Distribution and expression in vitro and in vivo of DNA vaccine against lymphocystis disease virus in Japanese flounder (Paralichthys olivaceus)

Lymphocystis disease,caused by the lymphocystis disease virus (LCDV),is a significant worldwide problem in fish industry causing substantial economic losses.In this study,we aimed to develop the DNA vaccine against LCDV,using DNA vaccination technology.We evaluated plasmid pEGFP-N2-LCDV1.3 kb as a DNA vaccine candidate.The plasmid DNA was transiently expressed after liposome transfection into the eukaryotic COS 7 cell line.The distribution and expression of the DNA vaccine (pEGFP-N2-LCDV1.3kb) were also analyzed in tissues of the vaccinated Japanese flounder by PCR,RT-PCR and fluorescent microscopy.Results from PCR analysis indicated that the vaccine-containing plasmids were distributed in injected muscle,the muscle opposite the injection site,the hind intestine,gill,spleen,head,kidney and liver,6 and 25 days after vaccination.The vaccine plasmids disappeared 100 d post-vaccination.Fluorescent microscopy revealed green fluorescence in the injected muscle,the muscle opposite the injection site,the hind intestine,gill,spleen,head,kidney and liver of fish 48 h post-vaccination,green fluorescence did not appear in the control treated tissue.Green fluorescence became weak at 60 days post-vaccination.RT-PCR analysis indicated that the mcp gene was expressed in all tested tissues of vaccinated fish 6-50 days post-vaccination.These results demonstrate that the antigen encoded by the DNA vaccine is distributed and expressed in all of the tissues analyzed in the vaccinated fish.The antigen would therefore potentially initiate a specific immune response.The plasmid DNA was injected into Japanese flounder (Paralichthys olivaceus) intramuscularly and antibodies against LCDV were evaluated.The results indicate that the plasmid encoded DNA vaccine could induce an immune response to LCDV and would therefore offer immune protection against LCD.Further studies are required for the development and application of this promising DNA vaccine.

Evaluation of the Protection against Infectious Bursal Disease (IBD) Challenge in Progeny Born to Parents Having Received a Vaccination Program Using a Herpesvirus of Turkey-Infectious Bursal Disease (HVT-IBD) Vector Vaccine

Broiler breeder vaccination against IBD is usually based on the injection of at least one inactivated vaccine in oil adjuvant, typically included in a combined vaccine. Priming using one or several IBD vaccine (s) has been the most common way to immunize the breeders so far. In summary, protection against vvIBD challenge in chicks of one commercial genetic line vaccinated in ovo with the HVT-IBD vector vaccine was demonstrated. The parents’ IBD vaccination program, using the HVT-IBD vector vaccine alone, the HVT-IBD vector vaccine plus IBD inactivated vaccine, and inactivated IBD vaccine alone, did not impair their progeny’s in ovo HVT-IBD vector vaccine take and subsequent protection against vvIBD virus challenge. An advantage in terms of immunization of the progeny against vvIBD was shown in the chicks born to breeders vaccinated with the HVT-IBD vaccine as a primer, as compared to breeders vaccinated with the inactivated vaccine alone. High level of IBD maternally-derived antibodies transmitted to the progeny by their parents induces together with an early onset of immunity by in ovo injection of a HVT-IBD vector vaccine clinical protection, as monitored on bursas, after vvIBD virus challenge.

Comparison of Three ELISA Kits for the Differentiation of Foot-and-mouth Disease Virus-infected from Vaccinated Animals

研究被执行验证工具包为 FMDV 的区别在中国开发了的 3 FMDV 3ABC-I-ELISA 感染并且种牛痘的动物。从天真、种牛痘的牛以及从被感染了的牛的 sera 的集合被商业诊断工具包对 FMDV 的 nonstructural 蛋白质(NSP ) 为抗体测试， Ceditest 吗？FMDV-NS (Ceditest？工具包) ， UBI？FMDV NONSTRUCTURAL 蛋白质 ELISA 方向插入(UBI？工具包) 并且一个 FMDV 3ABC-I-ELISA 工具包在 Lanzhou 发展了兽医研究院。三个工具包的测试参数(敏感和特性) 被决定，并且从 FMD 3ABC-I-ELISA 工具包获得的结果与从二个外国工具包获得了那相比。结果显示巧合在 FMDV 3ABC-I-ELISA 和 Ceditest 之间评价吗？工具包 98.05% 是，并且在 FMDV 3ABC-I-ELISA 和 UBI 之间的巧合率？工具包是 94.4% ；两 Ceditest 的敏感？并且 FMDV 3ABC-I-ELISA 工具包是 100% 。然而， UBI 的敏感？工具包仅仅是 81.8% 。与从天真或种牛痘的非感染的动物的 sera，所有测试的特性超过了 90% 。关键词 Foot-and-mouth 疾病病毒(FMDV )- 非结构的蛋白质 3ABC - ELISA CLC 数字 S852.65 基础项目：国家鈥 ? 73 鈥 ? 研究工程(G199901190， 2005CB23201 ) ；国际原子能机构(国际原子能机构)(10697/R2 )

Cloning and Prokaryotic Expression of VP1 Gene of Foot-and-Mouth Disease Virus (FMDV) Type O

According to the complete genome of foot-and-mouth disease virus(FMDV)type O,a pair of special primers was designed to amplify VP1 gene.The VP1 gene was amplified by RT-PCR and subsequently inserted into the expression vector pGEX-6p-1 and induced by IPTG.Then SDS-PAGE showed the expressed protein was 51 kD in molecular weight.Then the product was purified by GSTrap FF columns.The product was detected through Western-blot that showed the protein has antigenicity.It provided fundamental data and materials for further investigation on diagnosis method of FMDV.

PLASMID VACCINE ENCODING HN GENE FROM NEWCASTLE DISEASE VIRUS HAS MARKED ANTITUMORAL EFFECT IN VITRO

Objective: To explore the antitumor effects of hemaagglutinin-neuraminase gene (HN gene) from Newcastle disease virus. Methods: Plasmid vaccine of pIRHN was constructed and transfected into HeLa cells. The expression of HN was analyzed by Western blot analysis, and the mode of cell death was detected by fluorescence microscope, gel electrophoresis and TUNEL assay and the expression of p53 and bcl-2 was also analyzed in transfected Hela cells. The effect of pIRHN on sialic acid contents in the Hela cell was examined. Results: pIRHN nucleic acid vaccines could be expressed in eukaryotic cell. pIRHN could induce apoptosis after HeLa cells were transfected. The effect of antitumor responses of pIRHN was correlated with the contents of sialic acid in tumor cells, and there was no prominent evidence for the relatedness of the antitumor effect with the expression of p53 and bcl-2. Conclusion: pIRHN may become a new antitumor biological agent.

Effect of amino acid mutation at position 127 in 3A of a rabbitattenuated foot-and-mouth disease virus serotype Asia1 on viral replication and infection

An amino acid mutation(R127→I) in the 3A non-structural protein of an FMDV serotype Asia1 rabbit-attenuated ZB strain was previously found after attenuation of the virus. To explore the effects of this mutation on viral replication and infection, the amino acid residue isoleucine(I) was changed to arginine(R) in the infectious cDNA clone of the rabbit-attenuated ZB strain by sitedirected mutagenesis, and the R127-mutated virus was rescued. BHK monolayer cells and suckling mice were inoculated with the R127-mutated virus to test its growth property and pathogenicity, respectively. The effects of the R127 mutation on viral replication and virulence were analyzed. The data showed that there was a slight difference in plaque morphology between the R127-mutated and wild-type viruses. The growth rate of the mutated virus was lower in BHK-21 cells and its virulence in suckling mice was also attenuated. This study indicates that the R127 mutation in 3A may play an important role in FMDV replication in vitro and in pathogenicity in suckling mice.

Single amino acid substitution of VP1 N17D or VP2 H145Y confers acid-resistant phenotype of type Asia1 foot-and-mouth disease virus

Infection by foot-and-mouth disease virus(FMDV) is triggered by the acidic pH in endosomes after virus uptake by receptor-mediated endocytosis. However, dissociation of the FMDV 146S particle in mildly acidic conditions renders inactivated foot-and-mouth disease(FMD) vaccines much less effective. Type Asia1 FMDV mutants with increased resistance to acid inactivation were selected to study the molecular basis of viral resistance to acid-induced disassembly and improve the acid stability of FMDV. Sequencing of capsid-coding regions revealed four amino acid replacements(VP1 N17D, VP2 H145Y, VP2 G192D, and VP3 K153E) in the viral population of the acid-selected 10th passage. We performed single or combined mutagenesis using a reverse genetic system, and our results provide direct experimental evidence that VP2 H145Y or VP1 N17D substitution confers an acid-resistant phenotype to type Asia1 FMDV.

B Cell Epitopes within VP1 of Type O Foot-and-mouth Disease Virus for Detection of Viral Antibodies

In this study, the coding region of type O FMDV capsid protein VP1 and a series of codon optimized DNA sequences coding for VP1 amino acid residues 141-160 （epitopel）, tandem repeat 200-213 （epitope2 （＋2）） and the combination of two epitopes （epitopel-2） was genetically cloned into the prokaryotic expression vector PPRoExHTb and pGEX4T-1, respectively. VP1 and the fused epitopes GST-E1, GST-E2 （＋2） and GST-E1-2 were successfully solubly expressed in the cytoplasm of Escherichia coli and Western blot analysis demonstrated they retained antigenicity. Indirect VP1-ELISA and epitope ELISAs were subsequently developed to screen a panel of 80 field pig sera using LPB-ELISA as a standard test. For VP1-ELISA and all the epitope ELISAs, there were clear distinctions between the FMDV-positive and the FMDV-negative samples. Cross-reactions with pig sera positive to the viruses of swine vesicular disease virus that produce clinically indistinguishable syndromes in pigs or guinea pig antisera to FMDV strains of type A, C and Asia1 did not occur. The relative sensitivity and specificity for the GST-E1 ELISA, GST-E2 （＋2）, GST-E1-2 ELISA and VP1-ELISA in comparison with LPB-ELISA were 93.3% and 85.0%, 95.0% and 90%, 100% and 81.8%, 96.6% and 80.9% respectively. This study shows the potential use of the aforementioned epitopes as alternatives to the complex antigens used in current detection for antibody to FMDV structural proteins.

The Complete Genomic Sequence of a Foot-and-Mouth Disease Virus Isolated from the Swine

The complete genomic sequence of foot-and-mouth disease virus (FMDV) Chinese strain OH/CHA/99 was determined. The 8040 nt sequence and the deduced amino acid sequence werecompared with FMDV sequences published. The results showed that OH/CHA/99 shared highersequence homology with OTYTW/97, indicating their close genetic relationship. However,the strain had lower sequence identity with O1/Kaufbeuren/66 strain. Besides, largedeletions in 3A coding region were observed in OH/CHA/99. It was shown that the poly (A)tail of OH/CHA/99 had 56 As at least.

Amplification and Characterization of Bull Semen Infected Naturally with Foot-and-mouth Disease Virus Type Asia1 by RT-PCR

To investigate the security of semen biologically, 15 bull semen samples were collected (of which 5 exhibited clinical signs of Foot-and-mouth disease) and identified by RT-PCR and virus isolation. The results indicated that the semen of the infected bulls were contaminated by Foot-and-mouth disease virus (FMDV), but FMDV was not detected in semen samples from those bulls not showing clinical signs of Foot-and-mouth disease (FMD). This is the first report of the presence of FMDV in bull semen due to natural infection in China. The analysis of the partial sequence of the VP1 gene showed that the virus strain isolated from semen has 97.9% identity with the virus isolated from vesicular liquid of infected bulls showing typical signs of FMD and belonged to the same gene sub-group.

Adverse Effects of Inactivated Foot-and-Mouth Disease Vaccine—Possible Causes Analysis and Countermeasures

Foot-and-mouth disease (FMD) is an infectious and sometimes fatal viral disease that affects cloven-hoofed animals, and Chinese government adopts compulsory immunization measures for FMD. The adverse effects of FMD vaccine to pigs, cattle and goats have been reported increasingly frequent during the spring and autumn seasons when large numbers of farm livestock are vaccinated. The financial losses caused by vaccine adverse effects have been a serious concern for both farmers and primary prevention personnel. There are various causative factors reported to involve into adverse effect of FMD vaccine, including the inappropriate vaccine production, transportation and storage, livestock poor tolerance, and unqualified vaccinating manipulations. Symptomatic treatment and early drug prevention have a certain effect on the adverse effects. To analyze causes and propose countermeasures, in the current study possible reasons during the production and processing procedures of inactivated FMD vaccine were reviewed and corresponding countermeasures were recommended. The review may provide references for better use of vaccine to prevent FMD.

Review of Air Dispersion Modelling Approaches to Assess the Risk of Wind-Borne Spread of Foot-and-Mouth Disease Virus

Foot-and-mouth disease virus (FMDV) is one of the most economically serious veterinary pathogens due to its negative effects on livestock and its highly infectious nature via a variety of transmission paths through oral and inhalation routes. Measures to enhance outbreak management can be designed according to analytical results predicted by mathematical models for wind-borne dispersion, an important path of virus transmission. Accurate atmospheric dispersion models are useful tools for properly determining risk management plans, while inaccurate models may conversely lead to accidental loss in two possible ways. Overly strict measures, e.g., slaughter for too wide an area, can cause severe economic difficulties, including irreversible loss of business operations for a number of farms. On the contrary, inestimable loss potentially caused by lax controls is a persistent threat. In this paper, available modelling procedures for forecasting the spread of FMDV, which have been used since the 1970s, each having its advantages and limitations, are reviewed for the purpose of ensuring suitable application in various conditions of any future emergency cases.

Effect of Attenuated Highly Pathogenic Pig Reproductive and Respiratory Syndrome(HP-PRRS) TJM-F92 Strain Vaccine on Immune Antibody Levels against Classical Swine Fever(CSF) and Foot-and-Mouth Disease(FMD)

Effects of attenuated highly pathogenic pig reproductive and respiratory syndrome（HP-PRRS）TJM-F92 strain vaccine on immune antibody level against classical swine fever（CSF）and foot-and-mouth disease（FMD）were studied from October 8 to November 12 in 2014,in order to optimize vaccination program of CSF,HP-PRRS and FMD and to provide scientific guidance for animal disease control and prevention work.The results showed that attenuated HP-PRRS（TJMF92 strain）vaccine had no significant effect on immune antibody level of hog cholera lapinized virus（HCLV,ST passage cell vaccine）attenuated vaccine and FMD-O inactivated vaccines（OZK/93 strain）,and single or combined use of three vaccines received good immunization effects.

Preparation and Characterization of Monoclonal Antibodies against VP1 Protein of Foot-and-mouth Disease Virus O/China99

Monoclonal antibodies (McAbs) 1A9 and 9F12 against Foot-and-mouth disease virus (FMDV) serotype O were produced by fusing SP2/0 myeloma cells with splenocyte from the mouse immunized with O/China99. Both McAbs reacted with O/China99 but not with Asia 1, as determined by immunohistochemistry assay. The microneutralization titer of the McAbs 1A9 and 9F12 were 640 and 1 280, respectively. Both McAbs contain kappa light chains, but the McAbs 1A9 and 9F12 were IgG1 and IgM, respectively. In order to define the McAbs binding epitopes, the reactivity of these McAbs against VP1, P20 and P14 were examined using indirect ELISA, the result showed that both McAbs reacted with VP1 and P20. McAbs may be used for further studies of vaccine, diagnostic methods, prophylaxis, etiological and immunological researches on FMDV.

Generation of Monoclonal Antibodies against Non-structural Protein 3AB of Foot-and-Mouth Disease Virus

To identify linear epitopes on the non-structural protein 3AB of foot-and-mouth disease virus （FMDV）, BABL/c mice were immunized with the 3AB protein and splenocytes of BALB/c mice were fused with myeloma Sp2/0 cells. Two hybridoma monoclonal antibodies （mAbs） cell lines against the 3AB protein of foot-and-mouth disease virus （FMDV） were obtained, named C6 and E7 respectively. The mieroneutralization titer was 1：1024 for mAb C6, and 1：512 for E7. Both mAbs contain kappa light chains, and were of subclass IgG2b. In order to define the mAbs binding epitopes, the reactivity of these mAbs against FMDV were examined by indirect ELISA. The results showed that both mAbs can react with FMDV, but had no cross-reactivity with Swine Vesicular Disease （SVD） antigens. The titers in abdomen liquor were 1：5×10^6 for C6 and 1：2×10^6 for E7. In conclusion, the mAbs obtained from this study are specific for the detection of FMDV, can be used for etiological and immunological researches on FMDV, and have potential use in diagnosis and future vaccine designs.

Develope Monoclonal Antibody against Foot-and-mouth Disease Virus A Type

In order to develop an anti-FMDV A Type monoclonal antibody （mAb）, BABL/c mice were immunized with FMDV A type. Monoclonal antibodies （mAbs） 7B 11 and 8H4 against Foot-and-mouth disease virus （FMDV） serotype A were produced by fusing SP2/0 myeloma cells with splenocyte from the mouse immunized with A/AV88. The microneutralization titer of the mAbs 7Bll and 8H4 were 1024 and 512, respectively. Both mAbs contain kappa light chainS, the mAbs were IgG1. In order to define the mAbs binding epitopes, the reactivity of these mAbs against A Type FMDV, were examined using indirect ELISA, the result showed that both mAbs reacted with A Type FMDV. These mAbs may be used for further vaccine studies, diagnostic methods, prophylaxis, etiological and immunological research on FMDV. Characterization of these ncindicated that prepared anti-FMDV A mAbs had no cross-reactivity with Swine Vesicular Disease （SVD） or FMDV O, Asial and C Type antigens. Their titers in abdomen liquor were 1：5×10^6 and 1：2×10^6, respectively. 7B 11 was found to be of subtype IgGb 8H4 was classified as IgG2b subtype. The mAbs prepared in this study, are specific for detection of FMDV serotype A, and is potentially useful for pen-side diagnosis.

Expression,Purification and Activity Detection of Structural Protein VP1 of Foot-and-Mouth Disease Virus Serotype A

The paper was to obtain the VP1 protein of FMDV serotype A with high activity. With recombinant plasmid pMD19A-T-vp1 as the tem- plate, vpl gene fragment amplified by PCR was connected into prokaryotic expression vector pET28a to construct recombinant plasmid pET-A-vpl. The E. coli BL21 （DE3） strain containing recombinant plasmid pET-A-vpl were induced by IPTG. SDS-PAGE showed that VP1 protein was ex- pressed in the form of inclusion body, and its molecular weight was about 29 ku. Based on the optimizing IPTG concentration and expression time, the largest expression of VP1 protein was induced by 0.3 mmol/L IPTG for 6 h at 37 ℃. Western-Blot analysis indicated that the expression of VP1 protein could be specifically recognized by positive serum of FMDV serotype A. ELISA test showed that VP1 inclusion body protein had high activity after purification by washing and renaturation by urea concentration gradient dialysis.

Current updates on the epidemiology,pathogenesis and development of small molecule therapeutics for the treatment of Ebola virus infections

Ebola virus disease(EVD)is a rare,highly contagious and a deadly disease with a variable fatality rate ranging from 30%to 90%.Over the past two decades,Ebola pandemic has severely affected the sub-Sahara region including Democratic Republic of the Congo(DRC),and Uganda.The causative agents of the most EVD cases are three distinct species out of six Ebolaviruses namely Zaire Ebolavirus(ZEBOV),Sudan Ebolavirus(SUDV)and Bundibugyo Ebolavirus(BDBV).In recent years,significant strides have been made in therapeutic interventions.Notably,the US Food and Drug Administration has approved two monoclonal antibodies:InmazebTM(REGN-EB3)and Ansuvimab or EbangaTM.Additionally,many small molecules are currently in the developmental stage,promising further progress in medical treatment.Addressing the critical need for preventive measures,this review provides an in-depth analysis of the licensed Ebola vaccines-Ervebo and the combination of Zabdeno(Ad26.ZEBOV)and Mvabea(MVA-BN-Filo)as well as the vaccines which are currently being tested for their efficacy and safety in clinical studies.These vaccines might play an important role in curbing the spread and mitigating the impact of this lethal disease.The current treatment landscape for EVD encompasses both nutritional(supportive)and drug therapies.The review comprehensively details the origin,pathogenesis,and epidemiology of EVD,shedding light on the ongoing efforts to combat this devastating disease.It explores small molecules in various stages of the development,discusses patents filed or granted,and delves into the clinical and supportive therapies that form the cornerstone of EVD management.This review aims to provide the recent developments made in the design and synthesis of small molecules for scientific community to facilitate a deeper understanding of the disease and fostering the development of effective strategies for prevention,treatment,and control of EVD.

Public perceptions about the emerging human monkey pox disease and vaccination in twin cities of Pakistan: a cross-sectional study

Background:The monkey pox virus is caused by monkey pox(MPX),which is similar to both smallpox and cowpox.Near tropical rain forests,it usually occurs in isolated communities in Central and West Africa.The monkey pox virus,a member of the family Poxviridae and belongs to the genus Orthopoxvirus.Close contact with infected animals,sick people,or contaminated inanimate things can cause the virus to spread to humans.The illness typically takes 7 to 14 days to incubate and is characterized by fever,headache,lethargy,myalgia,generalized body pains,lymph node swelling,and skin lesions.Given the variety of illnesses that can result in skin rashes,it could be challenging to differentiate monkey pox solely based on clinical presentation,particularly for patients with an uncommon look.Objective:The main objective of the study is to evaluate public perceptions regarding the emerging human monkey pox disease and vaccination.Methods:The study was conducted using a cross-sectional study design.The sample size was 472 participants;however,10 questionnaires were excluded because of invalid data.The 462 questionnaires were included by expert validation from the general public of Rawalpindi and Islamabad,Pakistan.The data were analyzed using Chi-squared tests.Results:This questionnaire-based cross-sectional study was conducted from 15 Sep to 15 Oct 2022.The participants’perceptions,knowledge,and attitudes were collected via a 24-item-based questionnaire survey.The survey was based on 462 participants,196(42.4%)were females,and 266(57.6%)were males.The results reveal that out of 462 participants,clinical symptoms of monkey pox disease 82.7%(382),complications of monkey pox disease 81.2%(375),lymphadenopathy(swollen lymph nodes)is one clinical feature that could be used to differentiate between monkey pox and smallpox 81.2%(375)and monkey pox is common in Western and Central Africa 24.2%(112).Furthermore,the majority of participants(P≤0.05)agreed that health officials should start a vaccination campaign to combat monkey pox.Regarding preventive measures and vaccination campaigns,health officials should take public preventive measures 79.7%(368)and health officials start a vaccination campaign against monkey pox disease 56.3%(260).Conclusion:There was a significant difference seen in the public perception regarding monkey pox preventive measures and vaccination.The International health authorities must take priority-based preventative measures to prevent the spread of monkey pox disease around the world.

Prokaryotic Expression of P1 Gene of Type Asia1 Foot and Mouth Disease Virus(FMDV)and the Preparation of Its Antiserum

[Objective] The aim was to study the prokaryotic expression of P1 gene of foot-and-mouth disease virus（FMDV）type Asia 1and the preparation of its antiserum.[Method]The P1 gene of FMDV type Asia 1 was obtained by gene cloning techniques,and then cloned into pET-32a（＋）plasmid;subsequently the recombinant plasmid was transformed into E.coli BL21（DE3）;after the IPTG induction and protein purification,SDS-PAGE analysis was carried out;the ultrasonic wave was use to lyse the cultivated recombinant strain,and after the isolation and purification,this fusion protein was utilized to immunize New Zealand rabbits so as to prepare P1 protein antiserum.[Result]The positive clones were obtained;SDS-PAGE result showed that the target band was appeared at 105 kD;Western blot analysis showed that the antisera could bind to the expressed P1 fusion protein specifically;the ELISA titer of the rabbit anti-FMDV-P1 sera was approximately 1∶5 120.[Conclusion]This study had provided foundations for FMDV serological diagnostic methods and genetically engineered vaccine.