The SARS-CoV-2 (COVID-19) pandemic has highlighted the urgent need for strategies that rapidly inactivate airborne respiratory viruses and break the transmission cycle of indoor spaces. Air ions can reduce viable bacteria, mold, and virus counts, however, most studies use small test enclosures with target microbes and ion sources in close vicinity. To evaluate ion performance in real-world spaces, experiments were conducted in a large, room-size BSL-3 Chamber. Negative and positive ions were delivered simultaneously using a commercially available bipolar air ion device. The device housed Needle Point Bipolar ionization (NPBI) technology. Large chamber studies often use unrealistically high virus concentrations to ensure measurable virus is present at the trial end. However, excessively high viral concentrations bias air cleaning devices towards underperformance. Hence, devices that provide a substantial impact for protecting occupants in real-world spaces with real-world virus concentrations are often dismissed as poor performers. Herein, both real-world and excessive virus concentrations were studied using Influenza A and B, Human Respiratory Syncytial Virus (RSV), and the SARS-CoV-2 Alpha and Delta strains. The average ion concentrations ranged from 4,100 to 24,000 per polarity over 60-minute and 30-minute time trials. The reduction rate was considerably greater for trials that used real-world virus concentrations, reducing infectivity for Influenza A and B, RSV, and SARS-CoV-2 Delta by 88.3–99.98% in 30 minutes, whereas trials using in-excess concentrations showed 49.5–61.2% in 30 minutes. These findings strongly support the addition of NPBI ion technology to building management strategies aimed to protect occupants from contracting and spreading infective respiratory viruses indoors. Prior to the 2019 Covid pandemic, the Centers for Disease Control and World Health Organization recognized droplet and fomite transmission as primary modes of host infection . InfectiZero Ozone Emissions Validation | UL Solutions). For this study, the term “indoor” includes enclosed spaces such as houses, apartments, workplaces, schools, commercial units, airports, and hospitals [The goal of this study was to assess NPBI, a soft bipolar ionization technology, as an effective tool for inactivating respiratory viruses transmitted indoors via droplet nuclei. Bipolar ionization is effective at agglomerating ultrafine particles –10, inclospitals . The datospitals .The strain of SARS-CoV2 used was SARS-COV-2 USA-CA1/2020 and was obtained through BEI Resources, NIAID, NIH: SARS-Related Coronavirus 2, Isolate USA-CA1/2020, NR-52382. The SARS-CoV-2 Delta Variant was obtained through BEI Resources, NIAID, NIH: SARS-Related Coronavirus 2, Isolate hCoV-19/USA/PHC658/2021 , NR-55611, contributed by Dr. Richard Webby and Dr. Anami Patel. The Respiratory Syncytial Virus (RSV) was obtained through BEI Resources, NIAID, NIH: Human Respiratory Syncytial Virus, A2001/2-20, NR-28525. The Influenza A Virus (NR-31132) was obtained through BEI Resources, NIAID, NIH: Influenza A Virus, A/mallard/Wisconsin/2785/2009 (H2N3), NR-31132. The Influenza B Virus was obtained through BEI Resources, NIAID, NIH: Influenza B Virus, B/New York/1055/2003, NR-48660. Quality control data is listed in S1-S5 Tables in The most common method for studying virus viability is the Tissue Culture Infectious Dose 50 assay, or TCID50 which quantifies viral titer by determining the concentration at which 50% of the infected cells display a cytopathic effect (CPE) including changes in cell shape, fusion with surrounding cells, or inclusion body appearance in host cells. As long as the virus being studied results in cell death, this assay can be used when viral antibodies are not available, and little information on the virus is required. Serial dilutions of virus were plated onto VeroE6 host cell monolayers, left for 24 hours, and scored to find where 50% of host cells displayed CPE for quantitation of the original virus concentration; i.e. a TCID50. To calculate the actual amount of virus per ml, the TCID50 is multiplied by 0.7 according to the Poisson distribution . Once achttps://www.atcc.org/products/crl-1586), prepared in Dulbecco’s Modified Eagle Medium (DMEM) plus fetal bovine serum, 4mM Glutamine, and antibiotics, and incubated at 32°C overnight. Viral samples were serially diluted 1:10 (v:v) in Phosphate buffered Saline (PBS). The Vero E6 cells (0.1 mL) were mixed with 0.1 mL of each viral dilution in quadruplicate, and the virus allowed to absorb to the cells at 37°C for 2 hours. After absorption, 0.5 mL was added to each well and incubated at 37°C with the CPE monitored using an inverted microscope over one to four weeks with the number of positive and negative wells recorded. The dilution that caused a 50% infectivity rate of host cells was used in this study as the Tissue Culture Infectious Dose (TCID 50/mL) [One day before the Vero E6 cell host infection , 96 well plates were seeded with the Vero E6 cells  obtainedD 50/mL) . Once thhttps://gpsair.com/) was used to test NPBI against all viruses listed. NPBI is a soft ionization technology that incorporates emitters comprised of bundled microfiber carbon brushes. Large radius brush emitters ensure constant low voltage operation over the lifetime of the device. In contrast, steel needles erode and dull over time, leading to arcing and voltage fluctuations, requiring replacement at regular intervals. NPBI was designed for devices that are installed in typical HVAC systems or drop-ceiling destratification units. The technology produces an ion-rich cloud for duct distribution or ceiling distribution into building spaces. Soft ionization technology was initial developed as a toxicological tool to identify intact molecules in the 1970’s [2-, CO4-(H2O)n, and NO3-(H2O)n and are described in detail as the lower E e- ion-molecule interactions for water clusters Y-(H2O)n by Sekimoto and Takayama [The GPS-FC48-AC  manufacte 1970’s . Unlike Takayama . The devhttps://www.alphalabinc.com/, West Salt Lake City, UT, USA) AIC2 ion polarity Gerdien meters with USB datalogging confirmed negative and positive ion generation. The variable speed fan located behind the NPBI device  from CH Technologies USA  each, were positioned at 45-degree angles for homogenous mixing. For sampling, four probes were positioned along the centerline and protruded down 24” (0.61 m).Testing was conducted in a sealed 20’x8’x8’ (6.1x2.4x2.4m) BSL-3 chamber. The overall dimensions provided a total air volume of 36,245.6 L. A nebulizing port with a programmable compressor in the center of the 20-ft wall protruded 24 inches . At eachA GPS-FC48-AC ionizer was in the room’s centerline and elevated 6ft 1.8m; . During Control tests were conducted in triplicate with the fan turned on and the device present as in the test trials but turned off. The BLAM was loaded with 10 mL of viral suspension and operated as described above. Samples were taken at 0, 15, 30, 45, and 60 minutes. The control results showed a natural rate of loss and were used to assess the ability of the NPBI technology to reduce SARS-CoV-2 in the air.Before and after each trial, the UV system inside the testing chamber was activated for 30 minutes, followed by a 30-minute HEPA air purge. All test equipment was cleaned each day with 70% isopropanol. Collection lines were soaked in bleach for 30 minutes and rinsed repeatedly with distilled water. The nebulizer and vacuum collection pumps were decontaminated with hydrogen peroxide.Once the room was cleaned and baseline/background air measurements completed, 10 mL of virus in viral media was nebulized into the room via the dissemination port . The com30-T0/30). While all TCID50/mL data is in the supplemental, we calculated the viable virus particles/L of room air to present the data in a more relatable fashion; Eq .3 .V2 = the testing room air volume of 20’x8’x8’ or 1,280 ftE = collection filters 70% efficacy for capturing particles.0-T30/T0*100) for a given decay curve. The Net Virus Reduction Rate (NVRR) is the percent decrease in virus infectivity between the control decay and the experimental decay at the same time point to evaluate air cleaning device performance in decreasing airborne virus activity. The NVRR was calculated by subtracting the control virus concentration from the treatment virus concentration at a given time point and then dividing by the control virus concentration (eg. T30control-T30treatment/T30control). The Overall Virus Reduction Rate (OVRR) is the decrease in virus infectivity attributed to the air cleaning device per unit time and calculated by subtracting the elapsed time point virus concentration from the time zero virus concentration and dividing by the elapsed time in minutes (eg.T0-T30/30). Lastly, the Percent Improvement (PI) is a measure of the effectiveness of the device in removing the virus from the room air space, and was calculated by subtracting the treatment OVRR from the control OVRR, then dividing by the control OVRR and multiplying by 100 (eg. (((OVRRT30treatment-OVRRT30control)/OVRRT30control)*100).The extent of ion effects on viral viability was calculated using Gross Virus Reduction Rate, the Net Virus Reduction Rate, the Overall Virus Rate of Reduction, and the Percent Improvement. The Gross Virus Reduction Rate (GVRR) is a percent difference in virus infectivity for each sample period versus the starting virus concentration for a given decay curve calculated by subtracting the virus concentration at a given time point from time zero concentration and then dividing by the time zero concentration (eg. (Thttps://www.minitab.com/). Because each exposure time trial was conducted separately for each replicate, exposure time was classified as an independent factor in the analyses. Hence, a general linear model 2-way analysis of variance (ANOVA) was conducted for each virus trial, where Exposure Time with multiple levels and NPBI with two levels (Test/Control) were classified as categorical factors, and virus concentrations was classified as the continuous response variable. The model consisted of Exposure Time, NPBI, and Exposure Time by NPBI interaction. Post-hoc analysis to identify statistical differences associated with exposure time and ion level was conducted using Tukey pairwise comparisons [All statistical analysis was completed using Minitab statistical software version 21.3.1  minutes was the end of the nebulization period. The results were plotted to show a natural rate of loss and to demonstrate the high degree of similarity and repeatability between the control or test replicates. The rate and total difference in viral loss between the control and test trials were used to assess the NPBI’s ability to reduce the viral load in the room air.Overall, the control trial replicates were similar in terms of TCID50/mL and for the viable virus/L of room air as shown for individual trials Figs . The aveIn general, the average Gross Virus Reduction Rate (GVRR) ranged from 20.6–25.9% after 30 minutes and 41.1–67.5% after 60 minutes  for InflThe NPBI technology was assessed for its ability to effectively remove a variety of relevant viruses. For the test trials, as in the control trials, the 30 mL of virus preparation was divided into three 10 mL aliquots for the triplicate trials to be as similar as possible. In all cases, the operating device resulted in a substantial increase in loss of virus infectivity at 30 and 60 minutes . At 30 m3 and 12,415 particles/L  at 15 minutes, 5,347±298 particles/L (1.36x107±7.57x105 TCID50/mL) at 30 minutes, 2,646±172 particles/L (6.71x106±4.36x105 TCID50/mL) after 45 minutes, and 17±8 particles/L (4.42x104±1.94x103 TCID50/mL) by the trial end at 60 minutes  after 15 minutes, 744±43 particles/L (1.89x106±1.10x105 TCID50/mL) after 30 minutes, 396±16 particles/L (1.01x106±4.11x104 TCID50/mL) after 45 minutes, and 128±45 particles/L (3.25x105±1.15x105 TCID50/mL) after 60 minutes  after 30 minutes, to 441±48 particles/L (1.12x106±1.21x105 TCID50/mL) after 45 minutes, and to 23±14 particles/L (5.96x104±3.64x104 TCID50/mL) after 60 minutes  from the initial 9,735 particles/L (2.47x107 TCID50/mL), representing a 28.5±1.2% loss as compared to the 14.7±1.8% loss in the control (Tables 6±6.78x105 TCID50/mL), to 1,575±348 particles/L (4.00x106±8.83x105 TCID50/mL) after 45 minutes and finally to 1±0 (1.57x103±8.09x102 TCID50/mL) at 60 minutes  from a starting concentration of 380 particles/L  remained  from the starting concentration of 426 particles/L (1.08x106 TCID50/mL), a 47.2±4.7% loss as compared to 26.6±2.4% loss in the control (Tables 5±2.65x104 TCID50/mL) remained and corresponded to an 80.3±2.5% loss, nearly double the 45.4±3.7% in the control. At 25 minutes, only 11±9 particles/L (2.88x104±2.19x104 TCID50/mL) remained, representing a 97.3±2.0% clearance as compared to 54.7±2.0% in the control. Finally, at 30 minutes viral concentrations were below detection with a clearance of 99.9±0.1% as compared to the control of 68.4±3.6% , it was reduced to 152±6 particles/L (3.85x105±1.56x104 TCID50/mL) after 10 minutes, to 45±7 particles/L (1.15x105±1.81x104 TCID50/mL) at 20 minutes, to 1±0 particles/L (1.31x103±1.17x102 TCID50/mL) at 25 minutes and below detection at 30 minutes Click here for additional data file.


To (a) describe the demographics of opioid abusers; (b) compare the prevalence rates of selected comorbidities and the medical and drug utilization patterns of opioid abusers with patients from a control group, for the period from 1998 to 2002; and (c) calculate the mean annual per-patient total health care costs  from the perspective of a private payer.An administrative database of medical and pharmacy claims from 1998 to 2002 of 16 self-insured employer health plans with approximately 2 million lives was used to identify opioid abusers patients with claims associated with ICD-9-CM  codes for opioid abuse . A control group of nonabusers was selected using a matched sample  in a 3:1 ratio. Per-patient annual health care costs  were measured in 2003 U.S. dollars. Multivariate regression techniques were also used to control for comorbidities and to compare costs with a benchmark of depressed patients.740 patients were identified as opioid abusers, a prevalence of 8 in 10,000 persons aged 12 to 64 years continuously enrolled in health care plans for which 12 months of data were available for calculating costs. Opioid abusers, compared with nonabusers, had significantly higher prevalence rates for a number of specific comorbidities, including nonopioid poisoning, hepatitis , psychiatric illnesses, and pancreatitis, which were approximately 78, 36, 9, and 21 (P less than 0.01) times higher, respectively, compared with nonabusers. Opioid abusers also had higher levels of medical and prescription drug utilization. Almost 60% of opioid abusers had prescription drug claims for opioids compared with approximately 20% for nonabusers. Prevalence rates for hospital inpatient visits for opioid abusers were more than 12 times higher compared with nonabusers (P less than 0.01). Mean annual direct health care costs for opioid abusers were more than 8 times higher than for nonabusers . Hospital inpatient and physician-outpatient costs accounted for 46%  and 31%  of opioid abusers health care costs, compared with 17% ($310) and 50% ($906), respectively, for nonabusers. Mean drug costs for opioid abusers were more than 5 times higher than costs for nonabusers , driven by higher drug utilization (including opioids) for opioid abusers. Even when controlling for comorbidities using a multivariate regression model of a matched control of depressed patients, the average health care costs of opioid abusers were 1.8 times higher than the average health care costs of depressed patients.The high costs of opioid abuse were driven primarily by high prevalence rates of costly comorbidites and high utilization rates of medical services and prescription drugs.


Studies of ancient DNA have transformed our understanding of human evolution. Paleogenomics can also reveal historic and prehistoric agents of disease, including endemic, epidemic, and pandemic pathogens. Viruses—and in particular those with single- or double-stranded DNA genomes—are an important part of the paleogenomic revolution, preserving within some remains or environmental samples for tens of thousands of years. The results of these studies capture the public imagination, as well as giving scientists a unique perspective on some of the more slowly evolving viruses which cause disease. In this review, we revisit the first studies of historical virus genetic material in the 1990s, through to the genomic revolution of recent years. We look at how paleogenomics works for viral pathogens, such as the need for careful precautions against modern contamination and robust computational pipelines to identify and analyze authenticated viral sequences. We discuss the insights into virus evolution which have been gained through paleogenomics, concentrating on three DNA viruses in particular: parvovirus B19, herpes simplex virus 1, and smallpox. As we consider recent worldwide transmission of monkeypox and synthetic biology tools that allow the potential reconstruction of extinct viruses, we show that studying historical and ancient virus evolution has never been more topical. Ancient viral genomes are an important source of evidence of how viruses evolve in time and space. They provide a record of virus lineages that have gone extinct and help scientists understand changes in virulence or transmissibility. In this review article, we discuss how ancient virus genomes are extracted from ancient remains and how these data are analyzed. We give examples of unique insights into virus evolution that only ancient genomes can give, focusing on parvovirus, herpes simplex virus, and smallpox. Finally, we consider what ancient virus data might tell us about emerging infectious diseases.Paleogenomics is the study of the genetics of past or extinct populations at a genomic scale, utilizing ancient deoxyribonucleic acid (aDNA) from plants, animals, or pathogens. Paleogenomics and ancient virology are practiced on a range of time spans, ranging from historical samples from the 1970s all the way back to before the Last Glacial period, and have led researchers to scour desiccated rat middens and Siberian permafrost for traces of ancient viruses.In this review, we explore the history of research into viruses in historical and ancient material and how methodological developments have advanced our ability to identify and authenticate ancient virus sequence data. We then present case studies from three viruses where paleogenomics has made a major contribution to our understanding of virus evolution: parvovirus B19 (B19V), herpes simplex virus 1 (HSV-1), and variola virus (VARV). Finally, we look to the future of paleovirology.When we think of the evolutionary history of viruses, we often have questions around how long a given virus has been present in a particular host; whether modern disease presentations are the same as when viruses first emerged into a given population; and whether humans have always been the host of a given virus. For recently emerged zoonoses, we may able to answer these questions with contemporary human, environmental, and animal reservoir samples . Many viSudor Anglicus (variously known as the English sweat or the sweating disease) is thought to be a viral pathogen with a mortality rate of up to 50% . A summary of all published ancient RNA and DNA discoveries has recently been published elsewhere . InitialThe earliest studies of ancient viral nucleic acid were typically stored pathology specimens with contemporary diagnostic or symptom data, or from mummies with preserved soft tissue and visible lesions . Later, The success of the last decade in identifying ancient viruses in human remains has been driven not so much by a desire to detect pathogens within human remains, as by the increased use of metagenomic sequencing to acquire whole host genomes at a range of depths. Deeper host metagenomic data sets have been combined with robust computational tools for identifying the most likely taxonomic origin of reads. The combination of richer data, falling costs, improved aDNA extraction techniques, and new computational biology tools has allowed metagenomic sequencing to act as a screening tool for pathogens box 1). Samples with authentic viral reads can be prioritized for resampling, sequencing at greater depth, or target enrichment—or a combination of these approaches. These population screening approaches have allowed the identification of viruses such as hepatitis B virus , VARV M, and ade. SamplesBox 1:Clean excavation, avoiding contamination, and authentication of reads based on damage patterns.Because authentic ancient virus’ genetic data only represent a tiny fraction of the sequences in human remains, researchers should minimize possible contamination with modern viruses. This is accomplished by following a set of simple but effective guidelines:− At the excavation site:i. Handling of the samples with disposable gloves and other protective gear that should be replaced periodically.ii. Cleaning the samples with a brush and not water.iii. Using clean tools for samplings.iv. Storing the samples in a cold and dry place as soon as possible.− At the experimental laboratory:i. Performing experimental procedures in clean dedicated facilities, physically separated from facilities working with modern DNA/RNA.ii. Using positive air pressure to prevent exterior contamination from entering the lab.iii. Cleaning surfaces, incoming laboratory and archeological material using ultraviolet (UV) light, bleach, and/or detergent.iv. Wearing full protective gear including masks, goggles, hair covers, disposable overalls, shoes, and at least two layers of disposable gloves, which should be replaced periodically.− At the bioinformatic laboratory:i. Screening databases should contain a catalog of artificial sequences to reduce the number of spurious hits against viral species .ii. Screened sequences must be further validated using mapping and/or BLAST.iii. Mapped sequences should display a low average edit distance and an even distribution of coverage.iv. Sequences verified by mapping should be tested for the presence of aDNA post-mortem damage at the end of the reads .Metagenomics and/or target enrichmentGenetic data retrieved from ancient samples are metagenomic in nature, which may or may not contain virus. The samples are usually first sequenced in a non-targeted and non-specific way, followed by the metagenomic classification of the generated sequences . CaptureThe challenges of datingAncient viral sequences are of great interest because they allow us to date the emergence of viral lineages. For this to be possible, a correlation between genetic changes and samples’ age must exist . Nonetheless, this process is complex, and several factors can affect the dating:The number of genetic changes can be underestimated due to purifying selection and site saturation. There is no temporal signal .The selection of an inadequate substitution model to date the virus .The ancient virus lineage used to calibrate the phylogeny is too divergent from extant strains. The biological features of the ancient strains, the biological relationships with the host, and their evolutionary rates are different .Current methods for studying ancient virus evolution rely on the sequencing of viral nucleic acids from remains, preserved tissues, and other samples. It is therefore important to reflect that we can only sequence what is preserved, detectable, and classifiable. What do we mean by this?Nucleic acids in human remains degrade and are damaged over time. The damage results from microorganisms and environmental factors . Strand fragment bases are subject to damage by environmental factors such as UV light, heat, and DNases/RNases, which generates short fragments of genetic material which have often been deaminated in characteristic ways. Cool or cold, ideally dry, conditions with relatively constant temperatures are better for DNA and RNA preservation; warm, wet conditions and high levels of UV rays are worse for preservation. Soil pH also matters . DNA preIn an archeological context, mineralized tissues such as bone and teeth persist longer in the environment than soft tissues. The composition of the bone or tooth provides some protection for nucleic acid against degradation by soil fungi and bacteria or cyanobacteria in fresh and saltwater environments; there is also protection from damage by, for example, UV radiation . Soft tiWhen we consider the variety of viruses a human may be infected by during their lifetime, we face almost the full range of virus replication strategies: acute infection (influenza viruses and rhinoviruses), persistent infection (polyomaviruses and papillomaviruses), latent infection (herpesviruses), and genome integration —itself a latency strategy. Designing a single assay to detect all the viruses currently infecting an individually would be extremely challenging with current technology; adding the complete infection history would go beyond techniques currently available. Unsurprisingly, paleogenomics acts as a snapshot of some of the pathogens present with a body site or tissue at the time of death, biased by factors such as virus load, genetic material, and differential preservation of host and pathogen genetic material post-mortem.As previously mentioned, viruses do not leave unambiguous physical evidence of their presence in human bones. The detection of viral aDNA thus relies on the metagenomic screening of tens or hundreds of samples, to be able to detect and retrieve potential viral sequences. In this regard, archeological context can help to assess the existence of an epidemic episode in the past, typically with the presence of human bodies in mass burials or the historical documentation of epidemics. This approach to find samples suitable for screening has been successfully performed for pathogenic bacteria  and, in This is not a problem unique to paleogenomics. For example, in cases of meningitis in adults, an etiological infectious agent is identified in approximately 50% of cases . A studyIn order to identify viruses from ancient data, the virus reads must look like something we already know, something that is already in a database or with homology to known viral sequences. Viruses do not have regions such as 16S which help to identify new bacteria. Genomic markers are interesting from a metagenomic point of view because they are composed by a variable and highly conserved region, thus allowing for massive non-organism–specific sequence capture in a process known as metabarcoding. Regarding viruses, this means that most of the extant viral diversity has not been recorded . The lacRecent research on finding new RNA viruses by using the RNA-dependent RNA polymerase (RdRp) suggests these tools and approaches may be useful when applied to aDNA/ancient ribonucleic acid (aRNA) , but effMALT and KrakenUniq . Because it is an alignment-based method, it is robust to the possible bias induced by aDNA-intrinsic short sequences or aDNA damage (KrakenUniq is a classifier that relies on exact k-mer (subsequences of length k) matches against its database to assign a sequence to a specific taxon. It is computationally fast and efficient , genotype 2 , which is comparable to some RNA viruses  subs/site/year. This suggests that the MRCA of B19V is not in the 1800s, but is likely to be around 8000 Before Common Era (BCE). The MRCAs of genotypes 1, 2, and 3 are around 7, 2, and 2.5 thousand years before present, respectively.Analysis of ancient human remains from Eurasia and Greenland, dating back to 7 thousand years ago (kya), found five genotype 1 and five genotype 2 B19V genomes. B19V genomes from Colonial-era Mexico City  and one Studying ancient B19V genomes also shed light on the evolutionary history of genotype 2. By using ancient and modern genotypes to perform recombination analysis, aDNA confirmed inferences from modern B19V sequences : genotypIt was already known that estimating the substitution rate of B19V from plasma samples, or from DNA derived from autopsies and biopsies, led to differing estimations . By inclHSV-1 is a dsDNA virus that affects at least two-thirds of the adult human population. Most infections occur during infancy or childhood and are mild or asymptomatic; however, HSV-1 can lead to severe complications, especially in immune-compromised individuals. Following primary infection, the virus becomes latent in sensory neurons. When triggered by psychological or physiological stress, the virus can reactivate resulting in recurrent labial lesions. The host immune system in a healthy individual usually prevents a substantial viremia .Modern isolates of HSV-1 go back to 1925 (strain HF) . Many stBecause many species have their own species-specific alpha herpes viruses, a co-divergence scenario with anatomically modern humans seems likely; however, recent estimates of modern HSV-1 diversity dated to only the last 7,000 years . This coCurrently available ancient HSV-1 genomes come from four individuals from archeological sites across northern Europe, covering the last 2,000 years: a young adult male from an urban medieval hospital cemetery (1350–1450 CE) and an adult female from an early Anglo-Saxon cemetery (500–575 CE) in Cambridgeshire, United Kingdom; an adult male from a burial related to the Nevolino culture (253–530 CE) in Russia; and an adult male from the Netherlands (1600–1700 CE) . The indThe estimated age of sampled modern Eurasian HSV-1 diversity is 4.68 (3.87–5.65) kya, and extrapolation of estimated rates to a global data set (including African lineages) points to the age of extant sampled HSV-1 at 5.29 (4.60–6.12) kya, suggesting HSV-1 lineage replacement coinciding with the late Neolithic period and following known Bronze Age migrations. Unfortunately, the age of the recovered genomes precludes answering questions about what was circulating before the lineage replacement (if anything).It should also be noted that HSV-1 DNA was detected in the deciduous teeth of two children who lived over 30,000 years ago; however, there was insufficient HSV-1 DNA recovered to reconstruct a Pleistocene HSV-1 genome from the teeth. Thus, whereas this is further evidence that HSV-1 is an ancient human pathogen, it is not possible to use this ancient DNA to further reconstruct the deep history of HSV-1, for example, asking whether these ancient HSV-1 infections come from lineages that are extinct today or basal to current Eurasian diversity .Poxviruses have a reputation as notorious human pathogens, a reputation founded by the morbidity and mortality burden of two members of the orthopox genus: VARV, the causative agent of smallpox, and monkeypox virus (MPXV). Smallpox has been estimated to be the single most deadly infectious disease in human history, because of the cumulative death toll associated with infection and its complications . Indeed,As a family of viruses, the poxviruses are notable for their genome and viral particle size and their replication within the cytoplasm of infected cells, which is more typical of RNA viruses. Only VARV and molluscum contagiosum virus (MOCV) are obligate human pathogens , whereasBecause of the relatively distinctive skin rash and disease caused by VARV infection, we are fortunate to have a few different sources of evidence to help us understand the origins of this high-consequence human pathogen. Studies of ancient Egyptian mummies and historical medical textbooks and accounts have suggested that smallpox may have been a human disease since around 2000 BCE, although unambiguous written descriptions of smallpox symptoms only become common around 1600 CE . Ancient−6 subs/site/year and by Ferrari to be 10.67 × 10−6 subs/site/year. This suggested that VARV diversity sampled between 1944 and 1977 had a relatively recent common ancestor, in the sixteenth to seventeenth century, perhaps as the result of a population bottleneck. These studies of samples from the pre-vaccination era support a view that the diversity of VARV in Europe and North America before the twentieth century was considerably greater than in the post-vaccination period. This would seem difficult to reconcile with evidence of smallpox-like lesions on mummified remains and the presence of ancient written descriptions.Studies of VARV genomes derived from historical museum specimens point towards a relatively young age for smallpox. For example, the estimated substitution rates of VARV were estimated by Duggan to be 8.5 × 10−6 subs/site/year, suggests that different VARV lineages shared a common ancestor around 1,700 years ago.Metagenomic screening of ancient human remains has made an important contribution to how we view VARV genetic evolution. A study spanning the Viking era in northern Europe (600–1050 CE) revealed 11 individuals from this period with VARV DNA sequences preserved in their remains . Four seThe Viking-era VARV genomes also had a unique pattern of gene inactivation compared with later VARV strains, some of which imply that ancient VARV had the ability to infect a broader range of hosts than later, historical VARV strains. Other convergent gene inactivations suggest that both modern and ancient VARV lineages had a similar ability to induce a high fever in infected hosts. The phenotype of many of the differentially inactivated VARV genes between lineages is, however, unknown . This thUnderstanding the evolutionary history of VARV, despite its eradication, remains an important scientific goal. Between 2001 and 2018, cases of MPXV increased from 500 to 3,000 cases each year in the Democratic Republic of Congo (DRC), with case fatality rates estimated in a range from below 2% to as high as 11%, depending on MPXV genotype and the HIV status of the infected host . MPXV raOrthopoxvirus a successful human pathogen and how changes in particular regions of the genome may be associated with virulence, transmissibility, and host range. It also allows us to ask questions about what impact past selective pressures on the human genome from smallpox-associated mortality have had and whether this allows us to identify individuals at greater risk of severe outcomes following Orthopoxvirus infection. Studying recent MPXV evolution may in turn help to generate new hypotheses about early events in VARV evolution, which may become testable if even older VARV aDNA becomes available.Studying the ancient evolution of VARV alongside contemporary evolution of MPXV gives virologists vital insights into what makes an Ancient samples contain degraded and fragmented portions of viruses which are not replication competent . A samplIt is possible to create viral clones from synthetic genetic material which are unable to replicate their genomes (replication incompetent), clones which are able to replicate but not proliferate, or clones which replicate and proliferate in a manner akin to wild-type virus . These tThere are alternative techniques from molecular biology and viral immunology which allow high-risk pathogens such as Ebola virus to be studied more safely  and theyAncient viral nucleic acid is everywhere: human and animal remains, herbaria specimens, and environmental material such as middens, ice cores, and sediments. Our ability to sequence, classify, and analyze this ancient virome is growing with time, giving us fascinating insights into the tempo and dynamics of viral evolution across a range of time scales. The tools of molecular and synthetic biology are increasingly making it possible to study ancient and extinct viruses in the laboratory alongside circulating viruses. Even as we contemplate the possibilities and pitfalls of virus “de-extinction,” it is important to remember that researching ancient viral pathogens can also give us key insights into emerging infectious diseases. By learning about the viruses which afflicted our ancient ancestors, we may hope to reduce the burden of disease caused by their modern viral relatives.


Carios vespertilionis (Argasidae) is a common ectoparasite of bats in the Palearctic region and is suspected to be vector and reservoir of viruses and other microbial species in bat populations, some of which may act as zoonotic agents for human disease. The Soprano pipistrelle  is widely distributed in Europe, where it can be found inside or close to human habitation. We used meta-transcriptomic sequencing to determine the RNA virome and common microbiota in blood-fed C. vespertilionis ticks collected from a Soprano pipistrelle bat roosting site in south-central Sweden. Our analyses identified 16 viruses from 11 virus families, of which 15 viruses were novel. For the first time in Sweden we identified Issuk-Kul virus, a zoonotic arthropod-borne virus previously associated with outbreaks of acute febrile illness in humans. Probable bat-associated and tick-borne viruses were classified within the families Nairoviridae, Caliciviridae and Hepeviridae, while other invertebrate-associated viruses included members of the Dicistroviridae, Iflaviridae, Nodaviridae, Partitiviridae, Permutotetraviridae, Polycipiviridae and Solemoviridae. Similarly, we found abundant bacteria in C. vespertilionis, including genera with known tick-borne bacteria, such as Coxiella spp. and Rickettsia spp. These findings demonstrate the remarkable diversity of RNA viruses and bacteria present in C. vespertilionis and highlight the importance of bat-associated ectoparasite surveillance as an effective and non-invasive means to track viruses and bacteria circulating in bats and ticks.Ticks harbour a high diversity of viruses, bacteria and protozoa. The soft tick  The sequencing reads and viral sequences identified in this study were deposited at the Sequence Read Archive (SRA) database under accession numbers SAMN29627891–SAMN29627902 (Bioproject: PRJNA838788) and GenBank database .Carios vespertilionis. Given that both the bat and tick can be found in direct proximity to human habitation and are associated with disease-causing zoonotic agents, we used meta-transcriptomic sequencing to uncover the RNA virome and microbiota in ticks that had recently blood-fed off Soprano pipistrelle individuals. In addition to identifying 15 novel viruses and several abundant bacteria, we also detected Issuk-Kul virus, a zoonotic pathogen associated with human disease. Our study not only expands our knowledge of bat–tick-associated viruses and microbes, but also demonstrates the utility and importance of using ectoparasites to non-invasive survey bats for known and novel viruses and bacteria.Bats and ticks are known vectors and reservoirs of diverse pathogenic and non-pathogenic viruses, bacteria and protozoa. The Soprano pipistrelle is a common bat species in Europe that is often parasitized by the soft tick Carios vespertilionis, formerly known as Argas vespertilionis (Ixodida: Argasidae)  and the G-7-mTase [LSHP] domains. Similarly, the contig partially covering the RdRp was assigned with ~36 % similarity and 99.9 % confidence to the RdRp of the parainfluenza virus . The full diversity of RNA viruses characterized in this study included two negative-sense RNA viruses (-ssRNA), 12 positive-sense RNA viruses (+ssRNA) and one double-strand RNA (dsRNA) virus. Likewise, the virus prevalence ranged from six to ten viruses detected per tick library.We detected a high diversify of RNA viruses and microbiota, corresponding to bacteria and parasitic protozoa in the bat-ticks analysed. Overall, we identified 16 viruses based on the identification of RdRp sequences, including 15 putative novel viruses within the families oviridae . Among toviridae . The mosabundant . Also ofNairoviridae, including one novel virus. The novel virus corresponded to Gubbo nairovirus (GUBV) and exhibited the three segments typical to nairoviruses. GUVB shared a limited amino acid sequence in similarity with Artashat orthonairovirus based on comparison with the viral polymerase (aa %id=50.5) (We identified two viruses within the id=50.5) . We alsoid=50.5) . Both naid=50.5) . As expeid=50.5) . In partCaliciviridae provisionally referred to as Harg calici-like virus (HCAV), which was present in all the libraries at low to moderate abundance levels , including two ORFs encoding the RdRp and the major capsid protein VP1, respectively. Finally, among the most abundant viral families, we identified one novel virus – Valo virus (VALV) – belonging to the Hepeviridae that was well represented in all the libraries (RPM=200000–100000)  . Phyloge–100000) . As a caPicornavirales, we identified three iflaviruses , two polycipiviruses (Graso virus and Dudero virus), and one dicistrovirus (Aspo dicistro-like virus). Moreover, we identified two members of the Nodaviridae (Gravol virus and Bolka virus), one permutotretavirus (Ladskar virus) and one solemovirus (Ed virus). For all the viruses with the exception of Graso polycipi-like virus and Barko virus, we only detected the viral RdRp gene , including members of the Rickettsia, Delftia and Coxiella, which were present in all the libraries screened , including R. conorii, R. africae, R. slovaca and R. parkeri. Phylogenetic analysis based on the outer membrane protein A (ompA) gene suggested a close relationship to R. parkeri within the SFG (~ 99 % nt similarity)  . In the is ticks . C. vespTicks naturally harbour a highly diverse array of viruses, bacteria and protozoans. Since ticks are obligately haematophagous, these parasitic arthropods might also carry the viruses and microbiota of their hosts acquired during the blood meal . The natC. vespertilionis virome revealed the family Nairoviridae  as the most abundant and prevalent in the libraries of recently blood-fed ticks , they were not included in our analyses due to the limited length of the contigs. It should be noted, however, that paramyxoviruses have been reported in  species . That we species , and bat species .Caliciviridae and Hepeviridae is consistent with previous research on bat-borne and tick-borne viruses. A few bat caliciviruses (sapoviruses and unclassified viruses) have been discovered in European vespertilionid bats [P. pygmaeus [Caliciviridae, although establishing a definitive association with vertebrate/invertebrate hosts is uncertain. In contrast, the novel hepevirus VALV suspected to be associated with the tick virome was most closely related to Bulatov virus and Vovk virus that have been associated with the virome of Ixodes uriae ticks from the Antarctic peninsula [The presence of newly discovered +ssRNA virus members within the nid bats , althougpygmaeus . The caleninsula . In addieninsula , our stuIflaviridae, Nodaviridae, Solemoviridae and Partitiviridae, previously identified in the virome of different tick species [Dicistroviridae, Permutotetraviridae and Polycipiviridae that are probably infecting the bat-ticks. Indeed, the dominance of Graso polycipi-like virus (Polycipiviridae) and Ed virus (Solemoviridae) in the tick libraries might indicate the efficient replication of these viruses within this arthropod species, although this will need further research. Similarly, the occurrence of Partitiviridae in C. vespertilionis is compatible with studies suggesting that partitiviruses can possibly infect arthropods [C. vespertilionis has been largely focused on targeting tick-borne viruses of public health relevance [C. vespertilionis.As expected, a considerable fraction of the tick virome corresponded to viruses associated with invertebrates. This included virus families such as the  species . We alsothropods , as wellelevance , with thC. vespertilionis can harbour a repertoire of bacterial and protozoal species [Rickettsia spp. in C. vespertilionis collected in Sweden, corroborating previous reports in Europe [ompA genes, the close relationship to Rickettsia species, and in particular to R. parkeri, in the SFG might constitute a risk for vector-borne zoonotic disease. Rickettsial infections with some species within the SFG have been associated with pathogenicity in humans [R. parkeri is an emergent tick-borne pathogen and the causative agent of R. parkeri rickettsiosis in America [R. helvetica and R. felis have been associated with severe clinical manifestations, including meningitis [Rickettsia could play a role in the provision of folate in Ixodes pacificus ticks [Rickettsia and C. vespertilionis remains uncertain, as does their pathogenic potential for bat hosts [Rickettsia spp. have been reported in bat tissue samples collected from vespertilionid bats [Rickettsia spp. in nature merits investigation [It has previously been shown that  species . We idenn Europe . Althougn humans . For ins America . Similarningitis . In contus ticks . The ranat hosts . Likewisnid bats . Therefotigation .Delftia spp. and Coxiella spp. (Delftia spp. have been reported as core bacteria in the microbiome of Dermacentor variabilis [Delftia spp. in the tick libraries, a similar situation might exist for C. vespertilionis [Coxiella burnetii, the aetiological agent of Q fever, in C. vespertilionis ticks collected from Asia [Coxiella species are considered obligate and associated with nutritional and reproductive roles in ticks [We also reported the co-occurrence of bacteria such as a spp. . Delftriabilis . Given ttilionis . An earlrom Asia , althougin ticks . GeneralC. vespertilionis ticks in Sweden. The presence of dominant and underrepresented viruses and bacteria warrants further research into the nature of bat–tick interactions and how these impact viral and microbial transmission. Additional vector competence studies are required to demonstrate that C. vespertilionis ticks can become infected when feeding on an infectious host and maintain the pathogen such that it is capable of being transmitted to an uninfected, susceptible host [Overall, we provide new insights into the viral and bacterial diversity associated with ble host . Despiteble host .Click here for additional data file.Click here for additional data file.Click here for additional data file.Click here for additional data file.Click here for additional data file.Click here for additional data file.


The BID50 for the 2016 H5N8 virus was also slightly different in chickens and turkeys ; however, the BID50 for the 2014 H5N8 virus was higher for chickens than turkeys . With all viruses, turkeys took longer to die (MDTs of 2.6–8.2 days for turkeys and 1–4 days for chickens), which increased the virus shedding period and facilitated transmission to contacts. Highly pathogenic avian influenza viruses (HPAIVs) of subtype H5 of the Gs/GD/96 lineage remain a major threat to poultry due to endemicity in wild birds. H5N1 HPAIVs from this lineage were detected in 2021 in the United States (U.S.) and since then have infected many wild and domestic birds. We evaluated the pathobiology of an early U.S. H5N1 HPAIV  and two H5N8 HPAIVs from previous outbreaks in the U.S.  and Europe  in chickens and turkeys. Differences in clinical signs, mean death times (MDTs), and virus transmissibility were found between chickens and turkeys. The mean bird infective dose (BID Highly pathogenic (HP) avian influenza viruses (AIVs) of the H5 subtype A/Goose/Guangdong/1996 Eurasian lineage (Gs/GD) have caused multiple intercontinental outbreaks in domestic and wild bird populations through dissemination via migratory waterfowl ,3,4,5,6.In January 2022, an H5N1 HPAIV from this lineage was reported in a wild bird sample collected in December 2021 in South Carolina, United States (U.S.) ,27, becoThe reason for the unprecedented spread of the recent H5N1 clade 2.3.4.4b virus is unclear. To increase our understanding of the epidemiology and pathobiology of these viruses in gallinaceous species and to improve control of H5N1 HPAI in poultry, we characterized the infectivity, transmissibility, and pathogenicity of an early U.S. H5N1 isolate (clade 2.3.4.4b) in the two most important poultry species, turkeys and chickens, and compared such results to those for two H5N8 HPAIVs from previous outbreaks (clades 2.3.4.4c and 2.3.4.4b). Three H5 clade 2.3.4.4 HPAIVs were used in this study: A/American Wigeon/South Carolina/22-000345-001/2021 (H5N1) (AMWI/SC/21) (H5 clade 2.3.4.4b), A/Tufted duck/Denmark/11740-LWPL/2016 (H5N8) (TUDU/Denmark/16) (H5 clade 2.3.4.4b), and A/Gyrfalcon/Washington/41088-6/2014 (H5N8) (GYRF/WA/14) (H5 clade 2.3.4.4c). AMWI/SC/21 was kindly provided by the National Veterinary Services Laboratories (NVSL), Animal and Plant Inspection Service (APHIS), the U.S. Department of Agriculture (USDA). This virus was collected by the Wildlife Services National Wildlife Disease Program, APHIS, USDA, on 31 December 2021, and has also been named AMWI/SC/2022 based on the date of confirmation . TUDU/DeMeleagris gallopavo) were provided by a commercial producer and were reared at the USNPRC until three weeks of age. Four-week-old specific-pathogen-free white leghorn chickens  were obtained from the USNPRC in-house flocks. Birds were transferred to the animal BSL-3E facilities at the USNPRC, where each experimental group was housed in self-contained isolation units ventilated under negative pressure with inlet and outlet HEPA filtration. Chickens and turkeys had ad libitum access to food and water throughout the experiment. Housing and experimental procedures were reviewed and approved by the USNPRC Institutional Animal Care and Use Committee (IACUC). Day-old turkeys (50) of each virus for each bird species. The intended target doses were 2, 4, or 6 log10 50% embryo infective dose (EID50) per bird administered in 0.1 mL. The actual titers of the challenge viruses were directly determined via egg titration using standard methods as described above. In addition, to evaluate the transmissibility of each virus, three naïve hatch-mate birds from the same species were added to each dose group 24 h after inoculation (contact-exposed birds). A group of eight hatch mates of the chickens and turkeys served as non-inoculated controls for each species.A total of 80 chickens and 80 turkeys were used in this study. The experimental design was similar to previous studies ,35,36. BOropharyngeal (OP) and cloacal (CL) swabs were collected at 1, 2, 3, 4, 7, and 10 days-post-inoculation (dpi) from the inoculated birds, and at 1, 2, 3, 6, and 9 days post-contact (dpc) from the contact-exposed birds. Swabs were placed in 1 mL of BHI medium with penicillin (2000 units/mL), gentamicin (200 μg/mL), and amphotericin B (5 μg/mL) , and stored at −80 °C until they were processed to determine virus shed titers. Two birds from the groups that received the higher doses of the viruses were euthanized and necropsied at 2 dpi. Skeletal muscle, lung, spleen, heart, and brain tissues were collected from these birds and stored at −80 °C for virus detection and quantification. A full series of tissues were additionally collected for histopathology and fixed in 10% neutral-buffered formalin. Immunohistochemistry was also conducted to assay for avian influenza virus antigen in tissues using a previously described method . IdenticAll birds were observed for clinical signs and mortality from 0 to 11 dpi (direct inoculates) and 0 to 10 dpc (contacts). Birds showing severe clinical signs, including severe lethargy, prostration, neurological signs, or inability to eat or drink, were euthanized and counted as dead the next day for mean death time (MDT) calculations. At the end of the study, 11 dpi (or 10 dpc for contacts), surviving birds were bled and euthanized. 50 for each virus was calculated using the Reed–Muench method [Sera collected from all surviving birds was used to evaluate infection status by detecting AIV antibodies using the hemagglutination inhibition (HI) test and the agar gel immunodiffusion assay (AGID). Assays were performed using standard methods and homologous antigens ,39. The h method , using t®  [w/v) solution, and total RNA was extracted from the homogenates using Trizol LS reagent  and chloroform according to the manufacturer’s protocol. The resulting tissue RNA extracts were purified using an RNA Clean and Concentrator Kit , quantified with a NanoDrop™ 1000 Spectrophotometer (Thermo Fisher Scientific) following the manufacturer’s instructions, and accordingly diluted with Tris-EDTA buffer  to obtain 50 ng/µL of total RNA. Swab and tissue samples were processed for quantitative real-time RT-PCR (qRT-PCR) to determine viral titer equivalents. A standard protocol that demonstrated the high correlation between qRT-PCR and infectious titers determined in ECEs  was usedMA, USA) . For tis10 EID50/mL for AMWI/SC/21, 1.9 log10 EID50/mL for TUDU/Denmark/16 (H5N8) HPAIV, and 1.8 log10 EID50/mL for GYRF/WA/14. qRT-PCR was performed with the AgPath-ID one-step RT-PCR Kit  using a 7500 FAST real-time PCR system  as previously described . Standarhttps://www.gisaid.org/, (accessed on 15 April 2023)) by selecting the clade 2.3.4 and 2.3.4.4. After removing duplicated sequences, a total of 5327 sequences were subjected to the phylogenetic analysis. A streamlined sequence analysis including duplicate removal, alignment, and the maximum-likelihood (ML) phylogenetic tree was carried out using RAxML by utilizing the Galaxy web platform [Whole-genome sequences of the three H5 HPAIVs were compared . For each isolate, eight segments were concatenated in order of decreasing segment length. Pairwise sequence identities among the concatenated genomes of the three virus isolates were calculated in Geneious Prime 2023.1.1 . Full-length open-reading frame nucleotide sequences were aligned using MAFFT . Translaplatform ,45. The 50 for each virus.The results for virus infectivity, lethality, and transmission for chickens and turkeys are shown in 10 EID50 dose of AMWI/SC/21, and all chickens inoculated with the 5.6 or 7.6 log10 EID50 doses, became infected and died (MDTs between 1 and 2.7 dpi). In the two chickens in the lower-dose group that survived, the virus was detected in OP swabs at 2 dpi, but the birds were seronegative at the end of the study. With the given titers of the virus inocula, it was not possible to determine the exact 50% endpoint for BID50 determination. Assuming less than 50% infection at subsequent dilutions, the titer was around 2.6–3.3 log10 EID50; . The virus was detected in OP swabs from all contact-exposed chickens in all three dose groups, but none died or had antibodies against the virus at the end of the study. Three out of the five chickens inoculated with the 3.6 log10 EID50) became infected and died (MDT of 2 dpi). The virus was detected in OP swabs from all three contact-exposed birds in the high-dose group but only one died; the remaining two birds did not seroconvert. No chickens inoculated with the low dose (1.5 log10 EID50) or medium dose (2.8 log10 EID50) of this virus were infected except for one chicken in the medium-dose group, which died at 3 dpi. No contact-exposed birds in the low- or medium-dose groups were infected. The BID50 for TUDU/Denmark/16 in chickens was 4.2 log10 EID50. All five chickens inoculated with the high dose of TUDU/Denmark/16 (6.7 log10 EID50) or the contacts in this group became infected. All chickens from the medium-dose (4.6 log10 EID50) and high-dose (6.6 log10 EID50) groups became infected, and all, except one from the medium-dose group, died . The surviving bird did not seroconvert. The BID50 for this virus was 3.9 log10 EID50. No contact-exposed birds in the medium-dose group were infected. The virus was detected in OP swabs from all three contacts from the high-dose group and from the CL route in the bird that died. The remaining two birds did not seroconvert.No chickens inoculated with the low dose of GYRF/WA/14 . These birds also had ruffled feathers, periorbital swelling, and cyanotic combs. Some chickens died without showing clinical signs (peracute disease) (16.7%). Most of the surviving chickens did not show evidence of clinical disease. However, one and two contact-exposed chickens in the AMWI/SC/21 medium- and high-dose groups, as well as the two remaining contacts in the TD/Denmark/22 high-dose group, were less active and smaller than the rest of the birds. Non-inoculated chickens were clinically healthy throughout the experiment.50 for this virus in turkeys was 2.2 log10 EID50, assuming less than 50% survival (2/5 birds) at the next dilution; if all birds survived, the BID50 would be 2.6. All contact-exposed turkeys became infected and died (MDTs between 3.3 and 5.7 dpc).All turkeys inoculated with AMWI/SC/21, regardless of the dose given, became infected and died, with MDTs between 2.6 and 4.6 dpi depending on the virus dose; the higher the virus dose received, the shorter the MDT. The approximate BID50 for this virus was 4.7 log10 EID50. No turkeys in the low- and medium-dose groups of TUDU/Denmark/16 became infected. All turkeys inoculated with the high virus dose became infected and died (MDT of 3 dpi), as well as the contact-exposed birds in this group (MDT of 4.3 dpc). The BID50 for this virus was approximately 0.9 log10 EID50.All turkeys inoculated with GYRF/WA/14 and the contacts in these groups became infected and died. The MDT ranged from 3 to 8.2 days and correlated with dose. The MDT of the contact-exposed birds was between 5.7 and 7 dpc. The BIDOf the turkeys counted as dead, 35.7% showed no clinical signs before death. Mild lethargy was observed in the rest, which progressed to severe lethargy and prostration at which point they were euthanized . Some of the turkeys inoculated with AMWI/SC/21 also had ruffled feathers, and some inoculated with GYRF/WA/14 that were euthanized at the later timepoints had neurological signs consisting of torticollis and ataxia . OP and CL virus shedding was examined in inoculated and contact-exposed birds by qRT-PCR . 10 EID50, respectively), and all died that day  (All chickens from the TUDU/Denmark/16 high-dose group and one chicken from the medium-dose group shed high titers of the virus by both OP and CL routes (8.1 ± 0.6 and 5.7 ± 1.2 logt 1 dpi) B. Chicket 1 dpi) . 10 EID50, OP and CL, respectively, 1 dpi)  C. The vi, 1 dpi) . 10 EID50, respectively at 1 dpi) (10 EID50). Between 2 and 4 dpi, high virus titers were found in CL swabs from all inoculated turkeys in these two groups, which all died by 4 dpi. Shedding in the low-dose group occurred later, and birds died between 3 and 6 dpi. All contacts shed the virus by both routes and as early as 1 dpc in the medium- and high-dose groups (High virus titers were detected in OP swabs starting at 1 dpi from most turkeys inoculated with the medium and high dose of AMWI/SC/21 (4.7 ± 1.4 and 5.2 ± 0.4 logt 1 dpi) A. Moderae groups .10 EID50, respectively, at 2 dpi)  B. The cot 2 dpi) . 10 EID50), and all birds shed by the CL route by 2 dpi (5.3 ± 1.1 log10 EID50) (Turkeys inoculated with the high dose of GYRF/WA/14 shed high virus titers by the OP route as early as 1 dpi (6.1 ± 0.7 log0 EID50) C. All bi0 EID50) . Two birds from the groups of chickens and turkeys inoculated with the high dose of each of the viruses were euthanized and necropsied at 2 dpi. Because there were no survivors in the high-dose group of chickens inoculated with AMWI/SC/21 at this timepoint, one moribund chicken from the medium-dose group was examined. Similar gross lesions were observed in all the chickens and consisted of empty intestines, multifocal necrosis in the pancreas, congested lungs, petechial hemorrhages in the thymus, cecal tonsils and skeletal muscle, and splenomegaly with pale parenchymal mottling. The gross lesions observed in the turkeys included empty intestines and dehydration. Microscopic lesions and viral antigen staining were similar in severity and distribution among chickens and turkeys inoculated with AMWI/SC/21 and GYRF/WA/14 and were more severe and widespread in birds inoculated with TUDU/Denmark/16. Microscopic lesions consisted of multifocal necrosis in the parenchyma of several tissues, including the brain, spleen, adrenal gland, kidney, pancreas, bursa, thymus, cecal tonsils, harderian gland, and liver, which were similar to what was reported for other H5 HPAI viruses ,32,46,47The skeletal muscle, lung, spleen, heart, and brain were also collected from the birds necropsied at 2 dpi for viral quantification via qRT-PCR . ModeratA phylogenetic tree of Gs/GD-lineage H5 clade 2.3.4.4 HPAIVs was constructed including the three wild bird H5 HPAIVs used in this study . GYRF/WAWhole-genome sequence comparison between AMWI/SC/21 and the other two viruses showed 534 amino acid differences between AMWI/SC/21 and TUDU/Denmark/16 and 805 amino acid differences between AMWI/SC/21 and GYRF/WA/14. Most of these differences were present in the Neuraminidase (NA) protein due to the different NA subtypes. The similarity between the NA from AMWI/SC/21 and TUDU/Denmark/16 was 57.02% (347 differences), and with GYRF/WA/14, the similarity was 56.32% (346 differences) . Compared to the HA of AMWI/SC/21, the HA of TUDU/Denmark/16 was 97.2% similar, and the HA of GYRF/WA/14 was 92.6% similar, which could be attributed to the subclade differences, with AMWI/SC/21 and TUDU/Denmark/16 belonging to clade 2.3.4.4b and GYRF/WA/14 belonging to clade 2.3.4.4c . Except Pairwise amino acid comparisons between AMWI/SC/21 and the other two viruses showed that AMWI/SC/21 and TUDU/Denmark/16 had a smaller number of amino acid differences than AMWI/SC/21 and GYRF/WA/14. The amino acid differences between TUDU/Denmark/16 and GYRF/WA/14 were compared in a previous study, with some residues associated with biological changes of importance . Some ofSince 2014, Gs/GD lineage H5 clade 2.3.4.4 HPAIVs have rapidly emerged and spread from Asia to other parts of the world via migratory waterfowl, causing outbreaks in domestic and wild bird species ,53,54. T10 EID50), with two chickens surviving infection in the low-dose group. A difference was observed in MDTs, where chickens died 1.9, 1.4, or 1.6 days earlier than turkeys depending on the virus dose given; the higher the virus dose, the faster they died. Chickens also shed higher titers of the virus earlier than turkeys but for fewer days because of the earlier mortality. AMWI/SC/21 was detected in OP swabs of all contact-exposed chickens and turkeys, but only contact-exposed turkeys became infected and died. Contact-exposed turkeys shed viruses in moderate-to-high titers, whereas virus detection was minimal in contact-exposed chickens. As for TUDU/Denmark/16, the mean infective dose was slightly lower in chickens than in turkeys (4.2 vs. 4.7 log10 EID50), and the MDT for the high-dose groups was lower in chickens than in turkeys (2 vs. 3 dpi). The virus was detected in the OP swabs of the contact-exposed chickens in the high-dose group, but only one became infected and died; however, all contact-exposed turkeys became infected and died. Only the chicken that eventually died shed high titers by the CL route, compared to all contact-exposed turkeys that shed moderate-to-high titers by both routes. A major difference in BID50 was observed with GYRF/WA/14, with the virus being much more infectious for turkeys than for chickens (~0.9 vs. 3.8 log10 EID50). When comparing the MDTs in the medium- and high-dose groups, chickens died 2.6 or 0.4 days earlier, respectively, than the turkeys. The virus was detected in the OP swabs of the contact-exposed chickens in the high-dose group, but only one became infected and died; by contrast, all contact-exposed turkeys became infected and died. In the present study, we compared the infectivity, pathogenicity, and transmissibility in chickens and turkeys of one of the first 2021 H5N1 HPAIV wild bird U.S. isolates and two H5N8 HPAIV wild bird isolates from 2014 and 2016. We found that the 2021 H5N1 virus (AMWI/SC/21) was more infectious for chickens than the 2014 (GYRF/WA/14) and 2016 (TUDU/Denmark/16) H5N8 viruses. In turkeys, the 2021 H5N1 virus was more infectious than the 2016 H5N8 virus; however, the 2014 H5N8 virus was more infectious than the 2021 H5N1 virus. When comparing the results from chickens and turkeys, the approximate mean infective dose of AMWI/SC/21 was slightly lower in turkeys than chickens . This variable transmissibility of HPAIVs in chickens contrasts with the 100% transmission observed in turkeys. An important caveat is that laboratory studies do not perfectly simulate transmission in the field, and housing varies, so transmissibility may not always directly compare between studies.All the three H5 clade 2.3.4.4 HPAIVs used in this study caused systemic infection and high mortality in infected chickens and turkeys; however, similar to previous studies, the susceptibility to infection depended on the bird species, the virus strain, the exposure dose, and the virus’s degree of adaptation to gallinaceous species ,47,61,62In this study, birds were considered infected if virus shedding and seroconversion were detected. We found that birds that shed virus through both the OP and CL routes eventually died from infection. In contrast, the virus was only detected in OP swabs for 1–2 days in chickens that survived infection. One possible explanation for this observation is that the virus detected in OP swabs was virus contracted through environmental exposure  and may or may not be viable. Another explanation is that the virus replicated locally but did not spread systemically, and the bird’s innate immune system controlled the infection. In fact, some of the surviving chickens were smaller and less active than the non-inoculated birds, indicating possible subclinical or mild disease. The lack of detectable HI or AGID antibodies at days 10 or 11 after virus exposure suggests that the HPAIV did not replicate well, in contrast to prior studies with LPAIVs in chickens in which measurable antibodies were detected 7 days after inoculation . FurtherTurkeys are known to be generally more susceptible than chickens to infection with many LP and HPAIV isolates from wild waterfowl and poultry and to more effectively transmit the virus to direct contacts ,70,71,72One of the major differences in disease presentation between the chickens and turkeys in our study was the longer MDTs observed in the infected turkeys compared to chickens, which may be due to less severe vascular endothelial replication in turkeys than in chickens. Unusually long MDTs have also been reported in turkeys infected with other H5 clade 2.3.4.4 viruses ,65, and Another important difference observed in this study, apart from the earlier mortality observed in chickens compared to turkeys, was the clinical presentation of the disease. For example, all turkeys inoculated with AMWI/SC/21 became infected and died within 4.6 days regardless of dose. The chickens infected with this virus died within 2.7 days, and not all inoculated chickens died in the lower-dose group. This highlights the difference in clinical outcomes between chickens and turkeys, where chickens have a more rapid and severe response to HPAIV infection but need a higher initial dose of the virus to become infected. Blaurock et al. found that turkeys mount a different host immune response than chickens to HPAIV infection, which could explain these differences in infection outcomes . It is i50 of these viruses [50 values for the more gallinaceous-adapted 2015 H5N2 viruses were similar to what we found with the 2021 H5N1 wild bird isolate examined in this study, indicating that this virus can more easily infect poultry than the index wild-bird 2014 H5 HPAIVs. The clade 2.3.4.4b viruses have affected many bird species in their spread through different continents. It is not clear how the virus has adapted to replicate successfully in so many different bird species, both domestic and wild, but by doing so, it has facilitated its transmission and spread.The index H5N2 and H5N8 clade 2.3.4.4c HPAIVs detected in wild birds in the U.S. in 2014 were highly adapted to waterfowl and not yet well adapted to chickens and turkeys based on the comparatively high BID viruses ,46,47. H viruses ,46. The Although TUDU/Denmark/16 and AMWI/SC/21 are more closely related, the genetic comparison of the three viruses showed many amino acid differences between them, which complicates the determination of molecular markers associated with increased infectivity and transmission in birds. However, some of the amino acid changes associated with the increased pathogenicity in chickens or ducks found between TUDU/Denmark/16 and GYRF/WA/14  were alsThis study emphasizes the nuanced variations in pathobiology between different bird species and among different virus strains. These differences can play into the epidemiology and ecology of HPAIVs and have implications for the development of HPAIV outbreaks. The Gs/GD lineage H5 HPAIV outbreaks continue to occur, and thus, it is important to conduct pathobiology studies to inform and improve strategies to prevent and mitigate such outbreaks.


Arboviruses receive heightened research attention during major outbreaks or when they cause unusual or severe clinical disease, but they are otherwise undercharacterized. Global change is also accelerating the emergence and spread of arboviral diseases, leading to time-sensitive questions about potential interactions between viruses and novel vectors. Vector competence experiments help determine the susceptibility of certain arthropods to a given arbovirus, but these experiments are often conducted in real time during outbreaks, rather than with preparedness in mind. We conducted a systematic review of reported mosquito–arbovirus competence experiments, screening 570 abstracts to arrive at 265 studies testing in vivo arboviral competence. We found that more than 90% of potential mosquito–virus combinations are untested in experimental settings and that entire regions and their corresponding vectors and viruses are undersampled. These knowledge gaps stymie outbreak response and limit attempts to both build and validate predictive models of the vector–virus network. For the remaining 287 records, we undertook a second round of screening to determine suitability . After excluding one study for which we could not access the full text,Supplemental Table 1). For each of these, we recorded the species pairs of mosquitoes and arthropod-borne viruses that were experimentally tested together, regardless of how competence was measured  or whether the vector was found to be competent or not; the country from which wild mosquitoes were originally collected (even if populations were maintained in laboratory settings long-term); and when available, mosquito subspecies and (as applicable) dengue virus serotype. Using the taxize R package,,Culex sp.,” “Culex declarator/mollis”).We performed an initial screen of records based on abstracts, excluding reviews, methodology descriptions, and studies with no experimental infection  and mosquito species (N = 122) that have been tested experimentally, leaving the majority of all possible pairings of these specific viruses and mosquitoes untested , and most were untested in Anopheles (27 viruses)  . AlthougAe. aegypti (N = 47 studies) and Aedes albopictus (N = 19), Zika virus in Ae. aegypti (N = 35) and Ae. albopictus (N = 18), chikungunya virus in Ae. aegypti (N = 24) and Ae. albopictus (N = 23), and West Nile virus in Culex pipiens (N = 30), Culex quinquefasciatus (N = 16), and Culex tarsalis (N = 8). Most research has focused on flaviviruses  and alphaviruses , with less focus on bunyaviruses , rhabdoviruses , and orbiviruses (one study on Ife virus). Even within virus species, distribution of effort was often unequal; for example, dengue serotype 2 (DENV-2), the most readily available lineage for experimental work, is far better studied (N = 90 studies) than the other three serotypes . Focus on particular viruses over time tended to track the timing of major outbreaks , West Nile virus (N = 36), Venezuelan equine encephalitis virus (N = 31), Zika virus (N = 26), Eastern equine encephalitis virus (N = 24), chikungunya virus (N = 21), RRV (N = 19), DENV (N = 13), Japanese encephalitis virus (N = 13), and Barmah Forest virus (N = 8).The viruses studied in the widest range of mosquitoes were not always the most-studied viruses overall; the 10 viruses studied in the widest range of mosquito species were Rift Valley fever virus  have never been colonized, making vector competence experiments especially challenging and reliant on field-collected eggs). These species may be less important during epidemics, but they determine the risk of an enzootic virus reaching humans and, conversely, whether sylvatic cycles are established after an epidemic ends.,Our results suggest that research efforts in this field have largely been driven by the shifting priorities of arboviral outbreak response. As such, there are numerous gaps in viral taxonomy, vector taxonomy, and vector geography that limit the utility of available data for future outbreak response. Moreover, the vast majority of mosquito–virus pairs (> 90%) are simply untested within our sample of studies, indicating that network-level understanding of arbovirus ecology is incomplete. In particular, existing descriptions of the mosquito–virus network e.g., . In turnEfforts to fill these gaps could take several approaches. First, researchers can use network science to identify important vector–virus pairs and conduct vector competence experiments that would fill key knowledge gaps . Just as machine learning can be used to guide species sampling for viral discovery in nature,Ae. aegypti and Ae. albopictus and for vectors that are locally abundant or are known to feed on amplifying hosts.–Second, researchers can work toward a more cohesive geographic picture of how risk varies, both for cosmopolitan vectors such as Finally, our study highlights that a substantial breadth and depth of vector competence data are published every year in the peer-reviewed literature, but currently, the results of these experiments have no standardized home. Other recent studies highlight that synthesis of these data is possible, despite the complexity of metadata required to describe variation in experimental protocolsWolbachia have been considered as potential biological countermeasures to arboviral transmission in mosquitoes–As we show here, data science approaches can be useful to identify trends and gaps in scientific understanding of arboviral ecology and evolution.Viral Emergence Research Initiative , NSF BII 2021909. S. R. was additionally supported by NSF BII 2021909 and NSF BII 2213854.This work was supported by funding to the Supplemental materials


Monkeypox is a rare zoonotic DNA with lineage from the Poxviridae family, Chordopoxvirinae subfamily, and Orthopoxvirus genus. With a previous history of controlled and contained occasional outbreaks of the virus, currently, a widely erupted outbreak of monkeypox with progressively rising numbers has been reported since May 2022 in multiple countries of the western hemisphere that are not historically endemic for this infection, particularly the United Kingdom and European Union countries. We have written a comprehensive review article to help clinicians better understand the disease. The global cessation of smallpox vaccination has been hypothesized to cause the rise in monkeypox infections in recent years. Monkeypox, like any other viral infection, commences with prodromal symptoms; a maculopapular rash with centrifugal distribution usually follows. Polymerase chain reaction (PCR) confirms the diagnosis. Transmission in humans is possible through infected animals or humans. In the ongoing 2022 outbreak, the monkeypox virus has been undergoing novel mutations at an alarming rate. Treatment options for monkeypox are an area that still requires extensive research, and the utility of certain antiviral medications in treating monkeypox infection is currently being explored but is still controversial and debatable. Monkeypox is a rare zoonotic deoxyribonucleic acid (DNA) virus belonging to the same family as smallpox, the Poxviridae family, Chordopoxvirinae subfamily, and Orthopoxvirus genus . The virA massive shift in epidemiological trends has been seen after the year 2000; between the years 2010 and 2019, the average age of presentation increased from four years to 21 years. The first monkeypox outbreak outside the historically endemic areas of Africa occurred in 2003 in the United States of America (USA) by the West African clade, with around 71 confirmed and suspected cases. The outbreak was caused by some infected African rodents that were imported from Ghana to Texas and got domesticated with prairie dogs after arrival in the USA. This contact chain erupted a monkeypox outbreak in the USA, infecting a total of 81 humans, according to the Centers for Disease Control and Prevention (CDC) . The secWe performed a narrative review of the available literature on the monkeypox virus. During the literature search, we found that the majority of articles highlighted only the recent epidemiological trends, and very few articles discussed all aspects of this infection. Therefore, we decided to write a comprehensive narrative review inclusive of other aspects as well, such as clinical manifestations, mode of transmission, prevention, and potential treatment options.EpidemiologyAccording to the CDC website, a total of 88,549 confirmed cases of monkeypox, with 152 deaths, have been reported around the globe till July 19, 2023, 5:00 post meridiem Eastern daylight time (PM EDT), among which the United States is currently leading with 30,611 cases. A death toll of 152 out of 88,549 cases makes the global case fatality ratio (CFR) for monkeypox about 0.172% . For a more detailed breakdown of locations, please see Table The proportion of monkeypox cases in endemic versus non-endemic areas of the globe is shown in Figure TransmissionIn the endemic parts of Africa, the monkeypox virus has survived through some rodents and mammals acting as natural reservoirs, such as squirrels, African giant pouched rats, striped mice, non-human primates (NHPs), and dormice with animal to human transmission, possibly consequential to mutations in viral strains ,14,15-17Among the re-emergence of the monkeypox virus, data trends also show a probable sexual transmission hypothesized to be via direct skin-to-skin contact or through an exchange of genital secretions because of the presence of genital lesions in infected patients, since the majority of cases have been reported in individuals who identify as gay, bisexual, or, more precisely, MSM. Some patients may additionally have proctitis, in which case the clinical picture might get mistaken for a sexually transmitted infection (STI) especially if no preceding prodromal symptoms are present . The recAn infected individual can transmit infection up to 21 days before the onset of any symptoms ,19. MisdAlthough sufficient evidence exists in the literature that reports transmission of monkeypox infection from animals to humans and also among humans themselves, a unique case of human-to-animal transmission of the virus was reported in two men in Paris, who were inhabiting the same household under a polyandrous partnership, presented with anal ulceration and vesiculo-pustular body rash six days after having sex with other men; real-time PCR confirmed the diagnosis of monkeypox West African clade. Twelve days following their infectious manifestation, their domesticated dog, a male Italian and otherwise healthy greyhound, started to develop abdominal pustules with anal ulceration. A PCR test performed on the dog's sample revealed the monkeypox virus. The report described this case as the first ever known event of transmission of monkeypox infection from a human to an animal . AdditioA provisional diagnosis of monkeypox infection should be considered in patients with peri-genital rash, who have a recent travel history to affected areas, categorize as men who copulate with men, have a contact history with people having an identical rash, or those who are suspected or diagnosed cases of monkeypox infection . HoweverClinical featuresMonkeypox manifests in a way similar to smallpox, but with a less severe course, starting with a prodromal phase, which includes fever, flu-like symptoms, headache, and myalgia with the addition of lymphadenopathy, which is absent in the latter ,12,26. AA maculopapular rash with centrifugal distribution usually follows 3-4 days after the prodrome. The rash may initially be peri-anal or peri-genital and then spread out to the trunk, extremities, and face. The rash appears on the face in 95% of the cases, on palms and soles in 75%, mucous membranes in 70%, genitalia in 30%, and cornea and conjunctiva in 20% of cases . The infThe current outbreak of monkeypox shows several atypical features as compared to the past, and the prodrome has milder symptoms, which may even go unnoticed in some cases until the appearance of rash later on; in MSM or people with peri-genital rash, even the skin lesions manifest a limited distribution ,27,30-31HIV is common in the MSM population; therefore, HIV patients can also get infected with monkeypox regardless of their CD4 T lymphocyte count. In some cases, co-infection with syphilis also exists. Monkeypox in HIV patients usually starts with genital ulcers, and febrile prodrome is less common. Morbidity and mortality are higher in HIV patients with lower CD4 counts .Complications include secondary bacterial infections that can cause bronchopneumonia, encephalitis, keratitis, diarrhea, and even sepsis  11]. Pe. Pe11]. DiagnosisDifferential diagnoses for monkeypox include chickenpox, measles, herpes, syphilis, scabies, and drug-induced allergy. Diagnosis is confirmed by polymerase chain reaction (PCR) ,11. MonkScabs, swabs, and aspirated fluids from lesions are preferred over blood samples for PCR testing due to shortened lifespan of the virus in the blood. These samples require room temperature, but no media for transportation, except for tissue biopsies, which require transportation in a frozen state on dry ice. Histopathology findings show ballooning and multinucleation of keratinocytes with full-thickness epidermal necrosis, nuclear enlargement with an eosinophilic center and basophilic halo, and orthopoxvirus-specific eosinophilic cytoplasmic inclusions, called "Guarnieri bodies" or "B-type inclusions" -35. ImmuOne would typically expect a rising pattern in the total leucocyte count (TLC) in the case of bacterial sepsis. However, a decline in the differential count of neutrophils or neutropenia can occur in fulminant monkeypox infections with bacterial sepsis and is related to higher morbidity and mortality . TherefoKey definitionsThe following provisional definitions have been proposed by the European Centre for Disease Prevention and Control (ECDC) and CDC  and the West African clade, reveal genes that determine virulence in the respective clades. Open reading frames of the West African clade contain deletions and fragmentations, which result in a relative decrement in its pathogenicity as opposed to the other clade -52. A stKindrachuk et al. proposed that the Congo Basin clade modulates host cell responses differently than the West African clade, since the former manifests increased disease severity with a higher case fatality rate. Their experiment demonstrated that the Central African clade downregulates apoptosis in infected cells. Moreover, different patterns of phosphorylation were observed in the two clades, which were proven as potential targets for pharmacological intervention, including excessive Akt S473 phosphorylation and deficient p53-Ser15 phosphorylation. The impedance of Akt S473 phosphorylation by pharmacologic intervention led to a 261-fold decline in the Central African clade yield; however, the West African monkeypox clade remained unaffected .Genetic analysis of the Central African strain (ZAI-96) and three West African strains  disclosed a difference of 0.55-0.56% in the nucleotides between the Central and West African strains, with 173 and 171 unique functional genes, respectively . Novel mutationsMonkeypox is a DNA virus, so it does not exhibit multiple novel mutations, such as RNA viruses, for instance, HIV or SARS-CoV-2 . NeverthA systemic analysis by Wang et al. of the ongoing, widespread monkeypox epidemic in historically non-endemic countries for this infection revealed that 2022 strains phylogenetically belong to the same lineage as 2018 strains. However, the strains from 2022 contain 46 new consensus mutations, inclusive of 24 nonsynonymous mutations. If enough of such mutations are triggered in this virus, this can get more detrimental, since nonsynonymous mutations change protein sequences and favor evolutionary progression via natural selection. A total of 187 proteins were found to be encoded by the mutations, of which 10 proteins were more susceptible to mutations. The exact effect of these mutations on viral functionality is still unknown . Based oPreventionVariola, cowpox, vaccinia, and monkeypox virus all belong to the Orthopox genus. Immunological cross-reactivity and cross-protection exist among the Orthopox species, and hence infection with any one of these species provides some extent of protection against the others . It has A live attenuated vaccine of Vaccinia virus with genetic modification, Ankara-Bavarian Nordic (MVA-BN strain), has been developed under brand names "JYNNEOS" and "ACAM2000®" in the USA, "IMVANEX" in Europe, and IMVAMUNE in Canada against smallpox and monkeypox . The FooThe percentage of various symptoms in smallpox-vaccinated versus unvaccinated groups is depicted in Figure Smallpox vaccination can be given prophylactically to prevent monkeypox infection before or after a suspected or confirmed viral exposure. Pre-exposure prophylaxis (Pre-EP) is indicated only in high-risk groups, such as immunocompromised persons or medical personnel who are frequently exposed to orthopoxviruses; it is indicated when prolonged high exposure contact has taken place. Exposures that warrant a Pre-EP include unprotected direct mucocutaneous exposure to infected patient’s skin, body fluids, contaminated fomites, or standing with no personal protective equipment within 6 feet radius of an infected patient during any procedure that may produce aerosol from patient’s secretions, body fluids, and dry exudates or presence within 6 feet of an unmasked patient for at least three hours . The CDCSexual transmission has been implicated in the Vaccinia virus following its administration as the smallpox vaccine and multiple cases have been reported so far. In one such case, a military man who was recently vaccinated with the smallpox vaccine removed his bandages covering the vaccination site and had unprotected sexual intercourse following digital vaginal contact. His female partner had atopic dermatitis and developed a vulvar lesion four days after this event, which tested positive for the Vaccinia virus on PCR assay . In anotNo doubt that there is sufficient literary evidence for the utility of the smallpox vaccine for Pre-EP and Post-EP, but care must be taken for not leaving the vaccination site uncovered and covering it with a bandage until the scab that forms after vaccination falls off on its own, which may take two to three weeks due to risk of viral shedding from the vaccine site and possible transmission to immunocompromised or atopic individuals. Other precautions for this include changing the bandage every one to two days, proper disposal of the removed bandages, and proper hand sanitization while handling the wound and bandages . AnotherTreatmentMonkeypox infection conventionally exhibits mild clinical manifestations, and the majority of patients can recuperate without any treatment; however, prompt diagnosis and treatment are still crucial as it is a contagious infection with extensive outbreaks in many countries and can otherwise turn into a pandemic. Testing suspected individuals, isolation and treatment of confirmed cases, contact tracing, use of PPE in healthcare workers, and reporting new cases to local public health authorities can limit infection and transmission rates. Morbidity is higher in elderly and immunocompromised groups, the current global CFR as of July 19, 2023, is about 0.172% , can be beneficial. Treatment options should only be considered for high-risk groups; these include immunocompromised or people having comorbidities, elderly, pregnant or breastfeeding individuals, children younger than eight years of age, people having skin conditions that cause a breach in mucocutaneous barriers, and patients experiencing severe symptoms for monkeypox infection that may or may not require hospitalization. Treatment options for monkeypox are an area that still requires extensive research as the utility of certain antiviral medications in treating monkeypox infection is still controversial and debatable. The evidence available in the literature is preclinical with an insignificant amount of data available on human disease models . LiteratTecovirimat, a 4-trifluoromethyl phenol derivative antiviral , has now been approved by the European Medicines Agency (EMA) for the treatment of monkeypox. Although a variety of animal species models have demonstrated the therapeutic effectiveness of Tecovirimat against all Orthopox virus infections, no significant data are available in the literature to establish its efficacy in humans; however, a clinical trial called STOMP is currently under progress to assess it -75. By tTecovirimat can be administered either orally or intravenously (IV). Fecal elimination is the major clearance route, and no fetal risks have been reported with gestational usage in animal models. Some major adverse reactions reportedly include headache, gastrointestinal upset, xerostomia, and hypersensitivity reactions. The recommended dosage regimen for monkeypox treatment is 14 days in animal models and 21 days in safety data, and further clinical trials are still ongoing to explore 28-day regimens. See Table An acyclic nucleoside phosphate, cidofovir, and its lipid-conjugated prodrug, brincidofovir , are antivirals that block viral DNA synthesis of orthopoxviruses, including monkeypox virus, by inhibiting the viral DNA polymerase. Cidofovir is a monophosphate nucleotide and needs to be phosphorylated by intracellular kinases to get activated. In contrast, brincidofovir is cleaved following its intracellular uptake to release the phosphorylated cidofovir molecule already present in the drug. This explains brincidofovir’s higher cellular toxicity and stronger antiviral activity, greater selective index, and 25-fold efficacy than cidofovir against monkeypox, vaccinia, variola, and cowpox viruses . BrincidBrincidofovir is available in oral formulation only; 51% of the drug undergoes urinary clearance, while 50% of it is excreted in feces. The risk of teratogenicity has been reported in animal models, and partners should use contraception during and after the treatment till at least four months after the last dose. Nausea, vomiting, diarrhea, abdominal pain, and elevation of liver enzymes are the major known adverse effects. Brincidofovir is recommended to be taken in two doses, one week apart (days one and eight), with the following weight-adjusted dosage: “<10 kg: 6 mg/kg (suspension); 10 kg to <48 kg: 4 mg/kg (suspension); and 48 kg and above: 200 mg (20 mL or one tablet)” .Cidofovir is available in IV formulation only. It is known to be teratogenic, hence is not recommended in pregnant patients. Up to 75-80% of the drug undergoes renal clearance. Adverse effects include neutropenia, decreased ocular pressure, and nephrotoxicity. Limited data are available on its dosage regimen, but the available literature recommends a single dose of 5 mg/kg. About 2 gm probenecid is given three hours prior to cidofovir to prevent potential nephrotoxicity, followed by 1 L 0.9% NS prior to administration of 5 mg/kg cidofovir, diluted in 100 ml NS, and infused IV over 1 hour. If a higher volume can be tolerated, an additional 1 L NS can be given over one to three hours, starting with cidofovir infusion. Finally, 1 g probenecid should be repeated two and eight hours post-cidofovir infusion. The goal is to reduce the nephrotoxic potential of cidofovir by the administration of probenecid to reduce the tubular secretion of the antiviral along with good IV hydration to reduce the renal contact time ,86.Among the aforementioned three antiviral drugs, tecovirimat is considered the first-line and the best treatment option for monkeypox infection because of an established safety profile in humans, great antiviral response in both prophylactic and therapeutic terms, broad antiviral spectrum, and a more tolerable adverse effects profile ,87. VIG LimitationsWe have quoted the latest global statistics available for the number of cases and deaths; however, they are being updated every two weeks on the CDC website, and this can be a limitation to the current statistics mentioned in our review article.Monkeypox has now become a globally emerging public health challenge, with low statistics in historically endemic areas and rampant growth throughout non-endemic areas. Global cessation of the smallpox vaccine has been hypothesized to be the cause of recurrent monkeypox outbreaks across various parts of the globe, from time to time. Previous outbreaks outside the endemic areas were usually limited to single countries, but the ongoing 2022 outbreak has hit multiple countries in the Western Hemisphere simultaneously. Many atypical features have been observed in this outbreak, including sexual transmission among men who copulate with men, along with genital rash. Two clades with several mutated strains have been identified so far. The current outbreak is of the West African clade, which is less virulent than the Central African clade. The infection is usually self-limiting, with low death tolls; the current global CFR is about 0.712%. Like any other viral illness, the treatment is mainly symptomatic; however, severely immunocompromised groups or those developing severe complications can be administered antiviral drugs such as tecovirimat, brincidofovir, and cidofovir. The FDA and CDC have recommended the smallpox vaccine for Pre-EP and Post-EP of monkeypox as it significantly reduces morbidity and mortality rates and is now commercially available in the USA, Europe, and Canada. These treatment options have shown promising results, but further research is still needed in this area to establish solid treatment guidelines in order to abolish the current monkeypox outbreak.