Prevalence of Three Shrimp Viruses in Zhejiang Province in 2008

White spot syndrome virus (WSSV),Taura syndrome virus (TSV) and Infectious hypodermal and haematopoietic necrosis virus (IHHNV) are three shrimp viruses responsible for major pandemics affecting the shrimp farming industry. Shrimps samples were collected from 12 farms in Zhejiang province,China,in 2008 and analyzed by PCR to determine the prevalence of these viruses. From the 12 sampling locations,8 farms were positive for WSSV,8 for IHHNV and 6 for both WSSV and IHHNV. An average percentage of 57.4% of shrimp individuals were infected with WSSV,while 49.2% were infected with IHHNV. A high prevalence of co-infection with WSSV and IHHNV among samples was detected from the following samples:Bingjiang (93.3%),liuao (66.7%),Jianshan (46.7%) and Xianxiang (46.7%). No samples exhibited evidence of infection with TSV in collected samples. This study provides comprehensive information of the prevalence of three shrimp viruses in Zhejiang and may be helpful for disease prevention control in this region.

The high diversity of SARS-CoV-2-related coronaviruses in pangolins alerts potential ecological risks

Understanding the zoonotic origin and evolution history of SARS-CoV-2 will provide critical insights for alerting and preventing future outbreaks.A significant gap remains for the possible role of pangolins as a reservoir of SARS-CoV-2 related coronaviruses(SC2r-CoVs).Here,we screened SC2r-CoVs in 172 samples from 163 pangolin individuals of four species,and detected positive signals in muscles of four Manis javanica and,for the first time,one M.pentadactyla.Phylogeographic analysis of pangolin mitochondrial DNA traced their origins from Southeast Asia.Using in-solution hybridization capture sequencing,we assembled a partial pangolin SC2r-CoV(pangolin-CoV)genome sequence of 22895 bp(MP20)from the M.pentadactyla sample.Phylogenetic analyses revealed MP20 was very closely related to pangolin-CoVs that were identified in M.javanica seized by Guangxi Customs.A genetic contribution of bat coronavirus to pangolin-CoVs via recombination was indicated.Our analysis revealed that the genetic diversity of pangolin-CoVs is substantially higher than previously anticipated.Given the potential infectivity of pangolin-CoVs,the high genetic diversity of pangolin-CoVs alerts the ecological risk of zoonotic evolution and transmission of pathogenic SC2r-CoVs.

Immunogenicity of the Spike Glycoprotein of Bat SARS-like Coronavirus

A group of SARS-like coronaviruses(SL-CoV)have been identified in horseshoe bats.Despite SL-CoVs and SARS-CoV share identical genome structure and high-level sequence similarity,SL-CoV does not bind to the same cellular receptor as for SARS-CoV and the N-terminus of the S proteins only share 64%amino acid identity,suggesting there are fundamental differences between these two groups of coronaviruses.To gain insight into the basis of this difference,we established a recombinant adenovirus system expressing the S protein from SL-CoV(rAd-Rp3-S)to investigate its immune characterization.Our results showed that immunized mice generated strong humoral immune responses against the SL-CoV S protein.Moreover,a strong cellular immune response demonstrated by elevated IFN-γand IL-6 levels was also observed in these mice.However,the induced antibody from these mice had weaker cross-reaction with the SARS-CoV S protein,and did not neutralize HIV pseudotyped with SARS-CoV S protein.These results demonstrated that the immunogenicity of the SL-CoV S protein is distinct from that of SARS-CoV,which may cause the immunological differences between human SARS-CoV and bat SL-CoV.Furthermore,the recombinant virus could serve as a potential vaccine candidate against bat SL-CoV infection.

Indirect Enzyme-Linked Immunosorbent Assay Based on the Nucleocapsid Protein of SARS-like Coronaviruses

The nucleocapsid protein(N) is a major structural protein of coronaviruses. The N protein of bat SARS-like coronavirus(SL-CoV) has a high similarity with that of SARS-CoV. In this study,the SL-CoV N protein was expressed in Escherichia coli,purified and used as antigen. An Indirect Enzyme-Linked Immunosorbent Assay(indirect ELISA) was developed for detection of SARS-or SL-CoV infections in bat populations. The detection of 573 bat sera with this indirect ELISA demonstrated that SL-CoVs consistently circulate in Rhinilophus species,further supporting the proposal that bats are natural reservoirs of SL-CoVs. This method uses 1-2 μl of serum sample and can be used for preliminary screening of infections by SARS-or SL-CoV with a small amount of serum sample.

Development of a new field-deployable RT-qPCR workflow for COVID-19 detection

Background:Outbreaks of coronavirus disease 2019(COVID-19)have been recorded in different countries across the globe.The virus is highly contagious,hence early detection,isolation,and quarantine of infected patients will play an important role in containing the viral spread.Diagnosis in a mobile lab can aid to find infected patients in time.Methods:Here,we develop a field-deployable diagnostic workflow that can reliably detect COVID-19.Instruments used in this workflow can easily fit in a mobile cabin hospital and also be installed in the community.Different steps from sample inactivation to detection were optimized to find the fastest steps and portable instruments in the detection of COVID-19.Each step was compared to that of the normal laboratory diagnosis setup.Results:From the results,our proposed workflow(80 min)was two times faster compared to that of the normal laboratory workflow(183 min)and a maximum of 32 samples could be detected at each run.Additionally,we showed that using 1%Rewocid WK-30 could inactivate the novel coronavirus directly without affecting the overall detection results.Comparison of our workflow using an in-house assay to that of a commercially acquired assay produced highly reliable results.From the 250 hospital samples tested,there was a high concordance 247/250(98.8%)between the two assays.The in-house assay sensitivity and specificity were 116/116(100%)and 131/134(97.8%)compared to that of the commercial assay.Conclusion:Based on these results,we believe that our workflow is fast,reliable,adaptable and most importantly,field-deployable.

Ecological study of cave nectar bats reveals low risk of direct transmission of bat viruses to humans

Bats are reservoirs of various viruses.The widely distributed cave nectar bat(Eonycteris spelaea)is known to carry both filoviruses and coronaviruses.However,the potential transmission of theses bat viruses to humans is not fully understood.In this study,we tracked 16 E.spelaea bats in Mengla County,Yunnan Province,China,using miniaturized GPS devices to investigate their movements and potential contact with humans.Furthermore,to determine the prevalence of coronavirus and filovirus infections,we screened for the nucleic acids of the Měnglàvirus(MLAV)and two coronaviruses(GCCDC1-CoV and HKU9-CoV)in anal swab samples taken from bats and for antibodies against these viruses in human serum samples.None of the serum samples were found to contain antibodies against the bat viruses.The GPS tracking results showed that the bats did not fly during the daytime and rarely flew to residential areas.The foraging range of individual bats also varied,with a mean cumulative nightly flight distance of 25.50 km and flight speed of up to 57.4 km/h.Taken together,these results suggest that the risk of direct transmission of GCCDC1-CoV,HKU9-CoV,and MLAV from E.spelaea bats to humans is very low under natural conditions.

Expression and Assembly Mechanism of the Capsid Proteins of a Satellite Virus (XSV) Associated with Macrobrachium rosenbergii Nodavirus

The extra small virus （XSV） is a satellite virus associated with Macrobrachium rosenbergii nodavirus （MrNV） and its genome consists of two overlapping ORFs, CP17 and CP16. Here we demonstrate that CP16 is expressed from the second AUG of the CP17 gene and is not a proteinase cleavage result of CP17. We further expressed CP17 and several truncated CP17s （in which the N-or C-terminus or both was deleted）, respectively, in Escherichia coli. Except for the recombinant plasmid CP17^AC10, all recombinant plasmids expressed soluble protein which assembled into virus-like particles （VLPs）, suggesting that the C-terminus is important for VLP formation.

Identification and Characterization of Nuclear Localization Signals within the Nucleocapsid Protein VP15 of White Spot Syndrome Virus

The nucleocapsid protein VP15 of white spot syndrome virus (WSSV) is a basic DNA-binding protein. Three canonical bipartite nuclear localization signals (NLSs), called NLS1 (aa 11-27), NLS2 (aa 33-49) and NLS3 (44-60), have been detected in this protein, using the ScanProsite computer program. To determine the nuclear localization sequence of VP15, the full-length open reading frame, or the sequence of one of the three NLSs, was fused to the green fluorescent protein (GFP) gene, and transiently expressed in insect Sf9 cells. Transfection with full-length VP15 resulted in GFP fluorescence being distributed exclusively in the nucleus. NLS1 alone could also direct GFP to the nucleus, but less efficiently. Neither of the other two NLSs (NLS2 and 3) was functional when expressed alone, but exhibited similar activity to NLS1 when they were expressed as a fusion peptide. Furthermore, a mutated VP15, in which the two basic amino acids (11RR12) of NLS1 were changed to two alanines (11AA12), caused GFP to be localized only in the cytoplasm of Sf9 cells. These results demonstrated that VP15, as a nuclear localization protein, needs cooperation between its three NLSs, and that the two residues (11RR12) of NLS1 play a key role in transporting the protein to the nucleus.

Production and Characterization of Monoclonal Antibodies of Shrimp White Spot Syndrome Virus Envelope Protein VP28

BALB/c 老鼠与净化的白点症候群病毒(WSSV ) 被使免疫。六根 monoclonal 抗体房间线被 ELISA 选择， VP28 蛋白质在 E 表示了。coli。在 vitro 中立化，实验证明他们中的 4 个能在喇蛄禁止病毒感染。西方污点建议所有这些 monoclonal 抗体对 VP28 的 conformational 结构。monoclonal 抗体 7B4 用胶体的金粒子被标记并且过去常由标记的 immunogold 在病毒信封上定位 VP28。这些 monoclonal 抗体能被用来为 WSSV 感染开发免疫学的诊断方法。关键词怀特点症候群病毒(WSSV )- Mab - 信封 - 本地化 - 中立化 CLC 数字 S945.

Development of a Mengla virus minigenome and comparison of its polymerase complexes with those of other filoviruses

Ebola virus(EBOV)and Marburg virus(MARV),members of the Filoviridae family,are highly pathogenic and can cause hemorrhagic fevers,significantly impacting human society.Bats are considered reservoirs of these viruses because related filoviruses have been discovered in bats.However,due to the requirement for maximum containment laboratories when studying infectious viruses,the characterization of bat filoviruses often relies on pseudoviruses and minigenome systems.In this study,we used RACE technology to sequence the 30-leader and 50-trailer of Mengla virus(MLAV)and constructed a minigenome.Similar to MARV,the transcription activities of the MLAV minigenome are independent of VP30.We further assessed the effects of polymorphisms at the 50 end on MLAV minigenome activity and identified certain mutations that decrease minigenome reporter efficiency,probably due to alterations in the RNA secondary structure.The reporter activity upon recombination of the 30-leaders and 50-trailers of MLAV,MARV,and EBOV with those of the homologous or heterologous minigenomes was compared and it was found that the polymerase complex and leader and trailer sequences exhibit intrinsic specificities.Additionally,we investigated whether the polymerase complex proteins from EBOV and MARV support MLAV minigenome RNA synthesis and found that the homologous system is more efficient than the heterologous system.Remdesivir efficiently inhibited MLAV as well as EBOV replication.In summary,this study provides new information on bat filoviruses and the minigenome will be a useful tool for high-throughput antiviral drug screening.

Individual virome analysis reveals the general co-infection of mammal-associated viruses with SARS-related coronaviruses in bats

Bats are the natural reservoir hosts for SARS-related coronavirus(SARSr-CoV)and other highly pathogenic microorganisms.Therefore,it is conceivable that an individual bat may harbor multiple microbes.However,there is limited knowledge on the overall co-circulation of microorganisms in bats.Here,we conducted a 16-year monitoring of bat viruses in south and central China and identified 238 SARSr-CoV positive samples across nine bat species from ten provinces or administrative districts.Among these,76 individual samples were selected for further metagenomics analysis.We found a complex microenvironment characterized by the general cocirculation of microbes from two different sources:mammal-associated viruses or environment-associated microbes.The later includes commensal bacteria,enterobacteria-related phages,and insect or fungal viruses of food origin.Results showed that 25%(19/76)of the samples contained at least one another mammal-associated virus,notably alphacoronaviruses(13/76)such as AlphaCoV/YN2012,HKU2-related CoV and AlphaCoV/Rf-HuB2013,along with viruses from other families.Notably,we observed three viruses co-circulating within a single bat,comprising two coronavirus species and one picornavirus.Our analysis also revealed the potential presence of pathogenic bacteria or fungi in bats.Furthermore,we obtained 25 viral genomes from the 76 bat SARSr-CoV positive samples,some of which formed new evolutionary lineages.Collectively,our study reveals the complex microenvironment of bat microbiome,facilitating deeper investigations into their pathogenic potential and the likelihood of cross-species transmission.

Nasal vaccination of triple-RBD scaffold protein with flagellin elicits long-term protection against SARS-CoV-2 variants including JN.1

Developing a mucosal vaccine against SARS-CoV-2 is critical for combatting the epidemic.Here,we investigated long-term immune responses and protection against SARS-CoV-2 for the intranasal vaccination of a triple receptor-binding domain(RBD)scaffold protein(3R-NC)adjuvanted with a flagellin protein(KFD)(3R-NC+KFDi.n).In mice,the vaccination elicited RBD-specific broad-neutralizing antibody responses in both serum and mucosal sites sustained at high level over a year.This long-lasting humoral immunity was correlated with the presence of long-lived RBD-specific IgG-and IgA-producing plasma cells,alongside the Th17 and Tfh17-biased T-cell responses driven by the KFD adjuvant.Based upon these preclinical findings,an open labeled clinical trial was conducted in individuals who had been primed with the inactivated SARS-CoV-2(IAV)vaccine.With a favorable safety profile,the 3R-NC+KFDi.n boost elicited enduring broad-neutralizing IgG in plasma and IgA in salivary secretions.To meet the challenge of frequently emerged variants,we further designed an updated triple-RBD scaffold protein with mutated RBD combinations,which can induce adaptable antibody responses to neutralize the newly emerging variants,including JN.1.Our findings highlight the potential of the KFD-adjuvanted triple-RBD scaffold protein is a promising prototype for the development of a mucosal vaccine against SARS-CoV-2 infection.

The protective nasal boosting of a triple-RBD subunit vaccine against SARS-CoV-2 following inactivated virus vaccination

Dear Editor,Though COVID-19 vaccines have been developed and clinically deployed rapidly,new variants of concern(VOCs)are still emerging frequently and escalating around the world.More breakthrough infections occurred even vaccination rates are high.For possible ending of the pandemic,curbing infection and stopping transmission are priority.

Serological Evidence of Bat SARS-Related Coronavirus Infection in Humans,China

Dear Editor,Severe acute respiratory syndrome coronavirus(SARSCo V)is the causative agent of the 2002–2003 SARS pandemic,which resulted in more than 8000 human infections worldwide and an approximately 10%fatality rate(Ksiazek et al.2003;Peiris et al.2004).The virus infects both upper airway and alveolar epithelial cells,resulting in mild to severe lung injury in

The First Disease X is Caused by a Highly Transmissible Acute Respiratory Syndrome Coronavirus

Based on the announcement of the World Health Organization(WHO) in 2018, the Wuhan pneumonia caused by an unknown etiology should be recognized as the first Disease X. Later, the pathogen was identified to be a novel coronavirus denoted 2019-nCoV, which has 79.5% and 96% whole genome sequence identify to SARS-CoV and bat SARS-related coronavirus(SARSr-CoV-RaTG13), respectively, suggesting its potential bat origin. With high human-to-human transmission rate(R0), 2019-nCoV has quickly spread in China and other countries, resulting in 34,953 confirmed cases and 725 deaths as of 8 February 2020, thus calling for urgent development of therapeutics and prophylactics. Here we suggest renaming 2019-nCoV as "transmissible acute respiratory syndrome coronavirus(TARS-CoV)" and briefly review the advancement of research and development of neutralizing antibodies and vaccines targeting the receptor-binding domain(RBD) and viral fusion inhibitors targeting the heptad repeat 1(HR1) domain in spike protein of 2019-nCoV.

Detection and characterization of three zoonotic viruses in wild rodents and shrews from Shenzhen city,China

Diverse species of rodents and shrews, which are abundant worldwide, harbor a variety of viruses;some of these are closely related to human viruses and possess zoonotic potential. Previously studies have demonstrated that the mammarenavirus and hantavirus carried by rodents or shrews could cause diseases in human population. To determine the distribution of zoonotic viruses in Shenzhen city, the major city in southern China with a high population density, we analyzed 225 rodents(Rattus norvegicus and Rattus flavipectus) and 196 shrews(Suncus murinus) from urban and rural districts for the presence of mammarenavirus, hantavirus, and hepatitis E virus(HEV) by RT-PCR targeting the conserved regions. The infection rates for mammarenavirus, hantaviruses,and HEV in rodents and shrews were 3.56%, 6.89%, and 1.66%, respectively. Partial genome fragment analysis indicated that mammarenavirus and hantavirus strains had more than 90% and 99% nucleic acid identity with Cardamones virus and Seoul virus, respectively, which cause diseases in humans. Although the present HEV strains identified are typically found worldwide,phylogenetic analysis demonstrated a divergence of 16%. To our knowledge, the present work is the first report of the prevalence of mammarenavirus, hantaviruses, and rat HEV strains in rodents and shrews from Shenzhen city, China. Our findings highlight the zoonotic potential of rodent-and shrew-borne mammarenavirus and hantavirus, and the biodiversity of rat HEV isolates in Shenzhen city. The present work suggests that utilization of good hygiene habits is important to minimize the risk of zoonosis.

SARS-CoV-2 triggers inflammatory responses and cell death through caspase-8 activation

Severe acute respiratory syndrome coronavirus 2(SARS-CoV-2)infection can lead to respiratory illness and multi-organ failure in critically ill patients.Although the virus-induced lung damage and inflammatory cytokine storm are believed to be directly associated with coronavirus disease 2019(COVID-19)clinical manifestations,the underlying mechanisms of virus-triggered inflammatory responses are currently unknown.Here we report that SARS-CoV-2 infection activates caspase-8 to trigger cell apoptosis and inflammatory cytokine processing in the lung epithelial cells.The processed inflammatory cytokines are released through the virus-induced necroptosis pathway.Virus-induced apoptosis,necroptosis,and inflammation activation were also observed in the lung sections of SARS-CoV-2-infected HFH4-hACE2 transgenic mouse model,a valid model for studying SARS-CoV-2 pathogenesis.Furthermore,analysis of the postmortem lung sections of fatal COVID-19 patients revealed not only apoptosis and necroptosis but also massive inflammatory cell infiltration,necrotic cell debris,and pulmonary interstitial fibrosis,typical of immune pathogenesis in the lung.The SARS-CoV-2 infection triggered a dual mode of cell death pathways and caspase-8-dependent inflammatory responses may lead to the lung damage in the COVID-19 patients.These discoveries might assist the development of therapeutic strategies to treat COVID-19.

A triple-RBD-based mucosal vaccine provides broad protectionagainst SARS-CoV-2 variants of concern

The rapid mutation and spread of SARS-CoV-2 variants urge the development of effective mucosal vaccines to provide broadspectrum protection against the initial infection and thereby curb the transmission potential.Here,we designed a chimeric tripleRBD immunogen,3Ro-NC,harboring one Delta RBD and two Omicron RBDs within a novel protein scaffold.3Ro-NC elicits potent and broad RBD-specific neutralizing immunity against SARS-CoV-2 variants of concern.Notably,intranasal immunization with 3RoNC plus the mucosal adjuvant KFD(3Ro-NC+KFDi.n)elicits coordinated mucosal IgA and higher neutralizing antibody specificity(closer antigenic distance)against the Omicron variant.In Omicron-challenged human ACE2 transgenic mice,3Ro-NC+KFDi.n immunization significantly reduces the tissue pathology in the lung and lowers the viral RNA copy numbers in both the lung(85.7-fold)and the nasal turbinate(13.6-fold).Nasal virologic control is highly correlated with RBD-specific secretory IgA antibodies.Our data show that 3Ro-NC plus KFD is a promising mucosal vaccine candidate for protection against SARS-CoV-2 Omicron infection,pathology and transmission potential.

Protective Efficacy of Inactivated Vaccine against SARS-CoV-2 Infection in Mice and Non-Human Primates

The ongoing coronavirus disease 2019(COVID-19)pandemic caused more than 96 million infections and over 2 million deaths worldwide so far.However,there is no approved vaccine available for severe acute respiratory syndrome coronavirus 2(SARS-CoV-2),the disease causative agent.Vaccine is the most effective approach to eradicate a pathogen.The tests of safety and efficacy in animals are pivotal for developing a vaccine and before the vaccine is applied to human populations.Here we evaluated the safety,immunogenicity,and efficacy of an inactivated vaccine based on the whole viral particles in human ACE2 transgenic mouse and in non-human primates.Our data showed that the inactivated vaccine successfully induced SARS-CoV-2-specific neutralizing antibodies in mice and non-human primates,and subsequently provided partial(in low dose)or full(in high dose)protection of challenge in the tested animals.In addition,passive serum transferred from vaccine-immunized mice could also provide full protection from SARS-CoV-2 infection in mice.These results warranted positive outcomes in future clinical trials in humans.

Chevrier＇s Field Mouse(Apodemus chevrieri) and Père David＇s Vole(Eothenomys melanogaster) in China Carry Orthohepeviruses that form Two Putative Novel Genotypes Within the Species Orthohepevirus C

Hepatitis E virus(HEV)is the prototype of the family Hepeviridae and the causative agent of common acute viral hepatitis.Genetically diverse HEV-related viruses have been detected in a variety of mammals and some of them may have zoonotic potential.In this study,we tested 278 specimens collected from seven wild small mammal species in Yunnan province,China,for the presence and prevalence of orthohepevirus by broad-spectrum reverse transcription(RT)-PCR.HEV-related sequences were detected in two rodent species,including Chevrier’s field mouse(Apodemus chevrieri,family Muridae)and Père David’s vole(Eothenomys melanogaster,family Cricetidae),with the infection rates of 29.20%(59/202)and 7.27%(4/55),respectively.Further four representative full-length genomes were generated:two each from Chevrier’s field mouse(named Rd HEVAc14 and Rd HEVAc86)and Père David’s vole(Rd HEVEm40 and Rd HEVEm67).Phylogenetic analyses and pairwise distance comparisons of whole genome sequences and amino acid sequences of the gene coding regions showed that orthohepeviruses identified in Chinese Chevrier’s field mouse and Père David’s vole belonged to the species Orthohepevirus C but were highly divergent from the two assigned genotypes:HEV-C1 derived from rat and shrew,and HEV-C2 derived from ferret and possibly mink.Quantitative real-time RT-PCR demonstrated that these newly discovered orthohepeviruses had hepatic tropism.In summary,our work discovered two putative novel genotypes orthohepeviruses preliminarily named HEVC3 and HEV-C4 within the species Orthohepevirus C,which expands our understanding of orthohepevirus infection in the order Rodentia and gives new insights into the origin,evolution,and host range of orthohepevirus.