The UVC treatment caused a decrease over 4 log10 in SwPV titer on the polystyrene surface, whereas it consistently reduced about 5 log10 in PPV and SVA samples. No viable virus was recovered from PRRSV, PEDV, SIV, and BVDV samples. In filter paper, conversely, the efficacy was reduced. This study provides essential information on the inactivation effectiveness of a specific dose of UVC on important veterinary viruses, further supporting the rational application and strategic guidance for UVC radiation use to disinfect materials.Many swine farms employ UVC treatment in employees’ personal belongings and small tools entering farms as part of the biosecurity protocol to decrease the risk of pathogen introduction into the operation. However, the UVC efficacy in some veterinary viruses is not fully evaluated. This study evaluated the efficacy of ultraviolet type C (UVC) radiation in inactivating seven relevant veterinary viruses: Swine Poxvirus (SwPV), Porcine Reproductive and Respiratory Syndrome Virus (PRRSV), Porcine Epidemic Diarrhea Virus (PEDV), Swine Influenza Virus (SIV), Bovine Viral Diarrhea Virus (BVDV), Porcine Parvovirus (PPV), and Senecavirus A (SVA). The experimentally contaminated materials included polystyrene and filter paper. The samples were exposed to UVC for 5 min (total dose of 360 mJ/cm Controlling the spread of diseases in densely populated swine farms represents a major challenge for the swine sector, requiring strict sanitary measures to support biosecurity and animal health. As part of the biosecurity protocol in many swine farms, UVC radiation chambers are used to treat objects entering farms to decrease the risk of pathogen introduction into the operation ,2. The tPestivirus genus, which also includes the Classical Swine Fever Virus (CSFV). Additionally, the non-enveloped virus, Porcine Parvovirus (PPV), and Senecavirus A (SVA) were used.The various methodologies and equipment employed in prior studies hamper comparisons of the UVC treatment efficacy in inactivating different viruses ,10,11,122 in inactivating seven viruses on experimentally contaminated surfaces. The results for virus inactivation on the polystyrene (non-porous) surface and the filter paper material (porous surface) are shown in The present study tested the efficacy of a UVC dose of 360 mJ/cmAsfarviridae) may be susceptible to UVC inactivation. However, specific studies using ASF are required.After exposure to UVC radiation, there was a consistent decrease in the SwPV titer of about 4 log10 and 3 log10, respectively, in the polystyrene and filter paper material A. Poxvir4.75 TCID50, was completely inactivated on a polystyrene surface subjected to UVC treatment. However, when present on a porous surface, the titer reduction was limited to about 3 log10 . After a 20 to 30 min drying time followed by 10 min UVC treatment, no viable virus was recovered [In the current study, no infectious PEDV was recovered from either of the tested surfaces following treatment with a UVC dose of 360 mJ/cm 3 log10 C. Similaecovered .5.7 TCID50 in the control samples. Conversely, in the porous material, the treatment led to partial inactivation with a ~ 2 log 10 titer reduction   E.Although non-enveloped viruses, represented in our study by SVA and PPV, were not completely inactivated in polystyrene and porous material, a significant titer reduction for both viruses was observed. A 5 log10 titer reduction was consistently achieved for SVA in the polystyrene surface, whereas at least ~ 2 log10 reduction was reached in paper material F. SimilaUVC treatment of PPV-contaminated surfaces led to significant titer reduction in both materials, with an approximate 5 log10 for contaminated polystyrene and 4 log10 for contaminated filter material G. PPV in2 applied over 5 min) on the inactivation of seven veterinary relevant viruses. Complete inactivation of enveloped viruses in the polystyrene material was noted. However, a viable virus was retrieved from non-enveloped SVA and PPV. Typically, most of the materials treated in farms using UVC are clean personnel belongings such as food containers, cell phones, and small tools. Whereas the materials used in the current study may represent a significant percentage of the surface types exposed to UVC in swine farms, it should be emphasized that the virus inactivation level may vary in the various surface types and materials. It is critical to emphasize that the treatment will likely be significantly less effective on surfaces containing organic matter, which hampers direct UVC light exposure. In addition, the porosity degree or absorbing characteristics significantly impact UVC treatment effectiveness. UVC as a standalone disinfection protocol for surfaces contaminated with organic matter or porous surfaces may increase the risk of virus introduction into farms. Therefore, using additional decontamination steps in these situations would be critical for farm biosecurity.The study provides information on the effectiveness of a UVC treatment  and supplemented with 10% fetal bovine serum , 2 mM L-glutamine (Corning®), 1% Antibiotic-Antimycotic 100 X  and gentamicin . The cell cultures were maintained in an incubator at 37 °C supplemented with 5% CO2. The media composition for virus amplification was as described above except for PEDV and SIV amplification. The media used for PEDV and SIV amplification was FBS-free and supplemented with 2 µg/mL of TPCK trypsin . The virus stocks were titrated in duplicate using the Reed and Muench method [50) (The cell culture lines to amplify the viruses used in this study are listed in h method . The virhod [50) .®, UVC pass-through germicidal chamber (PTC)  was used. Four 15 W UVC lamps were present in the chamber. The lamps were distributed in the upper and bottom sides of the units. Two different types of surfaces were evaluated: a polystyrene surface (plastic) and a porous surface . The UVC radiation dose applied in the samples was monitored using Lutron UVC 254 nm Ultraviolet Light Meter (UVC-254SD) Data Logging. The UVC meter probe was placed adjacent to the tested samples.The overview of the study design is depicted in 2 of the paper filter. The samples were allowed to dry for approximately 30 min in a biosafety cabinet type A2. Dry samples were placed in a biocontainment and moved to the UVC chamber. In the chamber, samples were removed from the biocontainment and placed in the center of the chamber, about 50 cm from the light bulb located in the upper part of the chamber. The samples were then treated for 5 min . A set of samples was kept UVC-untreated and served as a positive control. After treatment, the samples were resuspended in 1 mL of MEM, and a virus titration assay was conducted.Approximately 100 µL of each virus tested was applied to a flat plastic surface or on 1 cm®) and free of FBS. The samples were placed in the incubator at 37 °C, supplemented with 5% CO2, for 72–96 h post-inoculation. The plates were then read in an inverted light microscope. The results were obtained by the identification of the cytopathic effect, and the viral titer was calculated according to the methodology of Reed and Muench [The UVC-treated and the control samples were titrated using a limiting dilution assay in 96-well plates. Briefly, 50 µL of the viral suspension was added to the wells, with eight replicates of each dilution. Then, 100 µL of the cell suspension was added to all wells (approximately 30 thousand cells/wells). For the PEDV and SIV virus titration assay, plates were prepared 24 h prior to inoculation, and the MEM used for the virus dilution was supplemented with 2 µg/mL of TPCK trypsin (Sigma-Aldrichd Muench . The stu


To the Editor,Monkeypox is a rare zoonotic disease caused by the monkeypox virus (MPV), an enveloped double-stranded DNA virus that belongs to the Orthopoxvirus genus of the Poxviridae family. It was first discovered as an outbreak in a cluster of monkeys in a Danish research institute in 1958. The first case in a human was a 9-month-old child recognized in 1970 in the Democratic Republic of the Congo, DRC . In the Poxviruses are very stable and remain contagious for long periods of time. A study conducted in Germany provided evidence of surface contamination in hospital rooms, stating the importance of protection measures against the monkeypox virus. Healthcare providers must apply personal protective equipment (PPE) while regularly disinfecting the frequent contact spots on the body surface, double gloving, and proper hygiene of the hands, in addition to disinfection of the possibly contaminated surfaces . AvoidinPreventive measures in the setting of surgery in the time of COVID-19 as a recent experience of public health emergency could be employed in the context of monkeypox disease . Strict In addition to potential aerosol-generating procedures (AGPs) such as cardiopulmonary resuscitation, surgical smoke, suctioning, intubation, and extubation, the techniques and tools used, such as electric cautery, pulse washing, and laparoscopic suction devices in transoral robotic surgery (TORS), minimally invasive surgery (MIS), laparoscopic and video-assisted thoracoscopic surgery (VATS) procedures, and other surgeries also generate aerosols infected with the virus ,18.Among the measures that can be taken in the field of anesthesia to reduce aerosol production include the use of rapid sequence intubation (RSI), face mask ventilation, open airway suction, prioritization of using regional anesthesia if possible, and use of positive pressure ventilation ,20.2 plume, creation of a water-tight, sterile and isolated environment with sterile plastic drapes help to reduce the transmission of monkeypox and the formation of aerosol particles [Decreasing application time, improving tissue moisture to reduce surgical smoke, limitation of the use of equipment and procedures such as harmonic scalpels, use of smoke evacuation system and filtration of COarticles ,21. Restarticles . In concNot applicable.No funding was received.Mohammad Mobin Teymouri Athar, Nastaran Sarvi-Pour: Conceptualization, Investigation, Writing- original draft. Arman Shafiee: Conceptualization, Investigation, Project administration, Writing-review & editing.Not applicable.Arman Shafiee.Not commissioned, externally peer reviewed.Data sharing is available by contacting corresponding author.Not applicable.


As the fear of the coronavirus disease 2019 (COVID-19) pandemic subsides, countries around the globe are now dealing with a fear of the epidemic surrounding the prevalence of monkeypox cases in various regions. Previously endemic to regions of Africa, the majority of monkeypox cases associated with the 2022 outbreak are being noted in countries around Europe and in the western hemisphere. While contact-tracing projects are being conducted by various organizations, it is unknown how this outbreak began. Monkeypox virus is one of the many zoonotic viruses that belong to the Orthopoxvirus genus of the Poxviridae family. Monkeypox cases received global attention during the 1970s, after the global eradication of smallpox. The smallpox vaccine provided cross-immunity to the monkeypox virus. Upon the cessation of smallpox vaccine administration, monkeypox cases became more prevalent. It was not until the 2003 US outbreak that monkeypox truly gained global attention. Despite the virus being named monkeypox, monkeys are not the origin of the virus. Several rodents and small mammals have been attributed as the source of the virus; however, it is unknown what the true origin of monkeypox is. The name monkeypox is due to the viral infection being first witnessed in macaque monkeys. Though human-to-human transmission of monkeypox is very rare, it is commonly attributed to respiratory droplets or direct contact with mucocutaneous lesions of an infected individual. Currently, there is no treatment allocated for infected individuals, however, supportive treatments can be administered to provide symptom relief to individuals; Medications such as tecovirimat may be administered in very severe cases. These treatments are subjective, as there are no exact guidelines for symptom relief. With the first human case reported in 1970 in a nine-month-old boy from the Democratic Republic of Congo, monkeypox is once again causing reason for panic as outbreaks are occurring across the western hemisphere. Endemic to central and western Africa, human monkeypox is a rare viral zoonosis that has been associated with the 2003-04 outbreak in the United States (US) [Monkeypox is one of the many zoonotic viruses that belong to the Orthopoxvirus genus of the Poxviridae family, as presented in Figure The clinical presentation of the monkeypox virus is similar to that of smallpox . MonkeypGenome comparisons of the west and central African strains yielded a set of candidate genes that may be involved in the differentiating clade virulence . The opePoxviruses are large, linear, double-stranded DNA viruses with a genome size ranging from 130 to 360 kbp that replicate in the cytoplasm of vertebrate or invertebrate cells -16. DNA Due to the larger size of poxviruses, it makes it harder for viruses such as monkeypox to breach host defenses by passing through gap junctions. The larger size of the virus also makes it difficult for the virus to replicate rapidly and orthopoxviruses need a more comprehensive strategy to survive within the host . The larThese proteins that are responsible for modulatory actions against the host’s immune response can be categorized into two groups, as highlighted in Figure Poxviruses’ replication cycle provides an insight into how the replication cycle of the monkeypox virus functions. As with other viruses, poxviruses also have proteins that enable and aid the binding of the virus to a cell, membrane fusion, and entry into the host cell. In the case of poxvirus, the mature virion (MV), which has a single membrane, and the extracellular enveloped virion (EV), which has an additional outer membrane, are disrupted before the fusion . There aPoxvirus DNA replication progresses within cytoplasmic structures that originally were called Guarnieri bodies and are now commonly called factories . Each faEpidemiologyMonkeypox has presumably occurred in sub-Saharan Africa for thousands of years, ever since humans acquired the virus through direct contact with infected animals . MonkeypIn an epidemiological modeling study, the authors reported the R0 value of monkeypox to be between 1.10 and 2.40 in countries where exposure to Orthopoxvirus species is negligible, R0 is referred to as the reproductive ratio, or in other words, the degree of transmissibility of the disease . This vaAs of July 1, 2022, the Center for Disease Control and Prevention (CDC) confirms 5783 cases of monkeypox, distributed in 52 different countries around the globe . Figure Various orthopoxviruses share similar genetic and antigenic features and, as such, infection by any of these viruses may provide substantial protection against infection by the other viruses from the orthopoxvirus genus. Due to cross-immunity, an immune response against any illness caused by any orthopoxvirus will decrease the likelihood of an infection by a different orthopoxvirus. Historically, vaccination against smallpox had been shown to be protective against monkeypox . Since tEven though monkeypox was first identified in Denmark in a macaque colony, the exact animal reservoirs, and initial animal-to-human transmission of the virus remain unknown -31. It hMonkeypox virus is believed to have several modes of transmission, all of which are associated with direct contact with infected animals or the with infected humans, as depicted in Figure The most common cause of human-to-human transmission, as rare as it is considered, is respiratory droplets. Figure The common, nonspecific symptoms, as depicted in Figure The hallmark feature of monkeypox is a disseminated vesiculopustular rash . The rasFurthermore, gastrointestinal symptoms that arise by the second week of illness such as vomiting and diarrhea can contribute to severe dehydration in an infected individual . The mouAlthough bronchopneumonia is a complication of monkeypox infection, it is a complication that is more commonly noted in individuals that are co-infected with the influenza virus . The resClinical management and treatmentCurrently, there are no specific treatments for monkeypox disease, however, experience with smallpox suggests that the vaccinia vaccine, cidofovir, tecovirimat, and vaccinia immune globulin (IVG) may have a use in monkeypox treatment . As per Despite the suggested treatment interventions, supportive and symptomatic therapy has been deemed the basis of managing a monkeypox viral infection. Table Previously endemic to regions of Africa, the monkeypox virus is now becoming a global concern, with sporadic cases being confirmed in regions in the western hemisphere. With human-to-human transmission most commonly occurring via respiratory droplets or direct contact with the mucocutaneous lesions of an infected individual, social distancing and contact tracing is imperative. Monkeypox cases are being confirmed in mid-age individuals. This can be attributed to the loss of cross-immunity from the smallpox vaccine seen in older individuals. This virus replicates within the cytoplasm and matures to create a primary viremia in which the virus spreads to the local lymph nodes. Monkeypox infection is also associated with complications such as bronchopneumonia, dehydration, respiratory distress, encephalitis, etc. Of all the complications, the most feared complication is corneal scarring, as it can lead to vision loss. It is important to be able to provide the appropriate supportive treatment to ensure that the risk of these complications can be minimized as much as possible. Supportive therapy, such as applying moist occlusive dressings, may be applied in areas where the rash is highly concentrated. As cases of monkeypox cases are still being confirmed globally, organizations are focused on understanding how these cases are sporadically occurring across Europe and the western hemisphere. Investigation into any potential treatments is important, along with understanding the true extent of all the symptoms of monkeypox and the long-term effects of the virus and the symptoms.


Iflaviridae family in the order Picornavirales are frequently found in RNA sequenced from arthropods, who serve as their hosts. Here we implement a metagenomic deep sequencing approach followed by rapid amplification of cDNA ends (RACE) on viral RNA isolated from wild and captured bat guano in Washington State at two separate time points. From these samples we report the complete genomes of two novel viruses in the family Iflaviridae. The first virus, which we call King virus, is 46% identical by nucleotide to the lethal honeybee virus, deformed wing virus, while the second virus which we call Rolda virus, shares 39% nucleotide identity to deformed wing virus. King and Rolda virus genomes are 10,183 and 8934 nucleotides in length, respectively. Given these iflaviruses were detected in guano from captive bats whose sole food source was the Tenebrio spp. mealworm, we anticipate this invertebrate may be a likely host. Using the NCBI Sequence Read Archive, we found that these two viruses are located in six continents and have been isolated from a variety of arthropod and mammalian specimens.Arthropods are integral to ecosystem equilibrium, serving as both a food source for insectivores and supporting plant reproduction. Members of the  Chiroptera) regularly feed on arthropods, giving direct entry of any pathogen within these bugs to one of the largest known mammalian reservoirs of zoonotic disease [Iflaviridae [Native insectivorous bats , a leader-protein virulence factor crucial for viral replication, and a 3C-like protease for processing of viral capsid proteins VP3, VP2, VP1, and VP4 (variably present) [Iflaviruses are non-enveloped, positive-sense single stranded RNA viruses with an average genome size of 9–11 kilobases in the family of avirales . The Iflpresent) ,9,10,11.present) . Today, present) ,13,14,15Iflaviruses play an intriguing ecological role as both parasitic and commensal symbionts, and are recovered from a wide range of arthropods. Typically, this viral family is isolated from whole arthropod homogenates or environmental samples containing arthropod tissues. Recently, novel iflaviruses have been found in glandular tissues of the pink bollworm moth (a common pest of cotton fields), in whole leafhoppers that reside in rice crops, and in the midgut tissue of the Vietnamese walking stick bug ,16,17. TIxoodes holocyclus and Ixodes scapulares ticks [The majority of these iflaviruses are introduced to bats and other mammals through the consumption of infected insects. Vector born exposure however is also possible, as iflaviruses infect arthropods such as the es ticks ,21. The Iflaviridae, which we demonstrate in this study.These novel iflaviruses have largely been found through shotgun or transcriptomic sequencing. RNA metagenomic next generation sequencing (mNGS) returns rich data about the RNA virome of any given environmental or clinical specimen through random shotgun sequencing of RNA within a given sample. Increasing numbers of viral sequences are now available in the Sequence Read Archive (SRA), and a simultaneous emergence of cloud-based platforms that have made these data available to the scientific community at the petabase scale ,23,24. PIn this paper we performed mNGS on RNA extracted from bat guano in Washington State and discovered the full genomes of two novel iflaviruses. Complete genomes were resolved through rapid amplification of cDNA ends (RACE). Through data mining of the SRA, we extracted relevant information about their descriptive epidemiology and concluded that our viruses are distributed worldwide and have been isolated from a diverse set of candidate hosts.Myotis yumanensis were collected from PAWS  and frozen at −20 °C . For the 2021 samples, two fresh guano samples were collected over the course of three days from rescued bats at Happy Valley Bats in Bothell, Washington, and frozen immediately at −80 °C. All rescued bats were housed in the same shed and sequestered in nests with members of their own genus. While the bats were rescues and some sustained physical injury, to our knowledge there was no evidence of viral infection or illness. Based on anatomical study, the presumed species of each of the bats from which the guano samples originated were Myotis californicus and Eptesicus fuscus. After thawing guano at room temperature, a 1:10 dilution of each sample from each species was made by approximating one mg of the sample and submerging it in 1 mL of Roche STAR buffer. Prior to RNA extraction, samples were bead beat until completely homogenous. After bead beating, samples were spun down at 10,000 g for 60 s to pellet guano particles and 100 µL of supernatant was removed for RNA extraction. The E. fuscus sample was filtered with a 0.45 µM filter prior to extraction. Qiagen RNeasy kit was used for RNA extraction following the designated protocol with elution in 30 µL of molecular grade water [For the 2016 samples, two sets of guano samples from putative de water .Prior to cDNA synthesis, 18 µL of RNA was DNase treated using Invitrogen Turbo DNase following the recommended protocol with incubation at 37 °C for 30 min . cDNA waReads were uploaded to Chan Zuckerberg ID (CZ ID) Metagenomic Sequencing Pipeline  with bats selected as host . CZ ID iIflaviridae sequences available as of 10 February 2022 were selected for alignment using poliovirus reference sequence (NC_002058) as an outgroup [Iflaviridae ORFs were aligned in Geneious Prime through MAFFT with Blosum 30 scoring matrix, a gap penalty of 1.53, and offset value of 0.123. The RdRp sequences were then selected within this alignment based on the RdRp genome annotation from NC_027917 after validating the annotation as accurate using HHpred [Iflaviridae RdRp sequences were then again aligned with the annotated Poliovirus (NC_002058) RdRp. The Newick tree was made using a maximum likelihood distance method with IqTree multicore version 2.0.3 for Mac OS X (-m MFP -B 1000 -T AUTO) [Iflaviridae isolated from likely mixed samples such as bat guano were left unlabeled.NCBI RefSeq outgroup . The opeg HHpred . Extract-T AUTO) . Each trSELECT m.acc, m.sample_acc, m.biosample, m.sra_study, m.bioproject, tax.total_countFROM ‘nih-sra-datastore.sra.metadata‘ as m, ‘nih-sra-datastore.sra_tax_analysis_tool.tax_analysis‘ as taxWHERE m.acc = tax.acc and tax_id = 1911104BigQuery search engine version 2.34.2 was used for metadata extraction based on SRA files with reads indexed as containing King and Rolda viruses . QueriesThe BioSample accessions generated from this output were then put into Entrez Direct E-utilities efetch version 16.2 for data extraction of host and read counts for the calculation of RPM for King and Rolda virus in each sample . MetadatTo complete King and Rolda virus genomes, 5′ and 3′ rapid amplification of cDNA ends (RACE) system was implemented on the original extracted viral RNA samples we described above. All primers designed for this experiment are available in the To obtain the 5′ end of each sequence, 5′ RACE using a template specific oligo was performed following the protocol from New England Biolabs (NEB): “5′ RACE with the NEB template switching RT Enzyme mix” . All priRNA structures were generated for the first 99 nucleotides (nt) in the 5′ untranslated region (UTR) and the full length 3′ UTR of each virus using RNAFold software version 2.5.0 . The secAfter locating the longest ORF using an ATG start, segments of each translated ORF were queried in Hhpred and annotated according to the top hits. Smaller and smaller segments were interrogated in separate query submissions to annotate the remaining coding sequence to look for more distant alignments.Tenebrio molitor mealworm was used as an estimate of tissue mass (105 mg). A total of 10 whole mealworms were entirely submitted for RNA extraction in ten separate tubes. A negative control containing only Roche STAR buffer was extracted along with bat and mealworm samples to rule out contamination. Samples were bead beat until completely homogenized. A volume of 200 µL of completely homogenized guano and mealworm tissue supernatant was filtered through a 0.22 µM filter. The 200 µL filtrate was then extracted using Qiagen RNeasy protocol with an elution in 30 µL of water as described above. Single stranded cDNA was synthesized with SSIV RT as described above using a King_9223R or Rolda_8224R at a concentration of 0.2 µM. Internal qPCR standards were generated via amplification of leftover extracted bat guano RNA using Rolda 7685F-8224R and King 8812F-9524R. Amplicons were gel-purified using the Promega gel wizard kit and quantitated using the dsDNA HS kit on the Qubit 3.0 Fluorometer. Each standard amplicon was diluted down to 500,000 copies per µL  and subject to a ten-fold serial dilution. qPCR was performed using the PowerUp SYBR Green Master Mix following kit specifications using quarter reactions. An amount each of 1 µL of the standards, STAR buffer negative control, and experimental input cDNA was aliquoted into each condition with three replicates of each condition. The reaction was run at 50 °C for 5 min, 95 °C for 2 min, followed by 40 cycles of 95 °C for 1 min, 54 °C for 15 s, and 60 °C for 1 min with a final melt curve analysis at 95 °C for 15 s, 54 °C for 1 min, and a final temperature increase to 95 °C at 0.15 °C/s. The King virus reactions were Sanger sequenced for confirmation due to the low viral copy number measured. The final Sanger trace is available in the PowerUp SYBR Green Master Mix was used following the manufacturer’s protocol to approximate copy numbers of King and Rolda virus per mg of bat guano and mealworm tissue . Each baMyotis yumanensis from a local animal welfare society in July 2016. De novo assembly of 750,352 reads from specimen 16-2260 yielded 7550 nucleotide (nt) and 2897 nt contigs that further assembled to 10,193 nt contig containing a 9114 nt open reading frame (ORF) that most closely aligned at 32.7% by amino acid by blastp to deformed wing virus at the time. We named this sequence King virus strain UWV2, given the lack of definitive host species association, and deposited it in GenBank (NC_031749). Re-alignment of the 16-2260 sequencing reads to the contig revealed the likely presence of more than one strain of King virus in the specimen based on a number of mixed alleles present in the capsid region occurring at frequencies around 50%. De novo assembly of 234,984 reads from specimen 16-1811 yielded an 8593 nt contig containing an 8017 nt ORF truncated at the 3′ end that most closely aligned at 32.0% by amino acid by blastp to diaphorina citri picorna-like virus. We named this sequence Rolda virus strain UW1, again given the lack of definitive host species association, and deposited it in GenBank (KX779452). Direct alignment of 16-1811 sequencing reads to the contig also demonstrated the likely presence of more than one species of Rolda virus in the specimen based on the identification of numerous minor alleles. In addition, the second longest contig from the de novo assembly of specimen 16-1811 yielded a 6911 nt contig of King virus  that shared 89.6% nucleotide identity with King virus and we called this strain UWV2.We originally detected two novel iflaviruses while metagenomically sequencing nuclease-treated bat guano collected from two putative Myotis californicus and Eptesicus fuscus genuses from a local bat rescue operation and prepared mNGS sequencing libraries. We ran these samples through Chan Zuckerburg ID (CZ ID) which resulted in an average of 35.5 million non-host reads recovered for each of the samples after adapter/quality trimming and host subtraction. We observed between E. fuscus specimen had the greatest amount of King virus at 1240 reads per million (RPM) on target covering 95.4% of the previously deposited genome by NCBI NT GSNAP alignment, while M. californicus specimen had the greatest amount of Rolda virus at 1730 RPM on target and coverage breadth of 100% by GSNAP alignment to NCBI NT.Five years later, we returned to metagenomically sequencing bat guano from Washington State. We extracted RNA from two bat guano samples collected from Tenebrio molitor mealworm. Despite members of the Iflaviridae family often being discovered in mammalian fecal matter, it has been established that the definitive host of these viruses is arthropods. We thus suspected that both viruses would be detectable in the mealworms being fed to the bats, and attempted quantification of King and Rolda viral copy numbers in the mealworm tissue. Ten whole live blended mealworms and the bat guano homogenate were analyzed through RT-qPCR which confirmed King and Rolda viruses at detectable levels within the mealworms. The King virus had low copy numbers within the mixed mealworms at an estimated 6.6 copies per mg of mealworm tissue yielding an average cycle threshold (Ct) of 38 per replicate. Due to a high Ct and low copy number, we confirmed the amplicon specificity via Sanger sequencing. In contrast, Rolda virus was detected at substantially higher levels within mealworm tissue at 1250 copies per mg. In the guano, King virus was detected in the E. fuscus sample at a level of 250 copies per mg of guano while Rolda virus had an estimated 38 viral copies per mg of guano. These data suggest that the King and Rolda viruses we isolated from the bats were likely ingested from actively infected Tenebrio molitor mealworms.We detected these viruses in bats held as captive rescues whose sole food source was the E. fuscus and M. californicus samples to the 10,169 nt King virus and 8920 nt Rolda virus showed read coverage across nearly the entire span of the genome aside from a coverage drop off in the UTRs resolved through RACE  and a 60 nt long 3′ UTR for Rolda virus (OM670245) through Sanger sequencing of PCR amplicons. The 5′ RACE using a template switching oligo followed by deep sequencing of the gene-specific PCR amplicons resulted in 973 and 535 nt 5′ UTR regions in King and Rolda viruses based on distance to the ORF ATG start codon. Remapping of the original reads from ugh RACE . We namee family ,47. HowePicornavirales non-coding regions are integral for genome replication, translation, and viral assembly [Completion of King and Rolda virus genomes allowed for investigation of structural elements within 5′ and 3′ UTRs . Since Rassembly , we evalPicornavirales VP2, VP3, and VP1 capsid structures, in addition to 2C helicase, 3C protease, and 3D polymerase. Phylogenetic analysis of the conserved 3D polymerase compared with other Iflaviridae in NCBI RefSeq using poliovirus as an outgroup demonstrated that both King virus and Rolda virus clustered uniquely and separately with other moth iflaviruses  has been indexed for both viruses. We took advantage of this fact to understand the descriptive epidemiology of King and Rolda virus by using BigQuery to comprehensively search the SRA for these viruses. In total, we found 37 SRA records with reads per million (RPM) >10 for King virus and 16 records for Rolda virus . SimilarIflaviridae family, which, to date, lacks definitive genera. Additionally, we directly detected and quantified these viruses in a putative arthropod host, the Tenebrio molitor mealworm, and found additional potential hosts via mining of metagenomic and transcriptomic data in the Sequence Read Archive (SRA).We have reported and described the complete genomes of two novel iflaviruses identified in bat guano from Washington State. Phylogenetic placement of these viruses puts them as unique species within the Apis Mellifera honeybee—two phylogenetically distant arthropods [The early deposition of these viral sequences in 2016 allowed for rapid searching of SRA data that, to date, remains expensive and has been described sparingly for RNA viruses . From ththropods ,51. ThesIflaviridae species, including deformed wing virus and sacbrood virus. It has been established that Picornaviridae maintain proteinase genes in the 2A region (2APro) that cleave host translational initiation factors thus suppressing eukaryotic protein synthesis [Iflaviridae 2A may hinder translational inhibition by blocking GCN1-GCN20 complex formation, thus preventing activation of GCN2 and downstream phosphorylation of eIF2alpha. This would imply Iflaviridae 2A region has coding sequence responsible for decreasing host protein synthesis through eIF2alpha. The e-values for these models however are by no means definitive, and alternatively the overall homology may largely be driven by the alpha helical repeats found in HEAT repeat containing proteins.In addition to confirming canonical picorna-like virus genes within these two iflaviruses, protein homology search through HHpred identified structures resembling the GCN1 translational control protein within the 2A region. We noted similar HHPred scores in the 2A region from other ynthesis . GCN1 isynthesis ,54. The Finally, we found that folding of King and Rolda RNA in two and three dimensions resulted in identical hairpin motifs with analogous Watson–Crick base pairing between each 2D and 3D model in both 5′ and 3′ King and Rolda virus UTRs. These structures generated in silico provide insight to in vivo conformation of viral RNA in these regions which may have implicit function. Moving forward, greater use of 3D RNA structures in UTRs has the potential for revealing patterns of nucleic acid motifs that may serve as virulence factors.Picornaviridae and deformed wing virus, strongly indicating that this genome is complete. More work may be required for completing the Rolda virus genome, as the 5′ end lacks this signature. Our data generated through the SRA, that offers insight into the host range for King and Rolda viruses, will also require direct experimental confirmation to determine if they are associated with any pathogenicity in these candidate arthropod hosts. As more King and Rolda viruses and other sparsely documented members of Iflaviridae are discovered through mNGS, SRA data will clarify the significance of these arthropods as definitive hosts. In the wake of the SARS-CoV-2 pandemic and expanded viral discovery efforts in bats and other mammalian species, we anticipate widespread detection of King and Rolda viruses and other members of Iflaviridae over the coming years [Our study is chiefly limited by the lack of experimental and phenotypic data for these viruses. Certainly, more work is required to determine the definitive hosts for King and Rolda viruses, and confirmation of the completeness of these and other viral genomes relies on the reverse genetics system as performed for deformed wing virus. The 5′ end of King virus matched the canonical terminal uracil nucleotides described in ng years .


Amplification of the purified virus without DNA extraction by ddPCR presented similar results to that from the extracted DNA, confirming the good resistance against PCR inhibitors. With the ddPCR, viral titer could be monitored in real time during the production of oHSV; the optimal harvest time was determined for the best virus yield in each batch. The ddPCR can be used as a useful tool for the quantification of oHSV and greatly facilitate the manufacturing process of oHSV products.Oncolytic herpes simplex virus (oHSV) is a type of virus that selectively targets and kills cancer cells, leaving normal cells unharmed. Accurate viral titer is of great importance for the production and application of oHSV products. Droplet digital PCR (ddPCR) is known for having good reproducibility, not requiring a standard curve, not being affected by inhibitors, and being precise even in the detection of low copies. In the present study, we developed a droplet digital PCR assay for the quantification of HSV-1 and applied it in the oHSV production. The established ddPCR showed good specificity, linearity, a low limit of quantification, great reproducibility, and accuracy. The quantification result was well-associated with that of plaque assay and CCID Herpes simplex virus types 1 and 2  are ubiquitous and important human pathogens. According to the WHO report [50) is also widely used for virus titration [For oncolytic virus development and production, the accurate quantification of the virus is extremely critical because the quality of OV products is vitally associated with the titer, and they usually exert the therapeutic effects in a dose-dependent manner in both pre-clinical and clinical use . Althougitration .Quantitative PCR (qPCR) is well-established for measuring viral nucleic acid by providing absolute quantification of a genomic target when used with an appropriate standard of known concentration. It has been broadly used for virus detection and the quantification of the viral genomic copy number for numerous viruses, due to its rapid detection, high sensitivity, high throughput, and cost-saving by the reduction of labor and materials . A few dDroplet digital PCR (ddPCR) is an emerging quantitative method (known as the third generation of qPCR) that allows absolute quantification of the target nucleic acids by fractionating the sample into multiple parallel PCR reactions ,14. Ever50. The specificity, lower limit of quantification, linearity, and reproducibility were well-verified as well. The established ddPCR assay substantially facilitated the rapid virus titration, as well as the whole oHSV production process.oHSV has great potential in the treatment of cancer as an immunotherapeutic drug. One big challenge for oHSV development is the large-scale production of high-titer virus. In the present study, we established a ddPCR assay for the detection and quantification of HSV-1 and applied it into the virus titration for different processing stages during oHSV production, which demonstrated viral titer results comparable with the results of traditional plaque assay and CCID2, 37 °C incubator.The wild-type herpes simplex virus type 1 (wt HSV-1), isolated from a clinical swab sample, and the oncolytic HSV (oHSV) were obtained from Dr. Hongkai Zhang at the State Key Laboratory of Medicinal Chemical Biology at Nankai University. The HSV-2 genome was kindly provided by the Wuhan Institute of Virology, Chinese Academy of Sciences. The varicella zoster virus genome was extracted from the virus stock in our company. Virus-producing Vero cells, obtained from WHO, were cultured in DMEM  supplemented with 10% fetal bovine serum (FBS)  and stored in a 5% COgH (envelope glycoprotein H) and one of which was against ICP8 (infected cell protein 8) of HSV-1. All primers and probes were synthesized by Sangon, Shanghai, China, with their sequences and fluorophores listed in gH gene , following the manufacturer’s instructions.We used the QX200™ AutoDG™ Droplet Digital™ PCR system  to complete the assay. Briefly, the ddPCR mixture consisted of 1.1 μL of DNA template, primers, probe, and 2 × ddPCR Supermix for Probes (No dUTP) . In the final 22 μL quantification reaction system, the final concentrations of primers and probes were 900 nM and 250 nM, respectively. The sample reaction system was added to a new 96-well plate with 8 wells in a row. We turned on the automated droplet generator and put these consumable materials and reagents into order: DG32 automated droplet generator cartridges, pipet tips for the AutoDG™ system, 96-well plate sample plate containing the ddPCR reaction system, a clean 96-well plate for receiving microdroplets, and the automated droplet generation oil for probes. We ran the droplet generator system, sealed the 96-well plate containing microdroplets with pierceable foil heat seals, and ran the PCR system in any PCR machine. The PCR procedure was as follows: 95 °C for 10 min; 94 °C for 30 s, 60 °C for 60 s, and 98 °C for 10 min. This was repeated for 41 cycles, then held at 12 °C. Finally, the data was read in droplet reader with QuantaSoft .Bioreactor was used for the oHSV production. Briefly, Vero cells were cultured with M199  supplemented with 5% fetal bovine serum in bioreactors. When the glucose achieved the optimal concentration, the medium was drained, and cells were rinsed with M199 medium once, prior to the addition of new M199 medium. oHSV was then added at a multiplicity of infection (MOI) of 0.03. Cytopathic effects were frequently monitored, and supernatants were harvested every 12 h, starting from 72 h post infection (hpi), until complete CPE was observed. All the harvested supernatants were clarified, concentrated, and purified. In each process, a small volume of the sample would be kept for virus titration and genomic copy number quantification.50 were used to quantify the infectious titer of the viruses. For plaque assay, 600 μL of 10-fold serially diluted viruses were added into the 6-well plate containing approximately 1 × 106 Vero cells/well. After one hour of adsorption, the inoculum was removed and replaced with an overlay mixture of 1 mL of 1.2% agarose  and 1 mL of 2 × DMEM medium supplemented with 5% FBS. The plate was placed back to the cell incubator for 3 days. The cells were fixed with 10% formalin  for 20 min prior to the removal of the overlay and staining with crystal violet . Plaques were counted, and the titer was calculated based on the plaque numbers in the dilution well and the initial inoculum used. For CCID50, Vero cells were pre-seeded in a 96-well plate at a density of 3 × 104 cells/well. At the next day, the medium was removed, and 100 μL of 10-fold serially diluted viruses were added into each column of the 96-well plate, that is, one dilution for 8 replicates. After one hour of adsorption, an additional 100 μL of DMEM medium containing 5% FBS was added into each well. The plate was put back to the cell incubator for 5 days, followed by the counting of the wells, where cytopathic effects were observed. The virus titer was calculated using the Kärber method described elsewhere.The plaque assay and CCIDT-tests.The results and data of ddPCR were generated and analyzed by QuantaSoft software version 1.7.4.0917 . Statistical analysis was performed using the GraphPad Prism software version 8.0.2 . The significance was determined using gH (envelope glycoprotein H) and one of which was against ICP8 of oHSV-1. The sequences of primers and probes used in this study are listed in To accurately quantify the genome copies of oHSV-1, we first designed three sets of primer pairs and probes, two of which were against 2 > 0.99). In addition, there was little difference regarding the genomic copy numbers when different primer pairs were used to detect samples at each designated dilution (p > 0.05)  prior to the detection by the ddPCR with different sets of primers and probes. As shown in  > 0.05) D. It ind > 0.05) . TherefoAlphaherpesvirinae. The genomes of oHSV-1, HSV-2, and VZV were extracted and detected by the ddPCR. The result showed that the genomic copy number of the purified oHSV-1 was 2.16 × 107 copies/μL, while a positive signal was barely detectable for HSV-2 and VZV , which all belong to  and VZV C. The am2, being 0.9936. This suggested that circular DNA could also be suitable for detection by ddPCR.The gH plasmid DNA was serially diluted for the detection of the genomic copy number by ddPCR. As shown in To identify the lower limit of quantification (LOQ) of the ddPCR assay, the genomic copy number of oHSV-1 DNA was detected B. Then, To further confirm the reproducibility of the ddPCR assay, the same experiment was performed for a total of four times by two researchers on different days. A good linear correlation was found between the logarithm of the genomic copy number and the logarithm of the dilution ratio in each experiment, where there was little difference in terms of the slopes and intercepts of the linear equations . This inTo verify the accuracy of the ddPCR detection, the genomic copy number of the serially diluted viruses was determined by quantitative PCR (qPCR) and ddPCR in parallel, and the infectious titer was measured by plaque assay as well. The results showed that there was a good linear correlation between the logarithm of the infectious titer and the logarithm of the dilution ratio E. MeanwhWe next employed the ddPCR assay to detect the oHSV genomic copy number during virus production to monitor the virus titer for the best harvest time point. oHSV was added to the bioreactor at an MOI of 0.03 under three different conditions (batches S1~S3). Starting from 72 h post infection (hpi) of oHSV, supernatants were harvested for the genomic copy number detection by ddPCR every 12 h until complete cytopathic effects (CPE) were observed. As shown in The DNA extraction is usually necessary before PCR/qPCR. However, this potentially causes errors, especially when accurate quantification is required, due to a suboptimal recovery rate or technical mistakes during the DNA extraction. It has been extensively demonstrated that the ddPCR assay is barely affected by the various inhibitors, which allows the accurate quantification of samples without the DNA extraction. Therefore, we further investigated if the ddPCR could quantify the oHSV-1 genome directly without the DNA extraction. As presented in 50 or plaque assay, so it would be very convenient to use the genomic copy number to calculate the viral infectious titer using a certain formula. For this purpose, we measured the virus infectious titer by plaque assay and the genomic copy number of the harvested and purified virus without extraction of viral DNA by ddPCR  for the determination of the optimal harvest time and to facilitate the manufacturing process of oHSV products.


Cryphonectria parasitica fungus, causal agent of chestnut blight, by virus infection (hypovirulence) has been shown to be an effective control strategy against chestnut blight in Europe and some parts of North America. The most studied mycovirus is the Cryphonectria hypovirus 1 (CHV-1) type species of the Hypoviridae family. To efficiently provide biocontrol, the virus must be able to induce hypovirulence in its fungal host in chestnut trees. Here, two different CHV-1 subtype I virus strains (E-5 and L-18), gained by transmissions, were tested for their hypovirulence induction, biocontrol potential, and transmission between vegetatively compatible (VCG) and incompatible fungal isolate groups in sweet chestnut seedlings and branches. Both strains of CHV-1 showed great biocontrol potential and could protect trees by efficiently transmitting CHV-1 by hyphal anastomosis between fungal isolates of the same VCG and converting virulent to hypovirulent cankers. The hypovirulent effect was positively correlated with the virus concentration, tested by four different reverse-transcription PCRs, two end-point and two real-time methods, one of which represents a newly developed real-time PCR for the detection and quantification of CHV-1.Biological control of  Castanea species caused by the ascomycete Cryphonectria parasitica (Murrill) M. E. Barr. The fungus originates from Eastern Asia , where 6 × 1023 is the number of copies per mole, DNA concentration is given in grams per microliter, and the molecular weight of one plasmid is in grams per mole assuming a plasmid size of 2627 bp and a 1 bp molecular weight of 660 g/mole.A ten 1:10 points serial dilution of the 94 bp specific qPCR product synthesized de novo and cloned into a plasmid pUC-GW-Kan , and posterior regression equation analyses, permitted estimating the fragment copy number, actual number of the CHV-1 virus, in real samples depending on the mean threshold cycles number per triplicate wells . For calAnalyses were conducted in R (version 4.1.0), with graphics produced using ggplot2 in R ,20,21,22Considering assay I with seedlings, three initial models were fitted to the data using different virus fixed effects: (1) virus strain, (2) virus absence/presence, and (3) original virus concentration.Regarding assay I, using branches, the same three initial models were fitted to the data.Considering assay II with seedlings, three separate models were applied to the data, one including the interaction of primary and challenge inoculation, one with the interaction between the primary inoculation and challenge inoculation strain, and one with the interaction between the primary inoculation and control/virus, with all models including row and column position (numeric) and initial lesion area  as additional predictors.Regarding assay II using branches, the data indicated that most samples had reached the maximum achievable lesion size by the end of the experiment, therefore data were analysed in two ways: (1) using the binary response of not achieved/achieved maximum lesion size, and (2) the analysis of actual lesion size for the subset of samples that had not achieved maximum lesion size. The number of samples achieving maximum lesion size versus not was used as a two-column matrix response in a generalized linear model with binomial errors and a logit link function. Two separate models were applied to the data, one including the interaction of primary and challenge inoculation and one with the interaction between primary inoculation and challenge inoculation strain, with both models including challenge inoculation compatibility as an additional predictor. AIC values were used to determine the most efficient model, with the analysis of deviance  used toAll the co-culture hypovirus transmissions were successful, with gained virus concentrations ranging between 234.37 and 612.83 ng/µL of RNA extract after the virus-specific RT-PCR [Both glycerol and the paper disk methods worked nicely in general to preserve the CHV-1 strains in the infected fungal isolates, and although there were no significant differences, the glycerol method results were on average slightly better than the paper disks preservation method .2,8 = 40.1, p < 0.0001). Lesions caused by virus-free isolates were significantly larger than those caused by virus-infected isolates  positive correlation between all pre- and post-experimental viral concentrations. The most efficient model included the absence/presence of the virus only . Lesions caused by virus-free isolates were significantly larger than those with the virus or negative controls  along with a main effect of virus absence/presence . Challenge inoculation virus presence was associated with significantly smaller lesions . Those treatments where challenge inoculations were compatible were significantly less likely to achieve maximum lesion area versus incompatible and control challenge inoculations. The second and third most efficient models included challenge inoculation viral strain;  along with primary inoculation isolate . Challenge inoculation strain 2 (L-18) was associated with smaller lesions. Smaller lesion areas were related with higher post-harvest virus concentrations  cultures because those results indicate around four-fold times more amplification efficiency than the protocol normally used (End-point Qiagen Extract) and without the need to perform either RNA extractions or gel electrophoresis. The triplicate tests’ standard deviation was very low, giving very similar results per sample. Furthermore, the use of the ten 1:10 serial dilution of the cloned qPCR product permitted determining the detection limit of this PCR protocol in two viruses. This real-time PCR was tested against five different subtypes of the mycovirus  with positive results. However, the unique weak point of this method is that it could be more difficult to discriminate statistically significant differences among the different infected isolates in comparison with the other three methods, especially the end-point methods .C. parasitica by reducing pathogenic growth and sporulation, hence the virus is used in Europe for disease control [C. parasitica from countries such as Japan, China [C. parasitica and CHV-1 have spread widely [Phytophthora cinnamomi and P. cambivora. Six genetically distinct CHV-1 subtypes have been identified in Europe  [Cryphonectria hypovirus 1 is known to induce hypovirulence in  control . The virn, China  and Koren, China , which ad widely . Origina2 and G) ,28. Subt2 and G) ,7,13, is2 and G) , Bosnia 2 and G) , Croatia2 and G) , Sloveni2 and G) , Macedon2 and G) , Turkey 2 and G) , and now2 and G) ,7,13, wh2 and G) , and it 2 and G) . The oth2 and G) , Spain [2 and G) , eastern2 and G) , Georgia2 and G) . The CHV2 and G) ,40, Wisc2 and G)  or Maryl2 and G)  where it2 and G) . C. parasitica have been evaluated on both chestnut seedlings and cut branches [There have been relatively few studies on the impact of CHV-1 induced hypovirulence using plant material ,44,45. Tbranches ,16, althbranches ,42, or bOur findings showed that there is a considerable difference in mean lesion area between virus-free isolates and those harbouring a high concentration of the CHV-1 virus, both when using seedlings and branches. The consistency of the plant material and controlled conditions used in our four assays gave high confidence about our observations and the conclusions that could be drawn from the data in relation to the efficacy of those artificially (by co-culture) infected English isolates.C. parasitica populations at the end of 2017 but does not appear to be widespread based on the very low frequency of findings and the low concentration of detectable virus in affected isolates [C. parasitica in the UK. These fungal isolates, which are now virus infected, initially originate from the east London area and Devon, where 77 and 60% of the total isolates belong to single VC groups (EU-10 in London and EU-9 in Devon) [Following findings of chestnut blight in the wider environment in England from late 2016 onwards [isolates . The resn Devon) ,47. It aCryphonectria parasitica belonging to vc type EU10 and EU9, the predominant vc type in London and Devon, respectively.(1). Two CHV1 strains originating from Switzerland were successfully transferred by hyphal anastomosis to British isolates of (2). The biocontrol potential of the CHV1-infected British isolates was experimentally verified and found to be dependent on the inoculum compatibility and on the virus concentration, on both seedlings and cut branches.(3). Real-time TakaRa III Toothpick method data revealed that this new PCR will be faster (no need for RNA extractions or electrophoresis), and more sensitive (around four times) for detection and quantification of the CHV-1 virus.(4). Following a completed Pest Risk Assessment (PRA) and regulatory approval, the CHV1-infected isolates characterized in this study will be available for further biocontrol testing under field conditions.


NS3 and NS5 genes (591 of NS3 and 883 of NS5) were identified, which might have caused changes in the ability of the virus to replicate. Based on phylogeographic analysis, Malaysia was the most likely country of origin for TMUV, while Shandong Province was the earliest province of origin in China. This study has important implications for understanding TMUV and provides suggestions for its prevention and control.Tembusu virus (TMUV) can induce severe egg drop syndrome in ducks, causing significant economic losses. In this study, the possible origin, genomic epidemiology, and transmission dynamics of TMUV were determined. The time to the most recent common ancestor of TMUV was found to be 1924, earlier than that previously reported. The effective population size of TMUV increased rapidly from 2010 to 2013 and was associated with the diversification of different TMUV clusters. TMUV was classified into three clusters  based on the envelope (E) protein. Subcluster 2.2, within cluster 2, is the most prevalent, and the occurrence of these mutations is accompanied by changes in the virulence and infectivity of the virus. Two positive selections on codons located in the  Flavivirus within the family Flaviviridae [E gene is usually used for phylogenetic analyses of genetic variation and molecular epidemiology.Tembusu virus (TMUV), a member of the genus iviridae , was firiviridae . It is civiridae ,4,5,6. Tiviridae . As the iviridae ; thus, tE gene for classification. To date, there have been three confirmed clusters, clusters 1, 2, and 3 [Genotyping of TMUV is typically based on the use of the 2, and 3 , which m2, and 3 . Althoug2, and 3 . The ana2, and 3 ,12.TMUV can cause severe egg drop syndrome in infected ducks, which exhibit many clinical symptoms including lack of energy, decreased appetite, and elevated body temperature. The morbidity rate of infected ducks can reach 80–90%, and the mortality rate can reach 10–30% ,14. ReseAlthough some researchers have used bioinformatics methods to study the origin and evolution of TMUV, the many different inference methods lack uniformity in different studies. In this study, we obtained TMUV gene sequences reported worldwide from the National Center for Biotechnology Information (NCBI) and analyzed them using maximum likelihood (ML), Markov chain Monte Carlo (MCMC), and other phylogenetic tools to study the origin, evolution rate, transmission dynamics, and geographic distribution of TMUV. Our findings provide a new global view for the prevention and control of future TMUV epidemics.https://www.ncbi.nlm.nih.gov/, accessed on 1 May 2022), and all available TMUV strains were obtained (accessed on 1 May 2022) (All sequences used in the present study were downloaded from the NCBI GenBank database (ay 2022) . In addip-value cut-off of 0.05 [Multiple alignments were performed using the fast Fourier transform (MAFFT) algorithm to compare and sort the complete genome and viral gene sequences used in this study . SplitsT of 0.05 .9 generations, obtained every 1000 generations and combined using LogCombiner software. The results were estimated using Tracer software (V1.7.1) after burn-in (10%). Parameters with an effective sampling size of >200 were adopted. The final MCC tree was displayed using FigTree software (V1.4.4). A Bayesian skyline coalescent model was set to estimate the efficient population size. Three independent runs were operated with a chain length of 1 × 109 generations and sampled at every 10,000 generations, and the final tree was generated by TreeAnnotator (1.10.5). The final tree was input to Tracer to generate a Bayesian skyline plot (BSP).Bayesian Markov chain Monte Carlo (MCMC) methods within Bayesian evolutionary analysis sampling trees (BEAST) (V1.10.4) are typically used to investigate tMRCA and evolutionary rates ,29. The Bayesian stochastic variable selection (BSSVS) is typically used for spatial propagation ,31. AccoE gene using MrBayes software with the GTR + I + G + F model with two parallel runs of 2,000,000 generations; the initial 25% of sampled data were discarded as burn-in [E gene was constructed using RAxML software based on the GTRGAMMAI model with 1000 replicates.To understand the relationship between different species of the Flaviviridae family, a Bayesian inference (BI) tree was constructed with hosts of different species of the  burn-in . An ML thttp://www.datamonkey.org/, accessed on 1 May 2022). The methods used to estimate the positive codon sites included fixed-effects likelihood (FEL), fast unconstrained Bayesian approximation (FUBAR), single-likelihood ancestor counting (SLAC), and the mixed-effects model of evolution (MEME). Positive selection sites were confirmed by at least two methods, with p-values for both FEL and MEME of less than 0.05, p < 0.1 by SLAC, and a posterior probability > 0.9 by FUBAR.The TMUV genome was selected using DATAMONKEY  [A total of 110 complete genome sequences, representing different clusters of TMUV, were aligned to analyze the potential recombination events of TMUV. The results indicated a recombination event in KJ740746, after which it was removed from the BEAST analysis. To better determine the evolutionary relationships among the different TMUV strains, a Bayesian MCMC estimation of the tMRCA was performed. Under the best clock model, the tMRCA of the complete genome was 1924, with a 95% highest probability density (HPD) ranging from 1850 to 1983. The tMRCA for the other genes was concentrated between 1932 and 1982 a. The nuite/year b. Our prBayesian skyline plot (BSP) analysis results can indicate effective population size changes in TMUV. We identified that the total TMUV showed a constant population size until approximately 15 years ago (2008), when a sharp increase in population size was observed, lasting for approximately five years (2013), after which the population remained high . When thE gene to further analyze the trend and classification of TMUV. As shown in To better understand the epidemiology of TMUV, we constructed an MCC tree and an ML tree based on the To further analyze the evolutionary relationship between different types of TMUV, we aligned the sequences of E proteins of different clusters of TMUV. We identified three specific amino acid mutation sites that may be key sites participating in the evolution of TMUV from cluster 3 to clusters 1 and 2 . ClusterTo eliminate uncertainty in the estimation process, the relationships among the strains were estimated based on well-supported contacts (BF > 15 and PP > 0.5) in different countries.As shown in To further analyze the distribution of TMUV in Asia, an MCC tree was constructed b. In addTo understand the relationships among different members of the Flaviviridae family, we downloaded complete genomic sequences of Zika virus, dengue virus, WNV, Quang Binh virus, and mosquito flavivirus to reconstruct BI trees . We founNS3 and NS5 genes , as shown in A total of 110 coding sequences of TMUV isolates  were useTMUV was first identified in mosquitoes in Malaysia in the late 1960s . It was Currently, tMRCA is the most reliable method for calculating the time of origin . After tE gene was 1.68 × 10−3 substitutions/site/year (95% HPD = 9.44 × 10−4 − 2.59 × 10−3 substitutions/site/year)  b, fasterte/year) , which mE gene to divide TMUV into three clusters and found that cluster 2 is the most prevalent genotype in China at present and that the strains that can infect chickens, pigeons, and mice are all in cluster 2 , as of 2018 the annual output of commercial ducks in Shandong Province accounted for approximately one third of the national total, and the inventory of laying ducks accounted for approximately one tenth of the national total, occupying an important position in China’s duck industry. Several other provinces, including Jiangsu, Anhui, and Guangdong, have large poultry industries. This large-scale transportation of poultry and intensive farming has contributed to the transmission rate of TMUV, allowing it to quickly spread across the country. However, we cannot ignore the possibility that TMUV can be transmitted by birds, such as pigeons; there is already evidence that it can be transmitted in waterfowl and other birds [Tracing the source of the virus transmission route will help prevent and control its spread in the future ,50. To ber birds ,17, whicer birds . In addi−3 substitutions/site/year, which is relatively fast in virus evolution, and we identified potential positive mutation sites on the NS3 protein. The two viruses (Zika virus and WNV) most closely related to TMUV are zoonotic viruses that pose a threat to public health, and some serological investigations have found TMUV antibody-positive cases in humans. Because TMUV is still evolving, and different flaviviruses are constantly interacting in the natural environment, human infection by TMUV is probably inevitable. It is therefore vital that prevention and control measures against TMUV be improved [Members of the Flaviviridae family pose a huge threat to global public health, but their transmission and pathogenic mechanisms are not clearly understood ,23. Phylimproved ,56.In conclusion, we successfully calculated the tMRCA and base substitution rate of TMUV and confirmed that avian TMUV is formed after multiple spills of the mosquito flavivirus and that TMUV can be transmitted by birds. Malaysia is thought to be the origin of TMUV, and from there it subsequently spread to China. Although TMUV is currently defined as a virus that infects only avian species, it cannot be ruled out as a potentially zoonotic virus. Overall, we performed a comprehensive bioinformatics analysis of TMUV, which should facilitate the planning of effective control strategies for TMUV infections in the future.


ASEAN (Association of Southeast Asian Nations) is composed of ten Southeast Asian countries bound by socio-cultural ties that promote regional peace and stability. South Asia, located in the southern subregion of Asia, includes nine countries sharing similarities in geographical and ethno-cultural factors. Chikungunya is one of the most significant problems in Southeast and South Asian countries. Much of the current chikungunya epidemic in Southeast Asia is caused by the emergence of a virus strain that originated in Africa and spread to Southeast Asia. Meanwhile, in South Asia, three confirmed lineages are in circulation. Given the positive correlation between research activity and the improvement of the clinical framework of biomedical research, this article aimed to examine the growth of chikungunya virus-related research in ASEAN and South Asian countries.The Scopus database was used for this bibliometric analysis. The retrieved publications were subjected to a number of analyses, including those for the most prolific countries, journals, authors, institutions, and articles. Co-occurrence mapping of terms and keywords was used to determine the current state, emerging topics, and future prospects of chikungunya virus-related research. Bibliometrix and VOSviewer were used to analyze the data and visualize the collaboration network mapping.The Scopus search engine identified 1280 chikungunya-related documents published by ASEAN and South Asian countries between 1967 and 2022. According to our findings, India was the most productive country in South Asia, and Thailand was the most productive country in Southeast Asia. In the early stages of the study, researchers investigated the vectors and outbreaks of the chikungunya virus. In recent years, the development of antivirus agents has emerged as a prominent topic.Our study is the first to present the growth of chikungunya virus-related research in ASEAN and South Asian countries from 1967 to 2022. In this study, the evaluation of the comprehensive profile of research on chikungunya can serve as a guide for future studies. In addition, a bibliometric analysis may serve as a resource for healthcare policymakers. Chikungunya virus, which was first isolated in Tanzania in 1953 , has beeAedes species and non-human primate species, such as monkeys, baboons, and mandrills [Culex and Anopheles have also been recognized as vectors of the chikungunya virus [Aedes albopictus and Aedes aegypti, which are reported as the primary vectors of chikungunya virus in Asia, Oceania, Europe, the Americas, and Africa [A. aegypti also transmits other types of viruses, including dengue virus and zika virus, as supported by several studies reporting the occurrence of dengue/chikungunya/zika virus or dengue/chikungunya virus simultaneous infections [The transmission routes of the chikungunya virus mostly occur via urban and sylvatic cycles . The sylandrills –30. In rya virus . Transmid Africa –36. Notafections –39. At pfections , 41. Recfections –48.The primary goals of ASEAN, also known as the Association of Southeast Asian Nations, are to foster regional stability and economic progress among its members . ASEAN cAmong South Asian countries, India is estimated to contain the highest number of chikungunya virus infection cases , 61–63. Bibliometric methodology has been established as a primary tool to evaluate research performance in various fields, including health , 73, sciDocuments associated with the chikungunya virus from ASEAN and South Asian countries indexed in Scopus from 1967 to 2022, excluding erratum and retracted articles, were extracted for our bibliometric study. To avoid including duplicate or invalid documents in the analysis, erratum and retracted articles were excluded. In this study, the Scopus database served as our sole data source because it covered a large range of journals and a high number of articles relative to other libraries such as Web of Science or PubMed , 94. Scon = 4107). Our search results, focusing on ASEAN and South Asian countries, included 1011 (78.98%) articles, 103 (8.04%) letters, 92 (7.18%) reviews, 20 (1.56%) notes, 17 (1.32%) conference papers, and 39 (3.04%) other types of documents, including book chapters, editorials, notes, and short surveys. Among the published documents, English is the most common language, accounting for 1279 (99.92%) of the total documents.From 1967 to 2022, the Scopus algorithm identified 1280 documents published by ASEAN and South Asian countries in the chikungunya-related field, accounting for 31.11% of the global productivity (n = 12) builds collaborative studies with more countries compared to the Maldives (n = 2).The chikungunya virus publishing trend in ASEAN and South Asian countries exhibited a gradual increase from 1967 to 2022 , followed by PLoS ONE , and the Indian Journal of Medical Research . Also, the characteristics of papers published by ASEAN and South Asian countries with the highest number of citations in the last five decades were summarized (Table The productivity among ASEAN and South Asian countries in research documents related to the chikungunya virus was also ranked, with India having the highest percentage Table . The topAedes”, “disease outbreaks,” and “alphavirus infection” in 2013; “epidemic,” “reverse transcriptase polymerase,” and “antibodies” in 2014; “isolation and purification,” “arthralgia,” and “dengue” in 2015. In 2016, research on “virology,” “animal cells,” “mice,” and “immunology” gained a significant attention in the field of chikungunya studies. Importantly, from 2017 onwards, the published articles mainly reported on “virus replication,” “metabolism,” “physiology,” “antivirus agent,” and “genetics” chikungunya virus, indicating that these terms are emerging and gaining significant attention in the last few years. The institutions from ASEAN and South Asian countries with the highest productivity in the field of chikungunya from 1967 to 2022 were also listed, highlighting the prominent contributions of India, Singapore, and Thailand (Table A network visualization of terms used by authors from ASEAN and South Asian countries in the 55-year period of chikungunya virus-related research was constructed Fig. a. The exOur research provided new perspectives on the scientific contribution of ASEAN and South Asian countries to chikungunya virus-related research. In general, ASEAN and South Asian countries contribute 31.11% of the global production of chikungunya virus-related studies. In 2006, the number of articles published by ASEAN and South Asian countries increased dramatically, from 2 documents in 2005 to 19 documents in 2006. After 2006, a gradual increase in chikungunya-related documents produced by ASEAN countries was observed, indicating that chikungunya virus-related topics are gaining prominence as a human health concern. A closer examination of the documents published in 2006 reveals that India, with 15 documents, contributed the most articles. Articles published in 2006 mostly discussed the re-emergence of the chikungunya virus in India and Malaysia , 110. MeIn bibliometric analysis, directly evaluating the weight or effect of publications is difficult. However, a number of studies argued that important insights could be obtained by analyzing the relationship between article relevancy and journal significance , 122. InAfter 2017, the topics of antiviral activity and neutralizing antibodies are emerging as the new focus on chikungunya-related studies in ASEAN and South Asian countries. Effective antiviral strategies are required to combat the rising prevalence of chikungunya virus infection and reduce mortality. Because there are no effective vaccines, significant research has been conducted to discover potent antivirals against the chikungunya virus Table . In paraA. aegypti and A. albopictus [The majority of global efforts have been devoted to concentrating on treating symptoms and preventive measures, because there are no commercially available vaccines or medications to cure chikungunya infections . The mitbopictus , 162. Efbopictus , 164. AdThis study uses a bibliometric technique to assess the current state and trajectory of chikungunya virus-related research published by ASEAN and South Asian countries. However, a few limitations, which also appear in other bibliometric analyses, were identified. First, our study registered the key term “chikungunya” only for title searches. Our analysis may have overlooked any publications that utilized the keyword “chikungunya” in the abstract or within the publication. Second, this study focused on publications indexed in the Scopus library. Although Scopus is the most widely used and trusted database, a few outlier papers may have been left out. Despite these limitations, our bibliometric study is the first to provide a concise overview of the chikungunya virus-related research profile by ASEAN and South Asian countries. Our research also demonstrates how bibliometric analysis may be used to estimate research productivity in the field of chikungunya.Our study is the first to present the contribution of ASEAN and South Asian countries to chikungunya virus-related research. By integrating literature review and bibliometric analysis, the current study aimed to provide an outline of the progress, current trend, and emerging topics in the field of chikungunya in ASEAN and South Asian countries. Over the past two decades, the research productivity on chikungunya virus-related topics in ASEAN and South Asian countries has increased remarkably, with a total of 1280 articles published in prominent journals. Previously, researchers prioritized studies related to the vector, infection, and nucleotide sequence of the chikungunya virus. However, in recent years, the research topics have shifted to the development of antiviral drugs and the genetics of chikungunya virus. Out of ten ASEAN countries, Thailand contributes the highest number of articles related to chikungunya, followed by Singapore. Meanwhile, India ranks first among South Asian countries in terms of productivity. The findings of this study can serve as a reference for ongoing chikungunya virus-related research and policymakers working in the field of healthcare, particularly in ASEAN and South Asian countries.


The human monkeypox is an emerging zoonotic orthopoxvirus with a clinical presentation similar to that of smallpox. It is difficult to differentiate monkeypox from other orthopedic infections, and laboratory diagnosis is the primary component of disease identification and monitoring. However, current diagnostics are time-consuming, and new tests are needed for rapid and precise diagnosis. Most cases have been reported in Central Africa; however, an increasing number of cases have been reported in Europe, the United States of America (USA), Australia, and the United Arab Emirates. Although investigation of the current global outbreak is still ongoing, viral transmission seems to have occurred during crowded events in Spain and Belgium. New therapeutics and vaccines are being deployed for the treatment and prevention of monkeypox, and more research on the epidemiology, biology, and ecology of the virus in endemic areas is required to understand and prevent further global outbreaks. Monkeypox virus has a propensity to spread among mammals, including humans. The natural host of the monkeypox virus remains largely unknown, but it has been isolated from a wild animal, once from a ropy squirrel in the Democratic Republic of Congo and once from a sooty mangabey in Côte d’Ivoire. The incubation period of the monkeypox virus, as seen in human-to-human transmission, is 12 days.Monkeypox is an emerging zoonotic disease in humans that arises from an orthopoxvirus belonging to the Poxviradea family, which is known to have a complex double-stranded DNA.It is believed that the virus is transmitted through respiratory secretions and saliva, or through direct contact with the exudate or crust material of the lesion. Viral shedding through feces is another potential source for the transmission of the virus. The central region of the genome of the monkeypox virus is 96.3% similar to that of the variola virus, which codes for structural proteins and essential enzymes, and differs substantially from the region of the genome that codes for virulence factors and host range factors. The 3.4 to 10% case fatality rate of monkeypox lies between the case fatality rates of variola minor and variola major, which have case fatality rates of 1% and 30%, respectively.Monkeypox virus has morphologic features similar to other orthopoxviruses, with a size of 200–250 nm, a brick-shaped virus that is enveloped and contains surface tubules along with a dumbell-shaped core component. However, numerous cases of monkeypox have been reported in humans and wildlife in Central and West Africa. The number of cases of human monkeypox virus has surged in recent years along with an increase in the geographic spread of the disease, as immunity to smallpox vaccination is waning.,8 In 2017, Nigeria experienced the largest outbreak of monkeypox virus in the West African clade with a 6% fatality rate.,9,10 Two cases of monkeypox were imported to the United Kingdom (UK) through Nigerian individuals in September 2018, and 1 became the source of nosocomial infections affecting healthcare workers. In addition, the monkeypox virus was imported to the United States of America (USA) in 2003 through rodents that were shipped from Ghana and housed with prairie dogs, which became the source of infection in humans.,10,12,13The disease is indigenous to the Democratic Republic of Congo, where the first case was reported in 1970.,13 It has been observed that the Central African clade, also known as the Congo basin clade, is associated with increased morbidity and mortality, human-to-human transmission, and viremia as compared to the West African clade.,10,13 Moreover, in the US, adults were disproportionately affected, contrary to the norm, and no fatalities were reported. In Africa, 1.5 to 17% of mortality is recorded, predominantly in children, due to the unavailability of medical care. Figure Two clades of monkeypox viruses have been identified: the West African clade and the Central African clade. The former has a case fatality rate of <1 percent with no report of human-to-human transmission, whereas the latter has a case fatality rate of 11%, and human-to-human transmission has been documented in the Central African clade. The virus that was involved in the US outbreak was considered the West African variant, and the one that involved the Democratic Republic of Congo was the Central African variant. The case fatality rate is 11% in individuals not vaccinated against smallpox because the vaccine offers cross-protection against the monkeypox virus.It has been demonstrated that the majority of the cases of monkeypox occurred in children <10 years of age. The attack rate was noted to be significantly higher in individuals who had no previous vaccination mark (7.2%) as compared to the attack rate for individuals who had a vaccination mark. (0.9%). High risk is associated with the monkeypox virus, as it can cause a disease of public health significance. In 1980, the World Health Organization (WHO) identified the monkeypox virus as the most important orthopoxvirus infection in humans after the eradication of smallpox; hence, surveillance is warranted. Although there is no evidence currently indicating that human-to-human transmission can sustain monkeypox virus in local communities, a study suggests that repeated exposure to animals infected with monkeypox in a population with low herd immunity can result in large clusters of individuals infected with monkeypox virus in the African rainforest. Similar to smallpox, the monkeypox virus poses a potential threat to biological warfare; hence, various antivirals and therapeutic medications are being developed.Despite having little propensity to spread among humans, the monkeypox virus poses a serious threat to life in the region of the Democratic Republic of Congo, West and Central Africa, and possibly globally.Scopus, MEDLINE, and Web of Science databases were searched using various medical subject heading (MeSH) combinations to identify the studies of interest. Following MeSH headings were used: “Monkeypox” OR “Human monkeypox” OR “Monkeypox virus” AND “Public health” OR “Outbreak” OR “Clinical features” OR “Epidemiology”. The information gathered from the included studies was incorporated into a narrative style for an evidence-based and updated report on the human monkeypox virus.,17 The monkeypox virus has demonstrated infectivity among a wide variety of multiple mammalian species, and contrary to its name, it has never been isolated from any species of monkey, having only been isolated once from a wild animal, a Funisciurus squirrel in the Democratic Republic of Congo. In light of this, the WHO is considering measures to rename the disease to avoid stigma, similar to the renaming of COVID-19. Further research is needed to identify the exact reservoir of the virus and its circulation in the wild.The monkeypox virus has 2 clades, the Congo Basin and West Africa, with each causing disease; however, the West African clade is considered less virulent.Human infections have been linked to contact with wild animals, but the precise exposure mechanism has been difficult to triangulate, particularly in areas where contact with animals may have occurred through household rodent infestations or hunting or preparation of bushmeat for domestic consumption. Transmission has been shown to occur via saliva and respiratory excretions, or contact with lesion exudate, crust material, or feces of infected animals. Previous evidence indicates that household members or those who care for a monkeypox patient are at an increased risk of acquiring an infection; however, it is less efficient than that observed in smallpox. Household members who were not vaccinated for smallpox showed a greater risk of acquiring the infection than those who were not vaccinated.The first case of human monkeypox was reported in 1970 in West Africa. Subsequently, most reported infections have occurred in the Congo Basin of Central Africa. Mortality varies between 3–6% and 17%, depending on the clade, and children, pregnant women, and immunocompromised individuals are at a high risk for adverse outcomes.As of June 17, a total of 2103 laboratory-confirmed cases and 1 probable case, including 1 death, had been reported to the WHO  were noted to have hemorrhagic pustules. Individuals with a previous vaccination history of smallpox reported mild rash that was pleomorphic, lymphadenopathy was present in roughly half of these individuals, and no mortality was reported. In individuals who survive the illness, postinfection scarring is one of the most common long-term sequelae. Figure One to 3 weeks after the onset of fever and lymphadenopathy, a rash appears, with lesions that erupt simultaneously and evolve at a similar rate. The rash has a peripheral distribution, but can envelop the entire body in the setting of severe disease. Complications were more prevalent in individuals who were not previously vaccinated (74%) than in those who were vaccinated (39.5%). Abnormal laboratory findings were noted in the setting of this viral illness, including elevated transaminase levels, low blood urea nitrogen levels, hypoalbuminemia, leukocytosis, and thrombocytopenia. It took a period of 3 to 12 days from the onset of illness to examine abnormal laboratory findings.The duration of the infection was approximately 4 weeks, and desquamation of the lesion suggested that the infection was in the ending phase. Patients can suffer from a host of complications during this viral illness such as respiratory distress, secondary bacterial infection, gastrointestinal involvement, bronchopneumonia, sepsis, dehydration, corneal infection resulting in loss of vision, and encephalitis. One pediatric patient in the US outbreak underwent mechanical ventilation due to encephalitis, while the other suffered from a massive retropharyngeal abscess and cervical lymphadenopathy, which compromised the tracheal airway, both of which were treated in the intensive care unit.Pediatric patients  are prone to be admitted to an intensive care unit; however, they are not at an increased risk of developing severe illness. In the US, patients who had dysphagia and hypoxemia and presented with mouth sores were more likely to have severe illness.Approximately one-third of adults in the US infected with monkeypox were vaccinated against smallpox; however, no significant difference was observed in the clinical presentation of vaccinated and unvaccinated individuals.Often, a misdiagnosis of varicella is made in monkeypox infection because it is also a febrile rash. However, some distinguishing clinical features can distinguish these 2 diseases. Varicella rash is centripetal, unlike that of monkeypox, which has a centrifugal pattern of distribution. The lesions of varicella appear superficially with irregular borders similar to a “dew drop on a rose petal”; however, the lesions of monkeypox infection are deep and hard, with well-circumscribed margins and umbilication. The rash has a rapid progression in varicella with lesions in multiple stages of development on the body, whereas the monkeypox rash progresses slowly, and the lesions are in a similar stage of development throughout the body. Moreover, lymphadenopathy is not observed in varicella and is a telltale sign of monkeypox infection.The prognosis of human monkeypox infection can be estimated by considering whether the individual has presented with complications and analyzing a few factors, including the health and previous vaccination status of the individual and the presence of any illness such as malaria, measles, or diarrheal disease occurring concomitantly with monkeypox infection. Since most cases of monkeypox virus are endemic in rural areas where electricity is not available, advances should be made to develop assays that can be tested in a very basic setting with limited training of personnel. Table Diagnosis of monkeypox infection is made by subjecting the material collected from the lesion to various techniques, such as electron microscopy and immunohistochemistry, to observe the presence of the monkeypox virus. Techniques such as real-time PCR can also be used to trace the virus in a sample, as they are highly effective and sensitive for the detection of viral DNA. Serological markers such as antiorthopoxvirus IgM and IgG can also be used to assess previous exposure to an orthopoxvirus, either through infection or vaccination. Several compounds show therapeutic potential against orthopoxvirus species, including cidofovir, which is used against a range of viruses as it exerts its effect through inhibition of DNA polymerase. Since cidofovir causes nephrotoxicity, a modified form of this drug-CMX-001, an oral antiviral medication, without the adverse effect of cidofovir, is in the stages of development and acts by inhibiting DNA polymerase. Another orally administered drug, ST-246, inhibits the release of intracellular viruses and has shown encouraging results against different species of orthopoxviruses, including variola virus. It has also successfully treated the monkeypox virus in nonhuman primates and small mammals.,23At present, no evidence-based treatment for monkeypox infection exists; symptomatic treatment and supportive care are the only ways to manage the patient. Recent developments in nanotechnology have been observed in medical research, where metal-based nanoparticles are used in biological systems. Many questions regarding this sophisticated technique remain unanswered. Nevertheless, silver-containing nanoparticles with a 10 nm diameter (Ag-PS-10) proved to be the most effective at inhibiting monkeypox virus infectivity, as observed by the dramatic reduction in monkeypox plaque formation at all concentrations tested.Since the smallpox vaccine confers cross-protection against monkeypox, a single dose of a third-generation smallpox vaccine called Imvanex was offered as postexposure prophylaxis as an off-label indication to individuals at intermediate and high risk of becoming infected with monkeypox virus in the UK in 2018. This also indicates a glaring double-standard against the global concern for this disease. While monkeypox has remained an endemic problem in Central and West African countries, having limited healthcare resources for monitoring and surveillance of this disease, as well as facing disruption in the form of war and climate change, it has only been given global attention once it infected countries in the Global North. The emergence of COVID-19 has led to large-scale deforestation, migration, conflict, climate change, and the increasing encroachment of the human population into previously untouched habitats may create grounds for the reemergence of more diseases and even push their mutation into more virulent strains of previously benign infections, which may render our current therapeutic interventions and vaccinations redundant. Countries in the Global North must share better diagnostic tools and research with monkeypox-endemic countries to create better engagement with local communities. Once a more comprehensive One Health approach is implemented, research on vaccines and therapeutic agents for these reemerging zoonotic diseases will be more productive and allow for the scaling up of prevention strategies.The reemergence of monkeypox infection in the wake of healthcare system disruptions caused by the COVID-19 pandemic points to many areas of public health, particularly in countries with limited resources. Although smallpox vaccination provides partial immunity against monkeypox infection, its global cessation after the eradication of smallpox disease puts many younger people at risk of infection. Indeed, most of the present cases have been reported in individuals under the age of 50 years. However, long-lasting immunity (>25 years) against monkeypox infection has been demonstrated in communities vaccinated against smallpox in the DRC.The human monkeypox is an infection with the potential to spread via zoonotic reservoirs. The most important reason for the displacement of the virus in nonendemic areas seems to be civil conflict and the relaxation of international travel. Other causes include the movement of people in the natural habitats of animal reservoirs for this virus, which makes humans prone to interaction with wildlife and zoonoses. The latest outbreak in the incidence of human diseases requires further investigation to better understand the range of factors associated with disease transmission and spread. Epidemiological risk factors, animal reservoirs, and COVID-19 coinfection should be the focus of current investigations in endemic areas. Control and preventive measures, including education and personal cleanliness, should be implemented at all incident locations worldwide. Monkeypox should be included in standard disease monitoring protocols, with periodic genomic studies and serological surveys, and health professionals and high-risk populations should be evaluated for vaccination. Because monkeypox is no longer a rare disease, it is necessary to identify animals in Africa that harbor orthopoxviruses, develop a better algorithm for diagnosing the monkeypox clinical spectrum and disease severity, and assess the risk of transmission concerning various types of contact with clinical cases.