Ensitrelvir, an oral antiviral agent that targets a SARS-CoV-2 main protease (3CLpro or Nsp5), is clinically useful against SARS-CoV-2 including its omicron variants. Since most omicron subvariants have reduced sensitivity to most monoclonal antibody therapies, SARS-CoV-2 resistance to other antivirals including main protease inhibitors such as ensitrelvir is a major public health concern. Here, repeating passages of SARS-CoV-2 in the presence of ensitrelvir revealed that the M49L and E166A substitutions in Nsp5 are responsible for reduced sensitivity to ensitrelvir. Both substitutions reduced in vitro virus growth in the absence of ensitrelvir. The combination of the M49L and E166A substitutions allowed the virus to largely evade the suppressive effect of ensitrelvir in vitro. The virus possessing Nsp5-M49L showed similar pathogenicity to wild-type virus, whereas the virus possessing Nsp5-E166A or Nsp5-M49L/E166A slightly attenuated. Ensitrelvir treatment of hamsters infected with the virus possessing Nsp5-M49L/E166A was ineffective; however, nirmatrelvir or molnupiravir treatment was effective. Therefore, it is important to closely monitor the emergence of ensitrelvir-resistant SARS-CoV-2 variants to guide antiviral treatment selection. Ensitrelvir is an oral antiviral agent that targets SARS-CoV-2 and is clinically useful against infection with omicron variants. The authors characterized mutant viruses with reduced sensitivity to ensitrelvir in vitro and in vivo. The ongoing global epidemic has resulted in the accumulation of amino acid substitutions throughout the genome of the virus. Recent subvariants including XBB, XBB.1.5, BQ.1.1, and CH.1.1 are less susceptible to therapeutic monoclonal antibodies against the spike protein of SARS-CoV-24. In contrast, antiviral compounds against SARS-CoV-2 such as ensitrelvir (S-217622), nirmatrelvir (PF-07321332), and molnupiravir (EIDD-1931) remain effective against such omicron variants and their subvariants9. Ensitrelvir and nirmatrelvir interfere with 3CL protease , whereas remdesivir and molnupiravir target the viral RNA-dependent RNA polymerase. As these drugs are increasingly used in clinical settings, there is concern about the emergence of viruses with reduced sensitivity or resistance.Three years have passed since the emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). During the pandemic, many variants of concern (VOCs) appeared and caused several waves of infection. Among these variants, omicron (lineage B.1.1.529) was identified at the end of 2021 and became globally dominant10, has shown antiviral activity in cell culture, mice, and hamsters13. It has also been shown to prevent direct transmission from infected hamsters to naive hamsters12. In humans, ensitrelvir is well-tolerated and has favorable pharmacokinetics, including a long half-life14. In the Phase 2/3 study, ensitrelvir treatment of patients with mild to moderate COVID-19 resulted in rapid viral clearance16. When taken within 3 days of onset, ensitrelvir reduced the time to resolution of symptoms such as fever, runny nose, sore throat, cough, and fatigue by 24 h (from an average of 8 days to an average of 7 days)17. Now, ensitrelvir has received emergency regulatory approval from the Ministry of Health, Labour and Welfare of Japan and Fast Track designation from the U.S. Food and Drug Administration for the treatment of COVID-19.Ensitrelvir, which was discovered through a structure-based drug design strategy24. Similarly, mutations that afford resistance to ensitrelvir have been found in Nsp5: M49I/L, S144A, E166A/V, L167F, and P168del reduce the potency of ensitrelvir in vitro25. In addition, naturally occurring amino acid changes such as M49I/L, S144A, P168del, and A173V contribute to reduced sensitivity to ensitrelvir in vitro26. A mutant virus possessing the P168del mutation showed similar replication kinetics as wild-type virus in VeroE6 cells, whereas a mutant virus possessing the A173V substitution was replication defective26. The M49I, G143S, and R188S substitutions have also been linked to ensitrelvir resistance27. Screening using a VSV-based system showed that the L167F substitution confers resistance to both nirmatrelvir and ensitrelvir20. A mutant virus harboring the L167F substitution replicated more slowly than wild-type virus and produced smaller plaques in VeroE6 cells20. The triple substitution of L50F, E166A, and L167F, which was selected by passaging wild-type virus in the presence of an nsp5 inhibitor, confers resistance to both nirmatrelvir and ensitrelvir28. A mutant virus carrying the L50F, E166A, and L167F substitutions showed similar pathogenicity in hamsters and transmitted to co-housed hamsters29. The Pharmaceuticals and Medical Devices Agency (PMDA) of Japan, reported that the D48G, M49L, P52S, S144A, and M49L/S144A substitutions confer reduced sensitivity to ensitrelvir in VeroE6/TMPRSS2 cells (https://www.pmda.go.jp/files/000249828.pdf). A crystal structure of the M49I mutant revealed that the greater hydrophobicity and steric volume of the isoleucine residue compared to the methionine residue shifted the ligand orientation within the binding site27. The M49I substitution also caused the loss of hydrogen bonds between ensitrelvir and T26, S144, and C14527. These alterations reduce virus sensitivity to ensitrelvir. Although the amino acid changes mentioned above have been associated with resistance to ensitrelvir, mutant viruses possessing mutations at positions 49 and 166 have not been well characterized in vitro or in vivo; growth comparisons in the presence or absence of ensitrelvir in vitro, detailed pathological comparisons, and studies comparing the efficacy of ensitrelvir nirmatrelvir, and molnupiravir in hamsters have not been done.Amino acid substitutions in nsp5 associated with resistance to nirmatrelvir have been reportedAccordingly, here, we attempted to obtain viruses with reduced sensitivity to ensitrelvir by passaging wild-type SARS-CoV-2 in vitro in the presence of ensitrelvir. With the knowledge of the amino acid substitutions in Nsp5 involved in resistance, we generated mutant viruses possessing these substitutions by using reverse genetics. We characterized the in vitro replication capability and pathogenicity in hamsters of these mutant viruses. We also evaluated the efficacy of antiviral treatments against these mutant viruses by using a hamster model.30. The viruses were tested for sensitivity to ensitrelvir (S-217622) together with another nsp5 inhibitor, nirmatrelvir (PF-07321332), and the antiviral ribonucleoside analogs molnupiravir (EIDD-1931) and remdesivir (GS-441524). The IC50 value of ensitrelvir (S-217622) against wild-type virus was 0.19 μM , whereas the IC50 values against Nsp5-M49L, Nsp5-E166A, and Nsp5-M49L/E166A were increased to 11.6 μM , 1.72 μM , and 37.4 μM , respectively  and remdesivir (GS-441524) showed a slightly higher IC50 value against the three mutant viruses than against the wild-type virus, for reasons that remain unclear. These results indicate that Nsp5-M49L, Nsp5-E166A, and Nsp5-M49L/E166A show moderate, slight, and considerable resistance to ensitrelvir, respectively.We attempted to determine whether a virus resistant to ensitrelvir S-217622) would emerge upon serial passage. The wild-type delta variant was passaged twice in the presence of 0.25 μM ensitrelvir, twice at 0.5 μM, and then once at 1 or 5 μM. After a total of five passages, 6 clones (clones 11–16) from the samples at the 1 μM condition and 6 clones (clones 21–26) from the samples at the 5 μM condition were obtained by plaque purification and their nsp5 nucleotide sequences were analyzed by Sanger sequencing. Clones 11, 12, 13, 15, and 16 possessed the M49L substitution in nsp5, clones 14, 22, 23, 24, 25, and 26 possessed the E166A substitution in nsp5, and clone 21 possessed both substitutions. These substitutions have the potential to confer resistance to ensitrelvir. To confirm this, we generated mutant viruses possessing the M49L, E166A, or M49L and E166A substitutions in nsp5  based on the wild-type delta variant by using a BAC-based reverse genetics system7622 woul6 PFU/ml, whereas 20 μM ensitrelvir completely inhibited wild-type virus replication  or absence (solid lines) of 20 μM ensitrelvir , whereas the others were 5.14, 5.23, 4.62, and 5.23. Therefore, the E47K substitution does not appear to have a large effect on viral replication efficiency. Nevertheless, further analysis will be required to characterize this substitution because it is located close to the enzyme active site of nsp5.Virus titers in the nasal turbinates and lungs of infected hamsters were measured at 3 and 6 dpi. At 3 dpi, virus titers in the nasal turbinates were comparable for all viruses tested, whereas those in the lungs of hamsters infected with Nsp5-E166A or Nsp5-M49L/E166A were significantly lower than those of wild-type virus Fig.  At 6 dpiThe lungs of hamsters infected with each virus were histopathologically analyzed at 3 and 6 dpi Fig. . H&E staTaken together, these data demonstrate that Nsp5-M49L maintains virulence comparable to that of wild-type virus, whereas Nsp5-E166A and Nsp5-M49L/E166A are slightly attenuated.5 PFU of wild-type virus or Nsp5-M49L/E166A were treated with ensitrelvir at 60 mg/kg twice daily, nirmatrelvir at 250 mg/kg twice daily, or molnupiravir at 250 mg/kg twice daily for three days beginning 1 dpi. Because nirmatrelvir was not co-administered with ritonavir, the efficacy of nirmatrelvir appears to be low. Virus titers in the nasal turbinates and lungs of the hamsters were measured at 4 dpi. In the hamsters infected with wild-type virus, ensitrelvir, nirmatrelvir, and molnupiravir significantly reduced the virus titers in both the nasal turbinates and lungs  and 166 (E166A/V)27. Therefore, these positions might be hot spots for amino acid substitutions that reduce sensitivity to ensitrelvir. Since recent omicron variants and subvariants naturally tend to obtain additional amino acid substitutions in nsp5 compared with the delta variant, amino acid substitutions at other positions might be selected during ensitrelvir treatment of patients. Therefore, we must monitor the amino acid substitutions in nsp5 that are detected in patients treated with ensitrelvir and examine the ensitrelvir sensitivity of isolates from these patients in laboratory experiments.Viruses resistant to anti-SARS-CoV-2 drugs are a major public health concern. Indeed, monoclonal antibody therapies face increasing escape mutants with amino acid substitutions in their spike protein. In contrast, antiviral compounds targeting virus proteins other than the spike continue to be efficacious and remain important countermeasures. However, amino acid substitutions that reduce sensitivity to such compounds have been found in viruses isolated from COVID-19 patients35, E166V22, and E166A28, confer reduced sensitivity to nirmatrelvir, we confirmed that viruses with the E166A substitution, even in combination with the M49L substitution in the backbone of the delta variant, showed similar sensitivity to nirmatrelvir in vitro and in vivo. Since the efficacy of nirmatrelvir in this study was relatively low because a P-glycoprotein inhibitor or ritonavir was not co-administrated with nirmatrelvir32, nirmatrelvir could be a treatment option against these resistant viruses. However, several amino acid substitutions such as E166V and L167F similarly confer resistance to both nirmatrelvir and ensitrelvir28, indicating that both ensitrelvir and nirmatrelvir might fail to suppress the propagation of mutant viruses possessing such substitutions. Even so, molnupiravir and remdesivir likely remain as effective against such double-resistant viruses as against wild-type virus because the mechanism of action of both molnupiravir and remdesivir differs from that of the anti-nsp5 drugs such as ensitrelvir and nirmatrelvir. Further analyses are required to determine whether a multi-drug resistant virus maintains viral fitness. To combat these multidrug-resistant viruses, we hope that many novel antivirals targeting other viral proteins will be approved for clinical use.The M49L substitution reduced susceptibility to ensitrelvir and the in vitro growth of Nsp5-M49L was not completely suppressed by ensitrelvir. The combination of the M49L and E166A substitutions further reduced susceptibility to ensitrelvir, resulting in similar growth kinetics of Nsp5-M49L/E166A in the presence and absence of ensitrelvir. Furthermore, ensitrelvir treatment did not reduce virus titers of Nsp5-M49L/E166A in the lungs of hamsters, although nirmatrelvir treatment was effective. These results demonstrate that Nsp5-M49L and Nsp5-M49L/E166A maintain viral fitness to a certain extent in vitro and in vivo, meaning that they have the potential to be a threat to public health as ensitrelvir-resistant viruses. Although amino acid substitutions at position 166, including E166D/GAll animal experiments were conducted in accordance with the University of Tokyo’s Regulations for Animal Care and Use, which were approved by the Animal Experiment Committee of the Institute of Medical Science, the University of Tokyo. The committee acknowledged and accepted both the legal and ethical responsibility for the animals, as specified in the Fundamental Guidelines for Proper Conduct of Animal Experiment and Related Activities in Academic Research Institutions under the jurisdiction of the Ministry of Education, Culture, Sports, Science, and Technology of Japan.All experiments with SARS-CoV-2 were performed in enhanced biosafety level 3 containment laboratories at the University of Tokyo, which are approved for such use by the Ministry of Agriculture, Forestry, and Fisheries and the Ministry of Health, Labour and Welfare, Japan. All experiments were conducted by PhD-level scientists who are highly experienced in such studies. All individuals working with SARS-CoV-2 have been vaccinated multiple times with COVID-19 vaccines. Staff working in enhanced BSL-3 wear disposable overalls and powered air-purifying respirators. The enhanced BSL-3 facility at the University of Tokyo includes controlled access, effluent decontamination, negative air-pressure, double-door autoclaves, HEPA-filtered supply and exhaust air, and airtight dampers on ductwork connected to the animal cage isolators and biosafety cabinets. The structure is pressure-decay tested regularly. All personnel complete biosafety and BSL-3 training before participating in BSL-3-level experiments. Refresher training is scheduled on a regular basis. Virus inventory, secured behind two physical barriers, is checked regularly. Virus inventory is submitted once a year to the Ministry of Agriculture, Forestry, and Fisheries and the Ministry of Health, Labour and Welfare, Japan. Procedures in response to laboratory accidents are established.2. The cells were regularly tested for mycoplasma contamination by using PCR and confirmed to be mycoplasma-free.VeroE6/TMPRSS2 (JCRB 1819) cells were propagated in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% fetal bovine serum (FBS), 1 mg/ml geneticin , and 5 μg/ml plasmocin prophylactic (Invivogen). VeroE6-TMPRSS2-T2A-ACE2 cells were cultured in DMEM containing 10% FBS, 100 U/ml penicillin–streptomycin, and 10 µg/ml puromycin. HEK293T cells were cultured in DMEM supplemented with 10% FBS. All cells were maintained at 37 °C with 5% COEnsitrelvir (S-217622) was kindly provided by Shionogi Co., Ltd. Active components of remdesivir (GS-441524), molnupiravir (EIDD-1931), and nirmatrelvir (PF-07321332) were purchased from MedChemExpress. Compounds were dissolved in dimethyl sulfoxide for in vitro experiments or 0.5% methyl cellulose for in vivo experiments prior to use.SARS-CoV-2 delta variant (hCoV-19/USA/WI-UW-5250/2021) was sequentially passaged a total of five times in the presence of ensitrelvir at 0.25, 0.5, 1, or 5 μM in VeroE6/TMPRSS2 cells. In total, 12 clones were purified from the passaged viruses by plaque purification. The Nsp5 nucleotide sequence of each clone was determined by Sanger sequencing.30. The mutations responsible for the M49L, E166A, and M49L/E166A substitutions in nsp5 were introduced during the PCR step. To rescue these viruses, pBeloBAC11 encoding wild-type, Nsp5-M49L, Nsp5-E166A, or Nsp5-M49L/E166A was transfected into HEK293T cells. At 3 days post-transfection, the supernatant containing the viruses was collected and inoculated onto VeroE6/TMPRSS2 cells at 37 °C to prepare virus stocks. The stock viruses were deep sequenced to confirm the absence of unwanted mutations, and no position contained unwanted nucleotides that exceeded 10% of the population in all stock viruses.The full-genome nucleotide sequence of SARS-CoV-2 delta variant (hCoV-19/USA/WI-UW-5250/2021) was assembled into the pBeloBAC11 vector to generate infectious cDNA clones under the control of a cytomegalovirus promoter as described previously36. In brief, viral RNA was extracted by using a QIAamp Viral RNA Mini Kit (QIAGEN). cDNA was synthesized by using a Lunar-Script RT SuperMix Kit (New England BioLabs) and subjected to a multiplexed PCR in two pools using ARTIC-N1 primers v5 and Q5 Hot Start DNA polymerase (New England BioLabs). The DNA libraries for Illumina NGS were prepared from pooled amplicons by using a QIAseq FX DNA Library Kit (QIAGEN) and then analyzed by using the iSeq100 System in 150-bp paired-end mode using an iSeq 100 i1 Reagent v2 (300-cycle) kit (Illumina). The reads were assembled by CLC Genomics Workbench .The whole genome of SARS-CoV-2 was amplified by using a modified ARTIC network protocol in which some primers were replaced or added7. Briefly, VeroE6-TMPRSS2-T2A-ACE2 cells in 96-well plates were infected with the indicated virus at 100–400 focus forming unit/well. After incubation at 37 °C for 1 h, the inoculum was replaced with 1% Methyl Cellulose 400  in culture medium containing serial dilutions of the antiviral compounds. The cells were incubated for 18 h at 37 °C and then fixed with formalin. The cells were stained with a mouse monoclonal antibody against SARS-CoV-2 nucleoprotein, clone N45 , followed by a horseradish peroxidase-labeled goat anti-mouse immunoglobulin (Jackson ImmunoResearch Laboratories Inc.). Foci were visualized by using TrueBlue Substrate (SeraCare Life Sciences). The focus numbers were quantified by using an ImmunoSpot S6 Analyzer, ImmunoCapture software, and BioSpot software (Cellular Technology). The 50% inhibitory concentration (IC50) values and 95% confidence intervals were calculated by using GraphPad Prism 9.3.0 (GraphPad Software).Antiviral susceptibilities were determined by using a focus reduction assay as previously reportedVeroE6/TMPRSS2 cells were infected with the indicated virus at a multiplicity of infection (MOI) of 0.001. After incubation at 37 °C for 1 h, the inoculum was replaced with medium with or without 20 μM ensitrelvir. Cell culture supernatants were collected at 8, 16, 24, 32, 40, 48, 56, 72, and 96 h post-infection. Virus titers were determined by use of a plaque assay in VeroE6/TMPRSS2 cells.n = 5) were intranasally inoculated with 105 plaque forming unit (PFU) of the indicated virus. Body weights were measured daily before inoculation and for 10 days post-infection (dpi). Respiratory parameters [Penh (a nonspecific assessment of breathing patterns) and Rpef (a measure of airway obstruction)] were also measured by using a whole-body plethysmography system (PrimeBioscience) as previously described38.Five- to six-week-old male Syrian hamsters (Japan SLC) were used in this study. For the pathogenicity study, hamsters (n = 10) were intranasally infected with 105 PFU of the indicated virus. At 3 and 6 dpi, the animals (n = 5 per timepoint) were euthanized and nasal turbinates and right lungs were collected. The virus titers in these organs were determined by use of plaque assays on VeroE6/TMPRSS2 cells. The left lungs were fixed in 4% paraformaldehyde in PBS and processed for paraffin embedding. The paraffin blocks were cut into 3-μm-thick sections and mounted on silane-coated glass slides, and then haematoxylin and eosin (H&E) stained for histopathological examination. To detect SARS-CoV-2 RNA, in situ hybridization was performed using an RNA scope 2.5 HD Red Detection kit (Advanced Cell Diagnostics) with an antisense probe targeting the nucleocapsid gene of SARS-CoV-2 (Advanced Cell Diagnostics) as previously described39. Tissue sections were also processed for immunohistochemical staining with a rabbit monoclonal antibody for SARS-CoV nucleocapsid protein , which cross-reacts with SARS-CoV-2 nucleocapsid protein. Specific antigen-antibody reactions were visualized by means of 3,3’-diaminobenzidine tetrahydrochloride staining using the Dako Envision system (Dako Cytomation). Histopathological scores of inflammation in the alveolar regions were determined based on the percentage of alveolar inflammation in a given area of a pulmonary section collected from each animal in each group by using the following scoring system38: 0, no inflammation; 1, affected area (≤1%); 2, affected area ; 3, affected area ; 4, affected area (>50%). An additional point was added when pulmonary edema and/or alveolar hemorrhage was observed. Therefore, histopathological scores of inflammation in the alveoli for individual animals ranged from 0 to 5.For the virus titration and pathological analysis, hamsters (5 PFU was inoculated into hamsters (n = 20). At 1 dpi, treatment with ensitrelvir at 60 mg/kg twice daily (n = 5), nirmatrelvir at 250 mg/kg twice daily (n = 5), or molnupiravir at 250 mg/kg twice daily (n = 5) was initiated41 and continued until 3 dpi. The remaining hamsters (n = 5) were administrated 0.5% methyl cellulose. The animals were euthanized at 4 dpi and their nasal turbinates and lungs were collected for the virus titration on VeroE6/TMPRSS2 cells.For the treatment study, wild-type virus or Nsp5-M49L/E166A at 10P values. Virus growth kinetics in vitro, body weight, Penh, and Rpef were compared by using a two-way ANOVA followed by Dunnett’s multiple comparisons. Virus titers in hamsters were compared by using a one-way ANOVA followed by Tukey’s multiple comparisons. Differences between groups were considered significant for P values < 0.05.GraphPad Prism software version 9.3.0 was used to calculate Further information on research design is available in the Peer Review FileReporting Summary


The management of future pandemic risk requires a better understanding of the mechanisms that determine the virulence of emerging zoonotic viruses. Meta-analyses suggest that the virulence of emerging zoonoses is correlated with but not completely predictable from reservoir host phylogeny, indicating that specific characteristics of reservoir host immunology and life history may drive the evolution of viral traits responsible for cross-species virulence. In particular, bats host viruses that cause higher case fatality rates upon spillover to humans than those derived from any other mammal, a phenomenon that cannot be explained by phylogenetic distance alone. In order to disentangle the fundamental drivers of these patterns, we develop a nested modeling framework that highlights mechanisms that underpin the evolution of viral traits in reservoir hosts that cause virulence following cross-species emergence. We apply this framework to generate virulence predictions for viral zoonoses derived from diverse mammalian reservoirs, recapturing trends in virus-induced human mortality rates reported in the literature. Notably, our work offers a mechanistic hypothesis to explain the extreme virulence of bat-borne zoonoses and, more generally, demonstrates how key differences in reservoir host longevity, viral tolerance, and constitutive immunity impact the evolution of viral traits that cause virulence following spillover to humans. Our theoretical framework offers a series of testable questions and predictions designed to stimulate future work comparing cross-species virulence evolution in zoonotic viruses derived from diverse mammalian hosts. Bats are natural reservoir hosts for viruses that cause higher case fatality rates in humans than do zoonoses derived from any other source. This study builds a nested within-host, population-level model to offer a mechanistic explanation for this phenomenon. The devastating impact of the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) pandemic highlights the extreme public health outcomes that can result upon cross-species emergence of zoonotic viruses. Estimating the relative threats posed by potential future zoonoses is an important but challenging public health undertaking. In particular, efforts to predict the virulence of emerging viruses can be complicated since chance will always play a role in dictating the initial spillover that precedes selection , virulenκΒ pathway [Although the disproportionate frequency with which the Chiropteran order may source viral zoonoses remains debated ,10, the  pathway , dampene pathway , and dim pathway . In addi pathway  and tole pathway . Moreove pathway . The extModern theory on the evolution of virulence typically assumes, either explicitly or implicitly, that high pathogen growth rates should both enhance between-host transmission and elevate infection-induced morbidity or mortality, resulting in a trade-off between virulence and transmission –39. TheoαS, the virulence incurred by a reservoir-optimized virus immediately following spillover to a novel host. We expressed αS as a function of the reservoir-optimized virus growth rate (TvS), which we model as proportional to the phylogenetic distance between reservoir and spillover host  as:gS, TvS, and TwS represent the spillover host analogues of previously described within-host parameters in the reservoir host equations  or immunopathology (Tw). See Our nested modeling approach first follows Alizon and van Baalen 2005)  in deriv005  in ders Figs . We subsi (2002) , model tons Figs . We presμR), elevated constitutive immune responses (gR0), more rapid leukocyte activation upon infection (gR), and more rapid virus clearance by the host immune system (cR). Additionally, higher mR), resulting in longer-lived immune cells. Critically, higher reservoir host tolerance of both virus-induced pathology (TvR) and immunopathology (TwR) also select for higher TvR and TwR) decouple the relationship between transmission and virulence, permitting evolution of high αS). Intriguingly, the virulence that a virus incurs on its spillover host (αS) accelerates substantially faster than that incurred on its reservoir host , the reservoir host tolerance of immunopathology (TwR), and the reservoir host magnitude of constitutive immunity (gR0). Within-host immunological data needed to quantify these parameters is lacking for most taxa; thus, we use regression analyses to summarize these terms across mammalian orders from publicly available life history data. In particular, we use well-described allometric relationships between mammalian body mass and basal metabolic rate (BMR) with lifespan and immune cell concentrations [μR, TwR, and gR0 across mammalian orders  demonstrate significantly elevated annual mortality rates using the median order mortality rate (Diprotodontia) as a reference. By contrast, Carnivora, Cetartiodactyla, Primates, and Perissodactyla demonstrate significantly reduced annual mortality rates as compared to the same reference  as predicted by body size (in grams), again spanning data representing 26 diverse mammalian orders. From this, we identify 5 orders  with significantly longer lifespans than predicted by body size—which scale to enhanced estimates for TwR. By contrast, we identify 8 orders  with significantly shorter lifespans than predicted by body size, which scale to lower value estimates for TwR . However, because mass is not factored into estimation of μR, these parameters diverge in select cases: for example, we estimate mid-range mortality rates for order Chiroptera, as compared with other mammals, but very high values for TwR becase Chiropteran lifespans—though not remarkably long at face value—far exceed those predicted by body size. By contrast, we estimate low values for both μR and TwR for order Cetartiodactyla, as hosts in this order are long-lived but less long-lived than predicted for body size.Drawing on well-described allometric relationships between mass and lifespan  and moreammation . We hypoTwR Figs  Tables. 10 relationship of baseline circulating neutrophil concentration (in 109 cells/L) as predicted by mass (in grams) and BMR (W); these data spanned only 19 mammalian orders , differences in mortality rates across mammalian orders—though substantial on multiyear timescales of the host—have limited influence on downstream predictions of differences in the evolution of virus growth rates. This result echoes prior observations from μR, as compared with other parameters. As a result, we ultimately recover the highest predicted optimal growth rates for viruses evolved in the orders Chiroptera and Monotremata, which exhibit both long lifespans per body size (corresponding to high estimates for TwR) and high baseline neutrophil concentrations (corresponding to high estimates for gR0). Data are particularly sparse, however, for order Monotremata, for which complete records are only available for 2 species (the platypus and the short-beaked echidna). As a result, predictions for this order should be interpreted with caution. More data, particularly for baseline neutrophil concentrations, will be needed to evaluate the extent to which these predictions hold across all 5 extant species in the Monotremata order. Additionally, we predict the evolution of the lowest growth rate viruses in reservoir hosts of the orders Scandentia, Cetartiodactyla, Diprotodontia, and Dasyuromorphia, all of which demonstrate significantly low estimates for the magnitudes of constitutive immunity and 2 of which  also demonstrate significantly low estimates for tolerance of immunopathology.From life history-derived estimates for rameters . In keeprameters , we predαS) of these viruses following emergence into a human host. Zoonotic spillovers are modeled as acute infections in the human, and virulence is calculated while varying only the growth rate of the spillover virus (TvS) between viruses evolved in differing reservoir orders. We vary this last parameter, TvS, to account for any differences in virus adaptation to reservoir host immune systems that are not already captured in estimation of reservoir host TwR and gR0. TvS is thus computed as the inverse of the scaled time to MRCA for each mammalian reservoir host order from Primates  relative to eutherian orders with similar predicted After establishing optimal growth rates for viruses evolved in diverse reservoir host orders  by reservoir order in relative terms to compare with estimates gleaned from the zoonosis literature [2 value of 0.57 in the corresponding regression of observed versus predicted values (αS) than do viruses derived from all other eutherian mammals . Bats demonstrate uniquely long lifespans for their body sizes and uniquely enhanced constitutive immune responses [TwR and high gR0, these reservoir host traits elevate predicted αS beyond all other eutherian orders  and correspondingly low values for tolerance of immunopathology (TwR) in this order. In addition, our model underpredicts virulence for Carnivora-derived viruses, based on within-host parameter estimates largely inverse to those recovered for Eulipotyphla . We are able to resolve this underprediction of Carnivora virulence when excluding rabies lyssavirus from data comparisons [2 = 0.57).Evaluated against the data ,8, our mparisons ,8  and robust constitutive immunity highlight several mammalian orders—chiefly Chiroptera and Monotremata—which show synergy in both features, offering support for ongoing efforts to elucidate links between antiaging molecular pathways and antiviral immunity ,64. FurtAll told, this analysis highlights a critical need for compilation of a more complete comparative immunological database that would enable quantification of the many additional within-host parameters  represented in our model. Our relative success in recapturing broad patterns in spillover virulence, despite data constraints, suggests that improvements in parameter estimation will almost certainly yield gains in our nuanced understanding of the process of cross-species virus emergence. For example, we model host tolerance of direct virus pathology as proportional to phylogenetic distance between reservoir and spillover host, but this term will likely also be modulated by virus tropism—presenting yet another mechanism by which viral adaptation to reservoir hosts could enhance spillover virulence. Indeed, coronavirus tropism is thought to be localized largely in the gastrointestinal tract for bat hosts ,68: In oμR, TwR, and gR0, which we here model universally across the entire Chiropteran order. Thus, we can expect considerable variation in the evolution of virulence for viruses evolved in diverse species within a given order—which we largely disregard here. As more data becomes available, our modeling approach could be fine-tuned to make more specific, species-level predictions of highly virulent disease risk.By summarizing within-host traits across mammalian orders, we generalize substantial within-clade diversity that likely also contributes to heterogeneous patterns in available data. Bats alone make up more than 1,400 species and account for some 20% of mammalian diversity . Only a Our analysis does not consider the probability of cross species viral emergence, or the potential for onward transmission of a spilled-over virus in a human host—both of which have been shown to correlate inversely with phylogenetic distance across mammals ,8,9. Indin vitro and in vivo scales. Empirical work should aim to measure rates of immune cell activation, growth, and mortality across diverse mammalian orders and determine whether natural virus growth rates are truly higher when evolved in bat hosts. Additional studies should test whether anti-inflammatory mechanisms in bat cells of different tissues are equally effective at mitigating virus-induced pathology and immunopathology and whether comparative taxonomic predictions of virus tolerance, resistance, and virulence evolution apply to non-viral pathogens, too. In light of the emergence of SARS-CoV-2, the field of bat molecular biology has echoed the call for mechanistic understanding of bat immunology [Currently, our work emphasizes the uniqueness of bats as flying mammals and, in consequence, as long-lived, tolerant reservoirs for highly virulent viral zoonoses. For the first time, we formalize a mechanism for the evolution of bat-derived viruses that demonstrate significant pathology upon spillover to non-bat, particularly human, hosts. In providing a theoretical framework to explain this phenomenon, we generate a series of testable questions and hypotheses for future comparative immunological studies, to be carried out at munology , and themunology , solicitI1) in a hypothetical reservoir host, allowing for the introduction of a rare mutant virus strain which generates distinct infections in that same host (I2)  applied to the birth rate. The subscript “R” on the birth, death, and crowding terms emphasizes that these rates are specific to the reservoir host. Because we aim to model the evolution of rates that link to within-host dynamics for the reservoir host, we further represent transmission and virulence as functions of the virus causing the infection  and mutant (rR2) virus strains.Here, With the use of model , we perfrR), we express the between-host parameters of transmission  [R) and the leukocyte population (LR) within each infected reservoir host:Next, to evaluate the within-host selective conditions underpinning the evolution of the virus growth rate (n (2005)  to adaptn (2005) ), to oven (2005) ,85,86. TrR corresponds to the intrinsic virus growth rate, cR corresponds to the attack efficacy of the immune system upon contact with the virus, and gR signifies the recruitment rate of immune cells scaled to the virus growth rate. The parameter, gR0, describes the constitutive, baseline leukocyte recruitment in the absence of infection, and mR gives the natural leukocyte death rate, all in the reservoir host.Here, mRrR> cRgR0).Building from above, we express rates of population-level transmission and virulence known to depend on within-host dynamics  multiplied by the virus growth rate (w). The terms TvR and TwR correspond to host tolerance of virus-induced pathology and immunopathology, respectively, under assumptions of “constant” tolerance, by which both virus pathology and immunopathology are reduced by a constant proportion across the course of infection. For constant tolerance, we assume that TvR > 1 and TwR > 1. See In line with previous work, we assume transmission to be a linear function of viral load , which wNow, we rewrite the equations for transmission  and viruUsing these within-host expressions, we then recompute the expression for invasion fitness  in withiIn We next derive an expression to explore the consequences of spillover of a virus evolved to its optimal growth rate in a reservoir host population—which we term d Sasaki , who devVS and LS correspond, respectively, to the spillover host virus and leukocyte populations, cS is the virus consumption rate upon contact with leukocytes in the spillover host, and gS is the growth rate of the spillover host leukocyte population in response to virus. We express the model in units of τ, which we assume to be short in comparison to the t time units of dynamics in the reservoir host population. Rather than solving for virus and leukocyte populations at equilibrium (as in the reservoir host population), we follow Gilchrist and Sasaki [Here, d Sasaki  to insteIf we divide LS (0) = 1, VS (0) = 1, assuming that both virus and leukocyte populations will be small at τ = 0, we can integrate  to Then, extending previous work , we calcNow, with this established expression for v) and propensity to elicit an inflammatory immune response (w) remained unchanged from one host to the next. Inspired by findings in the literature that report higher virulence in cross-species infections between hosts separated by larger phylogenetic distances [TvS) as a decreasing function of increasing phylogenetic distance between the reservoir and secondary host. All other immune-related parameters assume characteristics of the spillover host: gS is the spillover host’s leukocyte growth rate, and TwS corresponds to the spillover host’s tolerance of immunopathology.This yields the above expression for “spillover virulence.” Here, the growth rate of the virus is expressed at its evolutionary optimum evolved in the reservoir host , the reservoir host tolerance of immunopathology (TwR), and the magnitude of reservoir host constitutive immunity (gR0).Next, we develop estimates for the optimal virus growth rate (μR we fit a simple linear regression to the response variable of the inverse of maximum lifespan (in days) with a single categorical predictor of host order, using data from Jones and colleagues [Yij corresponds to natural mortality rate observations for i species belonging to j orders; α0 is the overall intercept; k indicates the total number of available orders ; βj is an order-specific slope; δij indicates observations of i species grouped into j orders; and εij is a normally distributed error term corresponding to species i within order j. We estimate μR by simply generating predictions from this fitted model across all 26 mammalian orders represented in our dataset . Here, we fit a linear mixed effects regression to the log10 relationship of lifespan (in years) as predicted by body mass (in grams). This second model takes the form:Yij corresponds to the log10 value of record of maximum lifespan (in years) for i species belonging to j orders; α0 is the overall intercept; β1 indicates the slope of the fixed predictor of log10 mass (in grams), here represented as X1; uj0 is the order-specific random intercept; and εij is a normally distributed error term corresponding to species i within order j. To estimate TwR, we extract the relative partial effects of mammalian order on maximum lifespan per body size, and then rescale these effects between 1 and 2 for assumptions of constant tolerance and between 0 and 1 for assumptions of complete tolerance , we fit another linear mixed effects regression to the relationship between the predictor variables of log10 mass (in g) and BMR (in W) and the response variable of log10 baseline neutrophil concentration (in 109 cells/L), which offers an approximation of a mammal’s constitutive innate immune response. BMR data for this model are derived from Jones and colleagues [Yij corresponds to the log10 baseline neutrophil concentrations (in 109 cells/L) for i species belonging to j orders; α0 is the overall intercept; β1 indicates the slope of the fixed predictor of log10 mass (in grams), here represented as X1; β2 indicates the slope of the fixed predictor of BMR (in W), here represented as X2; uj0 is the order-specific random intercept; and εij is a normally distributed error term corresponding to species i within order j. Using a similar approach as that employed above for TwR, we estimate g0 by extracting the relative partial effects of mammalian order on neutrophil concentration per mass-specific BMR, then rescale these effects between 0 and 1. Because we estimate significantly positive partial effects between the orders Chiroptera and Monotremata and baseline neutrophil concentration  are held constant across all taxa at values listed in Using these order-level summary terms for cS, gS, and mS constant in humans  due to a lack of informative data to the contrary. Then, to generate an order-level estimate for virus virulence incurred on humans (αS), we combine our order-level predictions for TvS, the human tolerance of an animal-derived virus, which we scale such that viruses derived from more closely related orders to Primates are more easily tolerated in humans. Specifically, we represent TvS as the scaled inverse of the cophenetic phylogenetic distance of each mammalian order from Primates. No summarizing is needed for TvS because, following Mollentze and Streicker [TvS, we divide all order-level values for this distance by the largest observed (to generate a fraction), then subtract that fraction from 2 for assumptions of constant tolerance (yielding TvS estimates ranging from 1 to 2) and from 1 for assumptions of complete tolerance (yielding TvS estimates ranging from 0 to 1).Once we have constructed an order-level prediction for treicker , we use treicker , which pTvS) to generate predictions of the expected spillover virulence of viral zoonoses emerging from the 19 mammalian orders for which we possess complete data across the 4 variable parameters . We scale these αS estimates—and their 95% predicted confidence intervals—in relative terms (from 0 to 1) to compare with estimates from the literature.We then combine reservoir host order-level predictions for αS) with estimates from the literature, we follow Day (2002) [CFRS) reported in the literature [αS for each mammalian order, using data on the duration of human infection (DIS) for each viral zoonosis. For this purpose, we collected data on DIS by searching the primary literature; raw data for infection durations and associated references are reported in our publicly available GitHub repository . Briefly, Day (2002) [CFRS) takes the form:σS corresponds to the recovery rate from the spilled-over virus in the human host. We also note that the total duration of infection, DIS, is given by:To compare estimates of spillover virulence generated from our nested modeling approach (y (2002)  to conveterature  to empirCFRS) into estimates of spillover virulence (αS) using the following equation:Therefore, we translated human case fatality rates of viral zoonoses  from corresponding predictor variables of reservoir host order, virus family, virus species publication count (a measure of research effort), spillover type (via bridge host versus direct), and vector-borne disease status (yes or no). Following [αS at the order-level from the fitted GAM, excluding the effects of viral family, to yield a literature-derived value for αS across disparate mammalian orders, against which to compare predictions from our nested modeling approach. Because the majority of the within-host parameters underpinning αS predictions in our nested modeling approach  are quantified only in a relative fashion, we were unable to compare direct magnitudes of virulence . To account for this, we rescale αS estimates from both our nested modeling approach and from the empirical literature from 0 to 1 and compare the relative rank of virulence by mammalian order instead. We predict αS for 19 discrete mammalian orders, though data from the literature are available for only 8 orders against which to compare.To obtain a composite order-level prediction for orted in , we adoported in  used by orted in  to summaterature . This beollowing , we summαS by fitting the same GAMs  recovered from the literature, we fit a simple linear regression to the relationship between observed and predicted virulence across the 8 mammalian orders for which we possess comparative data  has minimal influence on optimal virus growth rates (TwR), reservoir host magnitude of constitutive immunity (gR0), and spillover host tolerance of reservoir-derived virus pathology (TvS) impacts the resulting prediction for spillover virulence (αS)  . To thisain text . To quanain text .S1 FigrR2) will displace resident growth rates (rR1) at values indicated by the shaded regions. Reservoir host tolerance of immunopathology (TwR) and tolerance of direct virus pathology (TvR) are both modeled as low in left column  and high in right column (0.97 and 100 for row 1 and 2), assuming a complete (row 1) or a constant form (row 2). For this visualization, rR1 and rR2 span from 3.18 to 3.5. All other parameters involved in computation of github.com/brooklabteam/spillover-virulence-v1.0.0; doi: 10.5281/zenodo.8136864).Invading growth rates ((PNG)Click here for additional data file.S2 FigαS) of a reservoir-optimized virus evolved to μR), extent of constitutive immunity (gR0), leukocyte activation rate upon viral contact (gR), virus consumption rate by leukocytes (cR), leukocyte mortality rate (mR). Darker colored lines depict outcomes at higher values for reservoir host tolerance of virus pathology  or immunopathology , assuming no tolerance of the opposing type. Heat maps demonstrate how TvR and TwR interact to produce each outcome. Outcome ranges differ between lineplots (y-axes) and heat maps . Parameter values are listed in github.com/brooklabteam/spillover-virulence-v1.0.0; doi: 10.5281/zenodo.8136864).Figure replicates (PNG)Click here for additional data file.S3 FigαS) across a range of values for 2 mechanisms of tolerance in the spillover host: tolerance of direct virus pathology  and tolerance of immunopathology . Results are expressed under assumptions of constant tolerance (top panels) and complete tolerance (bottom panels). In main text results, TwS is held constant for all predictions of spillover virulence but TvS is varied proportionally to the inverse time to MRCA between reservoir and spillover host. Data and code used to generate all figure panels are available in our publicly available GitHub repository .Virulence of spilled-over virus ((PNG)Click here for additional data file.S4 FigμR), (B) tolerance of immunopathology (TwR) , (C) magnitude of constitutive immunity (gR0), and (D) magnitude of human tolerance of virus pathology for a virus evolved in a disparate mammalian reservoir (TvS). Estimates are derived from (A) linear model predictions of maximum lifespan at the order level, (B) the scaled effect of order on a linear mixed model prediction of lifespan per body size, (C) the scaled effect of order on a linear mixed model prediction of neutrophil concentration for mass-specific BMR, and (D) the magnitude of human tolerance of virus pathology for a virus evolved in a disparate mammalian reservoir (TvS), corresponding to data presented in github.com/brooklabteam/spillover-virulence-v1.0.0; doi: 10.5281/zenodo.8136864).Model parameter estimates for (A) reservoir-host background mortality ((PNG)Click here for additional data file.S5 FigμR, TwR, and gR0; visualized in αS), which also relies on order-specific values for the spillover host tolerance of direct virus pathology (TvS). Taxa in panels (A) and (B) are arranged in descending order from highest to lowest predicted values for, respectively αS. This order varies slightly from panel (A) to (B), as highlighted by alluvial flows and discussed in the main text. See μR, TwR, gR0, and TvS and αS. Data and code used to generate all figure panels are available in our publicly available GitHub repository .Figure replicates (PNG)Click here for additional data file.S6 Fig2 value is shown as a quantitative evaluation of model fit to the data. Dashed lines give the residual of each data point from the regression line. Data and code used to generate all figure panels are available in our publicly available GitHub repository .Figure plots observed vs. predicted spillover virulence for 8 orders from (PNG)Click here for additional data file.S7 FigμR, TwR, gR0, and TvS are listed in αS are listed in github.com/brooklabteam/spillover-virulence-v1.0.0; doi: 10.5281/zenodo.8136864).Figure replicates (PNG)Click here for additional data file.S8 Figgithub.com/brooklabteam/spillover-virulence-v1.0.0; doi: 10.5281/zenodo.8136864).Plot recapitulates (PNG)Click here for additional data file.S9 FigαS) from case fatality rates reported in the literature [αS estimates from nested model using constant, universal values across all orders for all parameters excepting the parameter profiled in the corresponding column . When not profiled, TwR = 1.5 (constant) and 0.5 (complete); TvS = 1.5 (constant) and 0.5 (complete); and gR0 = 0.5 for simulations resulting in square points. Finally, triangles give αS estimates from nested model approach using parameters generated by profiling the parameter in the corresponding column , while pairing it with values recovered using regression analysis the literature for other variable parameters ; profiling TwR, TvS, or gR0 while holding constant all other parameters across orders (thin dashed line); and profiling TwR, TvS, or gR0 while using linear regression estimates from life history data for parameters not being profiled (thick dashed line). Data and code used to generate all figure panels are available in our publicly available GitHub repository .Figure replicates terature  depictedrameters . Lines a(PNG)Click here for additional data file.S1 Table(PDF)Click here for additional data file.S2 Table(PDF)Click here for additional data file.S3 Table(PDF)Click here for additional data file.S1 File(PDF)Click here for additional data file.


Borrelia burgdorferi sensu lato and transmitted to humans by the bite of infected Ixodes ticks. Jamestown Canyon Virus (JCV) is an emerging mosquito-borne pathogen found mostly in the upper Midwest and Northeastern United States. Co-infection between these two pathogens has not been previously reported since it would require the host to be bitten by the two infected vectors at the same time. We report a 36-year-old man who presented with erythema migrans and meningitis. While erythema migrans is a pathognomonic sign of early localized Lyme disease, Lyme meningitis does not occur in this stage but in the early disseminated stage. Furthermore, CSF tests were not supportive of neuroborreliosis, and the patient was ultimately diagnosed with JCV meningitis. We review JCV infection, LD, and this first reported co-infection to illustrate the complex interaction between different vectors and pathogens and to emphasize the importance of considering co-infection in people who live in vector-endemic areas.Lyme disease (LD) is the most common tick-borne illness across the United States, caused by the bacterium  Borrelia burgdorferi and is delivered through the bite of Ixodes ticks. Untreated LD progresses through three stages after an incubation period of about three to 30 days. In the first stage, erythema migrans (EM) develops at the site of infection most commonly five to seven days following the tick bite [Lyme disease (LD) is the most common tick-borne illness across the United States . Approxiick bite -3.Jamestown Canyon Virus (JCV) is an emerging arboviral pathogen transmitted to humans by mosquitos. The virus is found throughout the United States, most commonly in the Northeast and Upper Midwest surrounding the Great Lakes . There aWith changes in climate, continued increase in travel, and increases in population, the spread of these disease vectors and the diseases themselves are on the rise . A recenoC), and a 15 x 10 cm circular erythematous rash, without central clearing, over the right lower quadrant of his abdomen  visit were escitalopram 10 mg daily, fluticasone propionate inhaler 50 mcg as needed, lorazepam 1 mg as needed, and valacyclovir 1000 mg as needed. The patient did not smoke, or use any illicit drugs, and he drank socially. He denied any recent travels outside Wisconsin or any sick contacts. The patient presented to the ED with a five-day history of headache, fever, and nausea without vomiting. His headache was constant, frontal, and bilateral. He also reported new neck stiffness, diaphoresis, and chills. Upon further evaluation, he was found to have additional symptoms of fatigue, loss of appetite, recent unexpected weight loss, and mild photophobia. The patient was found to have tachycardia (120 beats per minute), fever  and immunoglobin G (IgG) antibodies, and CSF testing for meningitis/encephalitis panel  PCR panel all came back negative  with confirmation by plaque reduction neutralization test (PRNT) performed at Wisconsin State Laboratory of Hygiene. One week later, repeat blood work was obtained, and it returned positive for Meningitis is a medical condition defined by inflammation of the meninges or the outer covering of the brain . MeningiThe patient discussed here presented with many of the signs and symptoms typical for meningitis. He initially presented with a five-day history of headache, chills, neck stiffness, mild photophobia, and nausea, among other typical symptoms. Physical examination demonstrated EM, a pathognomonic rash of early localized LD. In someone who has a history of significant outdoor exposure and lives in an LD endemic area, EM is diagnostic of early localized LD. Erythema migrans usually appears within five to seven days of an infection and can last for up to three weeks . NeuroboLa Crosse orthobunyavirus, Inkoo orthobunyavirus, and snowshoe hare virus [JCV is an arbovirus (arthropod-borne virus) that belongs to the California serogroup together with the re virus ,11. Simire virus . Unusualre virus ,14. JCV re virus . A JCV ire virus . Due to re virus .In patients with viral meningitis, such as JCV meningitis, one would expect to find lymphocytic pleocytosis in CSF. However, CSF findings in patients with neuroinvasive JCV are various. In some cases of neuroinvasive disease CSF can be completely clear while in other cases, neutrophilic pleocytosis was demonstrated together with hypoglycorrhachia and increased proteinorachia mimicking bacterial meningitis ,16. Our Ixodes tick. JCV is not commonly associated with co-infections, although a case of encephalitis with a JCV and varicella zoster virus (VZV) co-infection has been reported [JCV has been found to affect all age groups with the majority of patients being immunocompetent individuals. While JCV in immunocompetent people is usually a self-limiting disease, and treatment is largely supportive, in immunocompromised patients, the clinical course might be more dramatic. A recent review of the literature showed that patients on rituximab  might have a particularly prolonged course, and worse outcomes, largely due to their inability to mount appropriate serologic response . In thesBorrelia burgdorferi through a tick bite and JCV through a mosquito bite. The overlapping geographical areas of these two vectors capable of transmitting infections to humans, and our patient’s significant outdoor exposure made this co-infection possible. This rare set of events, and clinical manifestations of both infections at the same time makes this presentation unique and quite challenging from a diagnostic perspective. Clinicians in areas that are endemic for tick-borne and mosquito-transmitted diseases need to maintain a high index of suspicion and be aware of the possibility of co-infection in order to make a timely diagnosis. Further research is needed to better understand the interaction of these pathogens with humans and to understand if co-infections lead to more severe clinical presentation.Our patient contracted


Paramyxoviridae, genus Henipavirus. NiV is a biosafety-level-4 pathogen that is mostly spread by Pteropus species, which serve as its natural reservoir host. NiV is one of the major public health challenges in South and South East Asia. However, few molecular studies have been conducted to characterise NiV in a specific region. The main objective of this review is to understand the epidemiology, pathogenesis, molecular surveillance, transmission dynamics, genetic diversity, reservoir host, clinical characteristics, and phylogenetics of NiV. South and South East Asian nations have experienced NiV outbreaks. Phylogenetic analysis confirmed that two primary clades of NiV are in circulation. In humans, NiV causes severe respiratory illness and/or deadly encephalitis. NiV is mainly diagnosed by ELISA along with PCR. Therefore, we recommend that the governments of the region support the One Health approach to reducing the risk of zoonotic disease transmission in their respective countries.Nipah virus (NiV) is a zoonotic, single-stranded RNA virus from the family  Paramyxoviridae, genus Henipavirus, subfamily Paramyxovirinae and order Mononegavirales, causing severe respiratory disease and encephalitis in South Asia, with high mortality [Nipah virus (NiV) disease is an emerging zoonotic disease. NiV is an RNA virus from the family ortality , 2. NiV ortality . NiV is ortality . The RNAortality –13 , recognised in Malaysia and Cambodia, and NiV-Bangladesh (NiV-BD), recognised in Bangladesh and India , 15. FurPteropus, also called flying foxes [Pteropus hypomelanus, Pteropus vampyrus, Cynopterus brachyotis and Eonycteris spelaea, as well as the insectivorous bat Scotophilus kuhlii [Pteropus giganteus, Pteropus lylei and Hipposideros larvatus were also verified as reservoir hosts of NiV in Bangladesh, Cambodia and Thailand [Pteropus giganteus, Rousettus leschenaultii, Eonycteris spelaea, Rhinolophus sinicus, R. affinis, R. ferrumequinum, Nyctalus noctula and Scotophilus sp.) reported that potential reservoir hosts for several viruses are often found in human proximity and some of them even enter human dwellings at night [P. giganteus and R. leschenaultii are even consumed as bushmeat by some communities in Nepal [The reservoir hosts of NiV are fruit bats belonging to the genus ng foxes . Sequencs kuhlii . LikewisThailand . SerologThailand . Likewisat night . Some frin Nepal . Nepal ain Nepal , 22. HowNiV encephalitis is an emerging and re-emerging infectious disease considered a major public health problem by the World Health Organization (WHO). NiV is present mainly in South East Asia and can cause severe respiratory illness and deadly encephalitis in humans. NiV can be transmitted to humans from animals (bats or pigs), contaminated foods or infected fluids and can also be transmitted directly from human to human. The main objective of this review is to understand the epidemiology, pathogenesis, molecular surveillance, transmission dynamics, genetic diversity, reservoir host, clinical characteristics, and phylogenetics of NiV.A literature search was performed using PubMed, Web of Science and Google Scholar. The following keywords were used: ‘Nipah virus infection’ or ‘epidemiology Nipah virus’, or ‘clinical features Nipah virus’ or ‘diagnosis Nipah virus’, or ‘surveillance Nipah virus’, or ‘prevention and control’, ‘South and South East Asia’. Research papers, review papers and case reports were included in the search.NiV was named after the village of Sungai Nipah (Nipah River Village) in Negeri Sembilan, Malaysia, where NiV was first identified in 1999. At that time, the virus crossed the species barrier from bats and infected humans, resulting in encephalitis with up to 40% mortality and residual neurological problems among survivors. The outbreak was first recognised in pigs that consumed fruit partially eaten by fruit bats and transmitted the infection to humans. In March 1999, an outbreak of acute NiV infection was confirmed in 11 male abattoir workers with an average age of 44 years in Singapore. One of these workers died because of speculated transmission of the virus during the import of pork from Malaysia. Between 1998 and 1999, both countries  reported 246 cases of febrile encephalitis by NiV in addition to pigs in farms showing neurological and respiratory symptoms. In 2004, Bangladesh confirmed its first NiV outbreak by the presence of anti-NiV antibodies in serum samples. Since 2004, researchers have detected the viral nucleic acid of NiV by genome analysis. From 2004 to 2010, nine outbreaks were documented in Bangladesh. Likewise, in 2011 another NiV outbreak was reported to cause 15 deaths in a remote town named Hatibandha in the Lalmonirhat district of northern Bangladesh . The resThe first outbreak of NiV in India was detected in Siliguri, West Bengal, in 2001, characterised by febrile illness in association with altered sensorium . Some SiNiV infection in humans has a range of clinical presentations from asymptomatic infection to acute respiratory infection, fatal encephalitis and severe neurological disease. The death rate is in the range of 40–75% according to reported outbreaks between 1998 and 2018 –31. Acco4 for the NiV-BD genotype, which diverged from 1995 into two sub-lineages. Furthermore, phylogenetic tree analyses from bat roost urine and human samples categorised these samples into the same genotype (NiV-BD). NiV sequences from Malaysia, Cambodia and Thailand were categorised into NiV-MY genotypes, whereas those from Bangladesh, India and Thailand were categorised into other NiV-BD genotypes  were positive for NiV RNA, with cycle threshold (CT) values ranging from 32.7 to 38.5 including NiV-N gene fragments (N ORF 188–1599) sequenced from 100 roost urine specimens [Nepal is a South Asian country located along the central foothills of the Himalayas, sharing porous borders with India in the south, east and west. Open borders can cause an unprecedented outbreak of NiV in Nepal through human transmission. Furthermore, some fruit bat colonies in the foothills of Nepal are speculated to adopt seasonal migration patterns from higher to lower elevations (cross-border migration as well), which represents a high risk of zoonotic virus spread in Nepal. Another important factor that contributes to the rapid transmission of infectious diseases in South Asian and South East Asian countries is the increase in the human population . PoultryPteropus) are the natural hosts of NiV, which is a virus of concern for future epidemics and also seems to be spilling over from its animal reservoir to humans. Sequencing analysis confirmed that fruit bats are responsible for the transmission of NiV to domestic animals and humans through contaminated food or indirectly from person to person. Thus, NiV infection in domestic animals such as pigs should be monitored routinely and the farm should be cleaned daily using appropriate disinfection agents or detergents to prevent an outbreak. At border checkpoints in South Asian countries, domestic animals should be quarantined and tested for zoonotic diseases. In addition, banning the movement of animals from infected farms to other areas can reduce the spread of disease. Moreover, the One Health approach should be strictly employed to detect new cases by performing surveillance and quarantining domestic animals for providing an early warning to veterinary and human public health authorities [Many studies have confirmed that fruit bat species . For instance, farm workers may be instructed to avoid direct contact with farm animals, especially during slaughter and disposal, and wear appropriate personal protective equipment and clothing. Villagers may be taught to avoid consuming date palm fruits and drinking date palm juice that may be contaminated if bats live on those palm trees. Furthermore, surveillance of NiV and other infectious diseases that could be at high risk of transmission from animals to humans should be carried out. High-risk areas in South and South East Asia along the NiV girdle should be identified and mapped, and then continual surveillance should be conducted to facilitate the early detection of virus outbreaks, identify circulating strains among fruit bats, and monitor environmental factors and the dynamics of wide-ranging transmission.To respond to an outbreak, specialised research teams should be established to identify suspected cases of infection in humans and further analyse the possibility of the emergence of new species as potential reservoir hosts of the virus that may be responsible for its transmission to humans from bats. In other words, establishing mechanisms for cross-border screening, laboratory testing, and diagnosis facilities at points of entry and exit would ensure that suspected cases are referred to facilities at the provincial level for confirmation of NiV. Furthermore, more reliable tests for the surveillance of communities and livestock will be vital to achieving a better understanding of the ecology of the fruit bat host and transmission risks to intermediate hosts, enabling the implementation of the One Health approach for outbreak prevention and the management of zoonotic diseases like that caused by NiV. In summary, the control and prevention of NiV would be possible only if strictly endorsed guidelines and control strategies are implemented by employing the One Health approach in all South and South East Asian countries , 43, 44.In the absence of specific antivirals and vaccines, medicinal plants can play an important role by providing therapeutic agents for the treatment of various viral diseases. Previous studies have revealed that medicinal plants have promising antiviral properties and are used to protect against various public health threats that affect humans and animals. South East Asian countries have medicinal plant candidates that could be screened for the treatment of viral diseases, such as NiV. The following steps would be taken for this purpose: evaluation of current treatment approaches for viral infection, screening of antiviral drug candidates from natural products and elucidation of mechanisms, inhibition of viral protease, viral replication, viral spike protein, and receptor, and finally, screening of medicinal plant target compounds to develop an antiviral drug against NiV. The best method will probably be the screening of compounds that are responsible for altering or disturbing any step of the virus’ replication. Studies on medicinal plants for the treatment of viral diseases have reported 93 antiviral drug candidates, which could be a potential area of research in antiviral drug discovery. Furthermore, these drug candidates might also show inhibitory effects against influenza, parainfluenza, and other respiratory viruses . TherefoSouth Asian and South East Asian countries should increase awareness of the NiV risk factors and educate their people on measures to reduce NiV exposure and risk of infection. By applying the strategies listed in , public


Monkeypox virus (MPXV) has spread globally. Emerging studies have now provided evidence regarding MPXV transmission, that can inform rational evidence‐based policies and reduce misinformation on this topic. We aimed to review the evidence on transmission of the virus. Real‐world studies have isolated viable viruses from high‐touch surfaces for as long as 15 days. Strong evidence suggests that the current circulating monkeypox (mpox) has evolved from previous outbreaks outside of Africa, but it is yet unknown whether these mutations may lead to an inherently increased infectivity of the virus. Strong evidence also suggests that the main route of current MPXV transmission is sexual; through either close contact or directly, with detection of culturable virus in saliva, nasopharynx, and sperm for prolonged periods and the presence of rashes mainly in genital areas. The milder clinical presentations and the potential presence of presymptomatic transmission in the current circulating variant compared to previous clades, as well as the dominance of spread amongst men who have sex with men (MSMs) suggests that mpox has a developed distinct clinical phenotype that has increased its transmissibility. Increased public awareness of MPXV transmission modalities may lead to earlier detection of the spillover of new cases into other groups. Orthopoxviruses are large (size range: 140–450 nanometers) viruses with a brick‐like structure and a genome of approximately 200–500 kbp kb that contains over 200 genes.The human monkeypox virus  belongs to the After the first human case, sporadic outbreaks were reported in some countries in west and central Africa, mainly among children in rural, rainforest areas. Several outbreaks subsequently occurred in the DRC with high fatality rates (1%–2%).Mpox reached international attention in 2003, when 71 human cases were reported in the United States.In May 2022, human mpox cases were reported in Portugal, Spain, Canada, Belgium Sweden, Italy, Australia, France, Germany, the United States, and the UK.Successful transmission of any pathogen requires a minimum dose of replication‐competent virus to be delivered to a vulnerable anatomical site in a susceptible host. A combination of viral, host and environmental characteristics affect transmission. We, therefore, undertook a review to assess the empirical evidence relating to the transmission of mpox, in light of the recent surge in a number of cases worldwide. In contrast to existing reviews, we focus on how mpox may have adapted or changed in the recent outbreak to increase its incidence globally.21.Environmental viability of MPXV.2.Virus and host factors relating to transmission.3.Population dynamics of the transmission of MPXV.We aimed to summarize the major studies that have investigated three main factors that contribute to transmission:medRxiv preprint server, for English‐language titles and abstracts published until November 30, 2022; we also searched reference lists of relevant articles and institutional or governmental reports of MPXV transmission. Articles were included if they provided relevant information on the aforementioned factors. Selected articles included laboratory‐based studies of the virus, instructive case and cluster reports, and other observational or modeling studies. In relation to the factors, we focused on studies that could help to explain why there has been rapid, expanding community transmission of MPXV in 2022.We manually searched electronic databases  and the 33.13.1.1Little has been published examining the environmental viability of MPXV under experimental conditions. However, Orthopoxviruses in general remain infectious under dry conditions and different temperatures in the environment for long periods of time. Since 1972, Sparkes and Fenje found dried vaccinia virus to be stable for up to 39 weeks (at 4°C) without loss of infectivity.3.1.25 viral copies/cm2 on inanimate surfaces was estimated by PCR and replication‐competent virus was successfully isolated from surfaces with more than 106 copies/cm2. All surfaces directly touched by the patients’ hands showed viral contamination with the highest loads detected in both bathrooms.In real‐world settings, multiple studies in the United States and UK have identified MPXV DNA from samples taken from contaminated environmental surfaces, most commonly, high‐touch surfaces. Pfeiffer and colleagues assessed the presence and degree of surface contamination of 30 household objects contacted by two mpox patients in Salt Lake City.Morgan et al.Gould et al.3.23.2.1MPXV falls into two distinct clades, based on genetic and geographic variation: formerly Central Africa (or Congo Basin) clade, now known as clade I and formerly the West African clade, now known as clade II. There is approximately 0.5% genomic sequence difference between the two clades, with the former appearing to be more virulent based on higher observed mortality rates.3.2.2In the current 2022 outbreak, a new lineage B.1, classified as clade IIb for its close relationship to clade II has been identified.Sequencing by Isidro and colleagues also found that the circulating MPXV appears to have descended from the clade sampled in cases from Nigeria, Singapore, Israel, and the UK between 2017 and 2019.In the United States, Gigante et al.3.33.3.1With regard to the human host, transmission of MPXV was classically thought to be modulated by contact with infectious bodily fluid, including virus present in the generalized monomorphic pustular rashes on the face and hands, with a small degree of aerosol and droplet spread. An infected patient is thought to be noninfectious when their lesions have crusted over.3.3.2In a retrospective observational study, Adler and colleagues report longitudinal virological findings of seven patients with mpox who were diagnosed in the UK between 2018 and 2021, including the one healthcare worker who acquired the virus nosocomially and the one patient who acquired the virus abroad and transmitted it to an adult and child within their household. In addition to PCR positivity for MPXV DNA in all pleiomorphic skin lesions in these patients , the authors found shedding of MPXV DNA in the upper respiratory tract swabs for at least three weeks in three patients, including two that were treated with off‐patent antivirals.4 plaque‐forming units (PFU), of Clade I. In respiratory challenge studies with the prairie dog animal model using the Congo Basin Strain, virus titers of 104 and 103 PFU in most cases caused infection, and in one study, 1 out of 4 prairie dogs infected with 6 × 102 PFU MPXV became infected, and showed development of disseminated lesions.A review of experimental and natural infections of animals with MPXV between 1958 and 2012 found that in nonhuman primates, infection could be initiated by intrabronchial application of 5 × 103.3.3p < 0.001) and more frequent proctitis . In contrast to cases within Africa, most cases in the current outbreak are characterized by the presence of ulcerating genital rashes, which precede the development of generalized pustular rashes. High viral loads as well as culturable MPXV have been successfully found in semen samples (see below). All this suggests that the genital area may be a primary site of infection. Sexual and close contact was suspected as a mode of transmission during the 2017 outbreak of human mpox in Nigeria, but this was never proven.One unique feature of the current mpox outbreak appears to be the high prevalence of human‐to‐human transmission following sexual intercourse, either indirectly through close contact, or directly as a sexually transmitted infection. Moreover, the viral load in skin samples from the current outbreak is consistently the highest compared to other body locations, with evidence of replication‐competent virus isolation reported more frequently from skin samples than throat swabs. Eloy José Tarín‐Vicente et al.Patel et al.Traditionally, patients with poxvirus infections are likely to be infectious from symptom onset  until the lesions scab and fall off, and a new layer of skin has formed.3.3.4In endemic countries, wild animals (rodents and primates) have been found to carry MPXV.3.3.5Evidence demonstrating vertical transmission of mpox is also emerging. Mbala et al.3.3.6Detectable viral DNA has been found in blood samples amongst infected patients in the UK and United States3.43.4.10 describes the average number of secondary cases generated from an index case in an entirely susceptible population. The R0 of mpox has previously estimated to be between 0.57 to a maximum of 1.25.0 estimates for three study populations in England, Portugal and Spain were all greater than 1, which is the condition for cases to increase, with estimates ranging from 1.40 to 1.80.In infectious disease transmission dynamics, the basic reproductive number, or R0 are a function of host susceptibility, as well as the environmental and social factors where the outbreaks occur. Therefore, several factors may elevate R0 estimates in the current outbreak, compared to estimates from previous outbreaks in Central and West Africa. First, the transmission of MPXV is likely to have increased over time, due to declining immunity in the population after the end of smallpox vaccination in the general population. Second, direct contact among young MSMs is a source of a significant number of infections in the current outbreak. Therefore the number of onward transmissions per case could be increased due to the setting in which the virus is currently being transmitted (high rates of close contact). Similarly, the number of secondary transmissions per index case can show different levels of heterogeneity. The number of household contacts, knowledge of the condition, and any public health interventions employed will have an impact on these estimates. A systematic review by Beer and Rao in 2019 of previous outbreaks in Central and West Africa found secondary attack rates ranging between 0% and 11% in household contacts that were not vaccinated against smallpox.Estimates of R3.4.20 of 3 would drop to less than 1.5.Traditionally, individuals infected with mpox appear to take a long time to develop symptoms, with a long infectious period.However, Ward and colleagues analyzed the transmission dynamics of the current mpox outbreak in the UK with a contact tracing study, linking data on case‐contact pairs and on probable exposure dates.Of particular concern, the authors found that short serial intervals were more common than short incubation periods suggesting considerable presymptomatic transmission, which was validated through linked patient‐level records. For patients who could be linked through personally identifiable data, four days was the maximum time that transmission was detected before symptoms manifested.detected rather than true asymptomatic disease, that is, individuals having mpox lesions in areas which they are unaware. Therefore, effective public health messaging must emphasis the importance of looking for lesions in genitalia and other unexpected areas of the body to have any effect on reducing spread.Previous research has not found evidence of transmission and substantial shedding of MPXV before symptom onset, which is reflected guidance from WHO and the European Centre for Disease Prevention and Control.3.4.3There may be considerable heterogeneity in infectious viral load between individuals, the relationship between symptoms and viral load, and the relationship between symptoms and behavior. For example, individuals with large numbers of lesions may be very infectious, but may not be engaging in risk behavior. Modeling studies have investigated some of these heterogeneities and how they affect the short and long‐term dynamics of these outbreaks. For example, Endo and colleagues used a branching process transmission model, fitted to empirically sexual partnership data in the UK and found that there was a heavy‐tailed nature of the sexual partnership distribution, where a small fraction of individuals have disproportionately large numbers of partners can explain the sustained growth of mpox cases among the MSM population, despite the absence of such patterns of spread in past outbreaks.It is also possible that the current circulating mpox variant represents a similar evolutionary consequence as has been posited for HIV, whereby the reproduction number has been optimized by infected patients having minor symptoms (compared with previous variants in Africa) but remaining highly infectious to close contacts.4Before the current outbreak, relatively little evidence exists regarding the transmission dynamics of MPXV. A summary of our appraisal of the evidence regarding various modes of transmission can be found in Figure 0 of mpox are likely to have underestimated the current outbreak, probably because they were in populations with much less close contact. Whether mutations that are driving the current variants of mpox circulating worldwide also contribute to increased infectivity remains unknown. However, compared to previous variants, most patients infected with the current variant are MSMs, engage in high‐risk sexual activity, and remain systemically well before, during, and after their infection, with significantly fewer pox lesions compared to previous clades and focused in the groin areas. This suggests that the current MPXV variants have adapted  to maximize the probability of onward transmission. Increased public awareness of MPXV transmission modalities may lead to earlier detection of the spillover of new cases into other groups.Previous estimates of the RDaniel Pan and Julian W. Tang conceived the idea of the manuscript. Daniel Pan and Shirley Sze wrote the first draft of the manuscript. Christopher A. Martin, Joshua Nazareth, Christopher A. Martin, Jonathan Decker, Eve Fletcher, T. Déirdre Hollingsworth, Michael R. Barer, Manish Pareek, and Julian W. Tang commented and edited subsequent versions of the manuscript. All authors have reviewed and approved the final version of the manuscript submitted to the journal.The authors declare no conflicts of interest.


The locus of control is associated with a variety of psychological concepts, theories and researches, including learned helplessness, which is explained in way that person has learned to act helpless even when they actually have control over their situation or the ability to change a circumstance or outcome. In this scientific work, the aim is to examine the corelation between the locus of control and stress coping strategies in a group of health care workers. A sample is 110 respondents, of different age, gender, ages and educational degrees.In this research, the aim is to examine the connection between locus of control and coping strategies among healthcare workers during the Covid-19 epidemic. The research results showed that there are no statistically significant differences in the relationship between stress coping strategies and locus of control in relation to men and women and age. Also, there is a statistically significant difference in the stress coping strategy focused on avoidance in relation to the locus of control, i.e. it turned out that the coping strategy focused on avoidance is more pronounced in respondents with an internal locus of control than in those with an external locus of control.The research was conducted at the Clinical Center of Montenegro, in Podgorica, in March 2022, through an online program, due to the epidemiological situation. The sample consists of 110 respondents, of both sexes and aged from 20 to 65 years old, who were chosen by the method of random selection. The CISS and RI-E scales were used.The research showed that there is a connection between the locus of control and strategies for overcoming stress, but that there are no statistically significant differences in the connection between strategies for overcoming stress and locus of control in relation to men and women and their age, as well as that there is a statistically significant difference in the strategy coping with stress focused on avoidance in relation to locus of control, i.e. it was shown that the coping strategy focused on avoidance is more pronounced in respondents with an internal locus of control than in those with an external locus of control.Image:Image 2:Image 3:This research actually confirmed the importance of individuality and various factors that can affect a person, and because of this, it was very likely that not all hypotheses could be answered in the way the author expected before the research began. With this, it can be assumed that the personality of a person can hardly be related to broad styles of coping with stress, and that generalizations regarding gender, age, education and work experience cannot be made, because there are predominantly individual differences in the development of an individual.None Declared