Real-time quantitative PCR assay for rapid detection of murine virus contamination in bioproducts

Dear Editor,For decades,contaminated and misidentified cell lines in biological research remains a serious problem which the researchers call it"identity crisis"(Masters J R,2009).As reported in March 15,2010 by U.S.Food

Molecular dissection of phage lysin PlySs2: integrity of the catalytic and cell wall binding domains is essential for its broad lytic activity

The novel phage lysin PlySs2, is reported to be highly active against various bacteria, including staphylococci, streptococci and Listeria. However, the molecular mechanisms underlying its broad lytic spectrum remain to be established. In the present study, the lytic activity of the catalytic domain(CD, PlySc) and binding specificity of the cell wall binding domain(CBD, PlySb) of PlySs2 were examined. Our results showed that PlySc alone maintains very limited lytic activity. Enhanced green fluorescent protein(EGFP)-fused PlySb displayed high binding affinity to the streptococcal strains tested, including S.suis, S.dysgalactiae, and S.agalactiae, but not staphylococci, supporting its utility as a good CBD donor for streptococcal-targeted lysin engineering. EGFP-fused intact PlySs2 similarly displayed high affinity for streptococci, but not staphylococci. Notably, four truncated PlySb fragments showed no binding capacity. These findings collectively indicate that integrity of the PlySc and PlySb domains is an essential determinant of the broad lytic activity of PlySs2.

Human Monoclonal Antibodies as Candidate Therapeutics Against Emerging Viruses and HIV-1

More than 40 monoclonal antibodies (mAbs) have been approved for a number of disease indications with only one of these (Synagis) - for a viral disease, and not for therapy but for prevention. However, in the last decade novel potent mAbs have been discovered and characterized with potential as therapeutics against viruses of major importance for public health and biosecurity including Hendra virus (HeV), Nipah virus (NiV), severe acute respiratory syndrome coronavirus (SARS-CoV), Ebola virus (EBOV), West Nile virus (WNV), influenza virus (IFV) and human immunodeficiency virus type 1 (HIV-1). Here, we review such mAbs with an emphasis on antibodies of human origin, and highlight recent results as well as technologies and mechanisms related to their potential as therapeutics.

Identification of Immunogenic Determinants of the Spike Protein of SARS-like Coronavirus

Bat SARS-Iike coronavirus （SL-CoV） has a genome organization almost identical to that of SARS-CoV, but the N-terminus of the Spike （S） proteins, which interacts with host receptor and is a major target of neutralizing antibodies against CoVs, of the two viruses has only 63-64% sequence identity. Although there have been reports studying the overall immunogenicity of SsL, knowledge on the precise location of immunodominant determinants for SSL is still lacking. In this study, using a series of truncated expressed SsL fragments and SsL specific mouse sera, we identified two immunogenic determinants for SSL. Importantly, one of the two regions seems to be located in a region not shared by known immunogenic determinants of the SSARS. This finding will be of potential use in future monitoring of SL-CoV infection in bats and spillover animals and in development of more effective vaccine to cover broad protection against this new group of coronaviruses.

Bayesian evolutionary analysis for emerging infectious disease:an exemplified application for H7N9 avian influenza viruses

Dear Editor,Ever since the first human infection with H7N9 avian influenza virus（AIV）was reported in China in March 2013,there have been five H7N9 AIV pandemics in humans.Wave5 began earlier than the previous four waves,spread to more districts and counties in affected provinces,and had more confirmed cases（Wang et al.,2017）.

Epidemiological,Clinical and Serological Characteristics of Children with Coronavirus Disease 2019 in Wuhan:A Single-centered,Retrospective Study

Dear Editor,In December 2019,SARS-Co V-2 was first detected in the samples obtained from three adult patients who suffered from an unknown viral pneumonia in Wuhan(Li et al.2020).This unknown viral pneumonia is further named as coronavirus disease 2019(COVID-19)by the World Health Organization.

Trans Complementation of Replication-defective Omsk Hemorrhagic Fever Virus for Antiviral Study

Omsk hemorrhagic fever virus(OHFV) is a tick-borne flavivirus classified as a biosafety level-4(BSL4) pathogen. Studies of OHFV are restricted to be conducted within BSL4 laboratories. Currently, no commercial vaccines or antiviral drugs are available against OHFV infection. In this study, we recovered a replication-deficient OHFV with an NS1 deletion(OHFVDNS1) and reporter virus replacing NS1 with the Gaussia luciferase(Gluc)(OHFV-ΔNS1-Gluc). Both the defective OHFVDNS1 and OHFV-ΔNS1-Gluc virus could only replicate efficiently in the BHK21 cell line expressing NS1(BHK21NS1) but not in na?ve BHK21 cells. The Gluc reporter gene of OHFV-ΔNS1-Gluc virus was maintained stably after serial passaging of BHK21NS1 cells and was used to surrogate the replication of OHFV. Using NITD008, OHFV-ΔNS1-Gluc virus was validated for antiviral screening, and high-throughput screening parameters were optimized in a 96-well plate format with a calculated Z0 value above 0.5. The OHFV-ΔNS1-Gluc reporter virus is a powerful tool for antiviral screening as well as viral replication and pathogenesis studies in BSL2 laboratories.

Nanopore sequencing: a rapid solution for infectious disease epidemics

Emerging and re-emerging infectious diseases have given rise to a large number of human infections,morbidity,and heavy economic burden,including the Middle East respiratory syndrome caused by a coronavirus in 2012,global influenza pandemic caused by the H7N9 influenza A virus in2013,Ebola epidemic in West Africa in 2014,and Lassa fever epidemic in Nigeria in 2019.The healthcare war against viruses deserves constant surveillance due to the continuous emergence of new viruses and rapid evolution of existing viruses(Liu and Liu,2017).

Transcriptomic characteristics and impaired immune function of patients who retest positive for SARS-CoV-2 RNA

Coronavirus disease 2019(COVID-19),caused by severe acute respiratory syndrome coronavirus 2(SARS-CoV-2)infection,has become a global public health crisis.Some patients who have recovered from COVID-19 subsequently test positive again for SARS-CoV-2 RNA after discharge from hospital.How such retest-positive(RTP)patients become infected again is not known.In this study,30 RTP patients,20 convalescent patients,and 20 healthy controls were enrolled for the analysis of immunological characteristics of their peripheral blood mononuclear cells.We found that absolute numbers of CD4^(+)T cells,CD8^(+)T cells,and natural killer cells were not substantially decreased in RTP patients,but the expression of activation markers on these cells was significantly reduced.The percentage of granzyme B-producing T cells was also lower in RTP patients than in convalescent patients.Through transcriptome sequencing,we demonstrated that high expression of inhibitor of differentiation 1(ID1)and low expression of interferon-induced transmembrane protein 10(IFITM10)were associated with insufficient activation of immune cells and the occurrence of RTP.These findings provide insight into the impaired immune function associated with COVID-19 and the pathogenesis of RTP,which may contribute to a better understanding of the mechanisms underlying RTP.

Development of a biosensor assessing SARS-CoV-2 main protease proteolytic activity in living cells for antiviral drugs screening

Dear Editor,The Coronavirus disease 2019(COVID-19)pandemic is caused by severe acute respiratory syndrome coronavirus 2(SARS-CoV-2).The genome of this virus encodes two overlapping polyproteins,pp1a and pp1ab(Wu et al.,2020;Zhou et al.,2020).The functional polypeptides are released from those two polyproteins by extensive proteolytic events.

An improved method for identifying SUMOylation sites of viral proteins

Dear Editor, In recent years, post-translational modifications （PTMs） by small ubiquitin-related modifiers （SUMOs） have emerged as an important regulatory mechanism for both cellular and viral processes （Ribet and Cossart, 2010）. Identifying the SUMOylation sites of the target protein is important to understand the molecular mechanism under- lying SUMO modification and virus-host interactions, as well as provide new insights into antiviral drug develop- ment （Wimmer and Schreiner, 2015）. Traditional site- directed mutagenesis for identifying viral protein SUMO- ylation sites lacks a specific aim and is laborious （McManus et al., 2016）. Recently, mass spectrometry （MS） has been employed as an accurate and sensitive tool to identify PTM sites, thereby greatly expanding the number of known SUMOylated proteins （Pedrioli et al., 2006）. However, during viral infection, SUMOylation is highly dynamic and SUMOylated viral proteins often have low abundance, which makes studying SUMOylation under natural conditions difficult.

Characterization of ACTN4 as a novel antiviral target against SARS-CoV-2

The various mutations in severe acute respiratory syndrome coronavirus 2(SARS-CoV-2)pose a substantial challenge in mitigating the viral infectivity.The identification of novel host factors influencing SARS-CoV-2 replication holds potential for discovering new targets for broad-spectrum antiviral drugs that can combat future viral mutations.In this study,potential host factors regulated by SARS-CoV-2 infection were screened through different high-throughput sequencing techniques and further identified in cells.Subsequent analysis and experiments showed that the reduction of m6A modification level on ACTN4(Alpha-actinin-4)mRNA leads to a decrease in mRNA stability and translation efficiency,ultimately inhibiting ACTN4 expression.In addition,ACTN4 was demonstrated to target nsp12 for binding and characterized as a competitor for SARS-CoV-2 RNA and the RNA-dependent RNA polymerase complex,thereby impeding viral replication.Furthermore,two ACTN4 agonists,YS-49 and demethyl-coclaurine,were found to dose-dependently inhibit SARS-CoV-2 infection in both Huh7 cells and K18-hACE2 transgenic mice.Collectively,this study unveils the pivotal role of ACTN4 in SARS-CoV-2 infection,offering novel insights into the intricate interplay between the virus and host cells,and reveals two potential candidates for future anti-SARS-CoV-2 drug development.

Genetic Variation of Multiple Serotypes of Enteroviruses Associated with Hand, Foot and Mouth Disease in Southern China

Enteroviruses(EVs)species A are a major public health issue in the Asia–Pacific region and cause frequent epidemics of hand,foot and mouth disease(HFMD)in China.Mild infections are common in children;however,HFMD can also cause severe illness that affects the central nervous system.To molecularly characterize EVs,a prospective HFMD virological surveillance program was performed in China between 2013 and 2016.Throat swabs,rectal swabs and stool samples were collected from suspected HFMD patients at participating hospitals.EVs were detected using generic real-time and nested reverse transcription-polymerase chain reactions(RT-PCRs).Then,the complete VP1 regions of enterovirus A71(EV-A71),coxsackievirus A16(CVA16)and CVA6 were sequenced to analyze amino acid changes and construct a viral molecular phylogeny.Of the 2836 enrolled HFMD patients,2,517(89%)were EV positive.The most frequently detected EVs were CVA16(32.5%,819),CVA6(31.2%,785),and EV-A71(20.4%,514).The subgenogroups CVA16B1 b,CVA6D3 a and EV-A71C4 a were predominant in China and recombination was not observed in the VP1 region.Sequence analysis revealed amino acid variations at the 30,29 and 44 positions in the VP1 region of EV-A71,CVA16 and CVA6(compared to the respective prototype strains Br Cr,G10 and Gdula),respectively.Furthermore,in 21 of 24(87.5%)identified EV-A71 samples,a known amino acid substitution(D31 N)that may enhance neurovirulence was detected.Our study provides insights about the genetic characteristics of common HFMD-associated EVs.However,the emergence and virulence of the described mutations require further investigation.

Nasal delivery of broadly neutralizing antibodies protects mice from lethal challenge with SARS-CoV-2 delta and omicron variants

Multiple new variants of severe acute respiratory syndrome coronavirus 2(SARS-Co V-2)have constantly emerged,as the delta and omicron variants,which have developed resistance to currently gained neutralizing antibodies.This highlights a critical need to discover new therapeutic agents to overcome the variants mutations.Despite the availability of vaccines against coronavirus disease 2019(COVID-19),the use of broadly neutralizing antibodies has been considered as an alternative way for the prevention or treatment of SARS-Co V-2 variants infection.Here,we show that the nasal delivery of two previously characterized broadly neutralizing antibodies(F61 and H121)protected K18-h ACE2 mice against lethal challenge with SARS-Co V-2 variants.The broadly protective efficacy of the F61 or F61/F121 cocktail antibodies was evaluated by lethal challenge with the wild strain(WIV04)and multiple variants,including beta(B.1.351),delta(B.1.617.2),and omicron(B.1.1.529)at 200or 1000 TCID_(50),and the minimum antibody administration doses(5-1.25 mg/kg body weight)were also evaluated with delta and omicron challenge.Fully prophylactic protections were found in all challenged groups with both F61 and F61/H121 combination at the administration dose of 20 mg/kg body weight,and corresponding mice lung viral RNA showed negative,with almost all alveolar septa and cavities remaining normal.Furthermore,low-dose antibody treatment induced significant prophylactic protection against lethal challenge with delta and omicron variants,whereas the F61/H121 combination showed excellent results against omicron infection.Our findings indicated the potential use of broadly neutralizing monoclonal antibodies as prophylactic and therapeutic agent for protection of current emerged SARS-Co V-2 variants infection.

Baculovirus Surface Display of Zika Virus Envelope Protein Protects against Virus Challenge in Mouse Model

Zika virus(ZIKV)is emerging as a significant pathogen worldwide and may cause severe neurological disorders such as fetal microcephaly and Guillain-Barre syndrome.No drug or listed vaccines are currently available for preventing ZIKV infection.As a major target of neutralizing,ZIKV envelop(E)protein usually used for vaccine development.Nevertheless,the immunogenicity of ZIKV envelop(E)protein expressed by baculovirus display system has never been assessed.In this study,we reported a new strategy for surface display of ZIKV E protein by a recombinant baculovirus vector derived from Autographa californica multiple nuclear polyhedrosis virus(AcMNPV)and assessed its immunogenicity in mice.We produced recombinant fusion ZIKV E protein linked with signal peptide(SP)and transmembrane domain(TM)of AcMNPV GP64.The results showed that the recombinant protein was easy to produce by baculovirus display system.BALB/c mice immunized with this recombinant E protein developed ZIKV specific serum antibodies.The anti-E protein sera from the mice were able to effectively neutralize ZIKV in vitro.More importantly,AG6(IFN-a/b and IFN-c receptor deficient)mice immunized with recombinant E protein were protected against lethal ZIKV challenge.Together,thesefindings demonstrated that the recombinant E protein displayed by baculovirus can be conveniently prepared and displayed good immunogenicity in immunized mice.It is a promising practical approach for prompting the development of vaccine and related immunology research.

Isolation and characterization of Zika virus imported to China using C6/36 mosquito cells

Dear Editor,Zika virus（ZIKV）is a mosquito-borne flavivirus that usually causes asymptomatic infections or mild illness in humans.However,the unprecedented epidemics of ZIKV in Latin America since early 2015 have made this flavivirus an international health risk（Liu and Zhang,2016）.

CASTING: A Potent Supramolecular Strategy to Cytosolically Deliver STING Agonist for Cancer Immunotherapy and SARS-CoV-2 Vaccination

Stimulator of interferon genes,namely STING,an adaptor protein located in the endoplasmic reticulum,has been recognized as a shining target for cancer and infection research.However,STING agonists cyclic dinucleotides(CDNs)have shown almost zero efficacy in phase I clinical trials as a monotherapy,likely due to poor cellular permeability and rapid diffusion despite intratumoral injection.These deficiencies further affect other applications of CDNs,such as pandemic SARS-CoV-2 prevention and therapy.Here,we rationally design a supramolecular cytosolic delivery system based on controllable recognition of calixarene,namely CASTING(CAlixarene-STING),to improve CDN druggability,including degradation stability,cellular permeability,and tissue retention.CASTING efficiently enhances the immunostimulatory potency of CDGSF[a chemically modified cyclic di-GMP(CDG)]to generate an immunogenic microenvironment for melanoma regression,anti-PD-1 response rate increase,and durable memory formation against tumor recurrence.More importantly,CASTING displays a superior adjuvant activity on SARSCoV-2 recombinant spike/receptor binding domain vaccines,inducing robust and coordinated T-cell and antibody responses against SARS-CoV-2 infection in vivo.Collectively,the CASTING design represents an innovative advancement to facilitate the clinical translational capability of STING agonists.

Detection of diverse viruses in alimentary specimens of bats in Macao

Bats carry a variety of viruses, and some of them cause public health problems. Macao, which is famous for its gambling industry, has a complex population structure. The globalization in such an international metropolis has enhanced the chance of disease transmission. Therefore, surveillance of zoonotic viruses is necessary for the early warning of potential emerging infectious diseases.Here, we report the first surveillance of bat viruses in Macao. In this study, we collected 1004 samples involving 10 bat species from 7 sites from April 2015 to May 2016, and examined the presence of viruses using nucleic acid-based methods. Coronaviruses, adenoviruses and paramyxoviruses were detected in these samples, with a high prevalence of coronaviruses. While,none was positive for hepatitis A virus, hepatitis E virus or hantavirus. Co-infections are not common in those bat species, but coronavirus HKU6 and adenovirus can be found commonly occurred in Myotis ricketti.

Detection, isolation, and characterization of chikungunya viruses associated with the Pakistan outbreak of 2016–2017

The chikungunya virus(CHIKV) is a mosquito-transmitted alphavirus, which has infected millions of people in Africa, Asia, Americas, and Europe since it reemerged in India and Indian Ocean regions in 2005–2006. Starting in the middle of November 2016, CHIKV has been widely spread, and more than 4,000 cases of infections in humans were confirmed in Pakistan. Here, we report the first isolation and characterization of CHIKV from the Pakistan outbreak. Eight CHIKV strains were newly isolated from human serum samples using a cell culture procedure. A full-length genome sequence and eight complete envelope(E1) sequences of CHIKV from Pakistan were obtained in this study. Alignment of the CHIKV E1 sequences revealed that the eight new CHIKV isolates were highly homogeneous, with only two nonsynonymous substitutions found at generally conserved sites(E99 and Q235). Based on the comparison of 342 E1 sequences, the two nonsynonymous mutations were located in well-recognized domains associated with viral functions such as the cell fusion and vector specificity, suggesting their potential functional importance. Phylogenetic analysis indicated that the CHIKV strains from Pakistan originated from CHIKV circulating in the Indian region. This study helps elucidate the epidemics of CHIKV in Pakistan and also provides a foundation for studies of evolution and expansion of CHIKV in South Asia.

NSUN2 mediates distinct pathways to regulate enterovirus 71 replication

Increasing evidences suggest that the methyltransferase NSUN2 catalyzes 5-methylcytosine(m5C)modifications on viral RNAs,which are essential for the replication of various viruses.Despite the function of m5C deposition is well characterized,other potential roles of NSUN2 in regulating viral replication remain largely unknown.In this study,the m5C modified residues catalyzed by NSUN2 on enterovirus 71(EV71)RNAs were mapped.NSUN2,along with m5C modifications,played multiple roles during the EV71 life cycle.Functional m5C modified nucleotides increased the translational efficiency and stability of EV71 RNAs.Additionally,NSUN2 was found to target the viral protein VP1 for binding and promote its stability by inhibiting the ubiquitination.Furthermore,both viral replication and pathogenicity in mice were largely attenuated when functional m5C residues were mutated.Taken together,this study characterizes distinct pathways mediated by NSUN2 in regulating EV71 replication,and highlights the importance of its catalyzed m5C modifications on EV71 RNAs for the viral replication and pathogenicity.