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1.
Protein & Cell ; (12): 360-378, 2022.
Article in English | WPRIM | ID: wpr-929162

ABSTRACT

Rice stripe virus (RSV) transmitted by the small brown planthopper causes severe rice yield losses in Asian countries. Although viral nuclear entry promotes viral replication in host cells, whether this phenomenon occurs in vector cells remains unknown. Therefore, in this study, we systematically evaluated the presence and roles of RSV in the nuclei of vector insect cells. We observed that the nucleocapsid protein (NP) and viral genomic RNAs were partially transported into vector cell nuclei by utilizing the importin α nuclear transport system. When blocking NP nuclear localization, cytoplasmic RSV accumulation significantly increased. In the vector cell nuclei, NP bound the transcription factor YY1 and affected its positive regulation to FAIM. Subsequently, decreased FAIM expression triggered an antiviral caspase-dependent apoptotic reaction. Our results reveal that viral nuclear entry induces completely different immune effects in vector and host cells, providing new insights into the balance between viral load and the immunity pressure in vector insects.


Subject(s)
Animals , Cell Nucleus , Hemiptera/metabolism , Insect Vectors/genetics , Insecta , Nucleocapsid Proteins/metabolism , Oryza , Plant Diseases , Tenuivirus/metabolism , Virus Replication
2.
Protein & Cell ; (12): 120-140, 2022.
Article in English | WPRIM | ID: wpr-929156

ABSTRACT

Ebola virus (EBOV) is an enveloped negative-sense RNA virus and a member of the filovirus family. Nucleoprotein (NP) expression alone leads to the formation of inclusion bodies (IBs), which are critical for viral RNA synthesis. The matrix protein, VP40, not only plays a critical role in virus assembly/budding, but also can regulate transcription and replication of the viral genome. However, the molecular mechanism by which VP40 regulates viral RNA synthesis and virion assembly/budding is unknown. Here, we show that within IBs the N-terminus of NP recruits VP40 and is required for VLP-containing NP release. Furthermore, we find four point mutations (L692A, P697A, P698A and W699A) within the C-terminal hydrophobic core of NP result in a stronger VP40-NP interaction within IBs, sequestering VP40 within IBs, reducing VP40-VLP egress, abolishing the incorporation of NC-like structures into VP40-VLP, and inhibiting viral RNA synthesis, suggesting that the interaction of N-terminus of NP with VP40 induces a conformational change in the C-terminus of NP. Consequently, the C-terminal hydrophobic core of NP is exposed and binds VP40, thereby inhibiting RNA synthesis and initiating virion assembly/budding.


Subject(s)
Ebolavirus/physiology , HEK293 Cells , HeLa Cells , Humans , Nucleocapsid Proteins/metabolism , RNA, Viral/metabolism , Viral Matrix Proteins/metabolism , Virion/metabolism , Virus Assembly
3.
Medicina (B.Aires) ; 80(2): 162-168, abr. 2020. ilus, graf
Article in Spanish | LILACS | ID: biblio-1125058

ABSTRACT

En abril de 2019, UNICEF denunció que más de 20 millones de niños en todo el mundo no habían sido vacunados y alertó sobre posibles brotes de sarampión que, por su alta contagiosidad, es la primera enfermedad en emerger entre las prevenibles mediante vacunación. De continuar el descenso en las vacunaciones, podrían reaparecer también pertussis, tétanos y otras enfermedades con menor requerimiento de cobertura para alcanzar protección poblacional. A fin de agosto de 2019 se inició en la Argentina el actual brote de sarampión. Este virus se transmite por vía respiratoria, infecta múltiples órganos e induce inmunosupresión. Su genoma consiste en ARN de cadena simple. La genotipificación se efectúa por secuenciación de un fragmento de 450 nucleótidos de la proteína N que contiene la mayor densidad de variación de nucleótidos del genoma. En Sudamérica circula el genotipo D8, y en Norteamérica hay, además, un 8% de genotipo B3. Cada persona con sarampión infecta, en promedio, otras 12-18 en una población susceptible. La vacunación confiere protección directa e indirecta, e induce tanto anticuerpos como inmunidad celular. Los recién nacidos tienen protección hasta los 6 meses por anticuerpos maternos transmitidos vía placentaria. En la Argentina, el Calendario de Vacunación incluye dos dosis de triple viral, a los 12 meses y a los 5 años, y una dosis cero (6- 11 meses de edad) en distritos con casos de enfermedad. Una dosis protege al 93% de los vacunados a los 12 meses y dos dosis al 97%, de por vida.


In April 2019, UNICEF denounced that more than 20 million children worldwide had not been vaccinated and alerted on possible outbreaks of measles which, due to the high transmissibility of this virus, is the first disease preventable by vaccination to emerge. If the decline in vaccinations continues, pertussis, tetanus and other diseases, which require less coverage to achieve population protection, may also reappear. In Argentina, the current outbreak began in late August 2019. Measles virus is transmitted by air, infects multiple organs, and is associated with immunosuppression. Its genome consists of single stranded RNA. Genotyping is carried out by sequencing a 450-nucleotide fragment of the N protein, which contains the highest density of nucleotide variation. In South America, D8 is the circulating genotype and in North America, B3 accounts for 8% of the cases. Each person with measles infects, on average, another 12-18 people in a susceptible population. Vaccination confers direct and indirect protection, and induces both antibodies and cellular immunity. Newborns are protected by maternal antibodies transmitted via the placenta, up to 6 months. In Argentina, the Vaccination Calendar includes two doses of triple viral vaccine, at 12 months and 5 years, and a zero dose (6- 11 months of age) in districts with disease cases. The protection conferred by the vaccine is 93% at 12 months with a dose, and with 2 doses 97% for life.


Subject(s)
Humans , Infant , Child, Preschool , History, 19th Century , Vaccination , Measles/prevention & control , Argentina/epidemiology , Viral Proteins , Disease Outbreaks , Nucleocapsid Proteins , Genotype , Measles/history , Measles/epidemiology , Measles/virology , Nucleoproteins
4.
Arq. bras. med. vet. zootec. (Online) ; 71(4): 1428-1432, jul.-ago. 2019. tab, ilus
Article in English | LILACS, VETINDEX | ID: biblio-1038620

ABSTRACT

A vacinação é a forma mais utilizada para prevenir a bronquite infecciosa causada pelo vírus da bronquite infecciosa das galinhas (IBV). Contudo, as vacinas convencionais são incapazes de diferenciar aves infectadas de vacinadas. No presente trabalho foi construído, caracterizado, e avaliado como candidato vacinal, um adenovírus recombinante expressando o gene N do IBV. O gene N foi clonado em um adenovírus humano tipo 5 defectivo e transfectado para as células HEK-293A para gerar rAd5_N. Após o vetor ser obtido como esperado e a confirmação da expressão da proteína N em HEK-293ª, foi realizada inoculação pela via oculo-nasal na dose de 10 7 TCID 50 /0,1mL para imunização de galinhas livres de patógenos específicos (SPF). A resposta imunológica do Ad5_N e a proteção contra o desafio ao IBV foram avaliadas e comparadas com uma vacina viva comercial. Não foram detectados anticorpos anti-IBV em aves vacinadas com o Ad5_N. A vacina comercial induziu anticorpos detectáveis a partir do 7º dia pós-vacinal. Em aves vacinadas com o Ad5_N não houve aumento na expressão de IFNγ. Neste estudo, o rAd5_N obtido não conferiu proteção contra desafio com IBV-M41. Os resultados indicam a necessidade de avaliar adenovírus recombinantes expressando outros genes do IBV.(AU)


Subject(s)
Animals , Vaccines, Synthetic , Chickens , Coronavirus Infections/prevention & control , Infectious bronchitis virus , Nucleoproteins , Nucleocapsid Proteins
5.
Article in English | WPRIM | ID: wpr-739633

ABSTRACT

To develop the large scale serological assay for severe fever with thrombocytopenia syndrome virus (SFTSV) infection, we evaluated two different enzyme-linked immunosorbent assay (ELISA) methods using nucleocapsid protein (NP) and Gn proteins of CB1 (genotype B) SFTSV strains. The NP-based ELISA tests showed more sensitive with broad cross-reactivity between two different genotype A and B strains compared with those of Gn-based ELISA tests. However, Gn-based ELISA showed more genotype specificity and specificity. These result suggested that NP-based ELISA test could be applicable for general sero-prevalence studies of SFTSV infections, while Gn-based ELISA could be applicable for a certain specific genotype sero-prevalence study.


Subject(s)
Diagnosis , Enzyme-Linked Immunosorbent Assay , Fever , Genotype , Nucleocapsid Proteins , Sensitivity and Specificity , Thrombocytopenia
6.
Article in English | WPRIM | ID: wpr-758832

ABSTRACT

Porcine reproductive and respiratory syndrome (PRRS) is recognized as one of the most important infectious diseases causing serious economic loss in the swine industry worldwide. Due to its increasing genetic diversity, a rapid and accurate diagnosis is critical for PRRS control. The immunochromatographic strip test (ICST) is a rapid and convenient type of immunoassay. In this study, an on-site immunochromatographic assay-based diagnostic method was developed for detection of PRRS virus (PRRSV)-specific antibodies. The method utilized colloidal gold nanoparticle-labeled dual-type nucleocapsid proteins encoded by open reading frame 7. We evaluated 991 field samples from pig farms and 66 serum samples from experimentally PRRSV-inoculated pigs. Based on true PRRSV-specific antibody-positive or


Subject(s)
Agriculture , Antibodies , Colloids , Communicable Diseases , Diagnosis , Enzyme-Linked Immunosorbent Assay , Fluorescent Antibody Technique , Genetic Variation , Gold Colloid , Immunoassay , Chromatography, Affinity , Immunoglobulin M , Methods , Nucleocapsid Proteins , Open Reading Frames , Porcine Reproductive and Respiratory Syndrome , Porcine respiratory and reproductive syndrome virus , Sensitivity and Specificity , Swine
7.
Article in English | WPRIM | ID: wpr-772765

ABSTRACT

Porcine epidemic diarrhea virus (PEDV) is a highly infectious pathogen that can cause severe diseases in pigs and result in enormous economic losses in the worldwide swine industry. Previous studies revealed that PEDV exhibits an obvious capacity for modulating interferon (IFN) signaling or expression. The newly discovered type III IFN, which plays a crucial role in antiviral immunity, has strong antiviral activity against PEDV proliferation in IPEC-J2 cells. In this study, we aimed to investigate the effect of PEDV nucleocapsid (N) protein on type III IFN-λ. We found that the N proteins of ten PEDV strains isolated between 2013 and 2017 from different local farms shared high nucleotide identities, while the N protein of the CV777 vaccine strain formed a monophyletic branch in the phylogenetic tree. The N protein of the epidemic strain could antagonize type III IFN, but not type I or type II IFN expression induced by polyinosinic-polycytidylic acid (poly(I:C)) in IPEC-J2 cells. Subsequently, we demonstrated that the inhibition of poly(I:C)-induced IFN-λ3 production by PEDV N protein was dependent on the blocking of nuclear factor-κB (NF-κB) nuclear translocation. These findings might help increase understanding of the pathogenesis of PEDV and its mechanisms for evading the host immune response.


Subject(s)
Active Transport, Cell Nucleus , Animals , Coronavirus Infections , Allergy and Immunology , Virology , Genes, Viral , Host-Pathogen Interactions , Allergy and Immunology , Interferons , Genetics , Interleukins , Genetics , NF-kappa B , Metabolism , Nucleocapsid Proteins , Genetics , Allergy and Immunology , Physiology , Porcine epidemic diarrhea virus , Genetics , Virulence , Physiology , Promoter Regions, Genetic , Swine , Swine Diseases , Allergy and Immunology , Virology
8.
Article in English | WPRIM | ID: wpr-65506

ABSTRACT

Canine respiratory coronavirus (CRCoV) is commonly associated with canine kennel cough worldwide. Clinically infected dogs present coughing, sneezing, and nasal discharge. Severe infections may progress to pneumonia. Through serological surveys, CRCoV has been identified as a worldwide pathogen found in the respiratory tracts of dogs suffering from mild or severe respiratory disease. In this study, three dogs were obtained from a dog kennel. Over the previous 5 days, the dogs showed coughing, sneezing, and nasal discharge. To detect the etiologic pathogen, we performed multiplex RT-PCR (mRT-PCR) to amplify the genes encoding canine influenza virus matrix protein, canine distemper virus nucleocapsid protein, and CRCoV spike protein. Dot blotting was achieved with a CRCoV-specific probe. Nasal-secreting CRCoV was detected by the 442 bp CRCoV-positive PCR reaction in the nasal swabbing samples from dogs. Further, CRCoV-positive reactions by dot blot hybridization were detected in the nasal swabbing samples from dogs. In conclusion, we detected CRCoV in kenneled dogs with respiratory disease in Korea. Multiplex RT-PCR was able to detect successfully CRCoV infection in dogs. We suggest that mRT-PCR would be useful and effective for monitoring CRCoV infection in various kinds of dogs.


Subject(s)
Animals , Coronavirus , Cough , Distemper Virus, Canine , Dogs , Korea , Nucleocapsid Proteins , Orthomyxoviridae , Pneumonia , Polymerase Chain Reaction , Respiratory System , Sneezing
9.
Chinese Journal of Virology ; (6): 387-390, 2014.
Article in Chinese | WPRIM | ID: wpr-280355

ABSTRACT

This article aimed to study the antigenicity of nucleocapsid proteins (NPs) in six pathogenic phleboviruses and to provide theoretical evidence for the development of serological diagnostic reagents. NPs of six pathogenic phleboviruses were expressed and purified using a prokaryotic expression system and rabbits were immunized with individual recombinant NPs. Cross-reactions among NPs and rabbit sera were determined by both indirect ELISA and Western blotting analyses, and the sera titer was determined by indirect ELISA. Furthermore, sera from SFTS patients were also detected by each recombinant NP as a coating antigen using indirect ELISA. The cross-reactions and the sera titer were subsequently determined. Both the concentration and purity of recombinant NPs of six pathogenic phleboviruses met the standards for immunization and detection. The results of indirect ELISA and Western blotting showed that each anti-phlebovirus NP rabbit immune serum had potential serological cross-reactivity with the other five virus NP antigens. Furthermore, the sera from SFTS patients also had cross-reactivity with the other five NP antigens to a certain extent. Our preliminary study evaluated the antigenicity and immune reactivity of six pathogenic phleboviruses NPs and laid the foundation for the development of diagnostic reagents.


Subject(s)
Animals , Antibodies, Viral , Allergy and Immunology , Antigens, Viral , Genetics , Allergy and Immunology , Cross Reactions , Humans , Nucleocapsid Proteins , Genetics , Allergy and Immunology , Phlebotomus Fever , Diagnosis , Allergy and Immunology , Virology , Phlebovirus , Classification , Genetics , Allergy and Immunology , Rabbits
10.
Braz. j. microbiol ; 44(3): 879-882, July-Sept. 2013. tab
Article in English | LILACS | ID: lil-699783

ABSTRACT

Rabies is a zoonotic disease that affects all mammals and leads to more than 55,000 human deaths every year, caused by rabies virus (RABV) (Mononegavirales: Rhabdoviridae: Lyssavirus). Currently, human rabies treatment is based on the Milwaukee Protocol which consists on the induction of coma and massive antiviral therapy. The aim of this study was to assess the decrease in the titer of rabies virus both in vitro and in vivo using short-interfering RNAs. To this end, three siRNAs were used with antisense strands complementary to rabies virus nucleoprotein (N) mRNA. BHK-21 cells monolayers were infected with 1000 to 0.1 TCID50 of PV and after 2 hours the cells were transfected with each of tree RNAs in separate using Lipofectamine-2000. All three siRNAs reduced the titer of PV strain in a least 0.72 logTCID50/mL and no cytotoxic effect was observed in the monolayers treated with Lipofectamine-2000. Swiss albino mice infected with 10.000 to 1 LD of PV strain by the intracerebral route were also transfected after two hours of infection with a pool 3 siRNAs with Lipofectamine-2000 by the intracerebral route, resulting in a survival rate of 30% in mice inoculated with 100 LD50, while the same dose led to 100% mortality in untreated animals. Lipofectamine-2000 showed no toxic effect in control mice. These results suggest that intracerebral administration of siRNAs might be an effective antiviral strategy for rabies.


Subject(s)
Animals , Cricetinae , Mice , Antiviral Agents/metabolism , RNA Interference , RNA, Small Interfering/metabolism , Rabies virus/drug effects , Rabies virus/physiology , Rabies/drug therapy , Virus Replication/drug effects , Cell Line , Disease Models, Animal , Nucleocapsid Proteins/antagonists & inhibitors , RNA, Small Interfering/genetics , Survival Analysis , Viral Load , Virus Cultivation
11.
Article in English | WPRIM | ID: wpr-320304

ABSTRACT

<p><b>OBJECTIVE</b>To investigate the virulence characteristics of two fixed strains (CTN and aG) and a street strain (HN10) of rabies viruses isolated in China.</p><p><b>METHODS</b>ICR mice of different age groups were inoculated with CTN, aG and HN10 rabies virus strains via the intracracerebral (i.c.) or intramuscular (i.m.) routes, and observed for 20 days.</p><p><b>RESULTS</b>The CTN strain was pathogenic to 7-day-old suckling mice that received i.c. inoculations and 3-day-old suckling mice that received i.m. inoculations. The aG strain was pathogenic to 4-week-old mice that received i.c. inoculations and 7-day-old suckling mice that received i.m. inoculations. The HN10 strain was pathogenic to mice of all age groups via both inoculation routes. In moribund mice, the viruses had spread to most regions of the brain. The CTN and HN10 strains had similar dissemination patterns in the brain; both viral antigens could be found in the dentate gyrus (DG), whereas few viral antigens were present in the DG from specimens that had been infected with the aG strain.</p><p><b>CONCLUSION</b>A comprehensive sequence analysis of the G protein suggested that differences in gene sequences may be responsible for producing strain-specific differences in pathogenicity and distribution in the brain.</p>


Subject(s)
Animals , Antigens, Viral , Allergy and Immunology , Brain , Allergy and Immunology , Virology , China , Mice , Mice, Inbred ICR , Nucleocapsid Proteins , Allergy and Immunology , Rabies , Virology , Rabies virus , Allergy and Immunology , Virulence
12.
Protein & Cell ; (12): 445-455, 2013.
Article in English | WPRIM | ID: wpr-757792

ABSTRACT

Severe fever with thrombocytopenia syndrome virus (SFTSV), a member of the Phlebovirus genus from the Bunyaviridae family endemic to China, is the causative agent of life-threatening severe fever with thrombocytopenia syndrome (SFTS), which features high fever and hemorrhage. Similar to other negative-sense RNA viruses, SFTSV encodes a nucleocapsid protein (NP) that is essential for viral replication. NP facilitates viral RNA encapsidation and is responsible for the formation of ribonucleoprotein complex. However, recent studies have indicated that NP from Phlebovirus members behaves in inhomogeneous oligomerization states. In the present study, we report the crystal structure of SFTSV NP at 2.8 Å resolution and demonstrate the mechanism by which it processes a ringshaped hexameric form to accomplish RNA encapsidation. Key residues essential for oligomerization are identified through mutational analysis and identified to have a significant impact on RNA binding, which suggests that correct formation of highly ordered oligomers is a critical step in RNA encapsidation. The findings of this work provide new insights into the discovery of new antiviral reagents for Phlebovirus infection.


Subject(s)
Binding Sites , Crystallography, X-Ray , Mutation , Nucleocapsid Proteins , Chemistry , Genetics , Metabolism , Phlebovirus , Metabolism , Protein Binding , Protein Multimerization , Protein Structure, Quaternary , RNA, Viral , Metabolism , Recombinant Proteins , Chemistry , Genetics
13.
Chinese Journal of Virology ; (6): 63-66, 2012.
Article in Chinese | WPRIM | ID: wpr-354770

ABSTRACT

To clarify the pathogenesis of Duck enteritis virus (DEV), the cDNA library of duck's liver infected by DEV and a bait plasmid containing DEV nucleocapsid protein (NP) gene were constructed, then the receptor was screened from the cDNA library plasmid by the yeast two-hybrid system and verified by GST pull-down test. The results showed that the capacity of the primary cDNA library was 1 x 106 CFU with insertion size from 0.5 to 1 kb, and the bait plasmid of pGBKT7-NP showed no self-activation. The receptor reacting with DEV NP in duck liver was initially confirmed as the protein kinase C inhibitor (PKCI). These results provide new clues for further investigation on pathogenesis of DEV.


Subject(s)
Alphaherpesvirinae , Virulence , Animals , Ducks , Virology , Gene Library , Liver , Virology , Nucleocapsid Proteins , Genetics , Plasmids , Receptors, Virus , Two-Hybrid System Techniques
14.
Protein & Cell ; (12): 893-902, 2012.
Article in English | WPRIM | ID: wpr-757847

ABSTRACT

Nucleocapsid protein (NPs) of negative-sense single-stranded RNA (-ssRNA) viruses function in different stages of viral replication, transcription, and maturation. Structural investigations show that -ssRNA viruses that encode NPs preliminarily serve as structural building blocks that encapsidate and protect the viral genomic RNA and mediate the interaction between genomic RNA and RNA-dependent RNA polymerase. However, recent structural results have revealed other biological functions of -ssRNA viruses that extend our understanding of the versatile roles of virally encoded NPs.


Subject(s)
Animals , Capsid , Metabolism , Humans , Lassa virus , Chemistry , Physiology , Nucleocapsid Proteins , Chemistry , Metabolism , Orthobunyavirus , Chemistry , Physiology , RNA Viruses , Chemistry , Physiology
15.
Article in Chinese | WPRIM | ID: wpr-246187

ABSTRACT

<p><b>OBJECTIVE</b>To determine the antigen characteristics of different fragments of SARS-CoV N protein expressed in E. Coli and their application in the serological diagnosis.</p><p><b>METHODS</b>Based on preliminary analysis of 39 different segments of the N protein, We choosed six purified N protein for further antigenicity characterization in this study, including that PN360 (1 -360aa), PN301 (1-301aa), PN199 (30-228aa), PN185 (30-214aa), PN155b (60-214aa), and PN125 (90-214aa). We developed Western-Bolt and ELISA to detect antibody reactivity between truncated N fragments with sera from SARS-CoV-negative normal adults or SARS-CoV patient convalescent sera.</p><p><b>RESULTS</b>Western-Bolt results show that all the six fragments have reacted with the SARS patient convalescent sera, but the PN360 and PN301 showed obvious cross-reaction with sera from SARS-CoV-negative normal adults; sensitivity analysis using an ELISA coating with PN199, PN185, PN155b, PN125 as antigen showed that the PN185 and PN155b are better than PN125.</p><p><b>CONCLUSION</b>Truncated N protein PN185 and PN155b expressed in E. Coli are better antigen candidates used for detection of SARS-CoV specific antibody.</p>


Subject(s)
Antibodies, Viral , Blood , Blotting, Western , Enzyme-Linked Immunosorbent Assay , Escherichia coli , Genetics , Humans , Nucleocapsid Proteins , Allergy and Immunology , Peptide Fragments , Allergy and Immunology , Recombinant Proteins , Allergy and Immunology , Serologic Tests , Severe Acute Respiratory Syndrome , Diagnosis
16.
Article in English | WPRIM | ID: wpr-145342

ABSTRACT

Rabies is a major zoonotic disease that causes approximately 55,000 human deaths worldwide on an annual basis. The nucleocapsid protein and glycoprotein genes of the Korean rabies virus (RABV) have been subjected to molecular and phylogenetic analyses. Although the phosphoprotein (P) has several important functions in viral infection and pathogenicity, the genetic characterizations of the P of Korean RABV isolates have not yet been established. In the present study, we conducted genetic analyses of P genes of 24 RABV isolates circulating in the Republic of Korea (hereafter, Korea) from 2008 to 2011. This study revealed that the P genes of Korean RABVs are genetically similar to those of RABV strains of lyssavirus genotype I including V739 (dogs, Korea), NNV-RAB-H (humans, India), NeiMeng925 (raccoon dogs, China), and RU9.RD (raccoon dogs, Russia). Among Korean isolates, the RABV P genes showed low variability in the variable domains among Korean isolates; they had specific consensus sequences and amino acid substitutions capable of identifying geographic characteristics and retained specific sequences thought to be important for viral function. These results provide important genetic characteristics and epidemiological information pertaining to the P gene of the Korean RABV.


Subject(s)
Amino Acid Substitution , Animals , Consensus Sequence , Dogs , Genotype , Glycoproteins , Humans , Korea , Lyssavirus , Molecular Epidemiology , Nucleocapsid Proteins , Rabies , Rabies virus , Republic of Korea
17.
Article in English | WPRIM | ID: wpr-65165

ABSTRACT

This study measured the clinical prevalence of peste des petits ruminants (PPR) among sheep and goats in India between 2003 and 2009 by analyzing clinical samples from suspected cases of PPR that were submitted to the Rinderpest and Allied Disease Laboratory, Division of Virology, IVRI, Mukteswar for PPR diagnosis. PPR outbreaks were confirmed by detecting PPR virus (PPRV)-specific antigen in the clinical samples. Clinical samples (blood, nasal swabs, spleen, lymph node, kidney, liver, intestine, and pooled tissue materials) were taken from a total of 592 sheep and 912 goats in different states of India and screened for the presence of PPRV antigen using a monoclonal antibody-based sandwich ELISA kit. A total of 20, 38, and 11 laboratory-confirmed PPR outbreaks occurred among sheep, goat, and combined sheep and goat populations, respectively. Our findings provide evidence of widespread PPR endemicity in India. The underlying reasons could be variations in husbandry practices in different geographical regions, agro-climatic conditions, and livestock migration. Furthermore, decrease in the number of PPR outbreaks over time might be due to the effectiveness of current live PPR vaccines and timely vaccination of target species. Vaccination against PPR has been practiced in India since 2002 to control this disease.


Subject(s)
Animals , Antibodies, Monoclonal/immunology , Antigens, Viral/blood , Disease Outbreaks/veterinary , Enzyme-Linked Immunosorbent Assay/veterinary , Goat Diseases/epidemiology , Goats , India/epidemiology , Nucleocapsid Proteins/immunology , Peste-des-Petits-Ruminants/epidemiology , Peste-des-petits-ruminants virus/immunology , Prevalence , Risk Factors , Seasons , Sheep , Sheep Diseases/epidemiology , Vaccination/veterinary , Viral Vaccines/immunology
18.
Chinese Journal of Virology ; (6): 244-249, 2011.
Article in Chinese | WPRIM | ID: wpr-286047

ABSTRACT

Prokaryotic expression plasmids carrying N-terminal(1-163aa) and C-terminal(141-306aa) gene of HCoV-NL63 nucleocapsid protein were constructed with pET-30a(+) vector. Consequently, we prepared two purified proteins, Np and Cp, respectively, and established a Western blotting-based line assay (WBLA) for detection of antibodies against HCoV-NL63 using three purified proteins: Np , Cp and Nf, a full-length HCoV-NL63 nucleocapsid protein as previously reported. We detected anti-HCoV-NL63 antibodies among 50 sera samples collected from adult for health-examination by WBLA. The results showed that: 25 (50%), 27 (54%), 36 (72%) of 50 sera were indentified as anti-HCoV-NL63 antibody positive when the antigen was from Nf, Np and Cp, respectively. Among these sera with positive anti-HCoV-NL63 antibody,Cp showed highest antibody positive rate in WBLA,and consistent rates of detection were 64% between Nf and Np, 54% between Nf and Cp, 54% between Np and Cp. Our study provides the foundation for development of HCoV-NL63 serological detection reagents and an experimental tool for immunological research of HCoV-NL63 infection.


Subject(s)
Adult , Antibodies, Viral , Blood , Blotting, Western , Coronavirus , Chemistry , Allergy and Immunology , Humans , Nucleocapsid Proteins , Genetics , Allergy and Immunology , Peptide Fragments , Genetics , Recombinant Proteins , Allergy and Immunology , Serologic Tests
19.
Article in English | WPRIM | ID: wpr-306842

ABSTRACT

<p><b>OBJECTIVE</b>To investigate the relationship between the molecular characteristics and phylogenetic evolution of rabies N gene.</p><p><b>METHODS</b>Saliva samples were collected from rabies cases, and RT-PCR was used to amplify the N gene of rabies virus with the specific primers. The amplifying product of RT-PCR was cloned to pUCm-T vector and transformed into E.coli XL1-Blue and then the blue-white selection, PCR screening and gene sequencing were carried out to identify the positive clones. Finally, ExPASy and other bioinformatics software were used to analyze and predict the structure and biological characteristics of the N genome.</p><p><b>RESULTS</b>The amplification product of RT-PCR was 1 353 bp, the recombinant plasmid pUCm-T/N was constructed, the whole length of the N gene open reading frame was composed of 1 353 nucleotide residues to code 450 amino acids (20 kinds), the accession number submitted to the Genbank was HM756692, its sequence homology of nucleotides and amino acids compared with the vaccine strain CTN-1-V was 90% and 99% respectively. The evolutionary analysis showed that the isolated strain belonged to genotype I with certain geographic regionality.</p><p><b>CONCLUSION</b>The characteristics investigation and bioinformatics analysis of Hunan0806 N gene will provide fundamental data to reveal the significance of the N gene characteristics for rabies epidemiology and its prevention & control.</p>


Subject(s)
Amino Acid Sequence , Gene Expression Regulation, Viral , Physiology , Humans , Models, Molecular , Molecular Sequence Data , Nucleocapsid Proteins , Genetics , Metabolism , Phylogeny , Protein Conformation , Rabies , Virology , Rabies virus , Genetics , Metabolism , Saliva , Virology
20.
Chinese Journal of Biotechnology ; (12): 1149-1157, 2011.
Article in Chinese | WPRIM | ID: wpr-324477

ABSTRACT

To evaluate the effectiveness of rabies vaccination, we developed the SPA-ELISA method to detect the antibodies against rabies virus (RV) using the main antigenic determinant of nucleoprotein (RV N1) as antigen. The complete Nucleoprotein (N) gene and the partial N1 gene (1 000-1 353 bp) of RV Flury LEP strain were amplified using RT-PCR and PCR approaches. The two fragments were inserted into pGEX-6P-1 respectively. Then we transformed the recombinant plasmids into Escherichia coli BL21(DE3) strain and expressed them by adding 1 mmol/L of IPTG (isopropyl-beta-D-thiogalactopyranoside). SDS-PAGE analysis showed that both of the two recombinant proteins were presented as inclusion bodies. Compared with the complete nucleoprotein, the partial protein (RV N1) was expressed at a much higher level in E. coli BL21(DE3). The antigenic specificity of the partial N1 protein was confirmed by Western blotting. By coating the plates with purified RV N1 as an antigen, an SPA-ELISA method for the detection of the antibodies against RV was established. By optimizing this method, the optimal concentration of RV N1 coating the ELISA plate was 2 mg/L. The optimal concentration of serum samples and SPA-HRP was 1:100 and 1:4 000 respectively. Compared with a commercially available ELISA kit coating RV as antigen, the coincidence rate of SPA-ELISA was 94.1%. Our results show that the developed SPA-ELISA based on the RV N1 was useful for the detection of the antibodies against RV in the sera of dogs.


Subject(s)
Animals , Antibodies, Viral , Allergy and Immunology , Dogs , Enzyme-Linked Immunosorbent Assay , Methods , Epitopes , Allergy and Immunology , Escherichia coli , Genetics , Genetic Vectors , Genetics , Nucleocapsid Proteins , Allergy and Immunology , Rabies virus , Allergy and Immunology , Recombinant Proteins , Genetics , Allergy and Immunology , Staphylococcal Protein A
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