The infection kinetics and transmission potential of two Guaico Culex viruses in Culex quinquefasciatus mosquitoes

Guaico Culex virus(GCXV)is a newly identified segmented Jingmenvirus from Culex spp.mosquitoes in Central and South America.The genome of GCXV is composed of four or five single-stranded positive RNA segments.However,the infection kinetics and transmission capability of GCXV in mosquitoes remain unknown.In this study,we used reverse genetics to rescue two GCXVs(4S and 5S)that contained four and five RNA segments,respectively,in C6/36 cells.Further in vitro characterization revealed that the two GCXVs exhibited comparable replication kinetics,protein expression and viral titers.Importantly,GCXV RNAs were detected in the bodies,salivary glands,midguts and ovaries of Culex quinquefasciatus at 4–10 days after oral infection.In addition,two GCXVs can colonize Cx.quinquefasciatus eggs,resulting in positive rates of 15%–35%for the second gonotrophic cycle.In conclusion,our results demonstrated that GCXVs with four or five RNA segments can be detected in Cx.quinquefasciatus eggs during the first and second gonotrophic cycles after oral infection.

PA-E18G substitution in influenza A virus confers resistance to ZX-7101,a cap-dependent endonuclease inhibitor

Cap-dependent endonuclease(CEN)in the polymerase acidic protein(PA)of influenza A virus(IAV)represents a promising drug target due to its critical role in viral gene transcription.The CEN inhibitor,baloxavir marboxil(BXM),was approved in Japan and the US in 2018 and several other countries subsequently.Along with the clinical use of BXM,the emergence and spread of IAV variants with reduced susceptibility to BXM have aroused serious concern.Herein,we comprehensively characterized the in vitro and in vivo antiviral activities of ZX-7101A,an analogue of BXM.The active form of prodrug ZX-7101 showed broad-spectrum antiviral potency against various IAV subtypes,including pH1N1,H3N2,H7N9 and H9N2,in MDCK cells,and the 50%effective concentration(EC_(50))was calculated to nanomole level and comparable to that of baloxavir acid(BXA),the active form of BXM.Furthermore,in vivo assays showed that administration of ZX-7101A conferred significant protection against lethal pH1N1 challenge in mice,with reduced viral RNA loads and alleviated pulmonary damage.Importantly,serial passaging of H1N1 virus in MDCK cells under selection pressure of ZX-7101 led to a resistant variant at the 15th passage.Reverse genetic and sequencing analysis demonstrated that a single E18G substitution in the PA subunit contributed to the reduced susceptibility to both ZX-7101 and BXA.Taken together,our results not only characterized a new CEN inhibitor of IAV but also identified a novel amino acid substitution responsible for CEN inhibitor resistance,which provides critical clues for future drug development and drug resistance surveillance.

Isolation, identification and genomic characterization of the Asian lineage Zika virus imported to China

Dear Editor,Zika virus(ZIKV)used to be an unknown mosquito-borne flavivirus,and maintained its limited sylvatic circulation in a few African and Asian countries(Enfissi et al.,2016).Based on available clinical data,the symptoms in human infections with ZIKV are supposed to be similar to other arbovirus infections such as dengue,and characterized by fever,skin rashes,conjunctivitis,muscle and joint pain,malaise,and headache(Duffy et al.,2009).However。

Impulse pressuring diffusion bonding of titanium to 304 stainless steel using pure Ni interlayer

In the present study,impulse pressuring diffusion bonding technology(IPDB)was utilized between commercially pure titanium and 304 stainless steel(SS)using pure nickel(Ni)as interlayer metal.The interfacial microstructures of the bonded joints were investigated by scanning electron microscopy(SEM)and energy dispersive spectroscope(EDS)analyses.It is found that with the aid of the Ni interlayer,the interdiffusion and reaction between Ti and SS can be effectively restricted and robust joints can be obtained.Intermetallic compounds(IMCs)including Ti_2Ni,Ti Ni,and TiNi_3 are detected at the Ti/Ni interface;however,only Ni–Fe solid solution is found at the Ni/SS interface.The maximum tensile strength of 358 MPa is obtained by IPDB for 90 s and the fracture takes place along the Ti_2Ni and Ti Ni phase upon tensile loading.The existence of cleavage pattern on the fracture surface indicates the brittle nature of the joints.

Long-term stability and protection efficacy of the RBD-targeting COVID-19 mRNA vaccine in nonhuman primates

Messenger RNA(mRNA)vaccine technology has shown its power in preventing the ongoing COVID-19 pandemic.Two mRNA vaccines targeting the full-length S protein of SARS-CoV-2 have been authorized for emergency use.Recently,we have developed a lipid nanoparticle-encapsulated mRNA(mRNA-LNP)encoding the receptor-binding domain(RBD)of SARS-CoV-2(termed ARCoV),which confers complete protection in mouse model.Herein,we further characterized the protection efficacy of ARCoV in nonhuman primates and the Iong-term stability under normal refrigerator temperature.Intramuscular immunization of two doses of ARCoV elicited robust neutralizing antibodies as well as cellular response against SARS-CoV-2 in cynomolgus macaques.More importantly,ARCoV vaccination in macaques significantly protected animals from acute lung lesions caused by SARS-CoV-2,and viral replication in lungs and secretion in nasal swabs were completely cleared in all animals immunized with low or high doses of ARCoV.No evidenee of antibody-dependent enhancement of infection was observed throughout the study.Finally,extensive stability assays showed that ARCoV can be stored at 2-8℃ for at least 6 months without decrease of immunogenicity.All these promising results strongly support the ongoing clinical trial.

Enhanced protective immunity against SARS-CoV-2 elicited by a VSV vector expressing a chimeric spike protein

SARS-CoV-2 and SARS-CoV are genetically related coronavirus and share the same cellular receptor ACE2.By replacing the VSV glycoprotein with the spikes(S)of SARS-CoV-2 and SARS-CoV,we generated two replication-competent recombinant viruses,rVSVSARS-CoV-2 and rVSV-SARS-CoV.Using wild-type and human ACE2(hACE2)knock-in mouse models,we found a single dose of rVSV-SARS-CoV could elicit strong humoral immune response via both intranasal(i.n.)and intramuscular(i.m.)routes.Despite the high genetic similarity between SARS-CoV-2 and SARS-CoV,no obvious cross-neutralizing activity was observed in the immunized mice sera.In macaques,neutralizing antibody(NAb)titers induced by one i.n.dose of rVSV-SARS-CoV-2 were eight-fold higher than those by a single i.m.dose.Thus,our data indicates that rVSV-SARS-CoV-2 might be suitable for i.n.administration instead of the traditional i.m.immunization in human.Because rVSV-SARS-CoV elicited significantly stronger NAb responses than rVSV-SARS-CoV2 in a route-independent manner,we generated a chimeric antigen by replacing the receptor binding domain(RBD)of SARS-CoV S with that from the SARS-CoV-2.rVSV expressing the chimera(rVSV-SARS-CoV/2-RBD)induced significantly increased NAbs against SARS-CoV-2 in mice and macaques than rVSV-SARS-CoV-2,with a safe Th1-biased response.Serum immunized with rVSV-SARS-CoV/2-RBD showed no cross-reactivity with SARS-CoV.hACE2 mice receiving a single i.m.dose of either rVSV-SARS-CoV-2 or rVSV-SARSCoV/2-RBD were fully protected against SARS-CoV-2 challenge without obvious lesions in the lungs.Our results suggest that transplantation of SARS-CoV-2 RBD into the S protein of SARS-CoV might be a promising antigen design for COVID-19 vaccines.

Generation and Characterization of a Nanobody Against SARS-CoV

The sudden emergence of severe acute respiratory syndrome coronavirus(SARS-CoV) has caused global panic in 2003,and the risk of SARS-CoV outbreak still exists. However, no specific antiviral drug or vaccine is available;thus, the development of therapeutic antibodies against SARS-CoV is needed. In this study, a nanobody phage-displayed library was constructed from peripheral blood mononuclear cells of alpacas immunized with the recombinant receptor-binding domain(RBD) of SARS-CoV. Four positive clones were selected after four rounds of bio-panning and subjected to recombinant expression in E. coli. Further biological identification demonstrated that one of the nanobodies, S14, showed high affinity to SARS-CoV RBD and potent neutralization activity at the picomole level against SARS-CoV pseudovirus. A competitive inhibition assay showed that S14 blocked the binding of SARS-CoV RBD to either soluble or cell-expressed angiotensinconverting enzyme 2(ACE2). In summary, we developed a novel nanobody targeting SARS-CoV RBD, which might be useful for the development of therapeutics against SARS.

Construction and characterization of UAA-controlled recombinant Zika virus by genetic code expansion

Dear Editor,Genetic code expansion based on orthogonal tRNA-aminoacyl tRNA synthetase is an emerging technology to sitespecifically incorporate unnatural amino acids(UAAs)into viral proteins to produce UAA-controlled replication-defective virus.

Tamoxifen and clomiphene inhibit SARS-CoV-2 infection by suppressing viral entry

Dear Editor,COVID-19 pandemic caused by severe acute respiratory syndrome coronavirus 2(SARS-CoV-2)is still a threat to millions of lives worldwide.Although SARS-CoV-2 vaccines have been approved to reduce the severity and death associated with COVID-19,the number of SARS-CoV-2-infected cases still remains high,especially with the appearance of various mutant strains such as P.1.351 and P.1.617(also known as South Africa strain and India strain,respectively),which may reduce the efficacy of vaccine protection.There is an urgent need to develop effective antiviral agents to treat C0VID-19 patients,especially with those infected with SARS-CoV-2 variants of concern.

A highly immunogenic live-attenuated vaccine candidate prevents SARS-CoV-2 infection and transmission in hamsters

The highly pathogenic and readily transmissible SARS-CoV-2 has caused a global coronavirus pandemic,urgently requiring effective countermeasures against its rapid expansion.All available vaccine platforms are being used to generate safe and effective COVID-19 vaccines.Here,we generated a live-attenuated candidate vaccine strain by serial passaging of a SARSCoV-2 clinical isolate in Vero cells.

Rapid development of double-hit mRNA antibody cocktail against orthopoxviruses

The Orthopoxvirus genus,especially variola virus(VARV),monkeypox virus(MPXV),remains a significant public health threat worldwide.The development of therapeutic antibodies against orthopoxviruses is largely hampered by the high cost of antibody engineering and manufacturing processes.mRNA-encoded antibodies have emerged as a powerful and universal platform for rapid antibody production.Herein,by using the established lipid nanoparticle(LNP)-encapsulated mRNA platform,we constructed four mRNA combinations that encode monoclonal antibodies with broad neutralization activities against orthopoxviruses.In vivo characterization demonstrated that a single intravenous injection of each LNP-encapsulated mRNA antibody in mice resulted in the rapid production of neutralizing antibodies.More importantly,mRNA antibody treatments showed significant protection from weight loss and mortality in the vaccinia virus(VACV)lethal challenge mouse model,and a unique mRNA antibody cocktail,Mix2a,exhibited superior in vivo protection by targeting both intracellular mature virus(IMV)-form and extracellular enveloped virus(EEV)-form viruses.In summary,our results demonstrate the proof-of-concept production of orthopoxvirus antibodies via the LNP-mRNA platform,highlighting the great potential of tailored mRNA antibody combinations as a universal strategy to combat orthopoxvirus as well as otheremerging viruses.