Louping ill virus, which infect grouse and sheep in upland Britain, lie entirely within the clade of the separate species Tick-borne encephalitis virus. The two were defined as separate species based on their marked differences in geographical range, host associations, and pathogenicity, and clearly not their genetic relationships in this case.The species definition is particularly variable between virus groups. This taxonomic level was originally used as the primary division of viruses showing distinct disease patterns, epidemiology and host range. It has been entirely possible to maintain what is essentially a phenotypically based system of species assignments for many decades – these assignments do, after all, divide viruses into groups that differ in important medical, veterinary and agricultural properties that serve a major purpose for classification. Flexibility in what defines species is implicit in the polythetic species definition , 28 deveAs HTS methods become increasingly used, and the catalogue of viruses known only by their sequence data continues to expand almost exponentially, genomics-based species assignments are clearly required to accommodate these into the ICTV taxonomy , 22, 31.Virologists should not be daunted by the scale of the task ahead in making species assignment to the vast number of viruses identified by deep-sequencing methods. Recent descriptions of potentially tens or hundreds of new species, genus and family assignments for viruses isolated from arthropod and lower vertebrate host taxa , 23, 24 While the species level of taxonomy is one that best describes what we would consider individual viruses in a broad sense, the family level is the fundamental taxonomic level by which viruses of different kinds are organised. Eukaryotic viruses are currently assigned into a total of 111 families, with distinct genome configurations, virion morphologies, replication strategies and host interactions. Many of the groupings first described in early ICTV reports, generally based on electron microscopy appearance and genomic material, have stood the test of time – herpesviruses, poxviruses and rhabdoviruses are instantly and recognisably distinct morphologically, and their separateness has been verified by detailed analysis of their genome structures and replication mechanisms.A family is a group of genera (whether or not these are organized into subfamilies) sharing certain common charactersThe ICTV taxonomy, however, provides little information that might guide decisions on what is needed to justify the creation of new virus families or orders. The ICTV Code provides this as a definition of a virus family:A genus is a group of species sharing certain common charactersbuilding on:which, frankly, is equally uninformative. Although new families have continued to be assigned in the last three decades, there is no real systematic information or guidance on what virological, structural or genomic attributes would support the family-level assignments that have been made, and what may be used in the future. For example, family members must presumably share homologous genes, but how many and how similar? Must they have related genome organisations? Should they look similar by electron microscopy? Most crucially in this “metagenomic” era, are there any family-defining attributes recoverable from a virus sequence alone – or is it essential to visualise particle morphology and to determine some of the physical attributes of the virus, such as host range, cell tropism and pathogenicity, as part of an extended family descriptive definition? The ICTV offers no guidance. Perhaps as a direct result of this uncertainty, picorna-like, flavi-like, circo-like and a vast range of other (family)–like virus descriptions are to be found in literally hundreds of papers indexed by PubMed. In many cases, it is as if their authors, too, lacked the information or confidence to assign new families for newly discovered viruses. Guidance on this issue and, ideally, a better-defined set of criteria for family assignments seems long overdue – this is indeed required if the ICTV is going to embrace the growing dataset of virus genome sequences from metagenomic datasets.It was with this background that we set out to examine whether there were any consistent genomic attributes of eukaryotic virus families that correlated with their current family-level ICTV taxonomy relationships . InvestiGenome Relationships Applied to Virus Taxonomy” (GRAViTy), was applied to the complete list of 3,854 classified eukaryotic viruses with complete genome sequence data. Information such as gene contents, orientations and protein profiles were extracted from each genome sequence and systematically compared through the calculation of a composite generalised Jaccard (CGJ) distance, a metric that captures and weights the contributions of different genome features between pairs of viruses. This use of multiple features extracted from viral sequences contrasts with traditional phylogenetic methods, in which virus relationships are often calculated from small, highly conserved portions of their genomes, such as the catalytic core of the RdRp gene.This method, “Rubivirus) from the rest of the family Togaviridae concurs with a variety of other evidence that it should be re-classified into a separate family. Some virus families turned out to be far more diverse and often polyphyletic, including Rhabdoviridae and Reoviridae, and these represent future candidates for reorganisation of their taxonomy.Pairwise distances between members of particular virus groups, such as dsDNA viruses , for example, can be visualised as a heat map Fig.  or as deWith this newly developed ability to predict family memberships from genomic sequences alone, we next analysed large datasets of virus sequences derived from metagenomic datasets to determine the extent to which they might be classified as new potential families, or as members of existing ones. Using the same metrics that differentiated virus families in each Baltimore group, we were able to provisionally assign viruses in this combined metagenomic dataset as four new families of ssDNA viruses, four dsRNA virus families, potentially as many as 101 new family-level groupings of (+)ssRNA viruses and 16 new (-)ssRNA families. Clearly, these results are preliminary, and conclusions about such a large number of new families require corroboration with other methods, including RdRp phylogenies in the case of future RNA viruses, as is the extension of classification to genus and species levels. What we can say, however, is that future assignments will certainly approximate to relationships that GRAViTy has shown. Furthermore, the new frameworks for each Baltimore group depict sets of virus relationships that are consistent with and extend virus taxonomy in a manner that effectively follows the rules used in the current classification.Myoviridae, Podoviridae and Siphoviridae [Viruses infecting bacteria and archaea are extraordinarily abundant and diverse. Almost all of the currently classified bacterial viruses are, however, assigned to just three families of tailed phages, namely the oviridae , a divisSiphoviridae, Myoviridae and Podoviridae  and those of eukaryotic viruses. Members of the same phage family were generally far more divergent from each other and, despite their morphological similarities, frequently possessed genes that showed no detectable homology with each other ssRNA) . This anher Fig. . If we wSpounavirinae and Vi1virus taxa, as new virus families based on genome relationships rather than phonotypic properties, such as virion appearance and presumed host [Examining sequence relationships a little further, it was apparent that the subfamily-level groupings among tailed phages were generally more consistent with family assignments of eukaryotic viruses where these have been made Fig. . These fmed host , 15, 16.The ICTV is committed to the incorporation of viruses known only by their genome sequence into the current taxonomy , 25. ThiThey also reveal gaping holes in how the various taxonomic levels are defined – the ICTV code provides no indication of how different viruses have to be, either in terms of their phenotypic properties or in their genetic relatedness, to be considered members of the same or different species, genus or family. Given the importance of the family level in virus taxonomy, it is remarkable that there is no information about what features might define a family and enable new families to be assigned in a consistent way. The application of bioinformatic programs like GRAViTy, vContact and others , 8, 17 w


In children suffering from severe lower airway illnesses, respiratory virus detection has given good prognostic information, but such reports in the elderly are scarce. Therefore, our aim was to study whether the detection of nasopharyngeal viral pathogens and conventional inflammatory markers in the frail elderly correlate to the presence, signs and symptoms or prognosis of radiographically-verified pneumonia.N = 382) were prospectively studied as a cohort. Standard clinical questionnaire was filled by the study physician. Laboratory analyses included PCR diagnostics of nasopharyngeal swab samples for 14 respiratory viruses, C-reactive protein (CRP) and white blood cell count (WBC). Chest radiographs were systematically analysed by a study radiologist. The length of hospital stay, hospital revisit and death at ward were used as clinical endpoints.Consecutive episodes of hospital care of patients 65 years and older with respiratory symptoms (9/L (P = .006) and a CRP value over 80 mg/l (P < .05). A virus was detected in 30% of pneumonia episodes and in 40% of non-pneumonia episodes, but this difference was not significant (P = 0.09). The presence of a respiratory virus was associated with fewer revisits to the hospital (P < .05), whereas a CRP value over 100 mg/l was associated with death during hospital stay (P < .05). Respiratory virus detections did not correlate to WBC or CRP values, signs and symptoms or prognosis of radiographically-verified pneumonia episodes.Median age of the patients was 83 years (range 76–90). Pneumonia was diagnosed in 112/382 (29%) of the studied episodes. One or more respiratory viruses were detected in 141/382 (37%) episodes and in 34/112 (30%) episodes also diagnosed with pneumonia. Pneumonia was associated with a WBC over 15 × 10Among the elderly with respiratory symptoms, respiratory virus detection was not associated with an increased risk of pneumonia or with a more severe clinical course of the illness. CRP and WBC remain important indicators of pneumonia, and according to our findings, pneumonia should be treated as a bacterial disease regardless of the virus findings. Our data does not support routine virus diagnostics for the elderly patients with pneumonia outside the epidemic seasons.The online version of this article (10.1186/s12877-019-1125-z) contains supplementary material, which is available to authorized users. The number of over 80-year-old patients with weaning immune system is rising rapidly in Western societies, but the clinical significance of respiratory virus infections among this group remains unclear. The burden of pneumonia among the elderly is high as it includes significant morbidity, mortality and costs around the world . In the As polymerase chain reaction (PCR) and rapid antigen detection tests are increasingly available for respiratory virus detection , currentAmong the elderly, the all-cause mortality rate associated with respiratory viral infection increases with age and is approximately 6–7% among the over 85-year-old subjects , 13. In All of the aforementioned findings support a clinically meaningful role of virus diagnosis among the frail elderly. Thus, the aim of this study was to investigate how viral pathogens detected in the nasopharynx and conventional inflammatory markers (WBC and CRP) correlate to signs, symptoms and prognosis of pneumonia among the age group of 65 years and over. We hypothesized that virus detection could give clinically relevant information in addition to conventional inflammatory markers and chest radiograph findings in treating frail elderly patients.This prospective follow-up study investigated the association between virus detection and predefined clinical outcomes in elderly hospitalized patients. STROBE criteria were respected.The study was carried out in geriatric wards of the Turku City Hospital between July 2007 and April 2009 as part of a previously introduced study [Patients were considered to have respiratory symptoms if they had coryza, cough, sore throat, hoarseness or nasal stuffiness. In pneumonia episodes, the need for oxygen was considered as a sign of dyspnea. At study entry patients or their trustees were interviewed using a standardized questionnaire  by study physicians within 24 h of admission. The swabs were then stored in dry tubes in a refrigerator for a maximum of 24 h before transportation to the laboratory where they were stored at − 80 °C. The swab samples were analysed at the Department of Virology, University of Turku, Turku, Finland by a multiplex reverse-transcriptase (RT-)PCR test  for adenovirus, coronavirus NL63 and OC43, human bocavirus, human metapneumovirus (MPV), influenza A and B, and PIV1–3, and by using an ‘in-house’ RT-PCR test for RSV, RV - including rhinovirus type C - and enteroviruses (EVs) . Based oBlood samples for CRP and WBC analysis were routinely collected from all the patients as part of hospital treatment. The serum samples were stored at − 80 °C and analyzed by the hospital laboratory. The highest values measured during the hospital stay were used in statistical analysis.t-test, χ2 test and Fischer exact test (when counts < 5) were used when appropriate. Logistic regression with full model was used to analyse the association between clinical outcomes and virus etiology, pneumonia, chronic illnesses , age, gender and laboratory findings . Statistical significance was established at the level of P < .05. For statistics SAS Enterprise Guide 4.3  was used.In basic statistics, two sample P > .08, data not shown).A total of 921 episodes of hospital care were screened . The weight of the patients seemed to be lower in the group diagnosed with pneumonia (P = 0.05), whereas heart dysrhythmia seemed to be more common among patients not diagnosed with pneumonia (P = .03). In connection with the study episodes without pneumonia, in 16% the patient had a history of a stroke or transient ischemic attack (TIA) and in 16% the patient smoked, compared to 9.2 and 25% of the study episodes diagnosed with pneumonia, respectively. These differences were, however, not statistically significant . Otherwise cardiovascular, respiratory and other diseases were equally common in the study episodes diagnosed with pneumonia and the study episodes not diagnosed with pneumonia .Mean age of the patients in the study was 82.9 (sd 7.2) years Table . A diagnP = 0.09). Virus detection was also found not to be associated with dyspnea (P > .5), nor were there virus-specific differences found between study episodes diagnosed with pneumonia and with dyspnea, and study episodes diagnosed with pneumonia but without dyspnea (P > .1).A respiratory virus was detected in 141/382 (37%) nasopharyngeal swabs of the study episodes . As a categorical variable, a CRP value over 100 mg/l, or even over 80 mg/l, was associated with a pneumonia finding in the chest radiograph .When comparing study episodes diagnosed with pneumonia and with one or more respiratory viruses, and study episodes diagnosed with pneumonia but without respiratory viruses, no differences between WBC or CRP values were found . Also in connection with study episodes diagnosed with pneumonia, there was no association between the above mentioned clinical outcomes and laboratory findings (WBC over 15 × 109/L/l or CRP over 100 mg/l) (P > .2). In study episodes diagnosed with pneumonia and with dyspnea, death at ward was seen in 15% of the study episodes, whereas in connection to study episodes diagnosed with pneumonia but without dyspnea, the same was true in 5.3% of the study episodes, although this difference was not statistically significant (P = 0.07). However, study episodes diagnosed with pneumonia and with dyspnea lasted longer than study episodes diagnosed with pneumonia but without dyspnea (P = .02).In study episodes diagnosed with pneumonia, the presence of a respiratory virus was neither associated with clinical outcomes  nor with WBC values over 15 × 10P < .05, Table P = .04. Table No difference in the number of deaths at ward was seen between study episodes diagnosed with pneumonia and study episodes not diagnosed with pneumonia. A negative association was found between hospital revisit and virus detection; a revisit was less probable when a virus was present than when a virus was not present; 43 (31%) revisits occurred among the virus-positive study episodes and 93 (39%) revisits among the virus-negative study episodes


Zika virus infections and suspected microcephaly cases have been reported in Angola since late 2016, but no data are available about the origins, epidemiology, and diversity of the virus. We aimed to investigate the emergence and circulation of Zika virus in Angola.Diagnostic samples collected by the Angolan Ministry of Health as part of routine arboviral surveillance were tested by real-time reverse transcription PCR by the Instituto Nacional de Investigação em Saúde . To identify further samples positive for Zika virus and appropriate for genomic sequencing, we also tested samples from a 2017 study of people with HIV in Luanda. Portable sequencing was used to generate Angolan Zika virus genome sequences from three people positive for Zika virus infection by real-time reverse transcription PCR, including one neonate with microcephaly. Genetic and mobility data were analysed to investigate the date of introduction and geographical origin of Zika virus in Angola. Brain CT and MRI, and serological assays were done on a child with microcephaly to confirm microcephaly and assess previous Zika virus infection.Serum samples from 54 people with suspected acute Zika virus infection, 76 infants with suspected microcephaly, 24 mothers of infants with suspected microcephaly, 336 patients with suspected dengue virus or chikungunya virus infection, and 349 samples from the HIV study were tested by real-time reverse transcription PCR. Four cases identified between December, 2016, and June, 2017, tested positive for Zika virus. Analyses of viral genomic and human mobility data suggest that Zika virus was probably introduced to Angola from Brazil between July, 2015, and June, 2016. This introduction probably initiated local circulation of Zika virus in Angola that continued until at least June, 2017. The infant with microcephaly in whom CT and MRI were done had brain abnormalities consistent with congenital Zika syndrome and serological evidence for Zika virus infection.Our analyses show that autochthonous transmission of the Asian lineage of Zika virus has taken place in Africa. Zika virus surveillance and surveillance of associated cases of microcephaly throughout the continent is crucial.Royal Society, Wellcome Trust, Global Challenges Research Fund (UK Research and Innovation), Africa Oxford, John Fell Fund, Oxford Martin School, European Research Council, Departamento de Ciência e Tecnologia/Ministério da Saúde/National Council for Scientific and Technological Development, and Ministério da Educação/Coordenação de Aperfeicoamento de Pessoal de Nível Superior. Aedes spp mosquitoes. It is classified into two distinct lineages, the African and the Asian genotypes. Serological studies suggest that Zika virus might be widespread across Africa,Zika virus is an RNA virus of the Flavivirus genus that is primarily transmitted by Hundreds of millions of people in sub-Saharan Africa live in areas with competent mosquito vectors and appropriate climatic conditions that render them susceptible to infection with Zika virus.Evidence before this studyNS5 gene from Angola. Additionally, although epidemiological data for Zika virus and suspected microcephaly cases in Cape Verde have been reported in detail, data from Angola are extremely scarce.We searched PubMed with the keywords “Zika” and “Africa” for papers published in any language up to Oct 31, 2018. We also checked available Situation Report publications from WHO up to the same date for evidence of Zika virus or congenital Zika disease in Africa. The African lineage of Zika virus has been detected in Africa since the mid 20th century, yet evidence for spread of the Asian lineage of the virus is scarce. Two countries in Africa (Cape Verde and Angola) have reported cases of Zika virus infection that are thought to have been caused by a newly introduced Asian lineage virus. Sequence data are crucial to confirm and understand the spread of Asian lineage Zika virus in Africa, but these data are currently limited to a single 193 bp fragment of the Zika virus Added value of this studyWe sequenced Zika virus genomes from three acutely infected cases, including one baby with confirmed microcephaly. These sequences represent the first three Asian lineage genomes available from Africa. Sequence analysis suggested that Zika virus was probably introduced from Brazil to Angola between July, 2015, and June, 2016, and then circulated for at least 17 months. Our study provides the first genomic confirmation of autochthonous transmission of the Asian lineage of Zika virus within Africa. We also report the second confirmed case of Zika-virus-associated microcephaly in Angola. Our analyses from Angola improve understanding of the extent and clinical effect of Asian lineage Zika virus in Africa.Implications of all the available evidenceThe circulation of the Asian lineage of Zika virus in Angola is concerning because of the potential for continued viral spread across Africa. Evidence suggests that Zika virus has circulated and caused microcephaly in Angola and Cape Verde. Detection of additional transmission based on clinical data for suspected cases of microcephaly or clusters of mild illness could be challenging in countries where systems for reporting birth defects are limited and the burden of infectious disease is high. Further spread of the Asian lineage of Zika virus is not likely to be detected unless routine molecular surveillance systems are implemented. Implementation of such a surveillance system is especially important in countries that are linked by high human mobility to areas that have experienced recent outbreaks of Zika virus.Obtaining accurate surveillance data for Zika virus is challenging because most cases are asymptomatic, symptoms (when present) are mild and non-specific, and infections are frequently misclassified.In this study, we aimed to investigate the outbreak of Zika virus in Angola. Because surveillance data from Angola are scarce, we considered multiple data streams  that together provide a more complete picture of the outbreak than would be possible had we used any one data type alone. Our goal was to provide the first cohesive insight into the introduction, circulation, and possible public health effects of Zika virus in Angola.We did a multi-component investigation into Zika virus and suspected microcephaly cases in Angola. We assessed surveillance data from the Ministry of Health in Angola to identify acute cases of Zika virus infection confirmed by real-time reverse transcription PCR (RT-PCR) between December, 2016, and October, 2018. To identify additional real-time RT-PCR samples positive for Zika virus that are appropriate for sequencing, we also screened samples from 349 people with HIV living in Luanda, Angola, in 2017. We sequenced Zika virus from three samples and did phylogenetic analysis to elucidate the origins and duration of the Angolan outbreak. We analysed human air travel and data on the global incidence of Zika virus infection to support our findings about the geographical source of the introduced strain. Finally, we assessed suspected cases of microcephaly notified to the Ministry of Health in Angola, including an extensive clinical and serological investigation into one case and sequencing of the Zika virus genome from a second (previously identified) case.The Instituto Nacional de Investigação em Saúde  tests cases of suspected acute Zika virus, chikungunya virus, and dengue virus infection. Routine surveillance and diagnostic testing of suspected cases of acute arboviral infections were initiated in late December, 2016. Guidance issued by the Ministry of Health suggests that patients should be suspected of acute Zika virus infection if they present with fever , conjunctivitis, rash, or arthralgia. Clinical guidance in Angola suggests that patients are suspected of acute infection with dengue virus if they have acute fever (2–7 days' duration), plus at least two of headache, retro-orbital pain, myalgia, arthralgia, exanthema, leucopenia, or haemorrhagic signs. Chikungunya virus infection is suspected if patients have acute fever and unexplained severe arthralgia or arthritis.Serum samples were collected in public health facilities throughout Angola and then transported to the Instituto Nacional de Investigação em Saúde  for centralised real-time RT-PCR . Viral RTo find more samples positive for Zika virus that would be appropriate for Zika virus genomic sequencing, we sought additional samples for real-time RT-PCR screening that were collected between December, 2016, and October, 2017 , and stored at −80°C since sample collection. An appropriate sample set was identified at the Instituto Nacional de Luta Contra SIDA . These samples were collected between April and November, 2017 as part of a separate study investigating antiviral drug resistance among patients with HIV. Participants in this study provided written informed consent for their samples to be used in subsequent studies. No attempt was made to select a demographically representative sample set. We tested these samples for Zika virus with Bio-Manguinhos ZDC real-time RT-PCR kits  on an Applied Biosystems 7500 Fast machine.Sequencing of the Zika virus genome of all positive real-time RT-PCR samples was attempted at Instituto Nacional de Investigação em Saúde with an Oxford Nanopore MinION  device per previously published protocols.For genomic sequencing of samples identified in Angola that were positive for Zika virus, we used residual samples without informed consent but with the approval of the Ethical Review Board at the Angolan Ministry of Health. Specifically, residual anonymised clinical diagnostic samples, with no or minimal risk to patients, were provided for research and surveillance purposes within the terms of the Parecer Comité de Ética 06 Julho 2017 .While investigations in Angola were underway, Zika virus RNA was detected in the urine of a neonate with microcephaly born in Portugal in October, 2017, to a resident of Angola. Details of this case and the appropriate consent and ethical approval have been described elsewhere.We did phylogenetic analyses to explore the probable geographical and temporal origins of the Zika virus outbreak in Angola, and the minimal duration of local transmission. We used Muscle to align the three new Zika virus genomic sequences with 390 other viral genomes publicly available from GenBank.An outbreak of Zika virus infection was reported in Cape Verde in 2015–16, from which no genetic sequence data are available.24From Jan 1, 2018, health providers in Angola were required to notify the Angolan Direcção Nacional de Saúde Pública of infants with suspected microcephaly , and official notification forms were issued. Per guidelines from the Direcção Nacional de Saúde Pública, microcephaly should be suspected in male neonates with head circumference of less than 32 cm and in female neonates with head circumference of less than 31·5 cm, irrespective of gestational age. These measures are equivalent to two SDs below the median head circumference expected for a full-term birth.Serum samples from suspected cases of microcephaly and mothers of suspected cases were tested for Zika virus, dengue virus, and chikungunya virus by real-time RT-PCR, irrespective of the time between the birth and sample collection, according to the same protocols used for other samples.Serum samples from eight infants with suspected microcephaly were also tested with the ViDAS ToRC panel  for serological responses to toxoplasmosis, rubella, and cytomegalovirus. The eight infants were randomly chosen from early cases where samples were taken more than 6 days after delivery. Only eight samples were tested due to limited reagent availability.Serological diagnosis of exposure to Zika virus could not be done in Angola because ELISAs frequently do not accurately discriminate between previous exposure to dengue virus and previous exposure to Zika virus and facilities for conducting more specific tests were unavailable.We were able to do additional follow-up and confirmation of one suspected case of microcephaly because the girl was identified in Brazil, where a specific serological assay, the plaque reduction neutralisation test, and brain CT and MRI could be done. She was born in Moxico province, Angola, in August, 2017. In November, 2018, she travelled with her mother to the Microcephaly Reference Centre IPESQ , where microcephaly was diagnosed. Before November, 2018, neither the child nor her mother had travelled outside Angola. Plasma samples were taken from both on Nov 30, 2018, and tested for Zika virus and dengue virus infection with the EuroImmun IgG ELISA . A plaque reduction neutralisation test was done according to standard protocols  to quantThe funders of the study had no role in study design, data collection, data analysis, data interpretation, or writing of the report. The corresponding authors had full access to all the data in the study and had final responsibility for the decision to submit for publication.Between December, 2016, and October, 2018, serum samples were collected from 54 patients with suspected Zika virus, who were notified to the Angola Ministry of Health after clinical examination .Three cases of Zika virus infection were detected by routine surveillance using real-time RT-PCR . The earOne additional case of acute Zika virus infection  was detected by screening an unrelated archive of serum samples from people with HIV using real-time RT-PCR . This peNo additional positive cases of Zika virus were detected among samples collected during 2018 from the 336 patients with suspected dengue virus or chikungunya virus infections. Of the 51 people with suspected Zika virus infection who tested negative for Zika virus, three tested positive for dengue virus infection, and seven tested positive for chikungunya virus infection. No samples were co-infected with more than one virus.We did genomic sequencing and phylogenetic analyses of three Zika virus infections acquired in Angola: from one patient notified for suspected Zika virus acute infection, the asymptomatic person detected in the sample from the trial of people with HIV, and a neonate with microcephaly who was previously identified in Portugal.Maximum likelihood phylogenies suggested that the three Zika virus strains formed a single, well supported monophyletic clade within the Asian lineage of Zika virus that is circulating in the Americas  according to her mother. Clinical examination suggested severe and disproportionate microcephaly. The 21-year-old mother was a long-term resident of Moxico province, but travelled to Luanda during the second and third months of pregnancy and developed a rash during this visit . Diagnostic surveillance data showed that Zika virus was present in Luanda and Bengo province when she had this rash . The girCT and MRI, which were done when the child was age 15 months, confirmed microcephaly via reduced cerebral volume, and showed abnormalities consistent with congenital Zika syndrome that had been noted in Brazil and elsewhere—eg, calcification areas, ventriculomegaly, brainstem hypoplasia, dysgenesis of the cerebellum, and pachygyria .29 At agIn this multi-component outbreak investigation, we characterised the first known outbreak of Asian lineage Zika virus in continental Africa. Although surveillance, genetic, and clinical data were individually scarce, together these independent lines of evidence provide a cohesive picture of the outbreak of Zika virus infection in Angola. We report data for five cases of Zika virus infection between December, 2016, and October, 2017, and confirmed by real-time RT-PCR. The three Zika virus genomes that we sequenced represent the first Asian lineage genomes reported from Africa, and the first genome isolated from a case with Zika-virus-associated microcephaly in Africa. Our phylogenetic analyses showed that Zika virus was probably introduced to Angola from Brazil and then circulated for 17–28 months. Finally, our serological investigations in one mother–child pair supported the view that the Zika virus outbreak caused microcephaly in Angola.Phylogenetic analysis showed that the three Angolan Zika virus genomes formed a single clade with a common ancestor in June, 2016. These findings suggest either a single successful introduction that initiated local Zika virus transmission in Angola that continued until at least June, 2017, or recurrent but later introduction to Angola of viruses belonging to a specific lineage present in Brazil. The latter explanation is much less likely, because it is improbable that three or more independent introductions of Zika virus to Angola would belong to only one of the many different lineages that circulated contemporaneously in Brazil.Zika virus probably circulated in Angola for 17–28 months—sustained transmission that implies that the outbreak was substantially larger than the small number of cases detected by surveillance. Low case detection has been reported previously .Zika virus was probably introduced to Angola from Brazil, but we cannot rule out the possibility that it could have spread to Angola from affected locations where genomic data are unavailable. However, data for human flight mobility and the global incidence of Zika virus support our phylogenetic conclusion that Brazil is the most likely origin of the Angolan outbreak. Transmission of mosquito-borne viruses between these two countries was previously shown by the spread of chikungunya virus from Angola to Brazil in 2014.The Asian lineage of Zika virus that circulated in Angola caused microcephaly in Brazil and elsewhere.To date, there have been only two confirmed reports of microcephaly in which the mother of the infant lived in Angola: one infant who was identified clinically in this study, and one who was identified by Sassetti and colleaguesToxoplasma gondii, rubella, cytomegalovirus, herpes simplex, and others) in infants with suspected microcephaly makes the cause of these cases difficult to establish. Widespread, routine screening of pregnant women for STORCH pathogen infections and follow-up of children born with brain abnormalities would improve understanding of the extent and causes of birth defects in Africa.We present several complementary lines of evidence that together form a cohesive insight into the origins and effects of Zika virus in Angola. Despite the overall concordance of these different data streams, several aspects of the study have additional limitations that should be considered. First, the proportion of diagnostic samples that were positive for Zika virus infection by real-time RT-PCR in Angola is probably an underestimate of the true numbers of infections. Suboptimum sample transport and lack of information about the date of onset of symptoms mean that samples could have degraded before testing or been collected after viraemia was no longer detectable.GitHub. Zika virus genome sequences are available on GenBank with accession numbers MK829152-MK829154.Sequence alignments, XMLs, and tree files are all publicly available on


Copious studies have indicated that virus invasion participates in the pathogenesis of vitiligo. IFIH1, encoding MDA5 which is an intracellular virus sensor, has been identified as a vitiligo susceptibility gene. However, the specific role of MDA5 in melanocyte death under virus invasion is not clear. In this study, we first showed that the expression of anti-CMV IgM and MDA5 was higher in vitiligo patients than healthy controls. Then, by using Poly(I:C) to imitate virus invasion, we clarified that virus invasion significantly activated MDA5 and further potentiated the keratinocyte-derived CXCL10 and CXCL16 which are the two vital chemokines for the cutaneous infiltration of CD8+ T cells in vitiligo. More importantly, IFN-β mediated by the MDA5-MAVS-NF-κB/IRF3 signaling pathway orchestrated the secretion of CXCL10 via the JAK1-STAT1 pathway and MDA5-meidiated IRF3 transcriptionally induced the production of CXCL16 in keratinocytes under virus invasion. In summary, our results demonstrate that MDA5 signaling orchestrates the aberrant skin immunity engaging in melanocyte death via mediating CXCL10 and CXCL16 secretion, which supports MDA5 as a potential therapeutic target for vitiligo under virus invasion.Vitiligo is a disfiguring disease featuring chemokines-mediated cutaneous infiltration of autoreactive CD8 Multiple studies showed that vitiligo is caused by specific environmental factors in the population with genetic predisposition3. Virus, as an important environmental factor, has been determined to play profound parts in the pathogenesis of many autoimmune diseases including type 1 diabetes6, rheumatic arthritis9, and autoimmune thyroid disease11. These autoimmune diseases all occur with increased frequency in vitiligo patients13. In vitiligo paraffin-embedding tissues, the detective rate of CMV DNA is markedly higher than that in healthy controls (38% vs 0)14. Besides, the case-control study based on the large sample size determines that hepatitis C virus (HCV)15 is associated with vitiligo. Additionally, A strong causative link between turkey herpesvirus and vitiligo has also been established in the Smyth line chicken16. Moreover, many cases embody the correlation between virus infection and vitiligo22, particularly the inflammatory raised borders in vitiligo with HCV infection19, the reactivation of herpes simplex virus in lesions of vitiligo after excimer laser treatment20 and vitiligo occurrence after the resolution of drug-induced hypersensitivity syndrome which is characterized by sequential reactivations of herpes viruses22. It is noted that interferon-induced helicase C domain 1 (IFIH1), encoding melanoma differentiation-associated 5 protein (MDA5) which could sense the invasion of virus, has been identified as a susceptible gene for vitiligo by several genome-wide association studies (GWAS)25. However, the specific role of MDA5 in the pathogenesis of vitiligo under virus invasion is not clear.Vitiligo is a disfiguring disease characterized by melanocyte death which is caused by autoreactive melanocyte-specific CD827, and Poly(I:C) is an analog of this kind of dsRNA28. After sensing invasion of viruses, MDA5 triggers potent signaling cascade to eradicate virus by initiating the type I interferon (IFNs)29. Meanwhile, the inappropriate activation of MDA5 increases the risk of autoimmune diseases33. Upon activated, MDA5 could exploit the longer dsRNA (>1 kb) as a scaffold to induce the polymerization of the adaptor mitochondrial antiviral signaling protein (MAVS), a protein anchored in mitochondrial outer membrane36. Polymerization of MAVS triggers a signaling cascade culminating in the activation of the interferon-regulating factor 3 (IRF3) and the nuclear factor-kappa B (NF-κB)38. These transcription factors induce the type I and III IFNs, as well as other cytokines or chemokines42.As a pathogen pattern recognition receptor (PRR), MDA5 could sense the virus replication intermediate dsRNA derived from no matter DNA or RNA virus44 and CXCL1645 in epidermis have been established to play key roles in the skin trafficking of autoreactive melanocyte-specific CD8+ T cells1, which has been recognized as the main cause of melanocyte death in vitiligo. Thus, we hypothesized that intracellular virus sensor MDA5 could exacerbate the melanocyte death in vitiligo by inducing the section of chemokines from keratinocytes under virus invasion.In vitiligo, the excessive CXCL10 (chemokine (C-X-C motif) ligand 10)+ T in the skin tissues. Then, we clarified that virus invasion significantly activated MDA5 and further potentiated the secretion of CXCL10 and CXCL16 in keratinocytes. Mechanistically, we revealed that IFN-β mediated by the MDA5-MAVS-NF-κB/IRF3 signaling pathway orchestrated the secretion of CXCL10 via the JAK1-STAT1 pathway and MDA5-meidiated IRF3 transcriptionally induced the production of CXCL16 in keratinocytes under virus invasion. Therefore, we supported the MDA5 signaling pathway as a promising therapeutic target in vitiligo under virus invasion.In the present study, we initially manifested that the expression of MDA5 and anti-CMV IgM was upregulated in some progressive vitiligo, which was accompanied by the increment of the mRNA levels of CXCL10, CXCL16 in the epidermis and the infiltration quantity of CD8The peripheral blood samples and skin samples of vitiligo were collected from patients given a diagnosis of vitiligo at a progressive stage according to clinical manifestation in the Department of Dermatology, Xijing Hospital of the Fourth Military Medical University. All subjects had not received any systematic or topical therapy within 3 months before serum and skin samples collection. The control blood samples were collected from age- and sex- matched voluntary staffs working in Xijing hospital and the control skin samples collected from the age- and sex- matched subjects during plastic surgery in Xijing hospital. The skin samples of atopic dermatitis and psoriasis were collected from the in-patient given a diagnosis of atopic dermatitis and psoriasis respectively in the Department of Dermatology, Xijing Hospital of the Fourth Military Medical University. All participants gave their written informed consents according to the Declaration of Helsinki and approval from Clinical Research Ethics Committee of Xijing Hospital, Fourth Military Medical University. All serum samples were stored at -80 °C without repeated freezing and thawing. All skin samples used for immunofluorescence assay were embedded in paraffin at 4 °C. The total protein for Western-blot assay was extracted from fresh skin samples immediately and was stored at −20 °C. The total RNA for qRT-PCR assay was extracted from fresh skin samples and reverse-transcribed to cDNA and stored at −20 °C.  kit  was used to detect the anti-CMV IgM in serum according to the manufacturer’s instructions. The details are in the TM  which are performed complexes between Poly(I:C) and the transfection reagent LyoVecTM which can ensure that it functions in cytosol. Combined with the characteristic that they have an average size of 1.5–8 kb, these complexes could imitate the virus infection and further be sensed by MDA5.Poly(I:C) is a synthetic analog of double-stranded RNA (dsRNA), a molecule pattern associated with viral infection. The Poly(I:C) used in this article is Poly(I:C)/LyoVecNormal Human keratinocytes (NHKs) were isolated from healthy subjects and cultured in keratinocyte-SFM . Cells were grown to 80% confluency and used in 3–5 passages. A human keratinocyte cell line was purchased from American Tissue Culture Collection (ATCC) and cultured in Modified Medium RPIM 1640 with 10% FBS and L-glutamine . Poly(I:C)  was added to culture at different concentrations  in dose-dependent assays and at 0.6 μg/ml in function and signaling pathway exploration assays. For experiments, supernatant was harvested for ELISA, and the cells were processed for quantitative real-time PCR (qRT-PCR), Western-blot and immunofluorescence assay at the indicated time points.5 cells/well and treated with Poly(I:C) within 1.5 μg/ml for 24 h. Cell apoptosis was detected by using the Annexin V-PE/7-AAD kit . Annexin V-PE and 7AAD fluorescence were measured using flow cytometry  and analyzed with Expo32 software .NHKs were plated into 6-well plates at the density of 3 × 10For skin samples embedded with paraffin of progressive vitiligo patients with positive or negative anti-CMV IgM and healthy controls, the samples were deparaffinized and boiled in antigen retrieval solution (PH = 9.0). Next, the sections were incubated with the normal goat serum  for 1 h after 3 times washing with PBS. Then, incubated with the primary antibody  overnight at 4 °C. After washing, the secondary antibody  was used for 1 h at room temperature in dark. The cell nuclei were stained with Hoechst 33258  for 10 min in dark. Immunofluorescence analysis was applied with laser scanning confocal microscopy .For cell immunofluorescence, the HaCaT cells or NHKs were cultured on culture plates and treated with 0.6 μg/ml Poly(I:C) for the indicated time. Then, they were fixed with 4% paraformaldehyde for 10 min. After washing, the cells were incubated with 0.1% Triton X-100 for 10 min at room temperature, and then were blocked with normal goat serum for 30 min. The following procedures were similar as the histological immunofluorescence assay with the use of primary antibodies  and the secondary antibodies .Roughly, the 50% inhibitory concentration (IC50) of Poly(I:C) on NHKs was determined by CCK8 kit  according to the manufacturer’s instructions. In general, seeded in 96-well culture plates, cells were treated with 0-4 μg/ml Poly(I:C) for 24 h. Next, incubated with 100 μl fresh medium with 10 μl CCK8 solution for 2 h at 37 °C and optical density (OD) was measured at 450 nm by Model 680 Microplate Reader .(Supplementary Table 2) overnight at 4 °C. After washing, the membranes were incubated with secondary antibodies  for 1 h at room temperature. The bands were detected by ChemiDocTM XRS + system . As for the determination of MDA5 expression in skin tissues by Western-blot, the skin samples were soaked in Dispase at 4 °C overnight, and then we removed the dermis and subcutaneous tissues to get the epidermis tissues. The remaining procedures were similar to the above description.After washed and lysed, the total protein was collected and the concentration was measured with BCA Protein Assay kit . Equal amounts of protein were separated by 10% SDS-PAGE  and transferred to Polyvinylidene difluoride membranes . Then the molecules were blocked with 5% non-fat dry milk for 2 h. After washed with TBST transitorily, the membranes were incubated with the corresponding primary antibodies 5 cells/well for 24 h before the transfection. The 6 μl corresponding siRNA  and 6 μl Lipofectamine 3000  were added to 250 μl 1640 medium without fetal bovine serum (FBS). After mixed evenly, it was placed at room temperature for 10 min. Then, the mixed liquid was added to each well with 2 ml FBS-containing fresh medium.Cells were seeded at 3×10-ΔΔCT method. The relative mRNA expression was normalized to β-actin. Primer sequences are listed in Supplementary Table Total RNA was extracted with TRIzol  and then reverse-transcribed to cDNA using a PrimeScript RT reagent kit . qRT-PCR was performed by using SYBR Premix Ex Taq II  with the iQ5 PCR Detection System . Threshold cycle (CT) values were used to calculate the fold change by the 2Human CXCL9, CXCL12, CCL26, CCL2, IL-15, IFN-β Quantikine ELISA kits  and CXCL10, CXCL16, CCL20 DuoSet ELISA kits  were used to analyze cell supernatant samples, according to the corresponding manufacturer’s instructions.36. Crude cell lysis was resuspended in 1×sample buffer  and loaded onto a vertical 1.5% agarose gel . After electrophoresis in the running buffer (1×TBE and 0.1% SDS) for 35 min with a constant voltage of 100 V at 4 °C, the proteins were transferred to Immobilon membrane  for immunoblotting and the following procedures are same as the Western-blot assay.Semi-denaturing detergent agarose gel electrophoresis (SDD-AGE) was conducted according to the published protocolChromatin immunoprecipitation analysis was performed by using the ChIP (Chromatin immunoprecipitation) Assay Kit , according to the manufacturer’s instructions. Briefly, HaCaT cells were cross-linked with DNA by using 1% formaldehyde at room temperature for 10 min. After washing, cells were lysed in SDS lysis buffer. DNA was sheared to small fragments of less than 500 bp in length by means of sonication. The recovered supernatant fraction was incubated with antibodies overnight on a rotor at 48 °C. After washing, the precipitated IRF3 protein-DNA complexes were recovered by using IRF3 antibodies and protein G–Sepharose beads at 48 °C for 16 h. DNA was then purified with phenol/chloroform. A fraction was used as the PCR template  to detect the presence of promoter sequences between −1253 and −1338 of CXCL16. After the PCR amplification, each PCR reaction was analyzed by 2% agarose gel electrophoresis with D2000 DNA ladder .*P < 0.05, **P < 0.01, ***P < 0.001, #P < 0.05, ##P < 0.01, ###P < 0.001, ns: not significant.Each statistical analysis was performed by GraphPad Prism 7 for Windows (USA) with two-tailed Student’s unpaired t-tests or one-way analysis of variance (ANOVA). These data are conformed to the normal distribution and variance similarity between the groups that are statistically compared. These data represent as mean ± SD for at least three independent experiments. 14. To further confirm the link between virus infection and vitiligo, in the serum of progressive vitiligo we first used ELISA to detect anti-CMV IgM, symbolizing acute CMV infection46. We found that the positive rate of anti-CMV IgM was 8.92% in 56 progressive vitiligo patients, while that in 26 healthy controls was 3.84%, and the anti-CMV IgM levels were significantly higher in vitiligo patients than that in healthy controls  in NHKs was 0.6 μg/ml ml Fig. S. Next, tml Fig. S. In addiml Fig. S. Resultsml Fig. S. Thus, t53 in response to Poly(I:C) by qRT-PCR assay. The preliminary results underlined Poly(I:C) induced drastic mRNA increments of CXCL10, CXCL16, CXCL9 and CCL20  stimulation  treatment, Western-blot results manifested that the expression of phospho-NF-κB P65 and phospho-IRF3 apparently increased in a time-dependent manner -stimulated supernatant could apparently promote the expression of MDA5 and activate JAK1-STAT1 signaling pathway featuring the upregulation of phospho-JAK1 and phospho-STAT1  and found the -2000 to +1 region of the CXCL16 promoter has 2 predicted binding sites for IRF3  stimulation. We proved Poly(I:C) potentiated the transcription of IFN-β in a dose-dependent way as shown by qRT-PCR assay and promoted the secretion of IFN-β in a time and concentration-dependent manner Fig. . InterfeAT1 Fig. . Either AT1 Fig.  in the sAT1 Fig. , impededAT1 Fig. . What’s AT1 Fig. . ConsistAT1 Fig. . To explF3 Fig. S. These dF3 Fig. S.Fig. 6MDIn the present study, we initially manifested that the expression of MDA5 and anti-CMV IgM was upregulated in some progressive vitiligo. Then in vitro assays verified that virus infection, simulated by Poly(I:C), could promote the secretion of CXCL10 and CXCL16. Mechanistically, we demonstrated that IFN-β mediated by the MDA5-MAVS-NF-κB/IRF3 signaling pathway orchestrated the secretion of CXCL10 via the JAK1-STAT1 pathway and MDA5-meidiated IRF3 transcriptionally induced the production of CXCL16 in keratinocytes under virus invasion. In short, we proved that targeting MDA5 signaling pathway has the potent therapeutic potential in retarding the aggravation of vitiligo under virus invasion.6, and the potential mechanisms embody adjuvant initiative effect of autoimmune reaction, molecule mimicry, bystander activation and epitope spreading56. Vitiligo, as an autoimmunity-involved disease, is thought to correlate with virus infection22. Recently, IFIH1, encoding MDA5 which is a virus-associated pattern recognition receptor (PRR), has been identified as a susceptible gene for vitiligo3. Herein, we found the positive rate and levels of anti-CMV IgM in the serum of progressive vitiligo patients were significantly higher than that in healthy controls. In addition, our data showed the expression of MDA5 and IFN-β was enhanced in keratinocytes of vitiligo patients with positive anti-CMV IgM. As the sporadic evidences indicated, although MDA5 could also be activated by some self-derived dsRNA58 in vivo, the human evolves many crucial mechanisms to prevent the activation of IFIH1 via inhibiting the production of endogenous recognizable RNA60. More importantly, given that the higher expression of MDA5 and IFN-β is along with the positive anti-CMV IgM in some progressive vitiligo patients, we attribute the higher expression of MDA5 in the vitiligo patients with positive anti-CMV IgM to virus infection. All these above strengthen that virus infection is involved in some progressive vitiligo pathogenesis and indicate that MDA5 plays a key role in vitiligo under virus invasion.Increasing evidences have indicated the correlation between virus infection and autoimmune diseases such as Coxsackie virus and type 1 diabetes5. Meanwhile, the inappropriate gain-of-function of MDA5 could efficiently eradicate virus, but at the cost of increasing incidence to suffer autoimmune disease such as type-1 diabetes (T1D)61 and systemic lupus erythematosus (SLE)62. This argues against that the persistent viral infection per se induces autoimmunity and indicates that MDA5-mediated aberrant skin immunity functions prominently63. Herein, we highlighted the impacts of MDA5 on the dysfunctional local immune microenvironment, particularly on the secretion promotion of CXCL10 and CXCL16 under virus invasion in keratinocytes. Previous studies have in succession clarified that CXCL1043 and CXCL1645 play key roles in the trafficking of melanocyte-specific autoreactive CD8+ T cells from periphery blood to skin, which contributed significantly to melanocytes death in vitiligo. Specifically, the in vivo assay conducted in a mouse vitiligo model verified that, mice received Cxcr3-/- T cells develop minimal depigmentation, as do mice lacking Cxcl10 or treated with CXCL10-neutralizing antibody. In addition, CXCL10 neutralization treatment in vitiligo mice with the established and widespread depigmentation induces repigmentation43. Besides, functional study unveiled that the IFN-γ-JAK-STAT signaling axis is required for CXCL10 secretion in epidermis of vitiligo44 and blockade of this pathway using tofacitinib or ruxolitinib which are all JAK inhibitor rapidly induces the repigmentation of vitiligo65. With respect to the important roles of CXCL16 in vitiligo, we previously demonstrated that compared with nonlesional and healthy control skin, more CXCR6+ CD8+ T cells are located at the basal epidermis and dermis of perilesions from vitiligo patients, which is consistent with the higher content of CXCL16 in epidermis. Besides, in vitro assay verified that neutralizing CXCL16 in the supernatant of H2O2-treated primary keratinocytes significantly decreases the migration of CD8+ T cells sorted from vitiligo patients45. All these above further underscore the significance of MDA5-mediated chemokines in the pathogenesis of vitiligo. In addition, Previous studies have proved that IFN-γ signature49 and oxidative stress45 play key parts in the secretion of chemokines in keratinocytes. Our data further complement the specific mechanisms of chemokines secretion from keratinocytes during the process of melanocytes death and extend the underlying mechanism by which virus infection engages in autoimmune disease.MDA5, as a cytosolic virus sensor, contributes significantly to the elimination of virus in end-organs not only through the induction of antiviral interferons and other pro-inflammatory cytokines, but also by facilitating cell death66. Besides, MDA5 belongs to the RIG-I-like receptor family (RLR family) which could initiate the production of pro-inflammatory factors including IL-6, IL-8, IL-23, and TNF-α68. Hence, whether other functions of MDA5 participate in the pathogenesis of vitiligo in response to virus invasion is worthy to be explored.Notably, previous studies also emphasized the roles of IFN-β in reinforcing and bridging the innate and adaptive immunology such as promoting dendritic cells (DCs) maturation, skewing of TH1 polarization and reactivation of CTLs (cytotoxic T lymphocyte)36. What’s more, our data validated that the Poly(I:C)-stimulated keratinocytes supernatant had no impacts on the CXCL16 secretion and IRF3 could directly bind to the promotor of CXCL16 in response to virus. Combined with our previous finding that NF-κB P65 could directly mediate the transcription of CXCL16 in keratinocytes through directly binding to the promotor of CXCL1645, we draw a conclusion that CXCL16 is mainly elaborated by IRF3 and NF-κB in the downstream of MAVS under virus infection. As for CXCL10, we exhibited that it was orchestrated to a large extent by IFN-β which was induced by the MDA5 canonic pathway. Notably, another study underscored that Hantaan virus, the pathogen of hemorrhagic fever with renal syndrome (HFRS), promotes the secretion of CXCL10 by facilitating NF-κB and IRF7 to directly bind to the promotor of CXCL10 in the downstream of MDA569. This supports that NF-κB might mediate the secretion of CXCL10 not merely through IFN-β but also via prompting the transcription of CXCL10 directly. Moreover, our data exhibited that IFN-β could significantly prompt the expression of MDA5 via the JAK1-STAT1 signaling pathway, thus forming a positive feedback loop to aggravate this pathogenesis.Mechanistically, we demonstrated that MDA5 exerted its function by inducing the formation of MAVS aggregates after the colocalization of MDA5 and MAVS in keratinocytes. This result parallels the canonic MDA5 signaling pathway that, MDA5 binds to viral dsRNA through the C-terminal RD domain and sequentially the exposed N-terminal CARD domains interact with MAVS which rapidly forms the prion-like aggregates56. Hence, identifying the pathological evidence of specific virus or the solid causative link with virus infection in vitiligo warrants further investigation. In addition, based on our present data, in vivo assay should be further performed to explore the remarkable contribution of virus invasion in the exacerbation of vitiligo via the MDA5-chemokines pathway. Aiming at individualized treatment, we think our research could provide the evidence to the MDA5-targeting therapy in vitiligo under virus invasion.The present study deciphers that MDA5 could exacerbate vitiligo by inducing the secretion of chemokines in keratinocytes under virus invasion. Even so, the specific causal relationship are still difficult to solidly identify due to the complex interaction between virus infection and vitiligo such as the “hit and run” hypothesis of virusSupplementary MaterialsFig. S1Fig. S2Fig. S3Fig. S4Fig. S5Fig. S6


Predicting the electrical behavior of the heart, from the cellular scale to the tissue level, relies on the numerical approximation of coupled nonlinear dynamical systems. These systems describe the cardiac action potential, that is the polarization/depolarization cycle occurring at every heart beat that models the time evolution of the electrical potential across the cell membrane, as well as a set of ionic variables. Multiple solutions of these systems, corresponding to different model inputs, are required to evaluate outputs of clinical interest, such as activation maps and action potential duration. More importantly, these models feature coherent structures that propagate over time, such as wavefronts. These systems can hardly be reduced to lower dimensional problems by conventional reduced order models (ROMs) such as, e.g., the reduced basis method. This is primarily due to the low regularity of the solution manifold (with respect to the problem parameters), as well as to the nonlinear nature of the input-output maps that we intend to reconstruct numerically. To overcome this difficulty, in this paper we propose a new, nonlinear approach relying on deep learning (DL) algorithms—such as deep feedforward neural networks and convolutional autoencoders—to obtain accurate and efficient ROMs, whose dimensionality matches the number of system parameters. We show that the proposed DL-ROM framework can efficiently provide solutions to parametrized electrophysiology problems, thus enabling multi-scenario analysis in pathological cases. We investigate four challenging test cases in cardiac electrophysiology, thus demonstrating that DL-ROM outperforms classical projection-based ROMs. The electrical activation of the heart, which drives its contraction, is the result of two processes: at the microscopic scale, the generation of ionic currents through the cellular membrane producing a local action potential; and at the macroscopic scale, the propagation of the action potential from cell to cell in the form of a transmembrane potential –3. This (i) emulators, obtained, e.g., via Polynomial Chaos Expansions or Gaussian process regression . The per-example loss function ‖2,with ωh The architecture of DL-ROM is the one shown in t, μtest), for each t ∈ . Numerical results are extremely accurate, the mean relative error is indeed below 1% , even if the biophysical dynamics underlying nμ + 1. From a computational perspective, remarkable gains and simplifications can be obtained against a linear ROM, since (i) no hyper-reduction is required to enhance the evaluation of any μ- or u-dependent quantity, and (ii) even more interestingly, there is no need to evaluate the dynamics of the recovery variable w if one is only interested in the electrical potential.We highlight that the DL-ROM technique does not require to solve a (reduced) nonlinear dynamical system for the reduced degrees of freedom as in ; rather,We now assess the computational performances of the proposed DL-ROM strategy on four relevant test cases in cardiac electrophysiology. Our choice of the numerical tests is aimed at highlighting the performance of our DL-ROM method in challenging electrophysiology problems, namely pathological cases in portion of cardiac tissues or physiological scenarios on realistic left ventricle geometries.η = 10−4. Moreover, we perform cross-validation by splitting the data in training and validation and following a proportion 8:2 and we implement an early-stopping regularization technique to reduce overfitting  ms, without considering the time window  mm for the testing-parameter instance testμ = 12.9 · 0.31 mm2/ms. For the sake of comparison, we also report the POD-Galerkin ROM approximation, with cN = 1, of dimension n = 10 and n = 120. Clearly, in dimension n = 10 the DL-ROM approximation is far more accurate than the POD-Galerkin ROM approximation; to reach the same accuracy , by using the time resolution each technique requires and by varying the FOM dimension N on a 6-core platform Numerical tests have been performed on a MacBook Pro Intel Core i7 6-core with 16 GB RAM. The FOM solution with N = 16365 degrees of freedom requires about 40 minutes to be computed, against 57 seconds required by the DL-ROM approximation, thus implying a speed-up almost equal to 41 times.In ndicator ) achieveN = 16365, training the DL-ROM neural network on a GTX 1070 8GB GPU requires about 160 hours, whereas training the POD-Galerkin ROM  on a full node (20 Intel®Xeon® E5-2640 v4 2.4GHz cores) of a HPC cluster requires about 28 hours; in both cases, the time needed to assemble the snapshot matrix S is not included. We report also the GPU testing computational time of DL-ROM which is equal to 0.35 seconds thus obtaining a speed-up, with respect the POD-Galerkin ROM testing time with Nc = 4, equal to 172. The efficiency introduced at testing time justifies the higher training time of DL-ROM.Regarding the training (offline) times for this test case, featuring a FOM dimension μn = 3 parameters possibly reflecting intra- and inter- subjects variability. More precisely, the conductivity tensor now takes the formμ1 and μ2 consist of the electric conductivities in the longitudinal and the transversal direction to the fibers f0 = T, respectively, and μ3 regulates the action potential duration (APD) by definingTo take into account a case in which parameters also affect the ionic model, we finally focus on the solution of problem  in a squNt = 1000 time instances in the interval  and consider Ntrain = 5 × 5 × 5 = 125 training-parameters, i.e. μtrain = (12.9 ⋅ (0.06 + i0.035), 12.9 ⋅ (0.03 + j0.0175), 0.15 + s0.025) with i, j, s = 0, …, 4. For the testing phase, Ntest = 16 testing-parameter instances have been considered, each of them given by μtest = (12.9 ⋅ (0.0775 + i0.035), 12.9 ⋅ (0.0387 + j0.0175), 0.1625+ s0.025) with i, j, s = 0, …, 3. The maximum number of epochs is Nepochs = 10000, the batch size is Nb = 40 and, regarding the early-stopping criterion, we stop the training if the loss function does not decrease along 500 epochs.For the training phase, we uniformly sample n = 4, at μtest =  and μtest = , together with the relative error defined as in  cm, by considering the effect of the different parameters separately. More precisely, in μ3 vary, i.e. we take μ1 and μ2, i.e. we take In μn = 2 or 3 parameters:μn = 2, rainN = 5 × 5 = 25, tN = 1000, with μn = 3, rainN = 5 × 5 × 5 = 125, tN = 1000, with Finally, we report in tN time instances of the solution for a given testing-parameter instance. In this case, considering trainN = 125 training-parameter instances allows us to reduce the training computational time of a factor 3, even if more parameters are considered, a larger training set is provided. However, we highlight that stating general conclusions about the training complexity and costs, as a function of the number of parameters and the training set dimensions, is far from being trivial, and still represents an open issue in this framework.to analyze the effect of introducing an additional parameter on the training time for a prescribed level of accuracy, keeping the architecture of the network fixed. The training time refers to the overall training and validation time, while the testing one refers to the time needed by the DL-ROM to compute In this work we have proposed a new efficient reduced order model obtained using deep learning algorithms to boost the solution of parametrized problems in cardiac electrophysiology. Numerical results show that the resulting DL-ROM technique, formerly introduced in , allows The proposed DL-ROM technique provides ROMs that are orders of magnitude more efficient than the ones provided by common linear (projection-based) ROMs, built for instance through a POD-Galerkin reduced basis method, for a prescribed level of accuracy. Through the use of DL-ROM, it is possible to overcome the main computational bottlenecks shown by POD-Galerkin ROMs, when addressing parametrized problems in cardiac electrophysiology. The most critical points related to (i) the linear superimposition of modes which linear ROMs are based on; (ii) the need to account for the gating variables when solving the reduced dynamics, even if not required; and (iii) the necessity to use  hyper-reduction techniques to deal with terms that depend nonlinearly on either the transmembrane potential or the input parameters, are all addressed by the DL-ROM technique, which finally yields more efficient and accurate approximation than POD-Galerkin ROMs. Moreover, larger time resolutions can be employed when using a DL-ROM, compared to the ones required by the numerical solution of a dynamical systems through a FOM or a POD-Galerkin ROM. Indeed, the DL-ROM approximation can be queried at any desired time, without requiring to solve a dynamical system until that time, thus drastically decreasing the computational time required to compute the approximated solution at any given time.Outputs of clinical interest, such as activation maps and action potentials, can be more efficiently evaluated by the DL-ROM technique than by a FOM built through the finite element method, while maintaining a high level of accuracy. This work is a proof-of-concept of the DL-ROM technique ability to investigate intra- and inter- subjects variability, towards performing multi-scenario analyses in real time and, ultimately, supporting decisions in clinical practice. In this respect, the use of DL-ROM techniques can foster assimilation of clinical data with physics-driven computational models.So far, the training time required by the DL-ROM technique appears to be the major computational bottleneck, even if it is completely compensated by the great computational efficiency provided at testing time. Enhancing efficiency also during the training phase represents the focus of our ongoing research activity, and will be the object of a forthcoming publication.S1 AppendixWe provide the complete derivation of the spatial  discretization of system .(PDF)Click here for additional data file.S2 AppendixHere we report the configuration of the DL-ROM neural network used for our numerical tests.(PDF)Click here for additional data file.S1 FileIn the notes we report the feature maps of the DL-ROM neural network.(PDF)Click here for additional data file.S2 FileHere we report the resulting fibers distribution used for Test 3.(PDF)Click here for additional data file.S3 FileHere we show the ability of the DL-ROM approximation to replace the FOM solution when evaluating outputs of interest.(PDF)Click here for additional data file.S1 Code(TXT)Click here for additional data file.S1 Fig(EPS)Click here for additional data file.S2 Fig(EPS)Click here for additional data file.S3 Fig(EPS)Click here for additional data file.


A deeper understanding of mechanisms by which mammarenaviruses handle these signaling pathways is critical for understanding the virus/host interactions and potential diagnostic and therapeutic targets, through the inhibition of specific pathologic metabolic pathways.Mammarenaviruses are a diverse genus of emerging viruses that include several causative agents of severe viral hemorrhagic fevers with high mortality in humans. Although these viruses share many similarities, important differences with regard to pathogenicity, type of immune response, and molecular mechanisms during virus infection are different between and within New World and Old World viral infections. Viruses rely exclusively on the host cellular machinery to translate their genome, and therefore to replicate and propagate. miRNAs are the crucial factor in diverse biological processes such as antiviral defense, oncogenesis, and cell development. The viral infection can exert a profound impact on the cellular miRNA expression profile, and numerous RNA viruses have been reported to interact directly with cellular miRNAs and/or to use these miRNAs to augment their replication potential. Our present study indicates that mammarenavirus infection induces metabolic reprogramming of host cells, probably manipulating cellular microRNAs. A number of metabolic pathways, including valine, leucine, and isoleucine biosynthesis,  Bunyavirales: Arenaviridae: Mammarenavirus) are enveloped bi-segmented ambisense RNA viruses and each segment encodes for two oppositely oriented non-overlapping reading frames separated by an intergenic region, a small (S) segment that encodes the envelope glycoprotein precursor (GPC) and the nucleoprotein (NP) and a large (L) segment coding for the matrix protein (Z) and the RNA-dependent RNA polymerase (RdRp) [Mammarenaviruses (e (RdRp) ,2. Basede (RdRp) ,3,4.Some of these emerging, zoonotic viruses are pathogens of major clinical importance to humans, as (i) Lassa virus (OW) is endemic in West Africa and is estimated to cause up to 300,000 infections each year, (ii) Junín (Clade B NW) that periodically cause hemorrhagic fever (HF) outbreaks in Argentina, and (iii) LCMV (OW) which is distributed worldwide and is a neglected human pathogen of clinical significance, to immunocompromised individuals and pregnant women, due to teratogenicity ,6,7. AltMammarenavirus. Our research could help to further assess the roles of miRNAs in virus–host interactions, highlighting potential targets during infection, leading to a better understanding of the metabolic alterations required for the replication of each virus that may lead to novel therapeutic approaches through targeted inhibition of specific cellular metabolic pathways.MicroRNAs (miRNAs) are the smallest endogenous regulatory non-coding RNAs that play a central role in cell differentiation, proliferation, and survival by binding to complementary target mRNAs leading to translational inhibition or degradation. miRNAs are the crucial factor in diverse biological processes such as antiviral defense, oncogenesis, and cell development . There a® database of NCBI (http://www.ncbi.nlm.nih.gov/nuccore/), including all species of the genus Mammarenavirus officially recognized by the International Committee on Taxonomy of Viruses (ICTV) . Herein, for a more clear presentation of our results, we considered Clade D New World mammarenaviruses  as members of Clade A, as well as Xapuri virus that is associated with Clades B, and C was didactically classified here as belonging to Clade B NW mammarenaviruses.The genomic sequences used in the study were all retrieved from the GenBankhttp://www.mirbase.org). We used BLAST with the Geneious R11.1 software (https://www.geneious.com) to search for miRNAs that interact with the mammarenavirus genome with a perfect alignment of 11 nucleotides encompassing the 8 mer seed region, important specific gene silencing motifs, and regions from the viral RNA [Mammarenavirus genus.Currently, the human genome contains 2654 mature sequences of microRNAs identified in the miRBase database , Clade B (10), and Clade C (2) viruses and 19 OW species. Among the 2654 mature miRNAs identified in the miRBase database, we found that 566 miRNAs can bind to certain regions of the mammarenavirus genome in a total of 755 binding-sites  region for OW mammarenaviruses and 259 for NW . We found 27 binding-sites in the Z protein (Z), being 17 for OW and 10 for NW . We identified 19 binding-sites for the noncoding intergenic region of the L segment (L-IGR), with 13 for OW mammarenaviruses, 5 for Clade B, and 1 for Clade A. We have not identified any interaction to L-IGR of the Clade C. Regarding 5′UTR and 3′UTR untranslated region, we found four binding-sites to each region. In total, 233 binding-sites were found within the S segment, being 114 for glycoprotein precursor (GPC), 109 for nucleoprotein (NP), 9 for noncoding intergenic region of the S segment (S-IGR), and 1 bind in the 3′UTR untranslated region. Further information can be found in Fifty-two miRNAs were found binding to more than one virus in the same genomic segment. For example, the hsa-miR-2052 can bind to the S segment of Allpahuayo virus, Bear Canoyn virus, Pichinde virus, and Pirital virus, all belonging to Clade A NW . AnotherThirty-four miRNAs can bind to different regions of L and S segments. For example, hsa-miR-122b-3p can bind to two different positions of Aporé virus L segment, Flexal virus, Machupo virus, Paraná virus, and also to Alxa virus S segment. Another interesting miRNA was miRNA hsa-miR-9-5p that can bind in Pichindé virus L segment, Ryukyu virus, Mobala virus, and to Tacaribe virus S segment . The comTen miRNAs were predicted to bind different regions of the same virus, especially the miRNA has-miR-8485 binding in three different positions of Ryukyu virus L segment and five positions of Lunk virus L segment, both belonging to OW mammarenaviruses group .To further understand the biological implications of miRNAs binding to the mammarenavirus genome, miRabel was used to predict genes targeted by these miRNAs (data not shown). After genes prediction analysis, we performed pathway enrichment analysis of miRNAs target genes based on the KEGG database using miRabel and generated a report of the pathway mapping. Our analysis found a total of 75 pathways .d-Glutamine and d-glutamate metabolism, thiamine metabolism, and valine, leucine, and isoleucine biosynthesis were the main signaling pathways predicted to be regulated by miRNAs , 8 miRNAs that were found for Clade A, 10 for Clade B, 4 for clade C, and 27 for OW mammarenaviruses, including miRNAs that were found in more than one group of viruses. The thiamine metabolism was targeted 43 times, 3 miRNAs for Clade A, 13 for clade B, 6 for Clade C, and 21 for OW viruses. The results showed a total of 40 miRNAs interactions with the pathway with the valine, leucine, and isoleucine biosynthesis, 12 for Clade A, 12 for Clade B, 4 for Clade C, and 12 for OW viruses.The miRabel tool through the KEGG pathway analysis demonstrated that the targets of miRNAs were more associated with amino acid metabolism and metabolism of cofactors and vitamins. d-Glutamine and D-glutamate metabolism were the three main predicted pathways; for Clade B a total of 49 pathways were found with dorso-ventral axis formation first of all, and d-Glutamine and d-glutamate metabolism, Lipoic acid metabolism, valine, leucine, and isoleucine biosynthesis, and Thyroid cancer, equally represented, stand out in proportion. Twenty-two were found for Clade C with degradation of aromatic compounds and thiamine metabolism, biotin metabolism, adherens junction, long-term depression, and thyroid cancer, highlighted in miRNA proportion by pathway. For those miRNAs that were predicted in OW mammarenaviruses, we found a total of 59 pathways, especially dorso-ventral axis formation, degradation of aromatic compounds, and circadian rhythm.When considering pathways that have at least one experimentally validated interaction for miRNAs, we found a total of 69 pathways out of the 75 initials . Dorso-vAmong all predicted pathways, only seven showed no experimentally validated interactions for the set of miRNAs: caffeine metabolism; phenylalanine, tyrosine, and tryptophan biosynthesis; primary bile acid biosynthesis; proximal tubule bicarbonate reclamation; shigellosis; and synthesis and degradation of ketone bodies.We used KEGG pathway classification in order to predict biological relevance of each miRNA found for the different mammarenavirus included in this study. Target genes of miRNAs found were associated with six branches of KEGG pathways: cellular processes, environmental information processing, genetic information processing, human disease, metabolism and, organismal systems. Notably, target genes of miRNAs found are more frequently related to pathways that belong to the metabolism branch, followed by human diseases and organismal systems . StrikinWithin the KEGG metabolism classification, the predicted areas to be more affected by mammarenavirus infection are metabolism of cofactor and vitamins, metabolism of other amino acids, and amino acid metabolism. Pathways related to cancer studies are highlighted within human diseases group. For organismal systems, interactions occurs more frequently in the development and regeneration pathway. We also observed that the miRNAs found for the Junín, Machupo, Oliveros, Lassa, and Lymphocytic choriomeningitis viral genome were the ones with the greatest impact on different pathways . For exaIndividually evaluating each virus, we found different sets of mapped pathways. For example, for Pirital virus, miRNAs are mainly associated with the valine, leucine, and isoleucine biosynthesis. Olivero virus degradation of aromatic compounds followed by biotin metabolism were the main predicted pathways. MicroRNAs found for Lujo virus were mainly related to thiamine metabolism .MicroRNAs are noncoding RNAs which downregulate a large number of target mRNAs and modulate cell activity . MicroRNMiRNAs are characterized by variable expression in cells and tissues, which is influenced by the molecular cell environment. Some different miRNAs and miRNA families are predominantly expressed in certain tissues. However, the majority of miRNAs (>80%) is not specific for single tissues . A greatIn this article, we predicted several cellular miRNAs potentially regulated by mammarenavirus via direct binding to viral RNAs. Aside from virus specific binding sites, we also predicted cellular pathways that could be dysregulated during viral infection. A large intersection of predicted miRNA sets was found on miRNAs targeting cell metabolism for all mammarenavirus groups, including those considered as human pathogens. As known, viruses are entirely dependent on host metabolism to support its replication, and virally infected cells seem to require complex metabolic alterations in order to deal with the high anabolic demands essential during viral replication ,39,40.d-Glutamine and d-glutamate metabolism, thiamine metabolism, and valine, leucine, and isoleucine biosynthesis seems to be the most affected pathway during mammarenavirus infection. As previous studies have shown for other viral families, mammarenaviruses also could control host-cell metabolism via pos-transcriptional regulations to cope with the pace of the corresponding replication cycles [However, there are highly exclusive patterns of virus-induced remodeling of host cell metabolic machineries, and the mode of cell manipulation appears to be different between RNA and DNA viruses ,41. Metan cycles ,49.Glutamate metabolism plays a vital role in biosynthesis of nucleic acids and proteins . Glutami+ T cells. Glutamine concentrations are elevated in HIV-1-infected cells, implying that HIV-1 infection leads to considerable changes in the cellular glutamine metabolism [l-lysine amino acid play a limiting role in the synthesis of the virus proteins and in transcription initiation of the retrovirus life cycle, and the deficiency of this essential nutritional element can reduce viral load [l-lysine in HIV-infected patients, where it was found that plasma concentrations of l-lysine were negatively correlated with HIV-1 RNA levels and inversely with CD4 lymphocytes count. Therefore, an excess of l-lysine concentration leads to active HIV replication and reduces the concentration of plasma amino acid [Similar results have been reposted, where glutamine is important to support HIV-1 replication, when comparing HIV-1 infected with uninfected activated primary human CD4tabolism . In anottabolism . Lysine tabolism ,58,59. Tral load . Studiesino acid .In a recent review, Keshavarz and colleagues, based on available metabolomic studies, argued that influenza virus infection can affect with cellular metabolic pathways either directly or indirectly via stimulation of immune system mediators . CuriousVital metabolic pathways of host cells are one of the most widely used mechanisms targeted by viruses, resulting in extensive modifications. Some studies have revealed that many different human viruses, such as dengue , cytomegThe small molecule profile of serum from Lassa fever-infected patients indicated a physiological dysregulation affecting pathways mediating blood coagulation, and lipid, amino acid, nucleic acid metabolism changing the levels of numerous metabolites in the circulation . Serum lBiotin metabolism was among the most frequent pathways regulated by miRNAs that target mammarenavirus genome. Biotin may affect transcription of genes, biotinylation of proteins in cells, cellular growth, proliferation, and differentiation. The vitamin acts as a co-factor for five carboxylases that are critical for fatty acid, glucose, and amino acid metabolism ,76. BiotOne important pathway predicted to be associated with miRNAs targeting mammarenavirus genome was the circadian rhythm which is an internal biological clock that enables to sustain an approximately 24-h rhythm in the absence of environmental cues. The circadian clock system is a main regulatory factor for nearly all physiological activities and its disorder has severe consequences on human health ,85. InteSimilar results were found by Edgar and colleagues in infected wild-type mice with a strain of herpes virus and influenza A at different phases of the animals’ circadian clocks. These researchers demonstrated that the time of day of virus infection has an important impact on disease progression, in cellular models as well as in animals. In addition, they observed that clock disruption leads to increased virus replication and dissemination, indicating that severity of acute infections is influenced by circadian timekeeping . Since tThe identification of the molecular determinants underlying the distinct virulence of pathogenic and nonpathogenic arenaviruses is still a question of great importance for epidemiology and public health . In our Interaction between host and pathogen has a profound effect on the outcome of an infection. Knowledge about host–pathogen interactions is critical for understanding the pathogenesis of infection. However, it generally overshadows knowledge of metabolic cross-talk between host and pathogen ,94. At ad-Glutamine and d-glutamate metabolism, thiamine metabolism, and pools of several amino acids were impacted by the predicted miRNAs that would no longer regulate these pathways. The change caused by the effect of the mammarenavirus genome acting as a sponge to miRNAs probably allows activation of anabolic pathways necessary for the production of the viral nucleic acids, capsids, and eventually membrane envelopes. While some studies have demonstrated that viruses reprogram cell metabolism and rely on metabolic changes for optimal virus replication, significant work remains to determine as viral genome interact with host cell machinery to induce such alterations and characterize whether the same metabolic perturbations occur during infection. As well as other viruses, the mammarenavirus has likely evolved to modify the host metabolism for multiple purposes, facilitating viral replication and potentially also reflect antiviral defense mechanisms. The study of metabolism, including metabolomics approach, should not be restricted to energy supply and biosynthetic materials, but should also extend to a better understanding of its role during viral infection and pathogenesis. A deeper understanding of mechanisms by which mammarenaviruses handle these signaling pathways is critical for better understanding the virus/host interactions and potential targets for diagnostic and therapeutic, through inhibition of the specific pathologic metabolic pathways.Our present study indicates that mammarenavirus infection induces metabolic reprogramming of host cells, probably manipulating cellular microRNAs. A number of metabolic pathways, including valine, leucine, and isoleucine biosynthesis,