Among the plants mentioned, 11 plants showed the most promising medicinal plants against viral infections. Sambucus nigra and Clinacanthus nutans manifested antiviral activity against three different types of viral infections. Echinacea purpurea, Echinacea augustofolia, Echinacea pallida, Plantago major, Glycyrrhiza uralensis, Phyllanthus emblica, Camellia sinensis, and Cistus incanus exhibited antiviral activity against two different types of viral infections. Interestingly, Nicotiana benthamiana showed antiviral effects against mosquito-borne infections. The importance of phenolic acids, alkamides, alkylamides, glycyrrhizin, epicatechin gallate (ECG), epigallocatechin gallate (EGCG), epigallocatechin (EGC), protein-based plant-produced ZIKV Envelope (PzE), and anti-CHIKV monoclonal antibody was also reviewed. An exploratory approach to the published literature was conducted using a variety of books and online databases, including Scopus, Google Scholar, ScienceDirect, Web of Science, and PubMed Central, with the goal of obtaining, compiling, and reconstructing information on a variety of fundamental aspects, especially regarding medicinal plants. This evaluation gathered important information from all available library databases and Internet searches from 1992 to 2022.Exploration of the traditional medicinal plants is essential for drug discovery and development for various pharmacological targets. Various phytochemicals derived from medicinal plants were extensively studied for antiviral activity. This review aims to highlight the role of medicinal plants against viral infections that remains to be the leading cause of human death globally. Antiviral properties of phytoconstituents isolated from 45 plants were discussed for five different types of viral infections. The ability of the plants’ active compounds with antiviral effects was highlighted as well as their mechanism of action, pharmacological studies, and toxicological data on a variety of cell lines. The experimental values, such as IC Viral infections continue to cause health threats worldwide even with the development of various types of vaccines. The emergence of new diseases caused by viral infections, such as COVID-19, has eliminated people’s assurance that infectious diseases may be effortlessly eradicated. Viral infections are the most common cause of infectious disease transmitted within close personal contact in an indoor environment. These infections are airborne via droplet or direct and indirect contact transmissions . The demTraditional medicines are still widely used in most countries around the world, and their use is on the rise. Biologically active compounds isolated from medicinal plants are envisaged as new safe drugs and can be a key to finding low-cost treatments with fewer complications in curing many diseases. Traditional ethnomedicinal applications of a wide diversity of plants are now being used as a powerful tool for disease prevention . ApproxiThe antiviral properties of medicinal plants require extra exploration to be utilized as prevention in viral disease treatments . MedicinA virus is composed of DNA or RNA and surrounded by a protein coat. Viral infections occur when viruses invade and alter the genetic materials of healthy living cells, forcing them to manufacture more a-like viruses. People obtain viruses by swallowing, inhalation of them, through insects’ bites, or through sexual interaction. Commonly, viral infections are associated with respiratory organs , leading to common cold or influenza (flu) . VirusesRhinovirus (RV), respiratory syncytial virus (RSV), enterovirus, adenovirus, and human parainfluenza virus (HPIV) are some of the most important viruses that are generally considered to be the cause of common colds in humans ,25,26. ME. angustifolia, E. pallida, and E. purpurea) were reported to contain herbal remedy ingredients to treat common colds [E. purpurea showed a small reduction in virus titered with the highest dose of 50 μg/mL, including its root with a v/v 65% ethanol solution) to post-infection cell lines with HCoV-229E [50) 3.2 μg/mL, which was shown through MTT assay on Huh-7, Vero, and Vero E6 cells [E. purpurea to prevent infection caused by rhinovirus type 39 (RV-39) [E. purpurea placed in a 22% alcohol base developed cold, compared to 86% of the placebo recipients  [Echinacea pallida var. angustifolia displayed significant anti-rhinovirus (RV) activity. Extraction of E. pallida var. angustifolia root in ethanol 70%, hexane, and ethyl acetate showed a positive anti-RV activity with MIC100 (minimum concentration required to inactivate 100% virus) of 62, 69, and 85 μg/mL, respectively. This study concluded that alkamide constituents of E. pallida var. angustifolia were activated to some degree against rhinovirus as alkamide-rich hexane fraction showed a lower MIC100 than ethyl acetate fraction, which contained a relatively lesser alkamide [Echinacea or also known as coneflowers is the name of a flowering group of plants native that are to North America. Three out of nine commonly recognized types of Echinacea (on colds . The treCoV-229E . HCoV-22E6 cells . A rando (RV-39) . Accordict test) . On the alkamide .Sambucus nigra  is a species of flowering plant found in the family of Adoxaceae that are mostly found in Europe and North America. Recently, this plant was shown to reduce the incidence of cold as well as shorten the duration of cold and flu symptoms [S. nigra extract-treated cells showed no detectable viral cytopathic effect (CPE) at a multiplicity of infection (MOI) bronchitis virus  of 0.1 and a reduction in virus titers by six orders of magnitude. Although higher MOI (MOI = 1) had reduced the inhibition ability of S. nigra extract on the treated cells, viral titers were still significantly large by approximately four orders of magnitude. Virus titers decreased with increasing S. nigra concentrations in a dose-responsive manner [symptoms . There hsymptoms . Howevere manner . S. formosana Nakai stem ethanol extract and some phenolic acid had a potent effect against human coronavirus HCoV-NL63 with 50% inhibitory concentration (IC50) of virus yield, plaque formation, and virus attachment, which were 1.17, 4.67, and 15.75 μg/mL, respectively [S. formosana Nakai stem ethanol extract showed a significant reduction in HCoV-NL63 activity with IC50 of 3.54, 43.45, and 71.48 μM. Caffeic acid also significantly inhibited the replication and blocked the attachment of HCoV-NL63 to a host cell with IC50 = 8.1 μM. The study concluded that caffeic acid might be a vital component in anti-HCoV-NL63 activity. On the other hand, the antiviral activity of ectively . CaffeicP. lanceolata L. and in Kazeroun ethnobotany, P. coronopus is used to treat common colds [P. major was examined on a series of adenoviruses  that commonly cause cold-like symptoms [P. major possessed only a slight anti-herpes virus activity. However, certain pure compounds belonging to the five different classes of chemicals found in extracts of this plant exhibited potent antiviral activity. Among them is caffeic acid that exhibited the strongest activity against ADV-3 , whereas chlorogenic acid possessed the strongest anti-ADV-11  activity. Intriguingly, the effect of chlorogenic acid  possessed similar antiviral activity but a lower cytotoxicity than the standard drug  in inhibiting ADV-11 [P. asiatica extract (PAE) against the respiratory syncytial virus [Plantago is an important medicinal plant containing different compounds, such as flavonoids, alkaloids, terpenoids, vitamin C, and phenolic compounds (caffeic acid derivatives) with powerful antioxidant properties. The Plantago spp.’s herb tea is helpful for sore throat, dry cough, and stomach irritation . In souton colds . The antsymptoms . The resg ADV-11 . In addial virus .P. asiatica against RSV. The extractability was tested on GFP-tagged RSV on HEp2 cells and A549 cells, which was found to be dose-dependent as the extract at 50 μg/mL was the most effective at inhibiting viral replication and virus-induced cytotoxicity. For in vitro experiments, the cytotoxic concentration (CC50), effective concentration (EC50), and selectivity index (SI) of PAE were found to be 938.43, 39.82, and 23.5 μg/mL, respectively. In vivo experiments on BALB/c mice showed that PAE-treated mice significantly reduced viral mRNA in the respiratory tract at both 3 dpi and 5 dpi compared to the control phosphate-buffered saline (PBS)-treated group [ed group . Tilia cordata  and T. platyphyllus (known as large-leaved lime or linden) are found in warmer climates in Europe. The leaves, wood, volatile oil, and charcoal of the Tilia flowers are used for medicinal purposes [T. plathyphyllus and T. cordata are widely used traditionally as herbal tea. Commission E and British Herbal Compendium claimed that linden flowers are used for colds, any cold-related coughs, upper respiratory catarrh, common colds, irritable coughs, hypertension, and restlessness [purposes . Both T.lessness . T. flos from the family Malvaceae are taken internally as a tea to treat fever, flu, viral infections, and improved respiratory tract. The flavonoids contained in the flowers were reported to show antioxidant properties by acting as reactive oxygen species (ROS) scavengers, principally on hydrogen peroxide and the superoxide anion [T. cordata in human neutrophils ex vivo model. The results concluded that procyanidins were able to reduce inflammation and irritation of mucosa in common colds, pharyngitis, and tonsillitis. The investigated procyanidins (Epicatechin) possessed strong inhibition on the production of reactive oxygen species (ROS) by N-formylmethionyl-leucyl-phenylalanine (N-f-MLP) stimulated neutrophils at the lowest concentration of 1 μM [The flowers of de anion . Czerwiń of 1 μM . C. trichotomum is a plant belonging to the Lamiaceae (formerly Verbenaceae) family and grows wildly in fields and mountains in Korea, Japan, and China [C. trichotomum against the respiratory syncytial virus (RSV) in vitro cell cultures and an in vivo mouse model [C. trichotomum extract significantly reduced RSV replication, RSV-induced cell death, RSV gene transcription, RSV protein synthesis, and blocked syncytia formation  in the lungs of BALB/c mice. C. trichotomum extract significantly reduced RSV infection  by 50% (EC50) at a concentration of 27.95 μg/mL, whereas the cytotoxicity concentration (CC50) was at 764.17 μg/mL. Both values raised the selectivity index (SI) to 27.3, indicating the safety of a crude extract against RSV infection.nd China . Chathurse model . The treormation . IntriguInfluenza viruses consist of four distinct viruses: influenza A, B, C, and D, with influenza viruses A, B, and C commonly causing flu in humans . Flu freP. granatum, known as pomegranate, is a fruit-bearing deciduous shrub in the family Lythraceae, subfamily Punicoideae, that grows between 5 and 10 m tall. The pomegranate originated in the region, spreading from Iran to northern India, and has been cultivated throughout the Mediterranean since ancient times. The pomegranate peel’s ethanol extract and its n-butanol and ethyl acetate fractions have the highest inhibitory effect against influenza A virus, with IC50 values of 6.45, 6.07, and 5.6 μg/mL in MDCK cells, respectively. Upon treatment with the crude pomegranate’s polyphenolic extract (PPE) and its polar fractions in a dose-dependent manner, the production of virus significantly reduced (p < 0.05) [ < 0.05) . Antivir < 0.05) . The pot < 0.05) . Viral iG. sanguineum, also known as bloody crane’s bill or bloody geranium, is a hardy-flowering perennial herbaceous plant of the Geraniaceae family. It is also a Northumberland County flower. A polyphenol-rich extract isolated from the aerial roots (roots exposed to the air or above the soil) of the medicinal plant G. sanguineum had strong anti-influenza activity [n-BuOH extracts from aerial parts and roots of G. sanguineum were compared with the ethanol (EtOH) crude extract for their anti-influenza activities. Only the n-BuOH fraction  demonstrated significant in vitro antiviral activity in which out of four of the subfractions , only two of them  surpassed the overall effect of the total EtOH extract (SI > 50), whereas the EtOAc fraction was the most effective in an in vivo study [G. sanguineum on the proliferation of MDCK and CEF cells was determined using an MTT assay, which revealed IC50 values of 64 μg/mL and 72 μg/mL, respectively [activity . Using tvo study . The effectively .Echinacea purpurea roots was reported to show potent activity against the influenza virus. The ethyl acetate fraction of E. angustifolia root extract contained moderate activity against three viruses . In contrast, E. pallida root extracts were devoid of antiviral activity [E. purpurea required to inactivate 100% virus (MIC100) of tablets/capsules was found to be 2.5 μg/mL. E. purpurea teas have a 2.2 μg/mL concentration, which clearly showed that E. purpurea water extract taken as tea brewed at 80 °C possessed better antiviral activity than taken as capsules/tablets. Meanwhile, among a few extracts of the root of E. pallida var. angustifolia , only 55% ethanol extraction at 40 °C and ethyl acetate extract showed anti-influenza activity. However, ethyl acetate extract of E. pallida var. angustifolia possessed the best antiviral activity against influenza with MIC100 = 33.5 μg/mL than ethanol extract with 348 μg/mL [The aqueous fraction of activity ,68. The 48 μg/mL .Cistus incanus is rich in polyphenols that include many representatives with strong antioxidant and antiviral activities [C. incanus, rich in polymeric polyphenols exhibited antiviral activity against a highly pathogenic avian influenza A virus  in MDCK cell culture and a mouse infection model [C. incanus extract also prevented infection of cells by virus particles containing Ebola or Marburg virus envelope proteins, together with envelope components of filoviruses [tivities . A studyon model . MDCK ceoviruses .Glycyrrhiza uralensis, G. inflate, and G. glabra were prescribed as licorice in Chinese pharmacopeia, in which the roots and rhizomes are the main medicinal parts [G. uralensis was the most active compound in inhibiting the replication of SARS-associated viruses, including COVID-19 [al parts . Glycyrral parts . StudiesCOVID-19 ,73,74. GCOVID-19 .Chaenomeles sinensis is widely used as a traditional Chinese medicine to treat throat diseases [C. sinensis was pre-treated for A/Udorn/72 (H3N2) virus. Pre-treatment with CSD3 mildly reduced cell-binding, hemagglutination, and hemolytic activities by 70%. The main target step of CSD3 in the replication cycle is after cell-binding but before or at primary transcription, which increases the permeability of the virus envelope. CSD3 was suggested to be employed as a lozenge or mouthwash for daily use in preventing influenza virus infections [diseases . The polfections .Sambucus nigra juice (elderberry) with the addition of beta glucan 1,3/1,6, zincum gluconium, and acidum ascorbicum was studied against human pathogenic viruses: Influenza A H1N1 (FluA H1N1). S. nigra juice at 2.1% concentration showed a dose-dependent antiviral effect on FluA H1N1 with 2.5% concentration to exhibit the strong effect, which completely blocked the replication of the virus [50 = 770 ± 60 μg/mL on MDCK cells, CC50 = 810 ± 30 μg/mL on A549 cells) against influenza infection, causing flu (IC50 = 6000 ± 800 μg/mL). A stronger viral inhibition by elderberries was detected against the late-stage influenza cycle than in the early stage via blocking the virus glycoproteins. Elderberry acted on direct virus inhibition by suppressing viral entry, affecting the post-infection phase and viral transmission from cell to cell, together with indirect viral inhibition through modulating the release of cytokines, such as IL-6, IL-8, and TNF [he virus . This ju and TNF .O-galloyl-β-D-glucose or known as PGG was isolated from the branches and leaves of Phyllanthus emblica. PGG was tested for its cytotoxicity on MDCK cells in a cell-based screening assay to raise its CC50 value of 29.59 ± 4.32l μg/mL. PGG also showed potent inhibitory activity against influenza strain A/WSN/33(H1N1) with an EC50 value of 2.36 ± 0.29 μg/mL and selectivity index (SI) of 12.54 [P. emblica roots and showed potential antiviral properties against H3N2 virus strain with 50% inhibitory concentration (IC50) of 2.6 ± 0.7 μg/mL and 4.5 ± 0.6 μg/mL, respectively [1,2,3,4,6-penta-of 12.54 . Accordiectively .Camellia sinensis (green tea) extract (GTE) using the influenza A virus/Puerto Rico/8/34 (H1N1) as a model to examine the duration of the viral inactivating activity of the GTE that was stored at various temperature conditions [10 reductions, and 0.01% and 0.05% concentration reduced 2 log10 reductions, even though it was kept in storage for more than 2 months at different temperatures . The addition of ascorbic acid (as an antioxidant) to GTE was found to prolong the duration of the virucidal properties of GTE. Among three polyphenolic compounds of green tea viz. (−)-epigallocatechin gallate (EGCG), (−)-epicatechin gallate (ECG), and (−)-epigallocatechin (EGC), EGCG and ECG were found to be potent inhibitors of various influenza virus subtypes  replication in MDCK cell culture. The 50% effective inhibition concentration (EC50) of EGCG, ECG, and EGC for influenza A virus were 22–28 μM, 22–40 μM, and 309–318 μM, respectively. The study also suggested that the virucidal properties of catechin were due to the altering physical integrity of the membrane of host cells or the virus particles [Lee et al. (2018) evaluated the antiviral spectrum of nditions . GTE at articles .Sexually transmitted diseases (STDs) by viral infections are life-threatening without a proper cure. Sexually transmitted infections (STIs) caused by viruses include hepatitis B virus (HBV), herpes simplex virus (HSV), human immunodeficiency virus (HIV) and the human papillomavirus (HPV), which contribute to adverse effects on sexual and reproductive health, including infertility in women and several different types of cancers such as cervical cancer . Human pClinacanthus nutans is also known as snake grass from the Acanthaceae family. This plant is popular in Malaysia, Indonesia, Thailand, and China due to its diverse and potential medicinal uses in traditional herbal medicine. According to Kunsorn et al. (2013), three extracts of the C. nutans and C. siamensis  leaf extracts were found to exhibit potency of antiviral activity against HSV-1 strain KOS and HSV-2 strain Baylor 186 by inhibiting viral plaque formation. The results revealed that methanolic extract of C. nutans exhibited only a slight anti-HSV-1 activity (IC50 = 64.93 µg/mL) compared to the n-hexane (IC50 = 32.05 µg/mL) and dichloromethane extract (IC50 = 44.50 ± 2.66 µg/mL) of C. nutans. In contrast, methanolic extract of C. siamensis exhibited the strongest activity against HSV-1 (IC50 = 37.39 µg/mL), while the n-hexane and dichloromethane extracts exhibited only a slight anti-HSV-1 activity . Contrary to the C. siamensis, the n-hexane, dichloromethane, and methanol extracts of C. nutans exhibited a slightly weak anti-HSV-2 activity  [ctively) . C. incanus extract was demonstrated to inhibit HIV-1 and HIV-2 infections in vitro  [C. incanus were attributed to its antiviral properties as a polyphenol-enriched fraction (known as CiPP)  isolated from C. incanus extract possessed antiviral activity when tested on HIV-1LAI with lower cytotoxicity rate than C. incanus extract, whereas polyphenol-depleted fraction did not show any antiviral activity on HIV-1LAI [C. incanus against HIV via inhibition of viral attachment.CYSTUS052 substance derived from the 0 μg/mL) ,71. AntiHIV-1LAI . Figure Plantago major, a popular traditional Chinese medicine, has long been used for treating various diseases varying from cold to viral hepatitis [P. major possessed antiviral activity against HSV-2. Among the two main components found in P. major, caffeic acid  exhibited a stronger antiviral activity against HSV-1  and HSV-2  than chlorogenic acid (CC50 = 3995 µg/mL) against HSV-1  and HSV-2  [epatitis . A study = 46.2) .Polygonum minus Huds (Polygonaceae), commonly known as kesum or Persicaria minor, is considered one of the most important aromatic plants in Southeast Asia. The cytotoxicity and antiviral properties of methanolic extracts from the leaves and stem of P. minus were investigated. The LC50 value for leaf extract towards Vero cells was 875 µg/mL, while the LC50 value for stem extract was 95 µg/mL. Antiviral tests were performed on Vero cells infected with HSV-1 at three different concentrations of extract: 1.0 LC50, 0.1 LC50, and 0.01 LC50. The result showed that the treatment of cells with stem extract at the concentrations corresponding to 0.1 LC50 gave a higher cell survival compared to other concentrations [trations . This inC. incanus against HIV via inhibition of viral attachment. However, the mechanism of action from C. nutans and C. siamenensis plant extractions against HSV-1 and HSV-2 virus is via inhibition of viral plaque formation, whereas the mechanism of P.minus and P.major is via inhibition of viral attachment.Cutaneous Human papillomavirus (HPV), mainly from beta and gamma genera, is widely present on the surface of the skin and transmitted by skin-to-skin contact and enters the body via cutaneous layers . These sSarracenia purpurea (Pitcher plant), often referred to as the purple-colored pitcher plant, is a carnivorous plant of the family Sarraceniaceae. It is mainly found in the USA and Canada [S. purpurea on the causative agent of smallpox was pointed out by Arndt et al. (2012), where S. purpurea treatment worked on preventing early vaccinia virus protein synthesis and replication of monkeypox virus and variola virus [50 of S. purpurea against poxvirus was 10–15 µg/mL, while the dose that induced 50% cytotoxicity (CC50) was 70–75 µg/mL, resulting in an SI of approximately 5–7. According to a small pilot clinical trial conducted by Dah et al. (2017), the application of an aqueous extract of S. purpurea in gel base alleviates symptoms, supports healing, and improves the appearance of cold sores caused by herpes simplex virus [S. purpurea extracts are able to hinder the replication of HSV-1 by two mechanisms of action, which are through (1) inhibiting extracellular virions or viral attachment to the human host cell and (2) inhibiting the expression of viral immediate–early, early, and late genes when added at various times post-infection. This botanical has also previously been shown to inhibit the replication of poxviruses via the early inhibition of viral gene transcription [d Canada . Antipoxla virus . The dosex virus . Kannan cription .C. nutans possesses anti-inflammatory properties that are superior to benzydamine for the prevention of oral mucositis, which can be worsened with the presence of the herpes simplex virus [C. nutans orally as well as applied the extract on the lesion of oral mucositis of 60 patients. Pain severity of the patients was less on glycerin papayor extract treatments with later onset of oral mucositis than benzydamine used in the controlled group [C. nutans’ anti-VZV activity. In their clinical study, the C. nutans extract was formulated into a 5% C. nutans cream prior to testing its ability to combat VZV infections, where the organic extract exhibited anti-VZV activity through the direct inactivation stage and a positive curing effect [The whole plant of ex virus ,112. A sed group ,111. A cg effect .Matricaria recutita (German chamomile) is one of the commonly used medicinal plants. Its flower is externally used for skin inflammations and irritations, bacterial skin diseases, nappy rash and cradle cap, eczema, poorly healed infected wounds, abscesses, frostbite, and insect bites. Moreover, its flower is also used for baths, compresses, or rinses and poultices [Matricaria recutita consists of some of the largest group of medically important compounds -β-ocimene, (Z)-β-ocimene, -farnesol, and en-yn-dicycloethers) together with 0.75% of volatile oil [50) of 0.003% towards the virus [oultices . The esstile oil . The esshe virus .Aloe vera (Aloe) is an important and traditional medicinal plant belonging to the family Liliaceae, and it is indigenous to Africa and Mediterranean countries. The topical application of A. vera helps to prevent herpes ulcers and enhances the healing process of dermal injuries [A. vera gel is recognized as antiseptic as it was reported to kill or control mold, bacteria, fungus, and viruses with the ability to eliminate internal and external infections [A. vera gel extract, whereby it showed a significant inhibitory effect of 0.2–5% on HSV-1 growth in the Vero cell line [A. vera gel  exhibited even a more significantly anti-viral activity than 0.2% and 0.5% concentrations (p < 0.05), while 5% concentration had the maximum effect in reducing virus plaques presented after virus infection in the cells. A five percent concentration of A. vera gel extract was found to be nontoxic when tested on the Vero cell line. Zandi et al. (2007) tested the antiviral activity of a crude hot glycerin extract of A. vera gel against HSV-2 replication in the Vero cell line. The extract showed antiviral activity against HSV-2 before attachment, entry of virus to the Vero cells  and post attachment stages of virus replication  with CC50 = 3238 µg/mL. It was suggested that A. vera could be a good candidate as a natural source for antiviral drug development against HSV-2 [injuries . A. verafections . Rezazadell line . Higher st HSV-2 .Cornus officinalis  is a deciduous tree found mostly in China as well as in Korea and Japan. Other common names for this plant are Japanese cornelian cherry and Japanese cornel. The fruits of this plant have been used in all areas of its geographical distribution for a wide variety of diseases and complaints. The effect on CVA16 ) induced a cytopathic effect CPE, which was observed in the C. officinalis. The plant extract had shown significant cell viability of >50%, indicating antiviral activity against CVA16 [C. canadensis L. or known as bunchberry dogwood (from the family Cornaceae), is a plant used in Native American traditional medicine to treat possible antiviral infections [C. canadensis leaves, which was the most active extract that was able to inhibit virus absorption with EC50 of about 9 μg/mL [st CVA16 . Meanwhifections . The ext 9 μg/mL .Lysimachia mauritania is a biennial herb distributed worldwide, especially in the temperate and subtropical climate regions of both hemispheres, along coastal regions in East Asia, the Philippines, Micronesia, Polynesia, and the Indian Ocean islands. The data showed that L. mauritiana reduced lytic gene expression of the varicella-zoster virus and inhibited replication of the varicella-zoster virus (VZV). L. mauritiana mediated substantial downregulation of IE62 protein and major transactivation of IE, E, and L genes, which in turn resulted in the repression of E and L genes and inhibition of replication of the varicella-zoster virus. It was suggested that L. mauritiana might provide an important basis for the further development and tailoring of novel therapeutic agents to treat virus-associated diseases of varicella-zoster. The 50% inhibitory concentration (IC50) of L. mauritania extract for VZV was calculated as 26.09 µg/mL [09 µg/mL .Mentha haplocalyx is one of the most popular medicinal plants in the Lamiaceae family that is widely used in Chinese herbal medicine to treat various disorders and is also used as a supplement. M. haplocalyx was demonstrated to exert antiviral activity in blocking viral infection proinflammatory response. Its water extract was active against coxsackie A16 virus, the causative agent of hand, foot, and mouth disease (HMFD), with a 50% inhibitory concentration (IC50) value of 70.3 μg/mL. It significantly blocked the cytopathic effect of the coxsackie A16 virus [16 virus . In addi16 virus .C. sinensis (green tea) leaf contains large quantities of flavonoids called catechins, such as epigallocatechin and epigallocatechin gallate (EGCG). These polyphenolic compounds of green tea were reported to possess many health benefits, focusing specifically on the anti-HPV (human papillomavirus) effects of green tea, particularly after catechins were approved by the Food and Drug Administration (FDA) in 2006. The mechanism of action of green tea catechins against Human papillomavirus (HPV) is unclear; however, based on comprehensive clinical studies, they are unmistakably successful [ccessful . Polypheccessful .Hibiscus sabdariffa is a shrub belonging to the family Malvaceae. It is a popular vegetable in Indonesia, India, West Africa, and many tropical regions and it is well known as one of the ethnomedicinal plants in Nigeria [H. sabdariffa leaves (red and green leaves) antiviral activities against measles virus (MV) as well as the effects of the extracts on Hep-2 cells [H. sabdariffa had antiviral activities only at 10 and 15 mg/mL against MV. The result of this research suggested the promising antiviral activity of H. sabdariffa plant extracts. Nigeria . Sunday -2 cells . The preG. glabra roots or known as Licorice roots, is often used in ancient Siddha medicine and is approved by the German commission [G. uralensis Fischer and G. glabra L. are designated as G. radix (kanzo in Japanese) [G. uralensis against the etiological agents of HFMD through the identification of glycyrrhizic acid (GA) as the antiviral component of G. uralensis against enterovirus 71 (EV71) and coxsackievirus (CVA16) infection [G. uralensis possessed anti-EV71 infection at IC50 = 0.056 μg/mL [Extract of mmission . Tanemotapanese) . A studynfection . In addi56 μg/mL . A study56 μg/mL .Phyllanthus emblica that possess antiviral properties against the EV71 virus strain that mainly causes hand, foot, and mouth disease (HFMD) with IC50 values of 2.6 ± 0.7 and 2.6 ± 0.8 μg/mL, respectively [P. niruri Linn. could suppress the complications after the extract syrup was taken orally, three times per day for 5 days. Forty-six subjects (51.1%) had no new papules occur after 5 days of oral administration of P. niruri extract syrup [Glochicoccinoside D and phyllaemblicin C were identified as the main components of ectively . Sarisetct syrup .Matria recutita, Lysimachia mauritania, Menta haplocalyx, Camellia sinensis, Hibiscuss sabdariffa, Glycyrrhiza spp., Phyllantus spp., Cornus canadensis, Aloe barbadensis, Clinacanthus nutans, and Sarracenia purpurea that have antiviral activities against dermatology related viruses.Mosquito-borne viral diseases can be transmitted to humans and animals by viral-infected mosquitoes. Some of the common mosquito-borne viral diseases are dengue, Zika, Chikungunya fever, yellow fever, West Nile virus, eastern equine encephalitis, western equine encephalitis, St. Louis encephalitis, and La Crosse. Only certain types of mosquitoes can carry viruses, and very few are even infected by feeding on an infected host. Mosquito populations are the highest from spring to fall, causing a spike in mosquito-borne viral diseases or any mosquito-borne disease. Children below 12 and elderlies over 50 years old have higher chances of experiencing severe symptoms. The disease onset may be fever, headache, muscle aches, nausea, or vomiting, and may progress to seizures, paralysis, coma, and possibly death. Preventing mosquito bites is the most effective way of preventing the spread of these diseases . Table 6Dengue fever, with its severe manifestations in the form of Dengue Haemorrhagic Fever (DHF) and Dengue Shock Syndrome (DSS), has emerged as a great public health concern, spreading to all tropical and sub-tropical countries in the world, particularly important in Southeast Asia, which bear a high burden of dengue. The dengue virus (DEN virus) is a member of the family Flaviviridae with four antigenically related but distinct serotypes: DENV-1, DENV-2, DENV-3, and DENV-4. The incidence of disease and its severity varies between primary and secondary infections, possibly also across different dengue virus serotypes . Ooi andA. philoxeroids or also called “Alligators Weed’’, belongs to the family Amaranthaceae, which originated from South America but is currently invading Australia. Jiang et al. (2005) investigated an in vitro effect of A. philoxeroides extract against the dengue virus. An MTT assay was initially carried out to determine the cytotoxicity of A. philoxeroides extract on C6/36 cell lines. Coumarin-based extract of A. philoxeroides showed the lowest toxicity on cells (TD50 = 535.91), whereas a petroleum ether extract of A. philoxeroides had the strongest inhibitory effect on dengue virus (ED50 = 47.43) [An immersed aquatic plant, = 47.43) .Cladosiphon okamuranus belongs to the family Chordariaceae. It is a type of brown seaweed found naturally in Okinawa, Japan. Hidari et al. (2008) stated that a sulfated polysaccharide named fucoidan from C. okamuranus was found to potentially inhibit DENV-2 infection [nfection ,142. TheCladogynos orientalis belongs to the family Euphorbiaceae. This white stellate-hairy shrub is about 2 m high and is found in Southeast Asia, Malaysia, and Thailand. The dichloromethane ethanol extract of C. orientalis was tested in vitro for an anti-dengue activity against DENV-2 in Vero cells through the MTT assay. The results showed that the ethanol extract of C. orientalis at a concentration of 12.5 μg/mL exhibited 34.85% inhibitory activity against DENV-2. In addition, C. orientalis at a concentration of 100 μg/mL also exhibited an inactivated viral particle activity at about 2.9% [out 2.9% .C. nutans demonstrated a moderate anti-dengue virus activity with an IC50 value of 31.04 μg/mL [C. nutans was able to inhibit dengue viral 2 replication in the direct inactivation and post-incubation stages with a CC50 of 25 μg/mL [The 80% ethanol extract of the aerial part of 04 μg/mL . The phe25 μg/mL . Other eS. nigra was tested on dengue virus serotype-2 (DENV-2). A study showed that 400 µg/mL methanolic extracts of both plant parts possessed a protective effect against DENV-2 and an effective method to protect cell monolayers when the cells were treated with the extracts before they were infected [S. nigra flowers extract was more protective than pre-treatment with S. nigra leaf extract.The antiviral activity of the methanolic extracts (leaves and flowers) of infected . This inCarica papaya (papaya) is a popular and important fruit in tropical and subtropical regions, which is consumed worldwide. Thirty percent of the worldwide production of papaya is from Asia. Papaya is a family member of Caricaceae that is rich in vitamin A, vitamin C, and vitamin E and possesses antiviral, antibacterial, and antifungal properties [C. papaya’s leaves showed that it has a moderate inhibitory action against the dengue virus growth on LLC-MK2 cell line with EC50 of 1000 μg/mL and selective index of 1 [C. papaya’s leaves was also non-cytotoxic on LLC-MK2 cells (CC50 = 1000 μg/mL). Another study stated that aqueous extract of leaves from C. papaya had a promising anti-dengue activity on Vero cells infected with dengue virus 2  [C. papaya to significantly decrease dengue viral foci. An open-labeled randomized controlled trial was conducted on 288 patients with dengue fever and dengue hemorrhagic fever aged less than 60 years old to investigate the platelet-increasing property of C. papaya leaves juice. Platelet count of dengue patients significantly increased after 40–48 h of C. papaya leaves juice consumption during dengue infection [C. papaya twice per day for five days [operties . A studydex of 1 . The chl= 75.85) . The antnfection . The plaive days .Aedes aegypti and A. albopictus are the most common vectors of the chikungunya virus (CHIKV). CHIKV can be transmitted to a new, naïve host more quickly than for other mosquito-borne viruses as the viruses can be found in the saliva of the mosquitos. The viruses replicate in the midgut, disseminate into salivary glands, and can be found in saliva 2–3 days after the blood meal [Another mosquito-borne disease, Chikungunya virus (CHIKV), developed from a relatively obscure and geographically isolated pathogen into a serious public health hazard in recent years. It has now been discovered in Africa, Asia, Central, and South America, with the potential to spread to North America. ood meal . CHIKV iood meal . VariousPicrorhiza kurroa (rhizome part), Ocimum tenuiflorum (whole plant), and Terminalia chebula (fruit), showed anti-CHIKV activity by inhibiting viral attachment as well as replication inhibition by inhibiting helicase and protease activities [O. tenuiflorum and T. chebula aqueous extracts, the number of plaques also decreased as the extract concentration increased. Complete plaque inhibition (100% inhibition) was observed at 200 μg/mL. Meanwhile, P. kurroa aqueous extract showed only 67% inhibition at 200 μg/mL. The aqueous extracts of three plants, including tivities . All thrRhapis excelsa (family Arecaceae) and Vernonia amygdalina (family Compositae) could be the sources of anti-Chikungunya virus agents [R. excelsa resulted in the highest percentage of reduction in the viral load (98.1%), followed by the ethyl acetate extract of leaves of V. amygdalina (95.5%). In addition, the chloroform extract of R. excelsa and the ethyl acetate extract of V. amygdalina had the lowest EC50 values of 29.9 ± 0.9 and 32.4 ± 1.3 μg/mL, respectively. Cytotoxicity assay showed the CC50 values of each plant extracts of 161.5 ± 19.2 and 165.5 ± 9.2 μg/mL, respectively.Chan et al. (2016) revealed that s agents . Both plNicotiana benthamiana in treating CHIKV infection in vivo and in vitro [N. benthamiana (wild-type and glycoengineered-type of N. benthamiana) with the potential of neutralizing CHIKV in vitro and showed efficacy but with different potency against CHIKV infection in a murine model. The plaque reduction neutralization test (PRNT) was used to show strong neutralization activities of both mAb variants against CHIKV with statistically similar potency and EC50 of 130.5 μg/mL and 390.8 μg/mL for glycoengineered and wild type, respectively. An in vitro test of mAb from both types of N. benthamiana on five weeks old C57BL/6 mice showed a significantly lower viremia compared to PBS-treated mice.The viability of using an anti-CHIKV monoclonal antibody (mAb) expressed and assembled in wild-type and glycoengineered in vitro . CHKVmAbZika virus (ZIKV) is spread by mosquitoes, which is a mosquitoes-borne RNA virus of the Flavivirus genus that could cause congenital microcephaly and hemorrhage. This infection became a big global issue and a worldwide epidemic, which raised a huge concern. As the virus emerged, many tried to identify the plants that could be used as an alternative medicine against the Zika virus. Research studies showed positive results, which proved that medical plant-based extracts could be useful in treating the virus .Aphloia theiformis is a slender, evergreen tree with drooping branches, growing up to 20 m tall. This plant is well distributed around Reunion Island, a French department in the Indian Ocean. An analysis of A. theiformis showed its richness in phenolic compounds, mainly C-glycosylated xanthones, such as mangiferin and flavonoids, that possess many beneficial properties, including antiviral activities. Such antiviral properties are played by polyphenols, such as delphinidin, baicalein, and naringin, that were reported to inhibit the early steps of ZIKV replication [A. theiformis were stable, and the antiviral properties were shown by interfering with at least one of the early steps of ZIKV infection. The result revealed that it took 10–15 min to inhibit the Green Fluorescent Protein (GFP) expression by 50% at non-cytotoxic concentrations of A. theiformis extract [A. theiformis represented a high potential for prophylactic agents, targeting the entry of two medically relevant flaviviruses and could be used to treat patients. The concentrations of A. theiformis extract that inhibited 50% of cell viability (CC50) was at 3000 µg/mL, whereas the concentration that inhibited 50% of viral infectivity (IC50) was at 100 µg/mL, resulting in the selectivity index of A. theiformis extract of 30.lication . The ext extract . A. theiN. benthamiana is a plant producing ZIKV E (zE) that correlated with protective immunity against multiple ZIKV strains [N. benthamiana using SDS-PAGE analysis, Ni2+ affinity chromatography was effective in removing N. benthamiana host proteins and was able to enrich PzE to >90% purity. Yang et al. (2018) also stated that the treatment of C57BL/6 mice with PzE through injection had elicited a potent humoral response, which exceeded the required threshold that correlated with the protective immunity against ZIKV [ strains . The devnst ZIKV . This suPsiloxylon mauritianum fresh aerial aqueous extract [P. mauritianum extract were performed on mammalian cell lines using MTT assay, which resulted in the extract not exhibiting cytotoxic effects on Vero, A549 cells, human primary keratinocytes (HKPM), and fibroblast (FMa) cells with CC50 1044 ± 106.2 μg/mL, 657 ± 15.7 μg/mL, 353 ± 84.4 μg/mL, and 820 ± 26.5 μg/mL, respectively. Genotoxic effects of P. mauritianum were determined using COMET assay on mammalian cell lines and did not cause any damaging effects on the mammalian cell DNA. A viral inactivation assay was performed to determine if the P. mauritianum extract had the ability to neutralize ZIKV infectivity. The concentration of P. mauritianum that inhibited 50% of ZIKV infection (IC50) was estimated at 19.5 ± 4.8 μg/mL with a selectivity index calculated as 53.5. The time-of-drug-addition assays revealed that the P. mauritianum extract interfered with the attachment of the viral particles to the host cells.Clain et al. (2019) demonstrated the inhibition of different ZIKV African and Asian strains infection in vitro by  extract . CytotoxSumbucus nigra, Clinicanthus nutans, Carica papaya, and Cladosiphon okamuranus inhibited the replication of the DENV-2 virus. Ocimum tenuiflorum, Picrorhiza kurroa, and Terminalia chebula inhibited the viral attachment. Moreover, Rhapis excelsa and Vernonia amygdalina inhibited the viral load of the Chikungunya virus. Nicotiana benthamiana inhibited the early replication, whereas Aphloia theiformis inhibited the entry of the Zika virus.Humankind has dealt with various diseases for centuries. This brought the urge to utilize drugs from various parts of medicinal plants. Abundant sources, such as documents, preserved monuments, and original medicinal plants, are shown as evidence in the search for drugs from nature . AncientSambucus nigra and Clinacanthus nutans emerged as the most antiviral medicinal plants mentioned in this review against three viral infections. S. nigra significantly showed antiviral properties against common colds, influenza, and mosquito-borne infections, whereas C. nutans is effective against sexually transmitted diseases, dermatology, and mosquito-borne infections. Echinacea purpurea, Echinacea augustofolia, and Echinacea pallida are effective against common cold and influenza virus infections. Plantago major was reported to be effective against common colds and STDs. Glycyrrhiza uralensis, Phyllanthus emblica, Camellia sinensis, and Cistus incanus are effective against influenza and dermatology-related infections. Nicotiana benthamiana is effective against two types of mosquito-borne viral infections, which are Chikungunya and Zika viruses.Among all plants that were reviewed, 11 plants appeared to be the most promising plants to fight against viral infections. S. nigra fruits contain antiviral activity against infective bronchitis virus at an early point of infection and influenza A virus, while the leaves and flowers have antiviral properties against dengue virus type 2 by protecting monolayers cells and preventing viral infections [Streptococcus pyogenes and group C and G Streptococci) and Gram-negative bacteria  in liquid cultures. Intriguingly, elderberry does promote an indirect viral immune response in the infected body, whereas cyanidin 3-glucoside, a major bioactive compound, only promotes a direct response to the influenza infection [fections ,76,132. fections . Phenolifections . Sambucunfection .Clinacanthus nutans are known as snake grass belonging to the Acanthaceae family. This plant has diverse and potential medicinal uses in traditional herbal medicine for treating skin rashes, insects and snake bites, lesions caused by herpes simplex viral lesion, diabetes, as well as gout, where it is widely used in Malaysia, Indonesia, Thailand, and China [C. nutans’s leaves possesses anti-HIV-1 activity [C. nutans contained monogalactosyl diglyceride (MGDG) and digalactosyl diglyceride (DGDG) [2-hydroxy-(132-R)-phaeophytin b, 132-hydroxy-(132-S)-phaeophytin a, and 132-hydroxy-(132-R)-phaeophytin a of C. nutans were shown to have anti-herpes simplex activity. Their inhibitory activity affected the viral adsorption or penetration onto the host cells [C. nutans inhibited the production of dengue virus RNA as well as the protein of the infected cells [nd China . Accordiactivity ,155. Thest cells . Phaeophed cells .E. purpurea roots were known to have antiviral properties against human coronavirus HCoV-229E and influenza virus [E. pallida var. angustifolia root showed anti-rhinovirus (RV) and anti-influenza activity, and E. angustifolia root extract had moderate antiviral activities against influenza virus and rhinovirus. Some echinacea extracts were found to inhibit hemagglutinin and neuraminidase of influenza viruses [Subsequently, Echinacea was found to have benefits against antivirus, such as cold and flu. Echinacea is widely used as a preventive compound for infectious diseases in respiratory systems. Essentially, it is being utilized to decrease the side effects and time span of colds, flu, and upper respiratory tract diseases and help to stimulate the activity of the immune system. za virus ,37, whilza virus . In addi viruses ,37. Echi viruses .Glycyrhhiza urelensis, Phyllanthus emblica, Camellia sinensis, and Cistus incanus, showed antiviral activity against viruses that cause influenza and dermatology-related diseases. Roots of Glycyrrhiza plants are prescribed as licorices that contain medicinal compounds, glycyrrhizin. The glycyrrhizin has anti-influenza properties by inhibiting influenza virus replication, inhibiting influenza virus polymerase activity, and anti-varicella-zoster virus (VZV) [G. uralensis, which are glycyrrhizin, which inhibits replication of SARS-associated virus, and glycyrrhizic acid, which possesses antiviral activity against enterovirus (EV71) as well as coxsackievirus (CVA16) infections that cause hand, foot, and mouth disease [Phyllanthus plant is a flowering plant that can be found in tropical and subtropical regions. Branches and leaves of P. emblica showed potent inhibitory activity against influenza strain A/WSN/33(H1N1) [P. niruri extract syrup could help control the clinical manifestation of varicella-zoster virus infection. C. sinensis (green tea) is a shrub-type plant with fragrant, yellow-centered, and white flowers [C. sinensis extract contains catechins that possess antiviral properties against influenza A virus (H1N1), influenza A (H3N2), influenza B virus, and human papillomavirus (HPV) [Cistus incanus (family Cistaceae) is an evergreen dwarf shrub herb with hairy-sticky branches and leaves [C. incanus. CYSTUS052 protective effect was shown by the binding of polymeric polyphenol components to the influenza virus surface and inhibiting the binding of hemagglutinin to the cellular receptors [Four plants, us (VZV) ,124. The disease ,107. Phy33(H1N1) , while t33(H1N1) . Sariset flowers . C. sineus (HPV) ,77,156. d leaves . CYSTUS0eceptors . Accordieceptors . The mececeptors .Plantago major showed antiviral effects against viruses causing the common cold and sexually transmitted diseases. The phenolic compound, such as caffeic acid and chlorogenic acid of P. major had antiviral properties against a series of adenoviruses  and herpes simplex virus type 1 and 2. P. asiatica showed an antiviral effect against the respiratory syncytial virus [N. benthamiana. This plant expressed an anti-CHIKV monoclonal antibody (mAb) used in treating CHIKV infection in vivo and in vitro [N. benthamiana that provided treatment on mice against Zika viruses. It was revealed that PzE had the potential of inducing protective immunity of mice in vitro towards Zika virus infection. al virus ,43. Finain vitro . A plantThe antiviral properties of 45 different medicinal plants worldwide were discussed. Most of the reviewed medicinal plants exhibited antiviral effects against influenza infection and dermatology-related viral infections, such as enterovirus, coxsackie virus, varicella-zoster virus, monkeypox virus, variola virus, and herpes simplex virus. We identified the 11 most promising plants with the most effects against different types of virus infections. We also highlighted the bioactive compounds that claimed to be responsible for the respective plants, as well as the mechanisms of action against the virus infections.S. nigra and C. nutans were shown to possess the most potent antiviral effects. Echinacea spp  were found to be effective in treating colds and influenza. G. urelensis, P. emblica, C. sinensis, and C. incanus showed antiviral activity against viruses causing influenza and dermatology-related diseases. P. major revealed to own antiviral effects against viruses that cause the common cold and sexually transmitted diseases. Finally, N. benthamiana exhibits antiviral effects mostly against mosquito-borne viral diseases, such as chikungunya and Zika virus infection. These antiviral plants might provide a lead for the discovery of safe antiviral drugs to tackle various infections associated with different types of viruses. However, more preclinical studies of these medicinal plants are required to justify their efficacy against respective viruses. In general, this compilation will benefit the researchers and clinicians in narrowing down some targeted plant extracts and bioactive compounds for effective potential treatment against specific viral infections. More research is required to examine the efficacy of antiviral medications derived from traditional medicinal plant therapies, as most of these treatments have shown promising antiviral mechanisms of action.


Mammalian orthoreoviruses (MRVs) are increasingly reported to cause various diseases in humans and other animals, with many possibly originating from bats, highlighting the urgent need to investigate the diversity of bat-borne MRVs (BtMRVs). Here, we report the detection and characterization of a reassortant MRV that was isolated from a bat colony in Xinjiang, China. The BtMRV showed a wide host and organ tropism and can efficiently propagate the cell lines of different animals. It caused mild damage in the lungs of the experimentally inoculated suckling mice and was able to replicate in multiple organs for up to three weeks post-inoculation. Complete genome analyses showed that the virus was closely related to MRVs in a wide range of animals. An intricate reassortment network was revealed between the BtMRV and MRVs of human, deer, cattle, civet and other bat species. Specifically, we found a bat-specific clade of segment M1 that provides a gene source for the reassortment of human MRVs. These data provide important insights to understand the diversity of MRVs and their natural circulation between bats, humans, and other animals. Further investigation and surveillance of MRV in bats and other animals are needed to control and prevent potential MRV-related diseases. Orthoreovirus of the family Reoviridae. Currently, they are divided into ten species with Mammalian orthoreovirus as the prototype [Orthoreoviruses are a group of non-enveloped, segmented and double-stranded RNA viruses within the genus rototype . The virrototype . Their grototype .Mammalian orthoreoviruses (MRVs) have been isolated from diarrheal pigs in China, South Korea and the United States [Orthoreoviruses are commonly identified in animals and some of them are potential causative agents of neurological disease, pneumonia, respiratory illness and enteritis in a variety of animals including humans ,5,6,7. Od States ,9,10. Ind States . TherefoBats are considered important natural hosts for hundreds of viruses, including many important zoonotic pathogens such as the Marburg virus, Hendra virus and Lyssavirus ,13,14. DMammalian orthoreovirus (BtMRV) isolated from a bat colony in Xinjiang, China. The virus was prevalent in the intestine and lung tissues of the bat population and exhibited a close genetic relationship with MRVs from humans, deer, civets and cattle. The virus could efficiently replicate in cell lines of several animal species and showed mild pathogenicity in experimentally infected mice. These data provide important insights to understand the diversity of MRVs and their natural circulation between bats, humans and other animals.Here, we report the detection and characterization of a new reassortant bat-borne Pipistrellus pipistrellus bats were sampled at abandoned houses in Xinyuan and Qapqal counties in Ili Prefecture, Xinjiang Uygur Autonomous Region, China. The two counties were ~170 km apart. Lungs and intestines from sampled bats have previously been used for viral metagenomic analysis and the detection of parechovirus and polyomavirus [In 2016, 46 and 76 apparently healthy omavirus ,23. The omavirus . A semi-g for 10 min at 4 °C. The supernatants were passed through 0.22-µm filters and then were added to cells for 1 h incubation. After discarding the supernatants, cells were maintained in DMEM supplemented with 2% fetal bovine serum (FBS) and a 2-fold dose of 100,000 U/mL penicillin and 10,000 g/mL streptomycin . The infected cells were inspected daily for cytopathogenic effect (CPE). The indirect immunofluorescence assay (IFA) we previously established to detect bat rotavirus [50) of the virus.Virus isolation was initially carried out using the porcine kidney 15  cell line. Eleven MRV-positive intestines were individually homogenized with serum-free Dulbecco’s modified Eagle medium (DMEM)  and were centrifuged at 12,000× otavirus  was modiMyotis petax bat kidney cell line (BFK) established by our laboratory [Cell tropism of the virus was assessed using PK15, African green monkey kidney , baby hamster kidney-21 , Madin-Darby bovine kidney , human cervical carcinoma cell  and an immortalized embryonic boratory . After ig for 5 min. The supernatants were passed through a 0.22 μm filter, and 10 μL filtrates were negatively stained with 2% phosphotungstic acid and examined with a JEM-1200 EXII transmission electron microscope (TEM) . In addition, the PK-15 culture showing 50% CPE was scraped to prepare the ultrathin section. After centrifugation at 3000× g for 15 min at 4 ℃, cells were fixed overnight using 2.5% glutaraldehyde, and then using 1% OsO4 for 2 h. Fixed cells were dehydrated using acetone for 1 h and were impregnated with an acetone-epoxy resin mixture. After embedding, samples were polymerized at 60 ℃, cut into ultrathin sections, and stained using a mixture of 1% uranyl acetate and 1% lead citrate. Each slice was observed using a TEMH-7650 TEM .For electron microscopy, the PK-15 culture at 72 h.p.i. at passage 6 was harvested by three cycles of freezing-thawing, followed by centrifugation at 12,000× −5. The open reading frames (ORFs) were predicted using the ORF finder [To obtain the complete genomic sequence of the virus, we performed RNAseq of the three isolates at passage 6 on an Illumina NovaSeq sequencer using our MTT protocol . The cleF finder . The resF finder . The phyF finder .p-values < 10−10). Reassortment was further visualized using SimPlot v.3.5.1 [Potential genomic reassortment was initially identified based on the phylogenetic topology of the 10 segments. The sequences of the isolate QAPpC66 in this study and the potential reassortment parents were used for further validation. We trimmed the untranslated regions (UTRs) at both termini of each segment and concatenated them in the order of L1-L2-L3-M1-M2-M3-S1-S2-S3-S4. The MSA of tandem constructs was generated using ClustalW and then subjected to reassortment prediction using RDP v.4.95 with the algorithms of RDP, GENECONV, Chimaera, MaxChi, BootScan and SiScan . The rea v.3.5.1 .n = 5) were purchased from Liaoning Changsheng Biotechnology Co., Ltd, Shenyang, China. Three-day-old suckling mice were randomly divided into four groups with twelve animals in each. Two groups were either orally or intracranially inoculated with 3 × 105 TCID50 of the isolate QAPpC66. The other two control groups were mock-inoculated with uninfected PK-15 culture in the same way. Animals were weighed daily and monitored for clinical signs. Three animals in each group were euthanized at 7, 14, 21 and 28 days post-inoculation (d.p.i.) and were subject to examination of the organ lesions. The rectum, lung, liver, brain and kidney of each animal were collected for MRV detection. A quantitative RT-PCR (RT-qPCR) was designed to detect and quantify the genome copy of QAPpC66. The primer and probe sequences were Reo-QF1 (5′-ATCTTATAAGCGCGTGCCGA-3′), Reo-QR1 (5′-GGAGATATCCATGCAC GCCA-3′) and Reo-probe (FAM5′-TACACTTGATGGTGATACGGTCC-3′ TAMRA). These tissues were accurately weighed and completely ground before RNA extraction. The RT-qPCR program was performed using the MonAmp Taqman qPCR mix  under the following program: 15 s at 95 °C, then 40 cycles of 5 s at 95 °C, 30 s at 60 °C.Pregnant Kunming mice  . Values Pipistrellus pipistrellus bats were subjected to RT-PCR detection. The results revealed that the prevalence of MRV was higher in Qapqal  than in Xinyuan county   . For QapMyotis petax) were used to test the infectivity of the MRV. All these cells are susceptible and permissible to the virus and showed similar CPEs (6.67–7 TCID50/mL at 48 h.p.i. (6.00−6.83 TCID50/mL) in BHK, Vero, BFK and Hela cell lines, while its replication in the MDBK cell line (104.67 TCID50/mL) was less efficient , while the remaining samples were negative after five passages. Compared to mock-infected cells A, MRV-inlar CPEs B–G. The 8 h.p.i. H. The refficient I.Electron microscopy of the isolate QAPpC66 in PK-15 at passage 6 showed three morphotypes of virus particles A. The ma3 genome copies/g at 7 d.p.i., and were detectable until 14 d.p.i. with virus loads decreased to 9 × 102–2.1 × 103 genome copies/g in some liver and brain tissues. The virus was able to replicate in the brain and lung tissues of two of three OG mice with virus loads of 3.9–4.0 × 103 copies/g at 7 d.p.i., and the replication in lung continued until 21 d.p.i. with a virus load of 4.1 × 102 genome copies/g.To assess pathogenicity, the isolate QAPpC66 was orally and intracranially inoculated into suckling mice. Generally, these inoculated mice did not show overt clinical signs throughout the observation with a few animals at 2–5 d.p.i. demonstrating slight depression. The necropsy of animals at 7 d.p.i. showed that one mouse in the intracranial group (IG) had needle-like hemorrhages in the lung (data not shown), while two of the three mice in the oral group (OG) demonstrated thinner intestinal walls than those of the control group. qRT-PCR detection showed that the virus was positive in the brain, lung and kidney tissues of one of three IG animals with virus loads of 1.9–4.5 × 10Isolates QAPpC11, 15 and 66 were all subjected to RNAseq. After de novo assembly and blastx annotation, contigs corresponding to the 10 segmented genes were successfully obtained for the three isolates. They were 23,268 bp, 23,424 bp and 23,352 bp in length, all covering the CDS regions. Nevertheless, the UTRs of 5’ and 3’ ends could not be determined. They were 99.7–100% similar to each other at nt level with the same lengths of encoding genes, indicating that they were isolates of the same virus. Comparison with other known reference sequences available in GenBank showed that these segments were closely related to MRVs originating from human, cattle, deer, bat and civet, with as high as 98.2% nt similarities , and theTo reveal reassortment events among these viruses, a comprehensive reassortment analysis was conducted using BtMRV-QAPpC66 as a query. Several reassortment events were detected in the junction regions of these segments A. ConsisOrthoreoviruses have been increasingly detected in bats worldwide, with many of them capable of infecting humans and other animals ,44,45. HThe virus isolated here is closely genetically related to other bat-borne MRVs with as high as ~98% nt similarities in segments M2, M3, and S4 , but it The phylogenetic and reassortment analyses revealed an intricate reassortment network of MRVs among bats, humans and other animals. However, we realized that some reassortments did not occur recently, and even required other intermediate events. For example, the reassortment of S1 was predicted to occur between QAPpC66 and an American deer MRV strain OV24, but the two segments were just 92.4% identical and were from two different continents . Thus, tIn summary, these BtMRVs have extensive host tropisms, making them easily spread to different animal species by multiple transmission routes. With the advantage of their genome segmentation, they can exchange genomic segments as the co-infection of multiple viruses occurs in a cell or a host, resulting in a new reassortant ,39,47. M


Henipavirus [Henipaviruses, such as Nipah and Hendra, are enveloped, negative-strand RNA viruses belonging to the Paramyxoviridae family [After the two major viral outbreaks of COVID-19 and monkeypox, a new viral outbreak due to the Langya virus (LayV) was identified in febrile patients from the eastern Chinese provinces of Shandong and Henan [ipavirus . The Hene family . The Hene family . The Nipe family . Recent e family ,6. Also,e family .According to a recent sentinel surveillance report (April 2018–August 2021) from China, 35 febrile patients, mostly farmers and factory workers with a recent history of animal contact, tested positive for the LayV virus. Most patients were from the Shandong and Henan provinces in eastern China. A distinct phylogenetic LayV was isolated from a 53-year-old febrile patient’s throat swab utilizing a metagenomic approach. The acute and convalescent serum samples from 14 infected individuals demonstrated a fourfold rise in IgG antibody titer. The majority of patients presented with fever and other constitutional symptoms . SimilarMojiang henipavirus. The Mojiang virus is rat borne and was responsible for three deaths in Chinese miners in 2012 due to pneumonia [Henipaviruses [Henipaviruses that can be used for LayV. The only sensible public health interventions at present are surveillance  and restricting contact with affected animals.In a serosurvey of domestic animals, goats and dogs were seropositive at 2 and 5%, respectively. Out of the 25 species of wild small mammals that were examined, LayV RNA was primarily detected in shrews (27%) [neumonia . As we waviruses . There aTaiwan’s health department is currently monitoring the spread of LayV near the China–Taiwan border by instituting standardized molecular assay-based diagnostics . Nepal aGlobally, it is estimated that 70% of emerging infectious illnesses are transmitted to humans through interactions with animals as human populations expand into natural areas . AccordiTo put things in perspective, amidst the two major viral outbreaks plaguing the world at present time, the emergence of a new virus belonging to a genus that houses the Nipah virus, one of the most fatal infections of all time, can be a cause of concern. Despite the possibility of severe manifestations in immunocompromised, it must be noted here that based on the current knowledge, LayV is not fatal. Although current evidence suggests that LayV is not transmitted from human-to-human, there is a possibility that the virus may evolve and acquire this trait. Nipah, for example, was not transmitted from human-to-human in the first outbreak described from Malaysia, but a high percentage of human-to-human transmission was described in India and Bangladesh in the latter outbreaks . Therefo


Favipiravir is a nucleoside analogue that inhibits the replication and transcription of a broad spectrum of RNA viruses, including pathogenic arenaviruses. In this study, we isolated a favipiravir-resistant mutant of Junin virus (JUNV), which is the causative agent of Argentine hemorrhagic fever, and analyzed the antiviral mechanism of favipiravir against JUNV. Two amino acid substitutions, N462D in the RNA-dependent RNA polymerase (RdRp) and A168T in the glycoprotein precursor GPC, were identified in the mutant. GPC-A168T substitution enhanced the efficiency of JUNV internalization, which explains the robust replication kinetics of the mutant in the virus growth analysis. Although RdRp-N462D substitution did not affect polymerase activity levels in a minigenome system, comparisons of RdRp error frequencies showed that the virus with RdRp-D462 possessed a significantly higher fidelity. Our next generation sequence (NGS) analysis showed a gradual accumulation of both mutations as we passaged the virus in presence of favipiravir. We also provided experimental evidence for the first time that favipiravir inhibited JUNV through the accumulation of transition mutations, confirming its role as a purine analogue against arenaviruses. Moreover, we showed that treatment with a combination of favipiravir and either ribavirin or remdesivir inhibited JUNV replication in a synergistic manner, blocking the generation of the drug-resistant mutant. Our findings provide new insights for the clinical management and treatment of Argentine hemorrhagic fever. Development of antivirals requires cautious and extensive assessment of action mechanism as well as potential for emergence of resistant phenotype of the virus. In recent years, favipiravir has been put forward as a promising candidate for the treatment of Argentine hemorrhagic fever (AHF) caused by Junin virus (JUNV). We, therefore, aimed to provide experimental evidence on action mechanism of favipiravir to help guide its clinical use. Here we show that favipiravir causes lethal mutation that impairs virus infectivity. More importantly, we demonstrate that the virus has the capability to escape favipiravir selective pressure by acquiring two amino acid substitutions on glycoprotein precursor and polymerase proteins. This observation raises concern over the use of only favipiravir in therapeutic regimens. To overcome this risk, we show that combination of favipiravir with other nucleoside analogues demonstrates a synergistic effect and suppresses the ability of JUNV to escape drug pressure. Favipiravir, ribavirin, and remdesivir have a broad spectrum of antiviral activity. Therefore, combination therapies of these drugs would be expected to have potential therapeutic effects for not only AHF but also the diseases caused by a variety of viruses, including emerging RNA viruses. Argentine hemorrhagic fever (AHF) is a severe zoonotic disease that is endemic in Argentina and caused by Junin virus (JUNV). In addition to the intense clinical course of the disease, the lack of approved therapeutics and preventive countermeasures against JUNV highlights its significant threat to global public health –3. CurreFavipiravir  is a purine analogue originally developed as an antiviral agent for influenza and subsequently reported to inhibit the replication of broad spectrum of RNA viruses . FavipirThe emergence of drug-resistant mutants abolishes the effect of antiviral drugs in the short term and therefore imposing a public health risk. To date, experimental isolation of favipiravir-resistant mutants has only been reported for chikungunya virus  and ente50 value was determined by a dose-response experiment. 293T cells were infected with Candid #1 virus at a multiplicity of infection (MOI) of 0.1, in the presence of a series of favipiravir dilutions or DMSO. Quantification of viral titers at 48 hours post infection (hpi), showed favipiravir IC50 to be 4.9 μM with 95% confidence interval (CI) of 4.5 μM to 5.4 μM. This value is comparable with that of a previous study reporting favipiravir IC50 for JUNV in Vero cells ) and RdRp-D462 (IC50 = 289.8–362.1 μM [95% CI]) was observed, although RdRp-D462 showed a slightly increased resistance to favipiravir compared with that of RdRp-N462 . No cytotoxicity was observed in this assay . While lower concentrations of favipiravir only slightly inhibited MG expression, we observed that the inhibition of luciferase signal at higher concentrations (≥200 μM) is associated with the reduction of mRNA levels, suggesting favipiravir mainly inhibits our minigenome system via a chain termination-mediated mechanism. .Next, we investigated whether N462D substitution affects RdRp activity in Candid #1. A minigenome (MG) system based on the S segment of Candid #1 virus was constructed, and an MG assay in absence of favipiravir was performed. To ensure that both RdRp-N462 and -D462 plasmids had comparable expression levels, both proteins were tagged with a FLAG peptide and similar expression levels were confirmed by western blot analysis  the anti-JUNV effect of favipiravir was significantly higher when combined with ribavirin (ZIP synergy score: 14.02) or remdesivir (ZIP synergy score: 15.82) without any significant antagonistic effect. No cytotoxicity was associated with any of the tested drug combinations. Despite our attempt to isolate a resistant variant to combinational treatments, no resistant variant was generated even after 15 passages .Combination antiviral therapy is a promising approach to minimize the risk of the emergence of drug resistance and enhance the antiviral effect. Therefore, we investigated the inhibitory effect of favipiravir in combination with ribavirin and remdesivir. As shown in In this study, we attempted to understand the mechanism of antiviral action of favipiravir against JUNV by isolating resistant variants. In our approach, lower concentrations of favipiravir for three initial passages followed by higher concentrations for the remaining passages were used. This gradual increase in selective pressure allowed adequate levels of replication to continue without a sudden exposure of viral sub-populations to the lethal concentrations of favipiravir , thus enP = 0.125) reduction in reporter activity without any major impact on polymerase function . However, comparisons of mutation frequencies of Candid #1 and Candid #1-res virus revealed a significant reduction in polymerase error number in the case of RdRp-D642, suggesting an important role of this residue in polymerase fidelity  identified in this study was found to be within the GP1 subunit of the glycoprotein complex (GPC). Arenavirus GPC is a precursor protein that forms a trimer of stable signal peptides, GP1, and GP2 subunits, upon maturation by cellular enzymes. During virus entry, GP1 and GP2 are responsible for the recognition of receptors and the fusion with endosome membranes, respectively . The GPCstulated , 59. Whistulated , highlig. Furthermore, we showed that it was difficult to isolate JUNV variants that were resistant to the combination treatment . These findings suggest the potential of combination therapies for favipiravir with ribavirin or remdesivir.Here, we experimentally demonstrated that the potency of favipiravir could be significantly enhanced against JUNV if used in combination with ribavirin or remdesivir In conclusion, we described the isolation of a high replication fidelity variant of arenavirus with reduced susceptibility to favipiravir. More importantly, we provide experimental evidence that hyper-mutagenesis is the primary mechanism of favipiravir action against JUNV. Consistent with our observations, studies on favipiravir treatment of non-human primates infected with Lassa virus showed a reduction in virus infectivity without affecting viral load, providing evidence that favipiravir is primarily a mutagen against old world arenaviruses , 62. OurFurthermore, the antivirals used in this study, namely, favipiravir, ribavirin, and remdesivir, have been reported to have a broad spectrum of antiviral activity. Therefore, combination therapies of these drugs are expected to have a potential therapeutic effect for not only AHF but also the diseases caused by a variety of viruses, including emerging RNA viruses, as the antiviral regimen significantly decreases the possibility of drug-resistant mutant appearance.Human embryonic kidney (293T) and African green monkey kidney (Vero 76) cell lines were maintained in Dulbecco’s modified Eagle’s medium  with 10% fetal bovine serum (FBS) and 1% penicillin and streptomycin. The Candid #1 vaccine strain of JUNV was kindly provided by Dr. Juan C. de la Torre  . Favipir2. For the quantification of viral titers, a plaque assay was performed according to standard procedures using 10-fold dilutions of the samples in Vero 76 cells as previously described  A letter containing a detailed list of your responses to the review comments and a description of the changes you have made in the manuscript. Please note while forming your response, if your article is accepted, you may have the opportunity to make the peer review history publicly available. The record will include editor decision letters (with reviews) and your responses to reviewer comments. If eligible, we will contact you to opt in or out.[2] Two versions of the revised manuscript: one with either highlights or tracked changes denoting where the text has been changed; the other a clean version (uploaded as the manuscript file).Important additional instructions are given below your reviewer comments.Please prepare and submit your revised manuscript within 60 days. 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Ross, PhDSection EditorPLOS PathogensSusan RossSection EditorPLOS PathogensKasturi HaldarEditor-in-ChiefPLOS Pathogensorcid.org/0000-0001-5065-158XMichael MalimEditor-in-ChiefPLOS Pathogens <span style="font-family: "Noto Sans", sans-serif; font-size: 12px;"> orcid.org/0000-0002-7699-2064 </span> ***********************There are several issues that preclude acceptance of this manuscript in its current form, including placing the work in appropriate context of the field.Reviewer's Responses to QuestionsPart I - SummaryPlease use this section to discuss strengths/weaknesses of study, novelty/significance, general execution and scholarship.Reviewer #1: The manuscript covers ground that has been previously examined by a number of groups, many of which are referenced. Previous studies have shown that favipiravir acts as a purine analog (antagonized by authentic purine bases) that introduces mutations and, among other modes of action, pushes the virus to lethal mutagenesis. While the authors conclude that favipiravir resistance can be mediated through increased RdRP fidelity, the evidence for such, and the effect of the N462D mutation itself, is weak (see below). Additionally, others have reported the favipiravir will synergize with ribavirin, another purine analog whose mechanism of action is also complex. Prior studies have shown that ribavirin induces transition mutations, and the authors show the same for favipiravir. Indeed, transitions represent predominant mutations in various RNA viruses, even in the absence of base analogs.The authors description of the A168T mutation in the virus glycoprotein that arises in favipiravir-resistant virus suggests that they are unaware of the significant literature on this mutation. T168A is one of the important attenuating mutations that arose upon selection of the attenuated Candid#1 vaccine strain of JUNV. The mutation removes a glycosylation site and extensive studies from the Paessler lab suggest that the lack of glycosylation results in GPC instability, reduced GPC levels and induction of the unfolded protein response. The Nunberg lab has shown that this mutation in Candid#1 readily reverts back to 168T upon passage in cell culture as the virus is stressed, either by culture conditions or by unrelated mutation (or by favipiravir?). It is possible that by making the virus more fit, the A168T reversion event can contribute to overcoming the barrier imposed by favipiravir. As the effective load of the more-fit virus is greater, more virions will survive an antiviral effect that renders 90% of the virus population noninfectious. An analogous situation – where fitness is enhanced in a manner that overcomes the initial  defect - is described by Chen et al PPath 17(12) e1010191. The authors’ reported decrease in specific infectivity in the resistant virus may thus be the result of the N462D mutation in RdRP, as A168T is expected and indeed reported by the authors to increase infectivity .Although increased fidelity has been shown to mitigate the effects of base analogs in other systems, the authors’ evidence in this case is weak. The reported reduction in base substitutions in the resistant mutant vs wild-type in the presence of favipiravir is not obviously greater than that reported in the presence of DMSO , although the absolute numbers are greater in the wild-type than in the mutant . Firstly, these numbers are small and conclusions would be strengthened by using global RNA-Seq analysis rather than Sanger sequencing of some 50 individual 450-base clones of NP. . More importantly, the reduction in base substitutions may reflect the inhibitory effect of favipiravir treatment on virus replication  and the reduced specific infectivity of the mutant, which would combine to diminish the amount of infectious progeny at each round of virus replication and thereby the amount of RNA replication and the opportunity for mutation. Additional studies are needed to clarify the significance of both mutations. It would be interesting to see if N462D arises upon passage of recombinant A168T Candid#1, or if the A168T virus is itself less sensitive to favipiravir. If the mutation were to increase fidelity, then we might expect to see stronger antagonism by authentic purine bases  but this study was not performed. If RdRP fidelity is increased, it does not appear to extend to ribavirin or remdesivir as the favipiravir-resistant virus remains sensitive to these , suggesting that N462D, if indeed responsible for enhanced fidelity, may be specific to favipiravir. Taken together, it is difficult to assert that RdRP fidelity is increased or that N462D is responsible for this or favipiravir resistance.Specific scientific and editorial comments follow, in page order.1. line 58-59: might re-order to read “...disease that is endemic in Argentina and caused by...”2. line 61: rather than the “few reports” on the use of immune plasma therapy, the authors should know that this therapy is used routinely in Argentina.3. line 83: might replace “antiviral” with the more specific “favipiravir”, and “high” with “strong”.4. line 93: might replace “invalidates” with “abolishes”.5. line 107-109: please clarify – as Candid#1 vaccination is quite effective, why would one need to consider favipiravir therapy?6. para starting line 112: might introduce and provide background information on Candid#1.7. line 126: this is the first and only mention of “defective interfering particles”, a term that has specific meaning in virology (and in some arenavirus work). If the authors mean to refer to non-infectious genomes containing favipiravir-induced mutations, they might stick with the this nomenclature to avoid confusion with DIPs. Nowhere is “interference” with infection by noninfectious particles discussed.8. line 146: by “same mutations...as P0 parental virus”, are the authors referring to Candid#1-specific changes relative to pathogenic JUNV (including A168)? One wouldn’t expect too many other changes in a P0 stock. Please clarify.9. Fig 3: it is remarkable that the authors find extensive virus production at 8 hr post-infection (~3.5 log10 vs ~4.1 log10 at 24 hr). This is a considerably shorter eclipse period than commonly reported by others.10. line 179: please clarify or cite how the “tolerable threshold of error catastrophe” is “estimated”.11. line 192: the error bias is observed in the virus population and not the “transitional mutations”. Do the authors refer to “transition” substitutions, mutation that arise transiently during passage, or mutants (vs mutations) that arise on passage? Please clarify.12. line 208-210: It is likely that multiple rounds of infection take place in 48 hr after a low moi infection, and thus it likely that the lack of difference in final RNA copy number reflects more the duration of each cycle  than to any differential inhibition. Please clarify how RNA copy number is the same despite differential sensitivity to favipiravir.13. line 223, Fig 6A: it is unclear how much weight to place on the one point showing a difference (16 hr) when the two other points (8 and 24 hr) show no difference. Thus, the notion that entry is more rapid in the 168T pseudovirus is questionable.14. line 243: might specify that “no...effect” is in the absence of favipiravir.15. line 249-251 (and line 291): please explain how Fig 7F speaks to chain termination?16. line 267-269: might reword that it’s the gradual increase in selective pressure  that enables adequate levels of replication to support mutation and the emergence of resistant viruses. This is a typical design for selection.17. line 293: the authors claim ribavirin is non-mutagenic, yet literature claims otherwise? Please clarify.18. line 321-323, and Fig S4: the failure to identify resistance to the combination of favipiravir and ribavirin/remdesivir is rather unsurprising – at a minimum, one would need 2 (vs 1) mutation, and it is unclear whether the effective (synergistic) potency was considered in establishing selection conditions – selection may fail if replication is excessively reduced. However, Fig S4 appears to show no antiviral effect of the combination, as DMSO and combination tracks overlap . This too would explain the absence of selection. Please explain.Reviewer #2: In “Potential and action mechanism of favipiravir as an antiviral against Junin virus” Zadeh et al. describe the generation of favipiravir-resistant JUNV mutants, and further investigate the potential mechanisms of resistance. The objectives of the study are well defined, with a strong experimental method, clear figures, and well written results and discussion sections. The data presented is sound, and represents a thorough investigation into the resistance mechanisms conferred by the two mutations identified.The main comments I have on the data that would strengthen the manuscript are that: 1) relative appearance of the two identified mutations; 2) some data that has been generated is not included in this manuscript (data not shown); and 3) no data investigating these mutations in the more clinically relevant virulent JUNV strains.**********Part II – Major Issues: Key Experiments Required for Acceptanceabsolutely required to validate study conclusions.Please use this section to detail the key new experiments or modifications of existing experiments that should be Generally, there should be no more than 3 such required experiments or major modifications for a "Major Revision" recommendation. If more than 3 experiments are necessary to validate the study conclusions, then you are encouraged to recommend "Reject".Reviewer #1: The authors description of the A168T mutation in the virus glycoprotein that arises in favipiravir-resistant virus suggests that they are unaware of the significant literature on this mutation. T168A is one of the important attenuating mutations that arose upon selection of the attenuated Candid#1 vaccine strain of JUNV. The mutation removes a glycosylation site and extensive studies from the Paessler lab suggest that the lack of glycosylation results in GPC instability, reduced GPC levels and induction of the unfolded protein response. The Nunberg lab has shown that this mutation in Candid#1 readily reverts back to 168T upon passage in cell culture as the virus is stressed, either by culture conditions or by unrelated mutation (or by favipiravir?). It is possible that by making the virus more fit, the A168T reversion event can contribute to overcoming the barrier imposed by favipiravir. As the effective load of the more-fit virus is greater, more virions will survive an antiviral effect that renders 90% of the virus population noninfectious. An analogous situation – where fitness is enhanced in a manner that overcomes the initial  defect - is described by Chen et al PPath 17(12) e1010191. The authors’ reported decrease in specific infectivity in the resistant virus may thus be the result of the N462D mutation in RdRP, as A168T is expected and indeed reported by the authors to increase infectivity .Although increased fidelity has been shown to mitigate the effects of base analogs in other systems, the authors’ evidence in this case is weak. The reported reduction in base substitutions in the resistant mutant vs wild-type in the presence of favipiravir is not obviously greater than that reported in the presence of DMSO , although the absolute numbers are greater in the wild-type than in the mutant . Firstly, these numbers are small and conclusions would be strengthened by using global RNA-Seq analysis rather than Sanger sequencing of some 50 individual 450-base clones of NP. . More importantly, the reduction in base substitutions may reflect the inhibitory effect of favipiravir treatment on virus replication  and the reduced specific infectivity of the mutant, which would combine to diminish the amount of infectious progeny at each round of virus replication and thereby the amount of RNA replication and the opportunity for mutation. Additional studies are needed to clarify the significance of both mutations. It would be interesting to see if N462D arises upon passage of recombinant A168T Candid#1, or if the A168T virus is itself less sensitive to favipiravir. If the mutation were to increase fidelity, then we might expect to see stronger antagonism by authentic purine bases  but this study was not performed. If RdRP fidelity is increased, it does not appear to extend to ribavirin or remdesivir as the favipiravir-resistant virus remains sensitive to these , suggesting that N462D, if indeed responsible for enhanced fidelity, may be specific to favipiravir. Taken together, it is difficult to assert that RdRP fidelity is increased or that N462D is responsible for this or favipiravir resistance.Reviewer #2: Firstly, in figure 1 it is apparent that between P10 and p11 the resistant mutation (with the 2 mutations) was generated. However, the authors only present sequence data from the P0 and P11 strains. I think the manuscript would be strengthen if the viruses at some of the intermediate passage numbers were also sequenced, and therefore the relative appearance of each individual mutation could be elucidated. From a readers point of view, I would be interested in knowing  whether one came before the other, or did both appear at P11 together? This would also generate data which could strengthen the conclusions on the discussion, knowing which of the mutations (if any) is the more relevant for the resistance observed here.Secondly, one comment I had regarding the GPC-A168T mutation was regarding the effect this had on cellular entry, specifically attachment. Although the experiment was performed (with the pseudotype) these data were not included in the manuscript. I think however that, if possible, these data should be included as a panel in figure 6 as I believe, even if it is negative data, it is an important finding. It would also clearly demonstrate the thoroughness to which the authors have investigated the potential mechanisms of the GPC-A168T mutation.Finally, although the authors state that there is no reverse genetics system for JUNV, there is published data of a system . However, I understand that without a BSL4 lab this would be impossible to set up, I wondered if the authors had given any thought to collaborating with a lab that does have this facility to generate a JUNV with these mutations to look at resistance in more clinically relevant strains?**********Part III – Minor Issues: Editorial and Data Presentation ModificationsPlease use this section for editorial suggestions as well as relatively minor modifications of existing data that would enhance clarity.Reviewer #1: Specific scientific and editorial comments follow, in page order.1. line 58-59: might re-order to read “...disease that is endemic in Argentina and caused by...”2. line 61: rather than the “few reports” on the use of immune plasma therapy, the authors should know that this therapy is used routinely in Argentina.3. line 83: might replace “antiviral” with the more specific “favipiravir”, and “high” with “strong”.4. line 93: might replace “invalidates” with “abolishes”.5. line 107-109: please clarify – as Candid#1 vaccination is quite effective, why would one need to consider favipiravir therapy?6. para starting line 112: might introduce and provide background information on Candid#1.7. line 126: this is the first and only mention of “defective interfering particles”, a term that has specific meaning in virology (and in some arenavirus work). If the authors mean to refer to non-infectious genomes containing favipiravir-induced mutations, they might stick with the this nomenclature to avoid confusion with DIPs. Nowhere is “interference” with infection by noninfectious particles discussed.8. line 146: by “same mutations...as P0 parental virus”, are the authors referring to Candid#1-specific changes relative to pathogenic JUNV (including A168)? One wouldn’t expect too many other changes in a P0 stock. Please clarify.9. Fig 3: it is remarkable that the authors find extensive virus production at 8 hr post-infection (~3.5 log10 vs ~4.1 log10 at 24 hr). This is a considerably shorter eclipse period than commonly reported by others.10. line 179: please clarify or cite how the “tolerable threshold of error catastrophe” is “estimated”.11. line 192: the error bias is observed in the virus population and not the “transitional mutations”. Do the authors refer to “transition” substitutions, mutation that arise transiently during passage, or mutants (vs mutations) that arise on passage? Please clarify.12. line 208-210: It is likely that multiple rounds of infection take place in 48 hr after a low moi infection, and thus it likely that the lack of difference in final RNA copy number reflects more the duration of each cycle  than to any differential inhibition. Please clarify how RNA copy number is the same despite differential sensitivity to favipiravir.13. line 223, Fig 6A: it is unclear how much weight to place on the one point showing a difference (16 hr) when the two other points (8 and 24 hr) show no difference. Thus, the notion that entry is more rapid in the 168T pseudovirus is questionable.14. line 243: might specify that “no...effect” is in the absence of favipiravir.15. line 249-251 (and line 291): please explain how Fig 7F speaks to chain termination?16. line 267-269: might reword that it’s the gradual increase in selective pressure  that enables adequate levels of replication to support mutation and the emergence of resistant viruses. This is a typical design for selection.17. line 293: the authors claim ribavirin is non-mutagenic, yet literature claims otherwise? Please clarify.18. line 321-323, and Fig S4: the failure to identify resistance to the combination of favipiravir and ribavirin/remdesivir is rather unsurprising – at a minimum, one would need 2 (vs 1) mutation, and it is unclear whether the effective (synergistic) potency was considered in establishing selection conditions – selection may fail if replication is excessively reduced. However, Fig S4 appears to show no antiviral effect of the combination, as DMSO and combination tracks overlap . This too would explain the absence of selection. Please explain.Reviewer #2: Figure 1: I think it would be helpful to indicate on the x-axis where the dose of T705 was increased, or indicate the dose at each passage maybe?Figure 6A: I think y-axis should say normalized? And indicate % as a unit?Line 344: Please list Genbank number of Cd#1 strain used (if available)Line 733: “LOD” not shown on graph but listed in legend.Line 741: Should say “cells” and not “cell”.Line 747: LOD is shown on graph but not explained in legend.Line 761: Unsure how the n is 8 for this?Line 815: Unsure how the n is 6 for this?Lie 831: Should say “cells” and not “cell”.**********what does this mean?). 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Read more information on sharing protocols at  7 Jun 2022AttachmentResponse letter_Vahid et al-final.docxSubmitted filename: Click here for additional data file. 19 Jun 2022Dear Prof. Yasuda,We are pleased to inform you that your manuscript 'Potential and action mechanism of favipiravir as an antiviral against Junin virus' has been provisionally accepted for publication in PLOS Pathogens.Before your manuscript can be formally accepted you will need to complete some formatting changes, which you will receive in a follow up email. A member of our team will be in touch with a set of requests.Please note that your manuscript will not be scheduled for publication until you have made the required changes, so a swift response is appreciated.IMPORTANT: The editorial review process is now complete. PLOS will only permit corrections to spelling, formatting or significant scientific errors from this point onwards. 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All press must be co-ordinated with PLOS.Thank you again for supporting Open Access publishing; we are looking forward to publishing your work in PLOS Pathogens.Best regards,Amy L Hartman, PhDAssociate EditorPLOS PathogensSusan RossSection EditorPLOS PathogensKasturi HaldarEditor-in-ChiefPLOS Pathogensorcid.org/0000-0001-5065-158XMichael MalimEditor-in-ChiefPLOS Pathogens <span style="font-family: "Noto Sans", sans-serif; font-size: 12px;"> orcid.org/0000-0002-7699-2064 </span> ***********************************************************Reviewer Comments :Reviewer's Responses to QuestionsPart I - SummaryPlease use this section to discuss strengths/weaknesses of study, novelty/significance, general execution and scholarship.Reviewer #2: The authors have addressed all of my concerns and incorporated the edits/suggestions.**********Part II – Major Issues: Key Experiments Required for Acceptanceabsolutely required to validate study conclusions.Please use this section to detail the key new experiments or modifications of existing experiments that should be Generally, there should be no more than 3 such required experiments or major modifications for a "Major Revision" recommendation. If more than 3 experiments are necessary to validate the study conclusions, then you are encouraged to recommend "Reject".Reviewer #2: The authors have addressed all of my concerns and incorporated the edits/suggestions.**********Part III – Minor Issues: Editorial and Data Presentation ModificationsPlease use this section for editorial suggestions as well as relatively minor modifications of existing data that would enhance clarity.Reviewer #2: The authors have addressed all of my concerns and incorporated the edits/suggestions.**********what does this mean?). If published, this will include your full peer review and any attached files.PLOS authors have the option to publish the peer review history of their article  are generated on a different schedule and may not be made available as quickly.Soon after your final files are uploaded, the early version of your manuscript, if you opted to have an early version of your article, will be published online. The date of the early version will be your article's publication date. The final article will be published to the same URL, and all versions of the paper will be accessible to readers.Thank you again for supporting open-access publishing; we are looking forward to publishing your work in PLOS Pathogens. Best regards,Kasturi HaldarEditor-in-ChiefPLOS Pathogensorcid.org/0000-0001-5065-158XMichael MalimEditor-in-ChiefPLOS Pathogensorcid.org/0000-0002-7699-2064