RESUMO
Live attenuated vaccines have been used for control of the disease caused by goose parvovirus (GPV), but the mechanism involved in attenuation of GPV remains elusive. This report presents the complete nucleotide sequences of two live attenuated strains of GPV (82-0321V and VG32/1) that were independently developed in Taiwan and Europe, together with the parental strain of 82-0321V and a field strain isolated in Taiwan in 2006. Sequence comparisons showed that 82-0321V and VG32/1 had multiple deletions and substitutions in the inverted terminal repeats region when compared with their parental strain or the field virus, but these changes did not affect the formation of the hairpin structure essential for viral replication. Moreover, 82-0321V and VG32/1 had five amino acid changes in the non-structural protein, but these changes were located at positions distant from known functional motifs in the non-structural protein. In contrast, 82-0321V had nine changes and VG32/1 had 11 changes in their capsid proteins (VP1), and the majority of these changes occurred at positions close to the putative receptor binding sites of VP1, as predicted using the structure of adeno-associated virus 2 as the model system. Taken together, the results suggest that changes in sequence near the receptor binding sites of VP1 might be responsible for attenuation of GPV. This is the first report of complete nucleotide sequences of GPV other than the virulent B strain, and suggests a possible mechanism for attenuation of GPV.
Assuntos
Gansos/virologia , Parvovirus/genética , Vacinas Virais/imunologia , Sequência de Aminoácidos , Animais , Ásia , Sequência de Bases , Proteínas do Capsídeo/química , Proteínas do Capsídeo/genética , Europa (Continente) , Genoma Viral , Mutação , Proteínas não Estruturais Virais/química , Proteínas não Estruturais Virais/genéticaRESUMO
The method of reverse transcription (RT) followed by the polymerase chain reaction (PCR) was used to amplify two different fragments of the infectious bursal disease virus (IBDV) genome. Two sets of primer framed two different regions within the genes coding for proteins VP2 and VP3, respectively. Both sequences were detected in five strains of IBDV, whereas, none were obtained from uninfected control cells. The sensitivity of RT-PCR was carried out on nucleic acids from the IBDV infected cell cultures. The detection limit was 10(0) to 10(-1) TCID50 in ethidium bromide stained gels and could be enhanced further to 10(-1) to 10(-3) TCID50 by hybridization after southern transfer. In addition, detection of IBDV infection in 12 out of 14 bursal specimens examined by this technique was shown to be entirely consistent with the clinical history and an alternative diagnostic method. The speed, sensitivity, and specificity of this method is relevant for the diagnosis of infection with IBDV.
Assuntos
Vírus da Doença Infecciosa da Bursa/genética , Reação em Cadeia da Polimerase/métodos , Virologia/métodos , Animais , Sequência de Bases , Galinhas , Sondas de DNA , DNA Viral/genética , Estudos de Avaliação como Assunto , Vírus da Doença Infecciosa da Bursa/isolamento & purificação , Dados de Sequência Molecular , Reação em Cadeia da Polimerase/estatística & dados numéricos , Doenças das Aves Domésticas/diagnóstico , Infecções por Reoviridae/diagnóstico , Infecções por Reoviridae/veterinária , Sensibilidade e EspecificidadeRESUMO
The double-stranded RNA genome segment S3 of avian reovirus (ARV) S1133 was cloned following polyadenylation of both strands and cDNA synthesis of S3 RNA. The complete segment S3 nucleotide sequence was determined. S3 is 1196 base pairs long with one long open reading frame (ORF). The ORF possesses the AUG initiation codon in an optimum context for translation and starts at the first initiation codon (residue 24) and extends for 367 codons, sufficient to encode a protein of the same size as the known S3 gene product, protein sigmaB, one of the major outer capsid proteins of avian reovirus (Mr 41471). Protein sigmaB was subsequently expressed in Escherichia coli. The expressed protein sigmaB was indistinguishable from virion protein sigmaB as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, immunoblot assay, and N-terminal amino acid sequencing of several peptides generated by Staphyloccus aureus V8 protease digestion. ARV S3 genome segment possesses a pentanucleotide UCAUC at the 3'-terminus of its plus strand. The pentanucleotide sequence is common to the other genome segment S1 of ARV and to ten genome segments of mammalian reovirus at the 3'-terminus of their plus strands. Amino acid sequence analysis revealed that ARV sigmaB does not contain a repeated basic amino acid motif as do the three serotypes of mammalian reovirus. The results of amino acid sequencing suggest that the most susceptible cleavage sites of sigmaB to V8 protease are located in a hydrophilic area between amino acids 95 and 140.
Assuntos
Proteínas do Capsídeo , Capsídeo/genética , Orthoreovirus/genética , RNA de Cadeia Dupla/genética , RNA Viral/genética , Proteínas de Ligação a RNA , Animais , Capsídeo/química , Embrião de Galinha , Clonagem Molecular , Escherichia coli/genética , Genes Virais/genética , Dados de Sequência Molecular , Peso Molecular , Fases de Leitura Aberta/genética , Proteínas Recombinantes de Fusão , Análise de Sequência , Análise de Sequência de DNA , Homologia de Sequência de AminoácidosRESUMO
Liver tissues from animals that were suspected to have died of rabbit hemorrhagic disease (RHD) were used for isolation and characterization of the causative agent. Three strains of RHD virus were isolated as the supernatants of liver homogenates reacted positively by hemagglutination (HA) assays and were infective for rabbits after second passage in animals. Following extraction of liver homogenates from animals infected with each of three isolates, each virus strain was purified by CsCl density gradient ultracentrifugation for further characterization. In negative-stained preparations, the purified virions were icosahedral, measured approximately 40 nm in diameter, and were without an envelope. Morphologically, the three isolates were identical. By immunoblotting, a protein with a molecular weight of 60,000 was identified as the major structural protein in each isolate. Furthermore, two sets of primer framed two different regions within RHD virus genome and could amplify two fragments of the expected size, respectively, from each isolate, whereas, none were obtained from uninfected control samples. The identity of the amplified products was confirmed further using different restriction endonucleases. Among three isolates of RHD virus, neither protein migration patterns of the virions nor cleavage patterns of the amplified product by restriction enzymes were found to differ.
Assuntos
Vírus da Doença Hemorrágica de Coelhos/classificação , Vírus da Doença Hemorrágica de Coelhos/isolamento & purificação , Animais , Anticorpos Antivirais/sangue , DNA Viral/genética , Eletroforese em Gel de Ágar , Eletroforese em Gel de Poliacrilamida , Testes de Hemaglutinação , Vírus da Doença Hemorrágica de Coelhos/genética , Vírus da Doença Hemorrágica de Coelhos/imunologia , Vírus da Doença Hemorrágica de Coelhos/ultraestrutura , Immunoblotting , Fígado/virologia , Microscopia Eletrônica , Reação em Cadeia da Polimerase/métodos , Coelhos , Mapeamento por Restrição , Taiwan , Proteínas Virais/análise , Proteínas Virais/imunologiaRESUMO
Nine isolates of avian reovirus (ARV) from both healthy birds and birds with different clinical illness and one commercially available vaccine strain were selected and characterized by analysis of the migration pattern of their genomic double-stranded RNA (dsRNA) segments following separation by polyacrylamide gel electrophoresis. Different electropherotypes were observed and analyzed. The results show that the dsRNA segments of ARV were markedly polymorphic among isolates within the same serotype as well as among different serotypes. The results also show no correlation between electropherotype and disease state.
Assuntos
Genoma Viral , Doenças das Aves Domésticas/virologia , RNA de Cadeia Dupla/análise , RNA Viral/análise , Infecções por Reoviridae/veterinária , Reoviridae/genética , Animais , Galinhas/virologia , Codorniz/virologia , Reoviridae/classificação , Reoviridae/isolamento & purificação , Infecções por Reoviridae/virologiaRESUMO
Avian influenza viruses have 15 different hemagglutinin (HA) subtypes (H1-H15). We report a procedure for the identification and HA-subtyping of avian influenza virus by reverse transcription-PCR (RT-PCR). The avian influenza virus is identified by RT-PCR using a set of primers specific to the nucleoprotein (NP) gene of avian influenza virus. The HA-subtypes of avian influenza virus were determined by running simultaneously 15 RT-PCR reactions, each using a set of primers specific to one HA-subtype. For a single virus strain or isolate, only one of the 15 RT-PCR reactions will give a product of expected size, and thus the HA-subtype of the virus is determined. The result of HA-subtyping was then confirmed by sequence analysis of the PCR product. A total of 80 strains or isolates of avian influenza viruses were subtyped by this RT-PCR procedure, and the result of RT-PCR gave an excellent (100%) correlation with the result of the conventional serological method. The RT-PCR procedure we developed is rapid and sensitive, and could be used for the identification and HA-subtyping of avian influenza virus in organ homogenates.
Assuntos
Aves/virologia , Vírus da Influenza A/classificação , Vírus da Influenza A/genética , Animais , Primers do DNA/genética , Glicoproteínas de Hemaglutininação de Vírus da Influenza/genética , Humanos , Vírus da Influenza A/isolamento & purificação , Influenza Humana/virologia , Nucleoproteínas/genética , RNA Viral/genética , RNA Viral/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Sensibilidade e EspecificidadeRESUMO
A procedure was developed for differentiation of vaccine strains and field isolates of infectious laryngotracheitis virus (ILTV) by restriction fragment length polymorphism (RFLP) of DNA fragments amplified from the genome of ILTV by polymerase chain reaction (PCR). RFLP patterns of viral thymidine kinase (TK) gene, glycoprotein C (gC) gene, glycoprotein X (gX) gene and ICP4 gene amplified from different ILT viruses were compared. The results showed that the vaccine strain of tissue-culture-origin (TCO) could be readily distinguished from other ILT viruses. Moreover, two out of the four field isolates could be differentiated from vaccine strains of chicken embryo origin (CEO); but the remaining two field isolates were identical to the CEO vaccine strains. These results suggested that both vaccine-like and vaccine-unlike ILT viruses were involved in the field outbreak of this disease, and that the PCR/RFLP procedure could serve as a fast and sensitive method for the detection and differentiation of vaccine strains and field isolates of ILT viruses.
Assuntos
Alphaherpesvirinae/genética , Infecções por Herpesviridae/veterinária , Polimorfismo de Fragmento de Restrição , Doenças das Aves Domésticas/diagnóstico , Vacinas Virais/genética , Alphaherpesvirinae/imunologia , Alphaherpesvirinae/isolamento & purificação , Animais , Galinhas , DNA Viral/análise , Surtos de Doenças , Genes Virais/genética , Infecções por Herpesviridae/diagnóstico , Reação em Cadeia da Polimerase/métodos , Sensibilidade e EspecificidadeRESUMO
Two major outbreaks of parvovirus infection occurred in domestic waterfowls in Taiwan in the last two decades; the first was in 1982 and the second in 1989/1990. Parvoviruses isolated in the two outbreaks were sequenced between nucleotides 142 and 680 of the VP3 gene. Sequence comparisons reveal that these viruses could be divided into two groups respectively related to goose parvovirus (GPV) and Muscovy duck parvovirus (MDPV). Nucleotide differences between ''GPV'' and ''MDPV'' groups range from 16.2% to 19.4%. In comparison, the differences within the ''GPV'' group are only 0-6.5%, while those within the ''MDPV'' group are only 0.2-1.7%. Phylogenetic analysis reveals that parvoviruses isolated in the 1982 outbreak in Taiwan are all GPV-related, whereas those isolated in the 1989/1990 outbreak are all MDPV-related. GPV-related isolates from Taiwan were separated into two groups, Thai group and European group. In comparison, all MDPV-related isolates from Taiwan are clustered in a single group that is closely related to a French MDPV isolate. The MDPV-related infection in Taiwan occurred at almost the same time in 1989 as the MDPV outbreak in France. The close phylogenetic relationship between the ''MDPV'' Taiwanese isolates and the French MDPV isolate exhibited on the VP3 fragment investigated suggests that they should be compared more deeply, to look for a possible common origin. The MDPV-related 1985 isolate might be a candidate.
RESUMO
Portions of the hemagglutinin neuraminidase (HN) gene of Newcastle disease virus (NDV) isolates from two recent outbreaks were sequenced to investigate epidemiology of this disease in Taiwan. These NDV isolates were all viscerotropic velogenic according to the clinical lesions produced in chickens. Sequence data were obtained from 14 NDV isolates (12 from 1995 and 2 from 1984). All isolates differed in their nucleotide sequences (from 0.3 to 15.3%), and represented potentially different strains of NDV. Phylogenetic analysis revealed that these isolates are closely related to viruses isolated from Japan and Malaysia. Some viruses isolated in 1995 appeared to evolve from viruses isolated in 1984. The results suggest that the 1995 outbreak of Newcastle disease (ND) in Taiwan may have been caused by multiple strains of velogenic NDV that have cocirculated in Taiwan for some time. Moreover, NDV isolates from racing pigeons were very similar to isolates from chickens in the same period, suggesting that both domestic and free-living birds were involved in the spread of ND in Taiwan.
Assuntos
Surtos de Doenças/veterinária , Doença de Newcastle/epidemiologia , Vírus da Doença de Newcastle/genética , Filogenia , Sequência de Aminoácidos , Animais , Atrofia , Sequência de Bases , Medula Óssea/patologia , Linhagem Celular , Galinhas , Sequência Consenso , Primers do DNA , Suscetibilidade a Doenças , Hemaglutininas Virais/química , Hemaglutininas Virais/genética , Hematócrito , Dados de Sequência Molecular , Neuraminidase/química , Neuraminidase/genética , Doença de Newcastle/patologia , Doença de Newcastle/virologia , Vírus da Doença de Newcastle/classificação , Vírus da Doença de Newcastle/isolamento & purificação , Reação em Cadeia da Polimerase , Taiwan/epidemiologia , Timo/patologiaRESUMO
A homopolymer stretch composed of variable numbers of cytidine residues was found within the inverted terminal repeats of infectious laryngotracheitis virus (ILTV). A polymerase chain reaction procedure was developed to amplify a 750-bp fragment containing this homopolymer stretch. This fragment was then sequenced directly to determine the number of repeated cytidine residues in this homopolymer stretch, which could be used for strain differentiation. By this procedure, vaccine strains of tissue culture origin could be differentiated into two types: type I contains eight repeated cytidine residues, whereas type II contains 10 such residues. Vaccine strains of chicken embryo origin could also be divided into two types: type I contains mainly 11 repeated cytidine residues, whereas type II contains 15-21 such repeats. In comparison, two of the five field isolates examined contain 12-13 repeats; the other three field isolates contain 15-19 repeats, which were similar to the type II chicken-embryo-origin vaccines. The number of repeated cytidine residues described here could serve as a marker for the strain differentiation and epidemiologic study of ILTV.
Assuntos
Citidina/genética , Herpesvirus Galináceo 1/genética , Sequências Repetidas Terminais , Animais , Embrião de Galinha , Clonagem Molecular , Polímeros/química , Polimorfismo de Fragmento de Restrição , Análise de Sequência de DNA/veterináriaRESUMO
Three major outbreaks of Newcastle disease (ND) occurred in Taiwan in the last three decades (in 1969, 1984, and 1995). Newcastle disease viruses (NDVs) isolated in the three outbreaks, together with those isolated in 1998, were sequenced between nucleotides 47 and 435 of the fusion gene. A phylogenetic tree based on sequences obtained showed that the NDV isolated in 1969 was similar to the genotype III viruses. In contrast, all isolates in 1984 and seven of the eight isolates in 1995, together with all isolates in 1998, fell into the genotype VII. These results suggest that the 1969 outbreak of ND in Taiwan was caused by the genotype III virus, whereas the 1984 and 1995 outbreaks were caused by the genotype VII viruses. To date, the genotype VII viruses have caused many outbreaks in east Asia and western Europe. We suspect that these outbreaks have constituted the fourth panzootic of ND, which is distinct from the third panzootic caused by the "pigeon PMV-1 viruses." NDV isolated in Taiwan in 1984 was the earliest isolation of the genotype VII virus.
Assuntos
Surtos de Doenças/veterinária , Doença de Newcastle/epidemiologia , Vírus da Doença de Newcastle/isolamento & purificação , Sequência de Aminoácidos , Animais , Sequência de Bases , Columbidae , DNA Viral/química , Europa (Continente)/epidemiologia , Genótipo , Dados de Sequência Molecular , Filogenia , Alinhamento de Sequência , Taiwan/epidemiologia , Proteínas Virais/químicaRESUMO
Adult and 4-5-week-old rabbits were inoculated subcutaneously with rabbit haemorrhagic disease virus (RHDV). Samples were prepared from various tissues at intervals postinoculation (PI) for the detection of viral RNA and antigens. Using a haemagglutination test (HAT), viral antigens were detected in the liver, bile and spleen of the adult rabbits at and after 36 h PI. The reverse transcription-polymerase chain reaction (RT - PCR) showed that RHDVRNA was present in the liver, bile and spleen as early as 18 hours PI, whereas lung, kidney, thymus, mesenteric lymph node and buffy coat were found to be positive after more than 26 hours PI. In addition, viral RNA in urine and faeces showed a variable positivity at and after 36 hours PI. In the young rabbits, RT - PCR showed that RHDVRNA was present as early as 1 day PI in the liver, bile, spleen and buffy coat; whereas lung, kidney, thymus, mesenteric lymph node and faeces were found to be positive at and after 2 days PI. Bile and spleen were the only samples in which viral RNA was detected throughout the length of the experiment. Virus was not reactivated in six recovered virus-inoculated rabbits treated with dexamethasone or a classical swine fever virus vaccine. Using a haemagglutination inhibition test and an ELISA, antibody titres increased rapidly from one week PI onwards, peaked at approximately three weeks of age, and were maintained throughout the length of the experiment.
Assuntos
Infecções por Caliciviridae/virologia , Vírus da Doença Hemorrágica de Coelhos/isolamento & purificação , Reação em Cadeia da Polimerase Via Transcriptase Reversa/veterinária , Envelhecimento/imunologia , Animais , Anticorpos Antivirais/biossíntese , Antígenos Virais/análise , Vírus da Doença Hemorrágica de Coelhos/genética , Vírus da Doença Hemorrágica de Coelhos/imunologia , RNA Viral/análise , Coelhos , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodosRESUMO
A reverse transcription-polymerase chain reaction (RT-PCR) was established to amplify a 672-base pairs fragment on the segment S3 of avian reovirus (ARV). The amplified fragments were detected in nine strains of ARV as well as three tendon tissue specimens, indicating that the primer regions were well conserved. The RT-PCR was able to detect as low as 0.2 pg using an ethidium bromide stained gel. The detection limit could be enhanced further to 0.04 pg by hybridisation after southern transfer. The amplified DNA fragments from nine ARV strains and two tissue specimens showed different restriction enzyme cleavage patterns. Analysis of the data revealed that these 11 strains were classified into four groups. The results suggest that PCR followed by restriction enzyme analysis may provide a simple and rapid method for the characterisation of ARV isolates.
Assuntos
Doenças das Aves/virologia , Genoma Viral , Orthoreovirus/genética , RNA Viral/isolamento & purificação , Infecções por Reoviridae/veterinária , Animais , Embrião de Galinha , Eletroforese em Gel de Poliacrilamida , Orthoreovirus/classificação , Orthoreovirus/isolamento & purificação , Polimorfismo de Fragmento de Restrição , Doenças das Aves Domésticas/virologia , Infecções por Reoviridae/virologia , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Taipei China had been free from foot and mouth disease (FMD) over 68 years before the disease occurred in March 1997. The first suspected case was recorded on a pig farm in the Hsinchu Prefecture on 14 March 1997. Based on clinical signs, gross histopathological findings, and results of enzyme-linked immunosorbent assays and reverse-transcriptase polymerase chain reaction tests, diagnosis of FMD was confirmed by the Taiwan Animal Health Research Institute on 19 March 1997 and was reconfirmed by the FMD World Reference Laboratory in Pirbright (United Kingdom), on 25 March 1997. By the end of July 1997, 6,147 pig farms (about a quarter of the pig farms in Taipei China), were affected. The disease was well under control within two months by means of stamping-out and blanket vaccination. The Government purchased 21 million doses of inactivated oil-adjuvant FMD vaccine, which allowed for two injections per pig and one injection of other cloven-hoofed animals. Before the vaccine was used, the stamping-out policy was implemented, ensuring that all pigs in the affected farms were destroyed. After blanket vaccination, a partial stamping-out policy was adopted, i.e. only pigs showing clinical signs were destroyed.
Assuntos
Surtos de Doenças/veterinária , Febre Aftosa/prevenção & controle , Doenças dos Suínos/prevenção & controle , Vacinação/veterinária , Animais , Surtos de Doenças/prevenção & controle , Emergências/veterinária , Febre Aftosa/epidemiologia , Suínos , Doenças dos Suínos/epidemiologia , Taiwan/epidemiologiaRESUMO
Experiments were carried out to: 1) determine the antibody response of chickens to cell-free and attenuated preparations of JMV leukosis strain, 2) determine the differences in antibody response to these antigens between susceptible (P-line) and resistant (N-line) chickens by means of serum neutralization and indirect fluorescent antibody tests, 3) investigate the influence of maternal (passive) antibody on early (day-old) JMV vaccination and 4) investigate the influence of maternal antibody in chicks naturally exposed continuously to JM virus from day-old to 8 weeks of age on the pathogenesis of Type II leudosis (Marek's) infections and oncogenesis.
Assuntos
Formação de Anticorpos , Galinhas/imunologia , Herpesvirus Galináceo 2/imunologia , Doença de Marek/genética , Animais , Sistema Livre de Células , Efeito Citopatogênico Viral , Imunofluorescência , Herpesviridae/imunologia , Herpesvirus Galináceo 2/crescimento & desenvolvimento , Imunidade Materno-Adquirida , Injeções Intraperitoneais , Doença de Marek/imunologia , Doença de Marek/patologia , Testes de Neutralização , Perus , Vacinas Virais/administração & dosagem , VirulênciaAssuntos
Formação de Anticorpos , Galinhas/imunologia , Herpesviridae/imunologia , Doença de Marek/imunologia , Animais , Antígenos Virais , Sistema Livre de Células , Embrião de Galinha , Herpesviridae/isolamento & purificação , Infecções por Herpesviridae , Injeções Intraperitoneais , Rim/microbiologia , Doença de Marek/genética , Doença de Marek/microbiologia , Doença de Marek/patologia , Testes de Neutralização , Seleção Genética , Perus , Vacinas Virais/administração & dosagemRESUMO
While there are a number of methods available for detection of antibodies against waterfowl parvoviruses, none is able to differentiate responses against the capsid and non-structural proteins. To enable this, the capsid and non-structural proteins of goose parvovirus (GPV) and Muscovy duck parvovirus (MDPV) were expressed in Escherichia coli. These proteins were purified and used as antigens in western blotting assays of antibodies against GPV and MDPV. The results showed that 94.7% of the goose and 90.0% of the duck sera collected from the field contained antibodies against GPV or MDPV. Moreover, these sera could be classified into distinct groups based on differences in patterns of western blot reactivity. These different patterns might indicate different stages in infection. Western blotting assays of sera collected from experimentally infected ducks showed that antibodies against the non-structural protein appeared first after infection, followed by antibodies against the capsid protein. It was concluded that the recombinant capsid and non-structural proteins might serve as useful antigens for assays for antibodies against GPV and MDPV. Moreover, because these assays could discriminate between antibodies against the non-structural protein and those against the capsid protein, they may be useful in differentiating vaccinated from infected birds when recombinant capsid protein is used as the vaccine.
Assuntos
Anseriformes/virologia , Anticorpos Antivirais/imunologia , Proteínas do Capsídeo/metabolismo , Parvovirus/metabolismo , Testes Sorológicos/métodos , Proteínas não Estruturais Virais/metabolismo , Animais , Western Blotting , Proteínas do Capsídeo/imunologia , Primers do DNA , Escherichia coli , Parvovirus/imunologia , Proteínas não Estruturais Virais/imunologiaRESUMO
An assay protocol based on single-tube, noninterrupted reverse transcription-PCR (RT-PCR) for the detection of infectious bursal disease virus (IBDV) is described. After the conditions for RT-PCR had been optimized, a primer set framing a region within the gene coding for IBDV VP2 protein was used to amplify a 318-bp fragment of the IBDV genome. Amplified product was detected with three strains of IBDV, whereas none was obtained from uninfected bursal tissue or seven unrelated avian viruses. The sensitivity of this RT-PCR was tested with purified viral RNA from three strains of IBDV. The detection limit was 10 fg in an ethidium bromide-stained gel. In addition, this assay system was used to detect IBDV in bursal-tissue specimens from commercially reared chickens. The identity of the amplified products from the tissue specimen preparation was determined by using a rapid, simple procedure in which internally nested, end-labeled probes were used.
Assuntos
Infecções por Birnaviridae/veterinária , Vírus da Doença Infecciosa da Bursa/genética , Vírus da Doença Infecciosa da Bursa/isolamento & purificação , Reação em Cadeia da Polimerase/métodos , Doenças das Aves Domésticas/diagnóstico , Animais , Sequência de Bases , Infecções por Birnaviridae/diagnóstico , Bolsa de Fabricius/microbiologia , Capsídeo/genética , Proteínas do Capsídeo , Galinhas , Primers do DNA/genética , DNA Viral/genética , Estudos de Avaliação como Assunto , Vírus da Doença Infecciosa da Bursa/classificação , Dados de Sequência Molecular , Reação em Cadeia da Polimerase/estatística & dados numéricos , Sensibilidade e Especificidade , Sorotipagem , PerusRESUMO
Ten monoclonal antibodies (MAbs) were prepared against the nonstructural protein sigmaNS of avian reovirus S1133. Eight of them were selected for two-way competitive binding assay after coupling with horseradish peroxidase. The results allowed the definition of three epitopes, designated A, B, and C. Blocking assay of poly(A)-Sepharose binding activity of sigmaNS with MAbs indicated that MAb recognizing epitope B was able to block poly(A) oligomer binding, suggesting that epitope B is involved in ssRNA binding of sigmaNS. An immuno-dot binding assay was used to analyze the effect of denaturation on antibody recognition of the epitopes. All MAbs bound to protein sigmaNS in its native form. After denaturation by boiling in SDS and 2-mercaptoethanol, the binding of MAbs recognizing epitopes B and C was not affected. The reactivity of MAbs recognizing epitope A was fully abolished by denaturation. These results suggest that the binding of MAbs directed against epitope A is conformation-dependent; however, the recognition by MAbs of epitopes B and C is not conformation-dependent. In addition, the results from the cross-reactivity of MAbs to heterologous avian reovirus strains suggest that the three epitopes are highly conserved among these virus strains.
Assuntos
Anticorpos Monoclonais/imunologia , Anticorpos Antivirais/imunologia , Reoviridae/metabolismo , Proteínas não Estruturais Virais/imunologia , Animais , Afinidade de Anticorpos , Ligação Competitiva , Aves , Reações Cruzadas , Mapeamento de Epitopos , Epitopos/imunologia , Mercaptoetanol/farmacologia , Camundongos , Camundongos Endogâmicos BALB C , Desnaturação Proteica , RNA Viral/metabolismo , Reoviridae/genética , Dodecilsulfato de Sódio/farmacologia , Proteínas não Estruturais Virais/genéticaRESUMO
There are nine serotypes of avian paramyxovirus (APMV). Only the genome of APMV type 1 (APMV-1), also called Newcastle disease virus (NDV), has been completely sequenced. In this study, the complete nucleotide sequence of an APMV-6 serotype isolated from ducks is reported. The 16236 nt genome encodes eight proteins, nucleocapsid protein (NP), phosphoprotein (P), V protein, matrix protein (M), fusion protein (F), small hydrophobic (SH) protein, haemagglutinin-neuraminidase (HN) protein and large (L) protein, which are flanked by a 55 nt leader sequence and a 54 nt trailer sequence. Sequence comparison reveals that the protein sequences of APMV-6 are most closely related to those of APMV-1 (NDV) and -2, with sequence identities ranging from 22 to 44%. However, APMV-6 contains a gene that might encode the SH protein, which is absent in APMV-1, but present in the rubulaviruses simian virus type 5 and mumps virus. The presence of an SH gene in APMV-6 might provide a link between the evolution of APMV and rubulaviruses. Phylogenetic analysis demonstrates that APMV-6, -1, -2 (only the F and HN sequences were available for analysis) and -4 (only the HN sequences were available for analysis) all cluster into a single lineage that is distinct from other paramyxoviruses. This result suggests that APMV should constitute a new genus within the subfamily Paramyxovirinae.