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SARS-CoV-2, which likely spilled over from wildlife, is the third highly pathogenic human coronavirus. Being highly transmissible, it is perpetuating a pandemic and continuously posing a severe threat to global public health.

Previous studies have identified multiple viruses in dead or severely diseased pangolins, but descriptions of the virome in healthy pangolins are lacking. This poses a greater risk of cross-species transmission due to poor preventive awareness and frequent interactions with breeders. In this study, we investigated the viral composition of 34 pangolins with no signs of disease at the time of sampling and characterized a large number of arthropod-associated viruses belonging to 11 families and vertebrate viruses belonging to eight families, including those with pathogenic potential in humans and animals. Several important vertebrate viruses were identified in the pangolins, including parvovirus, pestivirus, and picobirnavirus. The picobirnavirus was clustered with human and grey teal picobirnaviruses. Viruses with cross-species transmission ability were also identified, including circovirus, rotavirus, and astrovirus. Our study revealed that pangolins are frequently exposed to arthropod-associated viruses in the wild and can carry many vertebrate viruses under natural conditions. This study provides important insights into the virome of pangolins, underscoring the importance of monitoring potential pathogens in healthy pangolins to prevent outbreaks of infectious diseases in domesticated animals and humans.先前的研究已经从死亡或严重患病的穿山甲中发现了大量病毒，但对健康穿山甲的病毒组的研究较少。而且由于缺乏健康动物携带病原体的防范意识，并且野生动物与饲养员频繁接触，正常野生动物携带的病毒存在更高的跨物种传播风险。在该研究中，我们调查了34只穿山甲的病毒组成，它们在被取样时没有疾病迹象，并对属于11个科的大量节肢动物相关病毒和属于8个科的各种脊椎动物病毒进行了鉴定，包括一些已知的对人类和动物有致病潜力的病毒。一些重要的脊椎动物病毒已在穿山甲中被发现，包括parvovirus（细小病毒），pestivirus（瘟疫病毒），和picobirnavirus（小双节RNA病毒）。其中我们发现的picobirnavirus与感染人类和灰鸭的picobirnavirus病毒聚集。我们还发现了许多具有跨物种传播潜力的病毒，包括circovirus（圆环病毒）、rotavirus（轮状病毒）和astrovirus（星状病毒）等。我们的研究表明，穿山甲在野外经常接触到节肢动物相关的病毒，并且在自然条件下可以携带许多感染脊椎动物的病毒。该研究提供了对穿山甲病毒组的重要见解，强调了监测健康穿山甲中潜在病原体的重要性，以防止在驯养动物和人类中爆发传染病。.

The ongoing outbreak of viral pneumonia in China and beyond is associated with a novel coronavirus, provisionally termed 2019-nCoV. This outbreak has been tentatively associated with a seafood market in Wuhan, China, where the sale of wild animals may be the source of zoonotic infection. Although bats are likely reservoir hosts for 2019-nCoV, the identity of any intermediate host facilitating transfer to humans is unknown. Here, we report the identification of 2019-nCoV related coronaviruses in pangolins (Manis javanica) seized in anti-smuggling operations in southern China. Metagenomic sequencing identified pangolin associated CoVs that belong to two sub-lineages of 2019-nCoV related coronaviruses, including one very closely related to 2019-nCoV in the receptor-binding domain. The discovery of multiple lineages of pangolin coronavirus and their similarity to 2019-nCoV suggests that pangolins should be considered as possible intermediate hosts for this novel human virus and should be removed from wet markets to prevent zoonotic transmission.

Motivation: When a temperate bacteriophage (phage) integrates into the chromosome of its host or to remain as latent episomal DNA, the prophage plays an important role in bacterial virulence acquisition and increase. With the recent release of thousands of bacterial high throughput sequencing data, there has been a growing interest in analyzing the impact of interdependency between a prophage and its host. One of the essential tasks is to acquire and identify the complete sequence of the prophage, i.e., the functional prophages. However, no existing tools can identify functional prophages from bacterial genomes. A few tools can predict putative prophage in bacterial genomes, but they are unable to distinguish functional prophages from defective prophages. To reduce the cost and to relieve the tedious biological experiments for functional prophage analysis, computational methods for predicting functional prophages based on bacterial high-throughput sequencing data would be an effective alternation. Results: This paper presents a method, LysoPhD, the first tool to predict functional prophage sequences using bacterial HTS data automatically and accurately. The main idea is to obtain whole genome of functional prophages from the bacterial genome based on phage gene annotation and reads-mediated contig connection. LysoPhD obtains whole genome sequences of functional prophages based on improved models. The obtained the sequences are verified by wet lab experiments. Specifically, LysoPhD is applied to a set of staphylococcus HTS data in the case studies. Ten functional prophages are predicted from 72 staphylococcus isolates. The 72 bacterial strains are then induced with Mitomycin C and subsequent deep sequencing verified of the 10 functional prophages. This shows that LysoPhD is a sensitive and accurate program for functional prophage prediction.

Adsorption of tobacco specific N-nitrosamines (TSNA) in industrial tobacco extract solution by ZnO modified activated carbon (AC) was deeply studied in order to discern the liquid adsorption. ZnO was coated on AC by wet impregnation and characterized with X-ray diffraction (XRD), X-ray photoelectron spectroscopy (XPS) and nitrogen-adsorption–desorption to evaluate its modification on the structure of sorbent. Apart from common tobacco extract solution, the industrial extract solution with a higher TSNA content and viscosity was used to assess the performance of ZnO modified AC with monitoring of LC–MS/MS technique. The influence of TSNA concentration, S/L ratio, adsorption time and recycle index was also systemically studied. To explore whether ZnO modification induces some selective adsorptive sites on AC, elution of TSNA from sorbent and the adsorption of main neutral aroma components in tobacco extract solution were performed. About 80% of TSNA could be trapped in the solution and the ZnO/AC sorbent showed a kind of selectivity for 4-methylnitrosamino-1-3-pyridyl-1-butanone (NNK).

In order to control the tobacco specific nitrosamines (TSNA) pollution caused by smoking, liquid adsorption of TSNA in tobacco-extract solution by modified activated carbon, zeolites and other porous materials was systemically studied with the monitor of Liquid Chromatography–Tandem Mass Spectrometry (LC–MS/MS) technique. The effect of solid–liquid ratio, the porous structure and surface acidic-basicity of sorbent on the liquid adsorption was assessed. Among various activated carbon (AC) samples modified with acid soak, ion-exchange or impregnation, the impregnated sorbent ZnAC could remove 73% of the TSNA in the solution, offering a cost-effective candidate for potential industrial application. Zeolite CaA was also efficient to trap more than 40% of TSNA in tobacco solution, and the influence of cation on the adsorption of TSNA by zeolite and the possible selectivity for the components of TSNA such as NNN, NAT and NNK were carefully examined. Besides, the fabrication of optimal micro-environment for liquid adsorption of TSNA was explored.

Zika virus (ZIKV) has caused significant outbreaks and epidemics in the Americas recently, raising global concern due to its ability to cause microcephaly and other neurological complications. A stable and efficient infectious clone of ZIKV is urgently needed. However, the instability and toxicity of flavivirus cDNA clones in Escherichia coli hosts has hindered the development of ZIKV infectious clones. Here, using a novel self-splicing ribozyme-based strategy, we generated a stable infectious cDNA clone of a contemporary ZIKV strain imported from Venezuela to China in 2016. The constructed clone contained a modified version of the group II self-splicing intron P.li.LSUI2 near the junction between the E and NS1 genes, which were removed from the RNA transcripts by an easy-to-establish in vitro splicing reaction. Transfection of the spliced RNAs into BHK-21 cells led to the production of infectious progeny virus that resembled the parental virus. Finally, potential cis-acting RNA elements in ZIKV genomic RNA were identified based on this novel reverse genetics system, and the critical role of 5′-SLA promoter and 5′-3′ cyclization sequences were characterized by a combination of different assays. Our results provide another stable and reliable reverse genetics system for ZIKV that will help study ZIKV infection and pathogenesis, and the novel self-splicing intron-based strategy could be further expanded for the construction of infectious clones from other emerging and reemerging flaviviruses.

Liquid adsorption of tobacco specific nitrosamines (TSNA) in the tobacco extracting solution by porous materials is reported, with the monitoring of standard LC–MS/MS technique, in order to reduce the environmental cancerogens in smoke at the source. Three types of adsorbent, zeolite, mesoporous silica and activated carbon are utilized to trap nitrosamines in the extracting solution of Chinese-Virginia type tobacco with low TSNA content, and their performance was measured by spectrophotometric and LC–MS/MS methods. Both measurements gave the same variation tendency on the nitrosamines analysis, enabling the photometric method to be the simple and feasible tool in preliminary tests. The influence of pore size and cations of zeolite along with the organic modification of mesoporous silica with chloroisopropyl triethoxysilane were carefully investigated, and the optimal liquid conditions were determined, in which CaA zeolite and modified mesoporous silica CAM48-10 sample can capture 34 and 46 % of TSNA in the tobacco extracting solution, offering the efficient candidate for controlling the carcinogen like nitrosamines in environment.

Bacteriophages constitute key gene transfer agents in many bacteria. Specifcally, they may confer gene mobility to Staphylococcus warneri that usually dwells in the skin of human and animals. In this study, we first extracted samples of bacteria from foot skin of foot ulcers sufferers and develop these bacteria. Then, our bioinformatic methods for prophage predicting were applied to the genome sequences of Staphylococcus warneri which are assembly from high-throughput sequencing data. Two novel sequences predicted to encode complete prophages were found in the bacterial genome. Using the inducing agent mitomycin C, we produced phage suspensions. These indeed encompassed the progeny of the two identifed prophages (denoted IME1367_01 and IME1367_02). From high-throughput sequencing data of the suspensions, we obtained the two complete sequence of phage IME1367_01 and IME1367_02. By electron microscopic, we observed these two different phages. The complete genomes of IME1367_01 and IME1367_02 are summited to NCBI: "https://www.ncbi.nlm.nih.gov/nuccore/KY653124.1"&"https://www.ncbi.nlm.nih.gov/nuccor e/KY653121.1". It will enrich our understanding of the lysogenic phages of Staphylococcus warneri.

The highly pathogenic avian influenza (HPAI) H5N1 virus has caused several outbreaks in domestic poultry. Despite great efforts to control the spread of this virus, it continues to evolve and poses a substantial threat to public health because of a high mortality rate. In this study, we sequenced whole genomes of eight H5N1 avian influenza viruses isolated from domestic poultry in eastern China and compared them with those of typical influenza virus strains. Phylogenetic analyses showed that all eight genomes belonged to clade 2.3.2.1 and clade 7.2, the two main circulating clades in China. Viruses that clustered in clade 2.3.2.1 shared a high degree of homology with H5N1 isolates located in eastern Asian. Isolates that clustered in clade 7.2 were found to circulate throughout China, with an east-to-west density gradient. Pathogenicity studies in mice showed that these isolates replicate in the lungs, and clade 2.3.2.1 viruses exhibit a notably higher degree of virulence compared to clade 7.2 viruses. Our results contribute to the elucidation of the biological characterization and pathogenicity of HPAI H5N1 viruses.

A novel Ebola virus (EBOV) first identified in March 2014 has infected more than 25,000 people in West Africa, resulting in more than 10,000 deaths. Preliminary analyses of genome sequences of 81 EBOV collected from March to June 2014 from Guinea and Sierra Leone suggest that the 2014 EBOV originated from an independent transmission event from its natural reservoir followed by sustained human-to-human infections. It has been reported that the EBOV genome variation might have an effect on the efficacy of sequence-based virus detection and candidate therapeutics. However, only limited viral information has been available since July 2014, when the outbreak entered a rapid growth phase. Here we describe 175 full-length EBOV genome sequences from five severely stricken districts in Sierra Leone from 28 September to 11 November 2014. We found that the 2014 EBOV has become more phylogenetically and genetically diverse from July to November 2014, characterized by the emergence of multiple novel lineages. The substitution rate for the 2014 EBOV was estimated to be 1.23 × 10(-3) substitutions per site per year (95% highest posterior density interval, 1.04 × 10(-3) to 1.41 × 10(-3) substitutions per site per year), approximating to that observed between previous EBOV outbreaks. The sharp increase in genetic diversity of the 2014 EBOV warrants extensive EBOV surveillance in Sierra Leone, Guinea and Liberia to better understand the viral evolution and transmission dynamics of the ongoing outbreak. These data will facilitate the international efforts to develop vaccines and therapeutics.

To confirm the hypothesis that the high frequency sequences of high throughput sequencing are the terminal sequences of the bacteriophage genome. An adaptor of specific sequence was linked to the end of the bacteriophage T3 genomic DNA, which was then subject to high throughput sequencing; as a control, the same T3 genomic DNA without adaptor was also analyzed by high throughput sequencing. The sequencing results were examined with bioinformatics software. Similar high throughput sequencing technique was applied to analyze the genomic sequence of N4-like bacteriophage IME11. Bioinformatics study showed that the sequences tagged with adaptors were consistent with the high frequency sequences without adaptor labeling. Our analysis also indicated that the end of the T4-like phage genome had specific sequences instead of random sequences, disagreeing with the previous assertion. Evidences were provided that N4-like bacteriophage had a particular terminal sequence: the left end of the genome was unique while the right end was permuted. The high throughput sequencing technique was convenient and practical to be used to simultaneously detect the terminal sequence and the complete sequence of bacteriophage genome.

To construct a stable HCV-producing cell model for anti-HCV drug research.The HCV-ribozyme recombinant plasmid pJFH1-Rbz was constructed to generate the exact 5' and 3' ends of HCV genomic RNA by placing two self-cleaving ribozymes at both ends of the HCV JFH-1 cDNA. The plasmid was then transfected into HepG2 cells and the resultant clones were screened with G418. Subsequently, immunofluorescence and Western blot were performed to detect the expression of HCV core protein, HCV RNA level was quantitated by TaqMan real-time PCR method and HCV particles was detected by electron microscopy.HCV core protein was detected in the screened cell clone, and the level of HCV RNA was up to 1000,0000 copies/ml in the culture medium. Electron microscopy showed the viral particles in the culture suspension were approximately 55 nm in diameter. IFN-treating experiment demonstrated that the HCV RNA level decreased with the increasing concentration of IFN alpha.We constructed a stable HCV-producing cell model which can be used for anti-HCV drug research.

To study the serological characterization of indeterminate Western blot (WB) results of HIV antibody and to find a new way to verify the HIV antibody indeterminate results and provide references for editing "National Guideline for Detection of HIV/AIDS".All of the 42 subjects who were confirmed as indeterminate HIV antibody in People' Liberation Army HIV Confirmation Laboratory from 2005 to 2006, were collected. Line immunoassay, HIV viral load test and HIV-1 p24 were tested and followed up for 3-6 months' to compare the changes of WB bands patterns.(1) For the 42 individuals with indeterminate HIV antibody, a total of 8 different patterns of bands were found in WB test including 45.2% of them were p24 mono band, 30.9% were gp160 mono band, 11.9% were gp160 with p24, 2.4% (only one case) were gp160gp120 +/-, gp41p24, p24p17, gp41 or gp120 respectively. It was noticed that the most patterns of common bands with indeterminate results were p24 mono band, gp160 mono band and gp160 with p24, which composed 88.0% of the whole indeterminate WB band patterns. (2) Twenty three cases had been followed up for more than 3 months with 22 giving no WB band image change and were confirmed as HIV sero-negative. The other one with case gp160 and p24 had developed to more bands in the period of 77 days follow-up with more bands, including gp160, gp120, p66, p31, p24 and p17,showed up and was confirmed as HIV primary infection. (3) Line immunoassay was applied to all of those 23 cases who had been followed up and the results showed that only one serological change was found and the case was confirmed to be HIV-positive. Among the other 22 cases without serological changes, 16 cases were proved to be HIV-negative, 6 cases were still indeterminate. The specificity was 72.7%. P24 antigen test showed negative in all the 23 cases, including the case which later was confirmed as HIV-positive. Of all the 23 originally indeterminate cases, viral loads were tested in 7 cases. Positive result was found in the case which was proved later to be HIV-positive. No viral loads were detected in the other 6 cases (LDL).The most common band patterns of indeterminate HIV antibody were mainly p24 monoband, gp160 monoband or with p24. Most of them (95.6%) were not infected by HIV, the bands showed up in WB test and demonstrated as non-specific reactions. Line immunoassay could determine about 70% of the indeterminate reactions. Results from viral load test also suggested that it was an efficient method to discriminate indeterminate results. With these two techniques, HIV serology could be diagnosed without 3 months' follow-up in primary infection which gave indeterminate WB results.

To construct phage antibody library with predetermined CDR3 and to screen humanized Fab of anti-human integrin alphanubeta(3) monoclonal antibody (mAb) by epitope guided selection.LCDR3 gene of mAb E10 was inserted into human light chain variable region gene library. Hybrid phage antibody library was constructed by cloning E10 chimeric Fd gene and human light chain variable region gene into pComb3. Humanized light chain gene was obtained by screening against human integrin alphanubeta(3). Likewise, humanized Fab were gained by panning human phage antibody library, which was constructed by cloning humanized light chain gene and human heavy chain Fd gene with E10 HCDR3 into pComb3.Three humanized Fab clones was obtained by screening hybrid phage antibody library and human phage antibody library, which contained 2.1x10(6), 2x10(7) colony forming units, respectively. Indirect ELISA and competitive inhibition ELISA analysis demonstrated that three humanized Fab antibody had specific binding activity with human integrin alphanubeta(3). The strongest anti-human integrin alphanubeta(3) reactive D5 clone was sequenced and sequencing analysis showed that the V(kappa) and V(H) were derived from VKIII and VHI, respectively.Humanized Fab of anti-human integrin alphanubeta(3) mAb has been successfully obtained by phage display technology which lays the foundation for further clinical research.

To obtain CHO/dhfr(-) cells line with integrated FRT sequence in the chromosome transcription active site and to express human-mouse chimeric antibody directed against Chikungunya Virus by using the cell line.The fusion gene of FRT and HBsAg was constructed by PCR and cloned into the MCS of pCI-neo to construct pCI-FRT-HBsAg. The pCI-FRT-HBsAg was transfected into CHO/dhfr(-) cells and cell clones with high expression of HBsAg were screened by detecting the amount of HBsAg with ELISA. A CHO cell clone with the highest expression was chosen and named as CHO/dhfr(-) FRT(+). pAFRT HFLF, a expression plasmid of chimeric antibody with RFT sequence was transfected into CHO/dhfr(-) FRT(+) cells and cell clones with high expression of the chimeric antibody were screened by increasing concentration of MTX. A CHO cell clone with high expression of the chimeric antibody was cultured in large scale and supernatant was collected from which the chimeric antibody was purified. The purified chimeric antibody was analyzed by SDS-PAGE, Western blot and IFA.A CHO/dhfr(-) cells line with integrated FRT sequence in the chromosome transcription active site was obtained successfully. A cell clone with yield of 5 mg/L of chimeric antibody was obtained, as compared with routine CHO cell expression system with a yield of 2 mg/L.A cell line with integrated FRT sequence in the chromosome transcription active site was obtained and with it human-mouse chimeric antibody directed against Chikungunya virus was expressed. This system lays a solid foundation which can be used for expressing antibodies and other proteins.

To construct single chain antibody (scFv) gene of mAb E10 against human integrin alphavbeta3.The VH and VL genes were amplified from hybridoma cells secreting mAb E10 by RT-PCR and connected with the use of linker (Gly4Ser)3 to assemble scFv gene. The scFv gene was cloned into prokaryotic expression vector pTIG-TRX and expressed in E. coli BL21 (DE3).SDS-PAGE analysis showed the expressed recombinant protein with relative molecular mass (Mr) being 31,000. Western blot confirmed that the protein was labeled with His6. scFv protein was expressed as soluble protein under the condition of a small amount of IPTG induction and culture at lower temperature. The purity of the protein purified through Ni-NTA agarose metal affinity resin column was over 91%. The purified protein could bind to the human integrin alphavbeta3 by ELISA confirmation.scFv against human integrin alphavbeta3 has been successfully constructed and expressed,which lays the foundation for further clinical research.

To express a human-mouse chimeric antibody against rh-bFGF antigen in eukaryotic cells.The VL and VH genes were amplified and cloned from the hybridoma 1F11 secreting anti-rh-bFGF mouse monoclonal antibody (mAb) and the CL and CH genes were amplified and cloned from the plasmid pMDHC and pMDLC. They were inserted into eukaryotic expression vectors after sequencing. The recombinant plasmids were then transformed into CHO-dhfr- cells for expression. The humanization and antigen specificity of expressed products were identified by indirect ELISA. The relative molecular mass (Mr) of expressed products was determined by SDS-PAGE.The cloned antibody genes were identified to be functional by sequencing. The chimeric antibody could be detected in the culture supernatant of transformed CHO cells. The humanization and specificity of the chimeric antibody to rh-bFGF were confirmed by ELISA.The mouse-human chimeric antibody against rh-bFGF has been expressed successfully in eukaryotic cells, which lays the foundation for its further clinical research.

To probe the possibility of screening tPA with higher activity through DNA shuffling.Human, rat and rhesus monkey tPA cDNAs were used as the templates of "DNA family shuffling". The diverse tPA library was expressed in CHO cells, then the screening was carried out.Two shuffled clones, t9 and t17 were selected. The activity of tPA expressed by t9 clone was 4-fold higher than the activity of human tPA. tPA expressed by t17 clone was found having a 88-amino-acid deletion, but still showed the same activity as human tPA. DNA sequencing demonstrated that the sequences of t9 and t17 were mainly derived from human and monkey tPA cDNAs.Above work has laid the foundation for further experiments in the screening for higher activity of tPA.

To construct an eucaryotic expression plasmid carrying the BMP7 gene and express in MSCs.The BMP7 gene was cloned into the eucaryotic expression vector pcDNA3.1. At the same time, mesenchymal stem cells (MSCs) were isolated and cultured in vitro. The plasmid carrying the BMP7 gene was transfected into MSCs.PCR and digesting demonstrated that the eucaryotic expression plasmid -pcDNA-BMP7 was obtained. RT-PCR and immunohistochemical methods showed that the BMP7 gene was expressed in MSCs.Construction of an eucaryotic expression plasmid carrying BMP7 gene and expression in MSCs provide a sound basis for gene therapy using the BMP7 gene and the ideal seeds for tissue engineering.

Messenger RNA was extracted from human peripheral lymphocytes and first strand cDNA was prepared by reverse-transciption. The cDNA of Fc fragment of human IgG1 was then obtained by PCR and was cloned into the pGEM T-vector. The DNA sequences encoding signal peptides of both light and heavy chains were synthesized and cloned respectively. For construction of the light chain expression plasmid, the light chain signal sequence was linked with the light chain variable and constant regions (VL-CL) which had been cloned previously by screening of phage display libraries with HBsAg. The resulting full-lenth light chain sequence was then inserted into pcDNA3.1, a mammalian expression vector. For construction of the heavy chain expression plasmid, the heavy chain signal sequence, the variable region, the first constant region (VH-CH1, cloned previously by screening of phage display libraries with HBsAg) and Fc fragment sequence were ligated to form a full-length heavy chain ORF, which was then cloned into another mammalian expression vector, pCI-DHFR1. CHO(dhfr(-)) cells were cotransfected with the above light and heavy chain expression plasmids, and cell clones expressing human anti-HBsAg antibodies were selected by G418 and methotrexate (MTX). The recombinant human antibodies were purified with protein L affinity chromatography from the cell culture medium. As human serum IgG, the recombinant IgG exhibited only one band with a molecular weight of more than 100 kD in non-reducing SDS-PAGE in reducing SDS-PAGE, however, it turned out to be two bands of approximately 50 kD and 25 kD respectively. Western-blot analysis demonstrated that the whole IgG in the non-reducing SDS-PAGE, and the heavy chain in the reducing SDS-PAGE both reacted with goat anti-human Fc antiserum.