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.

The First Imported Case of Monkeypox in the Mainland of China—Chongqing Municipality,China,September 16,2022

Monkeypox is a zoonotic viral disease caused by the monkeypox virus(MPXV),and historically,all outbreaks have been linked to Africa;however,monkeypox has been posing an alarming challenge to the world in 2022(1)as approximately 60,000 cases have been reported in more than 100 nations and regions worldwide(2).Currently,many cases of monkeypox were identified in many nonendemic countries outside of Central and West Africa,and human-to-human transmission has occurred frequently,especially among men who have sex with men(MSM)presenting new clinical symptoms similar to syphilis and other sexually transmitted infections(3).

Summary of the Detection Kits for SARS-CoV-2 Approved by the National Medical Products Administration of China and Their Application for Diagnosis of COVID-19

The on-going global pandemic of coronavirus disease 2019(COVID-19)caused by a novel coronavirus called severe acute respiratory syndrome coronavirus 2(SARS-CoV-2)has been underway for about 11 months.Through November 20,2020,51 detection kits for SARS-CoV-2 nucleic acids(24 kits),antibodies(25 kits),or antigens(2 kits)have been approved by the National Medical Products Administration of China(NMPA).Convenient and reliable SARS-CoV-2 detection assays are urgently needed worldwide for strategic control of the pandemic.In this review,the detection kits approved in China are summarised and the three types of tests,namely nucleic acid,serological and antigen detection,which are available for the detection of COVID-19 are discussed in detail.The development of novel detection kits will lay the foundation for the control and prevention of the COVID-19 pandemic globally.

A core-shell structured COYID-19 mRNA vaccine with favorable biodistribution pattern and promising immunity

Although inoculation of COVID-19 vaccines has rolled out globally,there is still a critical need for safe and effective vaccines to ensure fair and equitable supply for all countries.Here,we report on the development of a highly efficacious mRNA vaccine,SW0123 that is composed of sequence-modified mRNA encoding the full-length SARS-CoV-2 Spike protein packaged in core-shell structured lipopolyplex(LPP)nanoparticles.SWOT 23 is easy to produce using a large-scale microfluidics-based apparatus.The unique core-shell structured nanoparticle facilitates vaccine uptake and demonstrates a high colloidal stability,and a desirable biodistribution pattern with low liver targeting effect upon intramuscular administration.Extensive evaluations in mice and nonhuman primates revealed strong immunogenicity of SW0123,represented by induction of Th1-polarized T cell responses and high levels of antibodies that were capable of neutralizing not only the wild-type SARS-CoV-2,but also a panel of variants including D614G and N501Y variants.In addition,SW0123 conferred effective protection in both mice and non-human primates upon SARS-CoV-2 challenge.Taken together,SW0123 is a promising vaccine candidate that holds prospects for further evaluation in humans.

Eleven COVID-19 Outbreaks with Local Transmissions Caused by the Imported SARS-CoV-2 Delta VOC-China,July-August,2021

Starting from December 2019,Wuhan,China,encountered the first outbreak of coronavirus disease 2019(COVID-19)(1-2).The epidemic was successfully suppressed by strict containment so that the number of infected people was reduced to 0 on April 8,2020(3–4).After that,China experienced roughly 3 dozen outbreaks with local transmission caused by imported severe acute respiratory syndrome coronavirus 2(SARS-CoV-2).

Development and effectiveness of pseudotyped SARS-CoV-2 system as determined by neutralizing efficiency and entry inhibition test in vitro

With the development of the COVID-19 epidemic,there is an urgent need to establish a system for determining the effectiveness and neutralizing activity of vaccine candidates in biosafety level 2(BSL-2)facilities.Previously,researchers had developed a pseudotyped virus systemfor SARS-CoV andMERS-CoV,based onHIV-1 core,bearing virus spike protein.During the development of a pseudotyped SARS-CoV-2 system,a eukaryotic expression plasmid expressing SARSCoV-2 spike(S)protein was constructed and then co-transfectedwith HIV-1 based plasmid which containing the firefly luciferase reporter gene,into HEK293T cells to prepare the pseudotyped SARS-CoV-2 virus(ppSARS-2).We have successfully established the pseudotyped SARS-CoV-2 system for neutralization and entry inhibition assays.Huh7.5 cell line was found to be the most susceptible to our pseudotyped virus model.Different levels of neutralizing antibodies were detected in convalescent serum samples of COVID-19 patients using ppSARS-2.The recombinant,soluble,angiotensin-converting enzyme 2 protein was found to inhibit the entry of ppSARS-2 in Huh7.5 cells effectively.Furthermore,the neutralization results for ppSARS-2 were consistent with those of live SARS-CoV-2 and determined using the serum samples fromconvalescent patients.In conclusion,we have developed an easily accessible and reliable tool for studying the neutralizing efficiency of antibodies against SARS-CoV-2 and the entry process of the virus in a BSL-2 laboratory.

An Imported Case of BA.2 Lineage of Omicron Variant COVID-19—Guangdong Province,China,December 28,2021

On December 19,2021,a 31-year-old male boarded a flight from Bhubaneswar,India to Mumbai,India,and then another flight from Mumbai to Muscat,Oman.On December 24,he returned from Muscat to Guangzhou by airplane and was transferred to a centralized isolation hotel in Foshan.He was sampled on the third day of the quarantine,and the result returned to be coronavirus disease 2019(COVID-19)positive on December 27,2021.He was transported to the Guangzhou Eighth People’s Hospital for isolated treatment by negative pressure ambulance on the same day.The case had two lungs focal inflamed according to the chest CT examination.

Surveillance and Analysis of SARS-CoV-2 Variant Importation—China,January–June 2022

Introduction:The severe acute respiratory syndrome coronavirus 2(SARS-CoV-2)Omicron variant is the dominant circulating strain worldwide.To assess the importation of SARS-CoV-2 variants in the mainland of China during the Omicron epidemic,the genomic surveillance data of SARS-CoV-2 from imported coronavirus disease 2019(COVID-19)cases in the mainland of China during the first half of 2022 were analyzed.Methods:Sequences submitted from January to July 2022,with a collection date before June 30,2022,were incorporated.The proportions of SARS-CoV-2 variants as well as the relationships between the origin and destination of each Omicron imported case were analyzed.Results:4,946 sequences of imported cases were submitted from 27 provincial-level administrative divisions(PLADs),and the median submission interval was within 1 month after collection.In 3,851 Omicron sequences with good quality,1 recombinant(XU)and 4 subvariants under monitoring(BA.4,BA.5,BA.2.12.1,and BA.2.13)were recorded,and 3 of them(BA.4,BA.5,and BA.2.12.1)caused local transmissions in the mainland of China later than that recorded in the surveillance.Omicron subvariants dominated in the first half of 2022 and shifted from BA.1 to BA.2 then to BA.4 and BA.5.The percentage of BA.2 in the imported SARS-CoV-2 surveillance data was far higher than that in the Global Initiative on Sharing All Influenza Data(GISAID).The imported cases from Hong Kong Special Administrative Region,China,accounted for 32.30%of Omicron cases sampled,and 98.71%of them were BA.2.Conclusions:The Omicron variant showed the intra-Omicron evolution in the first half of 2022,and all of the Omicron subvariants were introduced into the mainland of China multiple times from multiple different locations.

First Imported Case of SARS-CoV-2 Omicron Subvariant BA.4-Guangdong Province,China,May 4,2022

On April 29,2022,a flight arrived at Baiyun International Airport,Guangzhou City,Guangdong Province,and departed from Amsterdam Schiphol Airport,Netherlands.After the first test of severe acute respiratory syndrome coronavirus 2(SARS-CoV-2)nucleic acid at the Baiyun International Airport,all passengers were admitted to a quarantine hotel for a routine 14-day medical observation.On April 30,one of the passengers(a 20-year-old Chinese female)was reported positive for coronavirus disease 2019(COVID-19),and then a nasopharyngeal swab sample was immediately retested on May 1 and reported positive.

Perspective on the application of genome sequencing for monkeypox virus surveillance

Monkeypox virus(MPXV),a DNA virus belonging to the Orthopoxvirus genus,causes a self-limiting zoonotic disease known as mpox.The human monkeypox infection was first recorded in a 9-month-old child in the Republic of Congo in the 1970s(Ladnyj et al.,1972).For a long time,mpox was mainly prevalent in the humid forests of Central Africa and parts of West Africa(Kabugae and El Zowalaty,2019).In recent years,MPXV has been spread to other countries through trade and travel.In 2003,a group of rare pets carrying MPXV was exported from Africa to the United States,and an outbreak of animal-to-human transmission occurred(Di Giulio and Eckburg,2004).In 2018,two cases of mpox were confirmed in travelers from Nigeria to the United Kingdom and one case was confirmed in Israel(Vaughan et al.,2018;Erez et al.,2019).Then,in 2019,one case was confirmed in Singapore(Ng et al.,2019).The outbreak of mpox in multiple non-endemic countries in North America and Europe started in May 2022(WHO,2022b).On July 23,2022,the WHO declared MPXV a Public Health Emergency of International Concern(PHEIC)(WHO,2022a).As of March 5,2023,86,309 confirmed mpox cases have been reported from 107 countries/regions worldwide(WHO,2023).In the mainland of China,the first imported case was confirmed by the Chinese Center for Disease Control and Prevention(Zhao et al.,2022),making this the fifth confirmed mpox infection in China.Neglected zoonotic mpox has been restricted in Africa but now it is back in the spotlight worldwide(Tan and Gao,2022).

Genomic Surveillance for SARS-CoV-2 Variants of Concern from Imported COVID-19 Cases-the Mainland of China,2021

Introduction:After the epidemic in Wuhan City was brought under control in 2020,local outbreaks of coronavirus disease 2019(COVID-19)in the mainland of China were mainly due to imported COVID-19 cases.The ongoing evolution of severe acute respiratory syndrome coronavirus 2(SARS-CoV-2)has continued to generate new variants.Some have been designated as variants of concern(VOCs)by the World Health Organization(WHO).To better assess the role of imported SARS-CoV-2 surveillance and the prevalence of VOCs in 2021,the genomic surveillance data of SARS-CoV-2 from imported COVID-19 cases of 2021 in the mainland of China were analyzed.Methods:The analyses included the number of sequence submissions,time of sequence deposition,and time of detection of the VOCs in order to determine the timeliness and sensitivity of the surveillance.The proportions of VOCs were analyzed and compared with data from the Global Initiative of Sharing All Influenza Data(GISAID).Results:A total of 3,355 sequences of imported cases were submitted from 29 provincial-level administrative divisions,with differences in the number of sequence submissions and median time of sequence deposition.A total of 2,388 sequences with more than 90%genomic coverage were used for lineage analysis.The epidemic trend from Alpha to Delta to Omicron in imported cases was consistent with that in the GISAID.In addition,VOCs from imported cases were usually identified after WHO designation and before causing local outbreaks.Conclusions:The global distribution of SARSCoV-2 VOCs changed rapidly in 2021.Robust genomic surveillance of the imported SARS-CoV-2 in the mainland of China is of great significance.Severe acute respiratory syndrome coronavirus 2(SARS-CoV-2),the etiological agent of coronavirus disease 2019(COVID-19),is constantly mutating under the different circumstances of global transmission(1).The emerging SARS-CoV-2 variants may have potential adverse impacts on epidemic traits and severity.To some extent,it is also capable of escaping natural and vaccine-induced immunity(2–3).Some of them were designated as variants of concern(VOCs)by the World Health Organization(WHO)(4).Therefore,robust surveillance is essential to assess the evolution of viruses in real time.After the epidemic in Wuhan City was brought under control in 2020,several COVID-19 outbreaks in the mainland of China have been proven to relate to SARS-CoV-2 contaminated cold-chain products(5–7),while most were caused by transmission through imported cases on flights,at isolation facilities,or in designated hospitals(8–9).Therefore,genomic surveillance for SARS-CoV-2 from imported cases is of great significance for monitoring the risk of different variants that were imported into the mainland of China,assessing the risk of importation-associated domestic spread,and helping guide public health interventions.On March 17,2020,the China CDC released a notice and launched genomic surveillance for SARS-CoV-2 from imported COVID-19 cases nationwide.The laboratories of provincial CDCs were required to conduct SARS-CoV-2 whole-genome sequencing for samples from imported cases and submit the genomic sequences to the China CDC in time.This study includes the analysis of genomic surveillance data of imported SARS-CoV-2 cases of 2021 from the mainland of China.

A novel luciferase immunosorbent assay performs better than a commercial enzyme-linked immunosorbent assay to detect MERS-CoV specific IgG in humans and animals

The Middle East respiratory syndrome(MERS)is a lethal zoonosis caused by MERS coronavirus(MERS-CoV)and poses a significant threat to public health worldwide.Therefore,a rapid,sensitive,and specific serologic test for detecting anti-MERS-CoV antibodies in both humans and animals is urgently needed for the successful management of this illness.Here,we evaluated various novel luciferase immunosorbent assays(LISA)based on nucleocapsid protein(NP)as well as fragments derived from spike protein(S)including subunit 1(S1),N terminal domain(NTD),receptorbinding domain(RBD)and subunit 2(S2)of S for the detection of MERS-CoV-specific IgG.Fusion proteins,including nanoluciferase(NLuc)and various fragments derived from the NP or S protein of MERS-CoV,were expressed in human embryonic kidney 293 T cells.LISAs that detected anti-MERS-CoV IgG were further developed using cell lysates expressing various fusion proteins.Panels of human or animal samples infected with MERS-CoV were used to analyze the sensitivity and specificity of various LISAs in reference to a MERS-CoV RT-PCR,commercial S1-based ELISA,and pseudovirus particle neutralization test(ppNT).Our results showed that the S1-,RBD-,and NP-LISAs were more sensitive than the NTD-and S2-LISAs for the detection of anti-MERS-CoV IgG.Furthermore,the S1-,RBD-,and NP-LISAs were more sensitive(by at least 16-fold)than the commercially available S1-ELISA.Moreover,the S1-,RBD-,and NPLISA specifically recognized anti-MERS-CoV IgG and did not cross-react with samples derived from other human CoV(OC43,229E,HKU1,NL63)-infected patients.More importantly,these LISAs proved their applicability and reliability for detecting anti-MERS-CoV IgG in samples from camels,monkeys,and mice,among which the RBD-LISA exhibited excellent performance.The results of this study suggest that the novel MERS-CoV RBD-and S1-LISAs are highly effective platforms for the rapid and sensitive detection of anti-MERS-CoV IgG in human and animal samples.These assays have the potential to be used as serologic tests for the management and control of MERS-CoV infection.

mNGS-based dynamic pathogen monitoring for accurate diagnosis and treatment of severe pneumonia caused by fungal infections

Metagenomic next-generation sequencing(mNGs)has been widely applied to identify pathogens associated with infectious diseases.However,limited studies have explored the use of mNGs-based dynamic pathogen monitoring in intensive care unit patients with severe pneumonia.Here,we present a clinical case of an 86-year-old male patient with severe pneumonia caused by a fungal infection.During the clinical treatment,four mNGS analyses were performed within two consecutive weeks.Various respiratory fungal pathogens,including Candida orthopsilosis,Candida albicans,and Aspergillus fumigatus were detected by mNGS of bronchoalveolar lavage fluid(BALF).Based on conventional pathogen identification and clinical symptoms,the patient was diagnosed with severe pneumonia caused by a fungal infection.The abundance of fungal species decreased gradually in response to antifungal and empirical therapies,and the fungal infections were effectively con-trolled.In summary,our results demonstrated that mNGS could effectively identify pathogens in patients with severe pneumonia.Additionally,dynamic pathogen monitoring based on mNGS could assist in the precise diag-nosis of complex infections and may facilitate rapid induction of the most appropriate therapy.

Detecting SARS-CoV-2 BA.2,BA.4,and BA.5 Variants Utilizing a Robust RT-RPA-CRISPR/Cas12a-Based Method-China,2023

Introduction:Since 2019,numerous variants of concern for severe acute respiratory syndrome virus 2(SARS-CoV-2)have emerged,leading to significant outbreaks.The development of novel,highly accurate,and rapid detection techniques for these new SARSCoV-2 variants remains a primary focus in the ongoing efforts to control and prevent the coronavirus disease 2019(COVID-19)pandemic.Methods:Reverse transcription-recombinase polymerase amplification combined with the clustered regularly interspaced short palindromic repeatsassociated protein 12a(CRISPR/Cas12a)system was used to validate the detection of the Omicron BA.2,BA.4,and BA.5 variants of SARS-CoV-2.Results:Our results demonstrate that the CRISPR/Cas12a assay is capable of effectively detecting the SARS-CoV-2 BA.2,BA.4,and BA.5 variants with a limit of detection of 10,1,and 10 copies/μL,respectively.Importantly,our assay successfully differentiated the three SARS-CoV-2 Omicron strains from one another.Additionally,we evaluated 46 SARS-CoV-2 positive clinical samples consisting of BA.2(n=20),BA.4(n=6),and BA.5(n=20)variants,and the sensitivity of our assay ranged from 90%to 100%,while the specificity was 100%.Discussion:This research presents a swift and reliable CRISPR-based method that may be employed to track the emergence of novel SARS-CoV-2 variants.