Analysis of Occurrence and Mixed Infection of Sugarcane Bacilliform Virus Disease in Hainan Sugarcane-growing Area

[Objectives]The paper was to explore the occurrence and mixed infection of sugarcane bacilliform virus disease in Hainan sugarcane-growing area.[Methods]A total of 348 sugarcane leaf samples were collected from 7 sugarcane-growing areas in Hainan Province.Molecular detection of sugarcane bacilliform virus(SCBV)was carried out by PCR using specific primers.[Results]SCBV was detected in 244 out of 348 sugarcane samples,with an average detection rate of 70.11%.The highest detection rate was 76.66%in the Danzhou sugarcane-growing area,while the lowest was 57.14%in the Baisha sugarcane-growing area.The SCBV-positive samples were subjected to testing for SCYLV,SCSMV,SrMV,and SCMV,respectively.The results indicated that 106 out of 244 positive samples exhibited a single infection with SCBV,while 138 samples exhibited mixed infections with SCBV and other sugarcane viruses.The proportion of mixed infections among the SCBV-positive samples was as high as 56.56%.Among the various types of mixed infections,two-virus and three-virus mixed infections were the most prevalent.[Conclusions]SCBV has emerged as a significant threat to the secure production of sugarcane in the Hainan sugarcane-growing region.It presents an explosive infection in the Hainan sugarcane-growing region and frequently combines with other sugarcane viruses to infect sugarcane.The findings of this study will provide a theoretical foundation for the prevention and control of sugarcane bacilliform virus disease.

Partial Fusion (F) Gene Analysis of Newcastle Disease Virus Detected in Pakistan during 2021-2022

Newcastle disease (ND) virus is a leading threat to commercial and domestic poultry in Pakistan. The virus infects and constitutes irreversible impairment to the nervous system, damages the respiratory system, and marks severe gastrointestinal lesions leading to heavy mortality in short-living birds and substantial losses in layers and breeders. The continuous emergence and evolution of the virus made it inclined to evade the humoral response and indirectly the circumvention of artificial active immunization. Newcastle disease is caused by the orthoavula genus of the paramyxoviridae family and has shown high genetic diversity even in their genotypes while information regarding enzootic trends of the virus is scanty in Pakistan. A total of 40 tracheal samples of NDV were collected from different commercial broiler farms and 11 isolates of NDV were identified. In the current study, we determined the genetic diversity of the Newcastle disease virus based on the partial sequencing of the fusion protein gene available in the NCBI database. Genetic analysis showed that seven isolates belonged to class I genotype VII and four belonged to class II genotype II. Interestingly, two isolates had epidemiological connections with vaccine-like class II genotype II. Our findings, concerning the recent outbreaks of class I genotype VII and class II genotype II of NDV in vaccinated commercial flocks, suggest possible potential partial mutations in the fusion protein gene. Genetic diversity and formation of the new cleavage site in an important neutralizing protein of wild strain are linked with the potency of artificial active immunization and a major cause of vaccine failure.

Genomic Sequencing and Molecular Characteristics of A Very Virulent Strain of Infectious Bursal Disease Virus Isolated in China

[Objective] The paper was to determine the genomic sequence of a very virulent strain of infectious bursal disease virus（IBDV）,and study its molecular characteristics.[Method] A very virulent strain（vvIBDV）（HLJ-0504） of infectious bursal disease virus（IBDV） with special characters was isolated in China and its genome was sequenced.[Result] Sequence analysis showed that segment A of HLJ-0504 was derived from vvIBDV,while segment B was from a distinct ancestor.The morbidity and mortality of HLJ-0504 was 100% and 86.7%to SPF chickens,respectively.[Conclusion] vvIBDV with distinct segment B were still circulating and the evolution of IBDV was diversified in China.Besides,it is hard to imagine that the virulence of IBDV is determined solely by segment A or B.

Genetic Analysis of the VP2 Hypervariable Region of Thirty-six Infectious Bursal Disease Virus Isolates in China during 2009-2012

[Objective] Infectious bursal disease （IBD） is a highly contagious immuno- suppressive disease caused by infectious bursal disease virus （IBDV）. IBDV is ge- netically prone to mutation, which results in challenges to the disease prevention and control. Thus, it is necessary to continuously monitor the prevalence of IBDV. [Method] 36 IBDVs were identified from ten provinces in China from 2009 to 2012. Partial fragments of VP2, including the hypervariable region （HVR）, from new iso- lates were sequenced and analyzed through comparisons with published sequences of IBDV, including 18 strains isolated previously by our lab and 24 reference strains from China and around the world. [Result] Phylogenetic analysis showed a co-exis- tence of IBDV strains belonging to classic, variant, attenuated, and very virulent IB- DV （wlBDV） in China. wlBDVs remain the predominant strains in China and the new subgroup was emerging. Alignment analysis revealed several distinct amino acid mutations that might be involved in virulence or antigenicity variation. [Conclu- sion] The results offered evolutionary clues showing the emerging trend of obvious variations and diversity of IBDV in major poultry-producing regions of China particu- larly in recent years. These findings will contribute to a better understanding of the genetic evolution of IBDV.

Effects of Biological Pesticides on Prevention and Treatment of Virus Diseases in Greenhouse Tomato in Autumn

To alleviate the damage of tomato virus diseases, reduce chemical pesticide consumption and residue, and prevent environmental pollution, forecasting, randomized block design and statistical analysis were adopted to conduct field efficacy tests for Lentinan ＋ Gibberellin-heteroauxin-brassinolide （GHB） and Ningnanmycin ＋ GHB. The results showed that when the virus diseases in greenhouse tomato were serious, 0.5% Lentinan AS 3 000 ml ＋ GHB WP 600 g and 8% Ningnanmycin AS 900 ml ＋ GHB WP 600 g per hectare were applied 4 times with an interval of 7 days, and the control effects on the 10th, 17th, 26th and 35th day were above 88%, 87%, 78% and 67%, respectively. The difference in control effect of the two biological pesticides was insignificant, while their control effects were all significantly better than that of moroxydine hydrochloride （CK） ＋ GHB. The obtained results indicated that Lentinan ＋ GHB and Ningnanmycin ＋ GHB were the ideal biological pesticides, which could be used not only for controlling tomato virus diseases, but also for realizing modern, pollution-free, green and organic agricultural production.

Screening of Anti-Newcastle Disease Virus Ingredients of Phyllanthus urinaria

[ Objective ] The aim of the study was to screen the antiviral ingredients of Chinese herbal medicine Phyllanthus urinaria in order to devel- op new drugs for the treatment of viral diseases of poultry. [ Method ] The whole grass of phyllanthus was respectively decocted in 75% ethanol （E） and pure water （PF）, and then the active ingredients were separately extracted in petroleum ether （PE）, ethyl acetate （EA）, and n-butyl alcohol （BU）. The extracts were inoculated on chicken embryo fibroblast （CEF） to observe their inhibitory effect on cytopathic effect （CPE） of Newcastle disease virus （NDV）, inoculated into chicken embryos to observe the changes of the hemagglutination titer of NDV, and inoculated in chickens to determine the mortality and evaluate their effect of immune protection. [ Result] The E -PE and E -BU extracts, especially E -BU extract, inhibi- ted CPE of NDV on CEF and significantly inhibited the proliferation of NDV in chicken embryo （P〈0.05） ; the survival rate of E-PE and E-BU group was extremely significantly higher than that of ribavirin group and the normal saline control （ P 〈0.01 ）, with E - PE group more higher. [ Condusion] The water extract of phyllanthus is less effective; E -PE extract and E -BU extract should effectively inhibit the CPE and proliferation in chicken embryo of NDV and also have better immune protection against NDV.

Ebola Virus Disease: General Characteristics, Thoughts, and Perspectives

In December 2013, a new round of Ebola virus disease （EVD） first occurred in a remote countryside of Guinea and then spread in Guinea, Liberia, Sierra Leone, and Nigeria of West Africa. EVD, caused by Ebolavirus and previously known as Ebola hemorrhagic fever, is an acute infectious disease with fatality rates up to 90%. As of August 22, 2014, the number of suspected and confirmed cases was 2615, causing 1427 deaths[I]. On August 8, 2014, World Health Organization announced the current outbreak in West Africa as an international public health emergency. The global epidemic tendency remains ambiguous to date. In recent years, China closely collaborates with West Africa in labor, business, overseas education, and also sends aid medical team there. Thus, the risk of importing the disease cannot be ignored. We conduct this literature review of epidemiology, pathogen, prophylaxis, and treatment to provide evidence for controlling the risk and carrying out effective interventions.

Effect of Astragalus polysaccharides on Erythrocyte Immune Adherence of Chickens Inoculated with Infectious Bursal Disease Virus

Two hundred and forty specific pathogen free leghorn chickens were randomly divided into four groups and reared in isolated pens. The tested chickens were negative to infectious bursal disease virus （IBDV） at 25 d old. Group 1 was treated with saline, whereas Groups 2, 3, and 4 were inoculated with 0.3 mL IBDV suspension intranasally the next day. Groups 3 and 4 were also administered with Astragalus polysaccharides （APS） intramuscularly twice daily at 5 or 10 mg kg-1 BW, respectively, until 31 d old. The erythrocyte-C3b receptor rosette rate （E-C3bRR） and the erythrocyte-C3b immune complex rosette rate （E-ICRR） were measured at 25, 29, 32, 35, and 38 d old. The results showed that IBDV significantly reduced E-C3bRR and E-ICRR when compared with the control group （P 〈 0.05）, while simultaneous administration of APS with 1BDV maintained E-C3bRR at similar levels to the control group （P 〉 0.05） and increased E-ICRR when compared with the control group and the group non-treated with APS （P 〈 0.05）. APS treatment reduced the morbidity and mortality of chickens inoculated with IBDV （P 〈 0.05）. The results suggest that APS may enhance the immune adherence of chickens erythrocytes by affecting the activity and/or the number of complement receptors on the erythrocyte membrane. These findings can be beneficial in providing an understanding of the basic mechanisms required for the rational application of APS in modern medicine.

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.

An improved scheme for infectious bursal disease virus genotype classification based on both genome-segments A and B

Infectious bursal disease(IBD)is caused by infectious bursal disease virus(IBDV),which has a genome consisting of two segments of double-stranded linear RNA.IBDVs have been traditionally divided into four phenotypes based on their pathogenicity and antigenicity,including classic,variant,very virulent,and attenuated IBDV.With the emergences of divergent molecular characteristics of novel strains produced by continuous mutations and recombination,it is increasingly difficult to define new IBDV strains using the traditional descriptive classification method.The most common classification scheme for IBDV with segmented genome is based solely on segment A,while the significance of segment B has been largely neglected.In this study,an improved scheme for IBDV genotype classification based on the molecular characteristics of both VP2(a viral capsid protein encoded by segment A)and VP1(an RNA-dependent RNA polymerase protein encoded by segment B)was proposed for the first time.In this scheme,IBDV was classified into nine genogroups of A and five genogroups of B,respectively;the genogroup A2 was further divided into four lineages.The commonly used phenotypic classifications of classic,variant,very virulent,and attenuated IBDVs correspond to the A1 B1,A2 B1,A3 B2,and A8 B1 genotypes of the proposed classification scheme.The novel variant IBDVs including the strains identified in this study were classified as belonging to genotype A2 d B1.The flexibility and versatility of this improved classification scheme will allow the unambiguous identification of existing and emerging IBDV strains,which will greatly facilitate molecular epidemiology studies of IBDV.

Immunogenicity of formaldehyde and binary ethylenimine inactivated infectious bursal disease virus in broiler chicks

Infectious bursal disease virus (IBDV) was inactivated by two different chemicals—formaldehyde and binary ethylenimine (BEI). Formaldehyde was used at 0.1% and 0.2%, while BEI was used at concentrations of 0.001 and 0.002 mol/L. These four vaccines were tested for their efficiency in generating humoral immune response in different groups of broiler chicks. Both BEI-inactivated vaccines gave relatively higher antibody titers and were almost twice as efficient as formaldehyde-inactivated ones.

Cytomegalovirus colitis in a patient with Behcet’s disease receiving tumor necrosis factor alpha inhibitory treatment

Anti-tumor necrosis factor alpha (TNF-α) inhibitors are effective in the treatment of various inflammatory rheumatic conditions. Increased risks of serious infections are the major issues concerning the long-term safety of these agents. We present a case of a young male Behcet’s patient whose disease was complicated by cytomegalovirus (CMV) colitis. Colitis started 10 d after the third Infliximab dose and responded to the cessation of TNF blocking treatment and administration of ganciclovir. Tumor necrosis factor alpha and interferon gamma act at several levels in combating viral infections.CMV infections should be kept in mind and included in the differential diagnosis of severe gastrointestinal symptoms in patients receiving anti-TNF agents.

Newcastle disease virus-based MERS-CoV candidate vaccine elicits high-level and lasting neutralizing antibodies in Bactrian camels

Middle East respiratory syndrome coronavirus （MERS-CoV）, a member of the Coronavifidae family, is the causative pathogen for MERS that is characterized by high fever, pneumonia, acute respiratory distress syndrome （ARDS）, as well as extrapul- monary manifestations. Currently, there are no approved treatment regimens or vaccines for MERS. Here~ we generated recombinant nonvirulent Newcastle disease virus （NDV） LaSota strain expressing MERS-CoV S protein （designated as rLa- MERS-S）, and evaluated its immunogenicity in mice and Bactrian camels. The results revealed that rLa-MERS-S showed similar growth properties to those of LaSota in embryonated chicken eggs, while animal immunization studies showed that rLa-MERS-S induced MERS-CoV neutralizing antibodies in mice and camels. Our findings suggest that recombinant rLa- MERS-S may be a potential MERS-CoV veterinary vaccine candidate for camels and other animals affected by MERS.

Inactivation of infectious bursal disease virus by binary ethylenimine and formalin

In this experiment conducted to study the inactivation dynamics of infectious bursal disease virus （IBDV） by binary ethylenimine （BEI） in comparison with formalin, IBDV was isolated from the bursa of infected chickens and its confirmation was done by agar gel precipitation test. Viral suspensions were subjected to inactivation with BEI and formalin for pre-set time in- tervals. BEI was employed at concentrations of 0.001 and 0.002 mol/L while formalin was used at 0.1% and 0.2%. Sampling was done at 6, 12, 24, 36 and 48 h of incubation and samples were tested for their inactivation status in 9-day-old embryonated eggs and 3-week-old broiler chickens. IBDV was completely inactivated by 0.001 and 0.002 mol/L BEI after 36 h of incubation at 37℃, whereas formalin at 0. 1% and 0.2% concentrations inactivated IBDV in 24 h.

Expression of putative zinc-finger protein lcn61 gene in lymphocystis disease virus China (LCDV-cn) genome

An open reading frame (lcn61) of lymphocystis disease virus China (LCDV-cn), probably responsible for encoding putative zinc-finger proteins was amplified and inserted into pET24a (+) vector. Then it expressed in E. coli BL21 (DE3), and His-tag fusion protein of high yield was obtained. It was found that the fusion protein existed in E. coli mainly as inclusion bodies. The bioinformatics analysis indicates that LCN61 is C2H2 type zinc-finger protein containing four C2H2 zinc-finger motifs. This work provides a theory for functional research of lcn61 gene.

Activity of T Cells Stimulated by Hemagglutinin-neuraminidase of Newcastle Disease Virus in vivo

To investigate the stimulated activity of T cells and the anti-tumor properties of hemagglutinin-neuraminidase（HN） of Newcastle disease virus（NDV） strain Changchun（NDVcc）, the expression of HN gene in hepatoma cells（human HepG-2 and mouse H22 cells） infected with the recombinant adenovirus（Ad-HN） was identified by Western blot analysis and flow cytometry. Sialidase activity of NDVcc HN expressed by Ad-HN was assayed by the periodate-resorcinol method. The in vivo anti-tumor effects of NDVcc HN were evaluated in the H22 solid tumor model. Regional lymph nodes of the mouse model treated with Ad-HN were removed to harvest T lymphocytes and evaluating the specific cytotoxicity of cytotoxic T lymphocyte（CTL） and natural killer（NK） cells by an L-lactate dehydrogenase（LDH） assay, in the mean time, the secretion of cytokines was analyzed by enzyme linked immunosorbent assays（ELISA）. The results show that NDVcc HN was effectively expressed by Ad-HN in HepG-2 and H22 cells. The sialidase activity assay showed that Ad-HN significantly reduced sialic acid level of the hepatoma cells compared with the cells infected the empty adenovirus vector（Ad-mock）. When treated with Ad-HN, the growth of subcutaneous H22 primary tumors in C57BL/6 mice was suppressed, and the mean mice survival increased. In addition, the treatment of Ad-HN elicited strong NK and CTL responses, and high levels of Th1 cytokines, such as IL-2 and IFN-γ. In conclusion, NDVcc HN effectively elicits T cell-mediate anti-tumor cytotoxicity via sialidase activity and may be a novel strategy for cancer immunotherapy.

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.

Ebola virus disease: From epidemiology to prophylaxis

The outbreak of Ebola virus disease(EVD) continues to spread through West Africa. Since the first reported EVD in March 2014, the number of cases has increased rapidly, with the fatality rate of >50%. The most prevalent Ebola virus belongs to the species of Zaire ebolavirus, with a mortality rate as high as 90%. Although there were introduced cases in other continents, Africa is the endemic area where fruit bats and apes are suspected to be Ebola virus carriers. The virus might be transmitted from the host animals to humans if humans consume relative raw and contaminated meats; however, human-to-human transmission via close contact is the major route of current outbreaks. EVD happens at any seasons and affected people of any race in any age groups. Direct contact with body fluids of EVD patients and living in the contaminated environment greatly increase the risk of being infected. Transmission viaaerosol is less possible but the transmission via droplet is possible in humans. Thus, health care providers are facing danger of getting Ebola virus infection. So far, there are limited vaccines, drugs and/or therapies to prevent Ebola virus infection or treat EVD. Medical workers should follow the current standard prophylactic procedures. Military forces can orchestrate efficient care to mass EVD casualties. Although it is necessary to speed up the pace of developing effective vaccine and therapeutics for the prevention and treatment of EVD, public health prophylaxis is the most important issue at present to control the spread of this disease cost-effectively.

Expression and Immunological Analysis of Capsid Protein Precursor of Swine Vesicular Disease Virus HK/70

VP1, a capsid protein of swine vesicular disease virus, was cloned from the SVDV HK/70 strain and inserted into retroviral vector pBABE puro, and expressed in PK15 cells by an retroviral expression system. The ability of the VP1 protein to induce an immune response was then evaluated in guinea pigs. Western blot and ELISA results indicated that the VP1 protein can be recognized by SVDV positive serum, Furthermore, anti-SVDV specific antibodies and lymphocyte proliferation were elicited and increased by VP1 protein after vaccination. These results encourage further work towards the development of a vaccine against SVDV infection.

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.