Optical biosensing of monkeypox virus using novel recombinant silica-binding proteins for site-directed antibody immobilization

The efficient immobilization of capture antibodies is crucial for timely pathogen detection during global pandemic outbreaks.Therefore,we proposed a silica-binding protein featuring core functional domains(cSP).It comprises a peptide with a silica-binding tag designed to adhere to silica surfaces and tandem protein G fragments(2C2)for effective antibody capture.This innovation facilitates precise site-directed immobilization of antibodies onto silica surfaces.We applied cSP to silica-coated optical fibers,creating a fiber-optic biolayer interferometer(FO-BLI)biosensor capable of monitoring the monkeypox virus(MPXV)protein A29L in spiked clinical samples to rapidly detect the MPXV.The cSP-based FO-BLI biosensor for MPXV demonstrated a limit of detection(LOD)of 0.62 ng/mL in buffer,comparable to the 0.52 ng/mL LOD achieved using a conventional streptavidin(SA)-based FO-BLI biosensor.Furthermore,it achieved LODs of 0.77 ng/mL in spiked serum and 0.80 ng/mL in spiked saliva,exhibiting no cross-reactivity with other viral antigens.The MPXV detection process was completed within 14 min.We further proposed a cSP-based multi-virus biosensor strategy capable of detecting various pandemic strains,such as MPXV,the latest coronavirus disease(COVID)variants,and influenza A protein,to extend its versatility.The proposed cSP-modified FO-BLI biosensor has a high potential for rapidly and accurately detecting MPXV antigens,making valuable contributions to epidemiological studies.

Introduction of monkeypox virus in Benin,2022

Dear Editor,Monkeypox is an infectious disease that is endemic in a dozen of African countries.Some imported cases have been also reported outside of Africa in the past[1].Since early May 2022,monkeypox infections including human-to-human transmission,were reported in a multi-country outbreak in non-endemic countries and declared Public Health Emergency of International Concern(PHEIC)by the World Health Organization(WHO)in July 2022[2].As of 20 September 2022,a total of at least 62,798 human cases of monkeypox with 20 deaths have been confirmed in 115 countries in five WHO regions[3].

Rapid identification of full-length genome and tracing variations of monkeypox virus in clinical specimens based on mNGS and amplicon sequencing

The monkeypox virus(MPXV)has triggered a current outbreak globally.Genome sequencing of MPXV and rapid tracing of genetic variants will benefit disease diagnosis and control.It is a significant challenge but necessary to optimize the strategy and application of rapid full-length genome identification and to track variations of MPXV in clinical specimens with low viral loads,as it is one of the DNA viruses with the largest genome and the most AT-biased,and has a significant number of tandem repeats.Here we evaluated the performance of metagenomic and amplicon sequencing techniques,and three sequencing platforms in MPXV genome sequencing based on multiple clinical specimens of five mpox cases in Chinese mainland.We rapidly identified the full-length genome of MPXV with the assembly of accurate tandem repeats in multiple clinical specimens.Amplicon sequencing enables cost-effective and rapid sequencing of clinical specimens to obtain high-quality MPXV genomes.Third-generation sequencing facilitates the assembly of the terminal tandem repeat regions in the monkeypox virus genome and corrects a common misassembly in published sequences.Besides,several intra-host single nucleotide variations were identified in the first imported mpox case.This study offers an evaluation of various strategies aimed at identifying the complete genome of MPXV in clinical specimens.The findings of this study will significantly enhance the surveillance of MPXV.

An MPXV mRNA-LNP vaccine candidate elicits protective immune responses against monkeypox virus

The monkeypox virus(MPXV)outbreak,declared a Public Health Emergency of International Concern(PHEIC)by the World Health Organization(WHO)in 2022,continues to pose a significant threat due to the absence of vaccines or drugs for MPXV infection.In this study,we developed an mRNA vaccine that expressing the A29L antigen,a specific protein of the intracellular mature virus.Our vaccine utilizes a thermostable ionizable lipid nanoparticle(iLNP)platform and has been administered to mice.Our find-ings demonstrate that the MPXV A29L mRNA vaccine candidate induces robust cross-neutralizing immune responses against both vaccinia virus(VACV)and MPXV live virus.Furthermore,immunization with the vaccine candidate provided protection against the VACV challenge in mice.These findings underscore the potential of mRNA-LNP vaccines as safe and effective candidates against monkeypox epidemics.Given the current absence of specific interventions for MPXV infection,our study represents a significant step forward in developing a viable solution to combat this ongoing public health threat.

Diagnostic approaches for monkeypox virus

Mpox(formerly Monkeypox)is a zoonotic infection caused by Monkeypox virus(MPXV).Since 2022,Mpox epidemics have occurred in many nonendemic countries and regions,leading the World Health Organization to declare a public health emergency of international concern.With the persistent transmission and evolution of MPXV,symptoms of Mpox have become milder,with some infections being asymptomatic.In addition,MPXV has become more contagious.Therefore,rapid and accurate diagnosis and screening of MPXV is vital to prevent and control MPXV epidemics.Here,we review and summarize the technical details,application scenarios,and the advantages and disadvantages of MPXV‐specific diagnostic methods.

Development of two multiplex real-time PCR assays for simultaneous detection and differentiation of monkeypox virus IIa,IIb,and I clades and the B.1 lineage

An ongoing multi-country outbreak of monkeypox was reported in May 2022 with several deaths,affecting 107 countries of all six World Health Organization(WHO)regions.The WHO has declared the current monkeypox outbreak to be a Public Health Emergency of International Concern.It is,thus,necessary to rapidly and accurately detect and distinguish different monkeypox virus(MPXV)clades.We designed primers and probes based on the alignment of 138 complete genomes of poxviruses.In Panel 1,we mixed one pair of primers and three probes to detect and differentiate the MPXV Western Africa(IIa,IIb clade)and Congo Basin(I clade)and other orthopoxviruses.In Panel 2,we mixed one pair of primers and two probes to detect the 2022 MPXV(B.1 lineage and its descendant lineages).In addition,we tested the specificity and sensitivity of the assay using real-time PCR.In Panel 1,the assay reproducibly identified various concentrations of two plasmids of the monkeypox virus,whereas other orthopoxviruses did not cross-react.In Panel 2,the probe annealed well to MPXV B.1 and showed the expected linearity.These two multiple real-time assays are inclusive and highly specific for identifying different clades of MPXV.

Monkeypox virus:a re-emergent threat to humans

Human monkeypox(MPX)is a rare zoonotic infection characterized by smallpox-like signs and symptoms.It is caused by monkeypox virus(MPXV),a double stranded DNA virus belonging to the genus Orthopoxvirus.MPX was first identified in 1970 and mostly prevailed in the rural rainforests of Central and West Africa in the past.Outside Africa,MPX was reported in the United Kingdom,the USA,Israel,and Singapore.In 2022,the resurgence of MPX in Europe and elsewhere posed a potential threat to humans.MPXV was transmitted by the animals-human or human-human pathway,and the symptoms of MPXV infection are similar to that of smallpox,but in a milder form and with lower mortality(1%–10%).Although the smallpox vaccination has been shown to provide 85%protection against MPXV infection,and two anti-smallpox virus drugs have been approved to treat MPXV,there are still no specific vaccines and drugs against MPXV infection.Therefore it is urgent to take active measures including the adoption of novel anti-MPXV strategies to control the spread of MPXV and prevent MPX epidemic.In this review,we summarize the biological features,epidemiology,pathogenicity,laboratory diagnosis,and prevention and treatment strategies on MPXV.This review provides the basic knowledge for prevention and control of future outbreaks of this emerging infection.

Efficient assembly of a large fragment of monkeypox virus genome as a qPCR template using dual-selection based transformation-associated recombination

Transformation-associated recombination(TAR)has been widely used to assemble large DNA constructs.One of the significant obstacles hindering assembly efficiency is the presence of error-prone DNA repair pathways in yeast,which results in vector backbone recircularization or illegitimate recombination products.To increase TAR assembly efficiency,we prepared a dual-selective TAR vector,pGFCS,by adding a PADH1-URA3 cassette to a previously described yeast-bacteria shuttle vector,p GF,harboring a PHIS3–HIS3 cassette as a positive selection marker.This new cassette works as a negative selection marker to ensure that yeast harboring a recircularized vector cannot propagate in the presence of 5-fluoroorotic acid.To prevent pGFCS bearing ura3 from recombining with endogenous ura3-52 in the yeast genome,a highly transformable Saccharomyces cerevisiae strain,VL6-48B,was prepared by chromosomal substitution of ura3-52 with a transgene conferring resistance to blasticidin.A55-kb genomic fragment of monkeypox virus encompassing primary detection targets for quantitative PCR was assembled by TAR using pGFCS in VL6-48B.The pGFCS-mediated TAR assembly showed a zero rate of vector recircularization and an average correct assembly yield of 79%indicating that the dual-selection strategy provides an efficient approach to optimizing TAR assembly.

Molecular phylogenomic analysis reveals a single origin of monkeypox virus transmission in Guangzhou,China in June 2023

The monkeypox(mpox)virus has caused worldwide transmission since its initial report in England in early May 2022.Available data from the World Health Organization(WHO)show that Europe and the Americas experienced a huge wave of mpox virus infection.Now the number of infected cases is on the rise in Asia.Several sporadic infections have been reported in China.In this study,we obtained high‐quality whole viral genomic sequences using a mpox virus‐specific amplicon‐based sequencing strategy.Our analysis of the phylogenomic characteristics indicated that all eight mpox virus sequences from Guangzhou belonged to the clade IIb lineage B.1.3 cluster.However,we could not locate the exact origins where the virus was imported,based on all the available mpox virus sequences from the Global Initiative on Sharing Avian Influenza Data(GISAID)database(https://gisaid.org/),except for their closest sequence similarity to that was reported from Japan.Novel amino acid mutations were found among the eight cases,suggesting that a local transmission may have occurred in Guangzhou,China.

Deep learning based detection of monkeypox virus using skin lesion images

As we set into the second half of 2022,the world is still recovering from the two-year COVID-19 pandemic.However,over the past three months,the outbreak of the Monkeypox Virus(MPV)has led to fifty-two thousand confirmed cases and over one hundred deaths.This caused the World Health Organisation to declare the outbreak a Public Health Emergency of International Concern(PHEIC).If this outbreak worsens,we could be looking at the Monkeypox virus causing the next global pandemic.As Monkeypox affects the human skin,the symptoms can be captured with regular imaging.Large samples of these images can be used as a training dataset for machine learning-based detection tools.Using a regular camera to capture the skin image of the infected person and running it against computer vision models is beneficial.In this research,we use deep learning to diagnose monkeypox from skin lesion images.Using a publicly available dataset,we tested the dataset on five pre-trained deep neural networks:GoogLeNet,Places365-GoogLeNet,SqueezeNet,AlexNet and ResNet-18.Hyperparameter was done to choose the best parameters.Performance metrics such as accuracy,precision,recall,f1-score and AUC were considered.Among the above models,ResNet18 was able to obtain the highest accuracy of 99.49%.The modified models obtained validation accuracies above 95%.The results prove that deep learning models such as the proposed model based on ResNet-18 can be deployed and can be crucial in battling the monkeypox virus.Since the used networks are optimized for efficiency,they can be used on performance limited devices such as smartphones with cameras.The addition of explainable artificial intelligence techniques LIME and GradCAM enables visual interpretation of the prediction made,helping health professionals using the model.

Monkeypox virus(MPXV):A Brief account of global spread,epidemiology,virology,clinical features,pathogenesis,and therapeutic interventions

The largest monkeypox virus(MPXV)outbreak of the 21st century occurred in 2022,which caused epidemics in many countries.According to WHO,physical contact with infected persons,contaminated surfaces,or affected animals might be a source of this virus transmission.A febrile sickness including few symptoms found in MPX disease.Skin rash,lesions,fever,headache,fatigue,and muscle aches symptoms were observed commonly for this disease.Animal and in vitro,studies have shown that the antiviral medications cidofovir and brincidofovir are effective against MPXV.The first-generation vaccinia virus vaccine was developed in 1960,and it helped to protect against MPXV with its side effects.A second-generation vaccination with limitations was launched in 2000.However,the CDC advised vaccinations for risk groups in endemic countries,including positive patients and hospital employees.The JYNNEOS vaccine,administered in 2 doses,also provides protection from MPX.This article presents concisely the most recent findings regarding epidemiology,genomic transmission,signs and symptoms,pathogenesis,diagnosis,and therapeutic interventions for MPXV,which may be helpful to researchers and practitioners.WHO declared that MPX was no longer a global health emergency due to its declining case rate,and a number of countries have reported new incidences.Further research-based investigations must be carried out based on the 2022 outbreak.

Monkeypox awareness,knowledge,and attitude among undergraduate preclinical and clinical students at a Malaysian dental school:An emerging outbreak during the COVID-19 era

Objective:To evaluate the awareness,knowledge,and attitude on monkeypox viral infection among preclinical and clinical dental students in Malaysia.Methods:A cross-sectional study was conducted among 229 preclinical and clinical dental students via an online self-reported questionnaire.The questionnaire included 3 items on awareness,15 items on knowledge,9 items on attitude and 2 open-ended questions.Data were presented as frequencies and percentages.Chi-square test was used to compare knowledge and awareness scores between preclinical and clinical dental students and content analysis was performed for open-ended responses.Results:Preclinical and clinical dental students were aware of the existence of monkeypox(89.5%and 94.4%,respectively),that the disease emerged in non-endemic countries(81.0%and 87.1%,respectively)and that it was declared a public health emergency of international concern by the World Health Organization(73.3%and 79.0%,respectively).Clinical dental students’overall knowledge level was significantly higher than preclinical dental students(P=0.014).Both preclinical(95.2%)and clinical(96.8%)dental students demonstrated positive attitudes toward monkeypox with no significant difference(P=0.736)noted between them.Three themes emerged from the open-ended questions:(1)reimplementation of nationwide lockdown,(2)impact on the economy and health,and(3)disruption to the educational system.Students also anticipated their face-to-face learning to be reduced should there be a new outbreak.Conclusions:Both preclinical and clinical dental students showed comparable awareness and attitudes,while the latter demonstrated greater satisfactory knowledge toward the re-emergence of monkeypox during the COVID-19 pandemic.Nonetheless,efforts to improve dental students’understanding of this alarming outbreak are required,to safeguard their health and minimise transmission.

An unfolding monkeypox outbreak in Europe and beyond

Dear Editor,Monkeypox is a zoonotic disease caused by the monkeypox virus(MPXV),a double-stranded DNA virus in the genus Orthopoxvirus,family Poxviridae.MPXV is known to be transmitted between animals such as rats and squirrels,but animal-to-human and human-to-human transmission has been known in Africa since 1970.Transmission is linked to direct contact with body fluids,skin lesions,and patient items as well as respiratory droplets in case of prolonged face-to-face contact[1].Until recently,only a few cases were associated with travel to endemic countries or contact with infected animals imported from endemic countries.

Monkeypox outbreak in the age of COVID‑19:a new global health emergency

Dear Editor,The World Health Organization recently declared monkeypox as a global emergency after reporting more than 57,000 new cases worldwide[1].Monkeypox is an orthopoxvirus similar in nature to the Variola virus,which is a causative agent for smallpox[2].Monkeypox virus presents as a unique challenge for global health and should be regarded with grave concern as the vast majority of cases are occurring in countries where the disease is not considered endemic.Furthermore,the spread of this pathogen is occurring concomitantly as the world is still engaged in a battle with the coronavirus disease 2019(COVID-19)pandemic,which has caused substantial damage to global healthcare infrastructure.If monkeypox continues its rapid spread,hospitals could be quickly overwhelmed by both COVID-19 and monkeypox cases.Now more than ever,early symptom recognition and use of all available treatments to contain monkeypox outbreaks are vital.Early precautions including early recognition of symptoms by physicians,use of currently available treatments,and promoting precautionary measures in at-risk populations may be vital in preventing hospital burden and further physician burnout.

Monkeypox:An emerging zoonotic pathogen

Monkeypox virus(MPXV),which belongs to the orthopoxvirus genus,causes zoonotic viral disease.This review discusses the biology,epidemiology,and evolution of MPXV infection,particularly cellular,human,and viral factors,virus transmission dynamics,infection,and persistence in nature.This review also describes the role of recombination,gene loss,and gene gain in MPXV evolvement and the role of signal transduction in MPXV infection and provides an overview of the current access to therapeutic options for the treatment and prevention of MPXV.Finally,this review highlighted gaps in knowledge and proposed future research endeavors to address the unresolved questions.

Review of the prevalence,diagnostics,and containment measures of the current mpox outbreak

Monkeypox(mpox),is a disease from the Poxviridae family that can cause several serious medical issues.This mini-review sought to analyze the existing literature regarding the current mpox outbreak with a focus on the prevalence,diagnostics,and containment measures.Mpox cases have been reported to World Health Organization(WHO)from 85 Member States in all six WHO regions during the period of January 1,2022,through August 3,2022.Standardized or optimized guidelines for the clinical care of patients with mpox are limited,particularly in low-resource settings.In an effort to achieve guidance and meet standards,special attention should be paid to this outbreak in order to eradicate such a rare infectious disease by analyzing prevention and control measures.Patient outcomes may also be poor,and their illnesses may last for a long time.The spectrum of clinical symptoms,including complications and sequelae,as well as aspects of the illness may be indicators of sickness severity and complications;therefore,its clinical presentation must be better understood to improve containment measures.In addition,it is important to create and evaluate a standard of care that takes a variety of parameters into account,including antiviral,immune therapies,and clinical metrics that are particular to mpox.The global emergence of mpox has presented new challenges for public health and has called for further investigation into its epidemiological profile across international contexts.

Evolution of Monkeypox C9L RNA G-Quadruplex Elevates the Translation of Its Only Kelch-like Protein by Accelerating G-Quadruplex Unfolding Kinetics

Genomic surveillance of monkeypox virus(MPXV)is essential to explore the reason of its unusual outbreak.Current phylogenomic analysis of the MPXV genome mainly focuses on the effect of amino acid mutations.Herein,we explore the evolutionary variation of RNA G-quadruplex(RG4)of MPXV and find that the genome evolution of MPXV can also produce new effects through changes in the RG4 structure.This RG4 is located in MPXV’s only Kelch-like C9L gene,which encodes for an antagonist of the innate immune response.The evolution of this virus increases the unfolding kinetic constant of C9L RG4 and promotes the C9 protein level in living cells.Importantly,all reported MPXV genomes in 2022 carry the C9L-RG4-5 pattern with the highest unfolding kinetic constant.Additionally,the RG4 ligand,RGB-1,can impede the unfolding of C9L-RG4-5 and thereby reduce the C9 protein level.These findings carve out a new path to comprehensively understanding MPXV virology.

Multi-valent mRNA vaccines against monkeypox enveloped or mature viron surface antigens demonstrate robust immune response and neutralizing activity

Monkeypox was declared a global health emergency by the World Health Organization,and as of March 2023,86,000 confirmed cases and 111 deaths across 110 countries have been reported.Its causal agent,monkeypox virus(MPV)belongs to a large family of double-stranded DNA viruses,Orthopoxviridae,that also includes vaccinia virus(VACV)and others.MPV produces two distinct forms of viral particles during its replication cycles:the enveloped viron(EV)that is released via exocytosis,and the mature viron(MV)that is discharged through lysis of host cells.This study was designed to develop multi-valent m RNA vaccines against monkeypox EV and MV surface proteins,and examine their efficacy and mechanism of action.Four m RNA vaccines were produced with different combinations of surface proteins from EV(A35R and B6R),MV(A29L,E8L,H3L and M1R),or EV and MV,and were administered in Balb/c mice to assess their immunogenicity potentials.A dynamic immune response was observed as soon as seven days after initial immunization,while a strong Ig G response to all immunogens was detected with ELISA after two vaccinations.The higher number of immunogens contributed to a more robust total Ig G response and correlating neutralizing activity against VACV,indicating the additive potential of each immunogen in generating immune response and nullifying VACV infection.Further,the m RNA vaccines elicited an antigen-specific CD4^(+)T cell response that is biased towards Th1.The m RNA vaccines with different combinations of EVand MV surface antigens protected a mouse model from a lethal dose VACV challenge,with the EV and MV antigens-combined vaccine offering the strongest protection.These findings provide insight into the protective mechanism of multi-valent m RNAvaccines against MPV,and also the foundation for further development of effective and safe m RNA vaccines for enhanced protection against monkeypox virus outbreak.

Biological characteristics, biosafety prevention and control strategies for the 2022 multi-country outbreak of monkeypox

Monkeypox is a zoonotic disease caused by the monkeypox virus(MPXV),which is a potential biological warfare agent of bioterrorism and poses the greatest threat to the world’s public biosafety and health after variola virus(VARV).While the coronavirus disease 2019(COVID-19)pandemic has not ended yet,monkeypox is spreading menacingly.The first case of monkeypox in a nonendemic country was confirmed on May 6^(th),2022,while the first imported case from Asia was found on June 21^(st).There were more than 16 thousand reported cases as of July 23^(rd),the day the World Health Organization(WHO)declared the global monkeypox outbreak a public health emergency of international concern(PHEIC)at the same level as smallpox and COVID-19;while there were more than 53 thousand cases as of September 1^(st).Therefore,we will propose relevant biosafety prevention and control strategies after analyzing the etiology of the 2022 multi-country monkeypox outbreak from the biological feature,transmissibility,epidemic,and variability of MPXV.

Preparedness for a monkeypox outbreak

In light of the ongoing COVID-19 pandemic,the unexpected outbreak and worldwide spread of monkeypox has gained global attention.As of June 22,2022,there were 3340 confirmed cases of monkeypox globally,which is the largest and most widespread monkeypox epidemic outside Africa.Monkeypox virus(MPXV)is transmitted from human-to-human through direct contact with infectious skin or mucosal skin lesions,respiratory droplets,or indirect contact with contaminated objects or materials,as well as mother-to-child vertical transmission.It is also possibly sexually transmitted through semen/vaginal fluid,and the possibility of community transmission cannot be ruled out.Monkeypox is a viral zoonotic disease caused by MPXV,which is an enveloped,linear,double-stranded DNA virus belonging to the Orthopoxvirus genus,of the Chordopoxvirinae subfamily,within the Poxviridae family.Monkeypox is usually a self-limiting infection,with symptoms lasting 2-4 weeks,and has a fatality rate that has historically fluctuated from 0%to 11%.Symptoms of monkeypox include intense headaches,fever,lesions,and lymphadenopathy.Although there is no specific treatment or vaccine for MPXV infection,antiviral drugs and vaccines for smallpox have been approved for use in several countries in response to the monkeypox outbreak.Before the virus can be allowed to establish efficient person-to-person transmission,rapid action must be taken to contain the local spread and,by extension,the multi-country outbreak of monkeypox.