In vitro antiviral activity of Brazilian plants (Maytenus ilicifolia and Aniba rosaeodora) against bovine herpesvirus type 5 and avian metapneumovirus.

CONTEXT: Medicinal plants are well known for their use in traditional folk medicine as treatments for many diseases including infectious diseases. OBJECTIVE: Six Brazilian medicinal plant species were subjected to an antiviral screening bioassay to investigate and evaluate their biological activities against five viruses: bovine herpesvirus type 5 (BHV-5), avian metapneumovirus (aMPV), murine hepatitis virus type 3, porcine parvovirus and bovine respiratory syncytial virus. MATERIALS AND METHODS: The antiviral activity was determined by a titration technique that depends on the ability of plant extract dilutions (25 or 2.5 µg/mL) to inhibit the viral induced cytopathic effect and the extracts' inhibition percentage (IP). RESULTS: Two medicinal plant species showed potential antiviral activity. The Aniba rosaeodora Ducke (Lauraceae) extract had the best results, with 90% inhibition of viral growth at 2.5 µg/mL when the extract was added during the replication period of the aMPV infection cycle. The Maytenus ilicifolia (Schrad.) Planch. (Celastraceae) extracts at a concentration of 2.5 µg/mL exhibited antiviral activity during the attachment phase of BHV-5 (IP = 100%). DISCUSSION AND CONCLUSION: The biomonitored fractionation of the active extracts from M. ilicifolia and A. rosaeodora could be a potential tool for identifying their active compounds and determining the exact mechanism of action.

Genetic diversity of avian infectious bronchitis virus isolated from domestic chicken flocks and coronaviruses from feral pigeons in Brazil between 2003 and 2009.

To detect the presence of infectious bronchitis virus or avian coronavirus, a nested reverse transcriptase PCR (RT-PCR) method was developed with the aim of amplifying a fragment of 530 bases, comprising the gene coding S1 protein. In the first step, all samples were submitted to RNA extraction, RT-PCR, and nested PCR. Next, only the positive nested-PCR samples were propagated in specific-pathogen-free (SPF) embryonated chicken eggs for virus isolation. Positive samples were then sequenced and analyzed using a molecular phylogeny approach. Tracheal swab samples were collected from 23 different domestic chickens distributed in three regions of Brazil, in the period between 2003 and 2009. Also analyzed were six swab samples (tracheal and cloacal) from asymptomatic pigeons (Columba livia), caught in an urbanized region in southeastern Brazil. The study revealed two major phylogenetic groups: one clustered with the Massachusetts vaccine serotype and another joined with the D207 strain. Interestingly, samples grouped with the Connecticut and Arkansas serotypes were also found. Pigeon isolates clustered with the Massachusetts serotype showed significant similarity (close to 100%) to those obtained from chickens. Only one pigeon isolate was seen to be grouped with the Connecticut serotype, and no correlation was observed between sample grouping and region origin. Understanding the diversity of genotypes and eco-epizootiology of the disease in different environments is expected to be helpful for vaccine production aimed at the main circulating variants. In this respect, one could also expect benefits in the management of other bird species that may act as avian coronavirus reservoirs.

Prevalence of newcastle disease virus in broiler chickens (Gallus gallus) in Brazil.

This study was carried out during 2002/2003, aiming to determine the prevalence of virulent Newcastle disease virus strains (NDV) in Brazilian commercial poultry farms. Clinical samples were obtained from the Southeastern, Southern and Central-Western regions, which comprise the main area of the Brazilian poultry production. Serum samples and tracheal and cloacal swabs of 23,745 broiler chickens from 1,583 flocks, including both vaccinated chickens and those with no vaccination information, were tested for NDV using a diagnostic ELISA kit. The seropositivity was 39.1%, and the isolation percentage by flock varied from 1.0 to 7.6%, and by region from 6.5 to 58.4%. Higher isolation rates (74.3-83.3%) were obtained after three passages in embryonated chicken eggs. All isolates preliminarily identified as NDV were characterized as nonpathogenic strains, as their Intracerebral Pathogenicity Index (ICPI) was below 0.7. Based on results of this study, Brazil can claim a virulent NDV-free status for commercial flocks.

A survey for maintenance of virulent newcastle disease virus-free area in poultry production in Brazil.

In 2003, Brazil was recognized as a pathogenic Newcastle Disease Virus (NDV) strain-free country for commercial poultry. This research was conducted in Brazil between December 2003 and March 2005 to verify the maintenance of this virulent NDV-free status. Serum samples from 5,455 flocks for commercial poultry farms were collected, comprising 81,825 broiler chickens. The farms were located in nine states of the country, grouped in three geographic regions. Serological evidence of NDV infection was detected in 28.8% of the surveyed farms. However, all fifteen viruses isolated and identified as Newcastle Disease Virus (NDV) were characterized as nonpathogenic strains, based on the Intracerebral Pathogenicity Index. These results showed that Brazil preserves the virulent NDV-free status for commercial flocks.

Phylogenetic comparison of the carboxy-terminal region of glycoprotein C (gC) of bovine herpesviruses (BoHV) 1.1, 1.2 and 5 from South America (SA).

Different types and subtypes of bovine herpesvirus 1 and 5 (BoHV-1 and BoHV-5) have been associated to different clinical conditions of cattle, in such a way that type/subtype differentiation has become an essential tool for understanding the pathogenesis and epidemiology of BoHV infections. In search for a genomic region that would allow a clear distinction between BoHV-1 and BoHV-5, the carboxy-terminal portion of glycoprotein C (gC), corresponding to residues 321-450 (BoHV-1) and 301-429 (BoHV-5) of 23 South American (SA) isolates (Brazil mostly) was amplified and sequenced. The nucleotide sequence alignments revealed levels of genomic similarity ranging from 98.7 to 99.8% among BoHV-1 isolates, 88.3 to 92% between BoHV-1/BoHV-5 and 96 to 99.7% among BoHV-5 isolates. At the amino acid level, sequence similarity varied ranging from 97.5 to 99.5% among BoHV-1, 77.5 to 84.4% between BoHV-1/BoHV-5 and 92.1 to 99.5% (BoHV-5/BoHV-5). The isolates could be clearly separated into BoHV-1.1, BoHV-1.2 and BoHV-5 after phylogenetic analysis. The results suggest that the phylogenetic analysis performed here can be used as a potential molecular epidemiological tool for herpesviruses.

An agar-overlay method for detection of toxins produced by Escherichia coli.

An agar overlay method, with Vero and HeLa cells, was used for detection of heat-labile enterotoxin and verotoxin from Escherichia coli. The method is more sensitive than the conventional cell culture assay, is rapid, easy to perform, and is suitable for epidemiological studies.

Molecular characterization of Brazilian avian pneumovirus isolates: comparison between immunochemiluminescent Southern blot and nested PCR.

Avian pneumovirus (APV) causes acute respiratory tract infection both in turkeys (turkey rhinotracheitis) and chickens (swollen head syndrome (SHS)) with sudden onset and rapid spread through the flocks. In this study, an immunochemiluminescent Southern blot RT-PCR assay was employed to detect a F gene transcript of the APV in two European turkey isolates and two Brazilian chicken isolates. Limiting dilution PCR was carried out to compare the sensitivity of immunochemiluminescent Southern blot assay and nested PCR assay (nPCR). The sensitivity and specificity of immunochemiluminescent Southern blot RT-PCR assay were comparable to that of nPCR, and at least 100 fold more sensitive than a single PCR amplification. Sequence analysis of the 175 bp product of the F gene revealed 100% identity with APV sequences described earlier.

Replication of classical infectious bursal disease virus in the chicken embryo related cell line.

Infectious bursal disease (IBD) is an acute, highly contagious viral disease. The diagnosis of IBD depends on time-consuming and costly procedures, like virus isolation on chick embryos and histopathological examination. A double antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA), immunoperoxidase and reverse transcription polymerase chain reaction (RT-PCR) were applied in this study to detect classical IBD virus (IBDV) after three blind passages of the Lukert strain on chicken embryo related (CER) cell monolayer after different periods of infection: 6, 12, 24 and 48h. Cytophatic effects were most evident 12 h post-infection (p.i.) but were observed at 6 h p.i. The maximum discrimination between IBDV-infected and uninfected cell suspensions obtained by the use of DAS-ELISA for virus detection corresponded to 0.597 +/-0.02 and 0.010 +/-0.01 after 12 h p.i., respectively. The RT-PCR was performed using the set of primers A3.1 and A3.2 to amplify the VP2 region of the IBDV genome. This molecular technique demonstrated that from 6h p.i., it was possible to detect the viral RNA. The results show that the CER cell line can be used for classical IBDV propagation, confirmed by the DAS-ELISA, immunoperoxidase and RT-PCR assay.

Restriction endonuclease and monoclonal antibody analysis of Brazilian isolates of bovine herpesviruses types 1 and 5.

Twelve Brazilian isolates and three reference strains of bovine herpesviruses (BHVs) were subjected to restriction endonuclease analysis (REA) and monoclonal antibody (MAb) analysis. Viral DNA was cleaved with BamHI, BstEII, EcoRI, HindIII and PstI. The monoclonal antibody panel allowed the differentiation between types 1 and 5 viruses, while REA with BstEII and HindIII showed the distinction between BHV-1 and -5 subtypes. Typical 1.1 and 1.2a patterns were observed with two isolates from respiratory disease. An isolate from semen of a clinically healthy bull displayed 1.2b profile, whereas another displayed a clear 5a pattern, which was never reported before. Seven out of nine Brazilian type 5 (BHV-5) isolates displayed REA patterns similar to the Australian BHV-5 strain N569 (BHV-5a), and differing from the Argentinean A663 strain (BHV-5b) virus. Another two BHV-5 isolates, which displayed an unusual MAb pattern of reactivity, showed a BstEII profile different from both reference strains of BHV-5. These two viruses were considered BHV-5 "non-a/non-b" subtype.

Characterization of bovine respiratory syncytial virus isolated in Brazil.

This paper presents the first isolation of bovine respiratory syncytial virus in Brazil and its physicochemical, morphological and molecular characterization. The virus was isolated from 33 samples of nasotracheal secretions, successively inoculated into a Madin-Darby bovine kidney cell culture, which was characterized by physicochemical tests and morphological observation by electron microscopy. The Brazilian sample is an RNA pleomorphic, enveloped, thermolabile and non-hemagglutinating spicular virus. Reverse transcription, followed by nested polymerase chain reaction (nRT-PCR) assay was carried out using oligonucleotides B1, B2A, B3 and B4 for the fusion proteins (F) and B5A, B6A, B7A and B8 for the attachment protein (G). The nRT-PCR-F amplified a fragment of 481 bp corresponding to part of the gene that codes for protein F, whereas nRT-PCR-G amplified a fragment of 371 bp, in agreement with part of the G gene. The virus isolated from Brazilian samples in this study corresponded to the bovine respiratory syncytial virus, and RT-PCR proved to be useful for the diagnosis of bovine clinical samples.

Molecular data of UL24 homolog gene (ORF37) from Brazilian isolates of equine herpesvirus type 1.

Equine herpesvirus type 1 (EHV-1) is associated with abortions, respiratory distress, and neurological disturbances in horses. The ORF37 of EHV-1 encodes a protein homolog to UL24 gene product of human herpesvirus that has been associated with neurovirulence. In the present work, ORF37 PCR fragments derived from two Brazilian EHV-1 isolates, a German isolate and an American reference strain were sequenced and characterized by molecular phylogenetic analysis. This genomic region is highly conserved an allowed to infer genetic distances between EHV-1 strains and other animal herpesvirus.

Clonagem das glicoproteínas transmembrana G e F de um isolado brasileiro do vírus respiratório sincicial bovino em sistema procarioto / Cloning of the transmembrane glycoproteins G and F from a Brazilian isolate of bovine respiratory syncytial virus in a prokaryotic system

The aim of this work was the cloning of those transmembrane glycoproteins G and F from an isolate bovine respiratory syncytial viruses (BRSV) - a Brazilian isolate of BRSV, named BRSV-25-BR in previous studies, in a prokaryotic system to proceed the sequencing of larger genomic fragments. The nucleotide substitutions were confirmed and these clones may also be used in further studies regarding the biological effects of those proteins in vitro and in vivo.

O objetivo deste trabalho foi a clonagem das glicoproteínas transmembrana G e F de um isolado de vírus respiratório sincicial bovino (BRSV) - um isolado brasileiro denominado BRSV-25-BR- que já demonstrou possuir mutações em regiões altamente conservadas do gene da proteína G - em sistema procariótico, com o intuito de sequenciar fragmentos genômicos maiores. As substituições de nucleotídeos foram confirmadas e tais clones podem ser utilizados em futuros estudos sobre os efeitos biológicos destas proteínas tanto in vitro como in vivo.

Prevalence of newcastle disease virus in broiler chickens (gallus gallus) in Brazil

This study was carried out during 2002/2003, aiming to determine the prevalence of virulent Newcastle disease virus strains (NDV) in Brazilian commercial poultry farms. Clinical samples were obtained from the Southeastern, Southern and Central-Western regions, which comprise the main area of the Brazilian poultry production. Serum samples and tracheal and cloacal swabs of 23,745 broiler chickens from 1,583 flocks, including both vaccinated chickens and those with no vaccination information, were tested for NDV using a diagnostic ELISA kit. The seropositivity was 39.1 percent, and the isolation percentage by flock varied from 1.0 to 7.6 percent, and by region from 6.5 to 58.4 percent. Higher isolation rates (74.3-83.3 percent) were obtained after three passages in embryonated chicken eggs. All isolates preliminarily identified as NDV were characterized as nonpathogenic strains, as their Intracerebral Pathogenicity Index (ICPI) was below 0.7. Based on results of this study, Brazil can claim a virulent NDV-free status for commercial flocks.

A survey for maintenance of virulent newcastle disease virus-free area in poultry production in Brazil

In 2003, Brazil was recognized as a pathogenic Newcastle Disease Virus (NDV) strain-free country for commercial poultry. This research was conducted in Brazil between December 2003 and March 2005 to verify the maintenance of this virulent NDV-free status. Serum samples from 5,455 flocks for commercial poultry farms were collected, comprising 81,825 broiler chickens. The farms were located in nine states of the country, grouped in three geographic regions. Serological evidence of NDV infection was detected in 28.8 percent of the surveyed farms. However, all fifteen viruses isolated and identified as Newcastle Disease Virus (NDV) were characterized as nonpathogenic strains, based on the Intracerebral Pathogenicity Index. These results showed that Brazil preserves the virulent NDV-free status for commercial flocks.

Virulence factors of avian Escherichia coli associated with swollen head syndrome.

Virulence characteristics of 50 strains of Escherichia coli isolated from chickens with swollen head syndrome were examined. The results were the following: in the absence of D-mannose, 74% of strains agglutinated guinea pig erythrocytes, but in the presence of D-mannose 32% agglutinated guinea-pig erythrocytes and 62% agglutinated human erythrocytes. When slide agglutination assays were carried out with antisera to adhesin of bovine and swine origin (K88, K99, F41, F42 987P and 2134P), only 14% of strains agglutinated with antiserum to F41. Colicin V was produced by 78% of the E. coli strains and 80% produced aerobactin. In the serum resistance test, 36 (72%) of strains showed resistance to normal chicken serum. Only seven (14%) strains expressed K1 capsular antigen, while motility was found in 62% of the strains.

Molecular characterization of Brazilian avian pneumovirus isolates using reverse transcription-polymerase chain reaction, restriction endonuclease analysis and sequencing of a G gene fragment.

A reverse transcriptase-polymerase chain reaction (RT-PCR) assay was employed to amplify a G gene transcript of the avian pneumovirus (APV) in two European turkey isolates and two Brazilian chicken isolates. The PCR products were digested using restriction endonucleases and the restriction patterns were compared with earlier reported isolate patterns. The same PCR products were cloned into pUC18 and sequenced. Restriction patterns and sequence analyses of the approximately 600 bp products from both Brazilian isolates revealed 99% identity with earlier reported subgroup A APV sequence, suggesting that these isolates belong to this subgroup.

Infection and apparent invasion of Vero cells by Paracoccidioides brasiliensis.

Paracoccidioides brasiliensis probably uses many different mechanisms to establish itself in the host and cause disease. In this work, we assess an in vitro model system which uses cultured mammalian cells to investigate the virulence factors of P. brasiliensis. We were able to demonstrate an invasion process of the yeast form of this fungus in Vero cell cultures. We deduced that the overall invasive process involved three steps: adhesion, followed by invasion of individual epithelial cells and spread to adjacent cells.

Bovine respiratory syncytial virus: immunohistochemichal detection in mouse and bovine tissues using a Mab against human respiratory syncytial virus

An immunoistochemical (IHC) test was developed to detect bovine respiratory syncytial virus (BRSV) in cell cultures and tissues of experimentally infected mice and calves, using a commercial monoclonal antibody (Mab) against human respiratory syncytial virus (HRSV), as a less expensive alternative, instead of producing specific monoclonal antibodies to BRSV. Clinical samples from calves suffering respiratory disease were also submitted to this test. IHC detected BRSV antigens in mouse tracheas (3, 5 and 7 days post-infection) and lungs (5 and 7 days post-infection), and in one of three lungs from experimentally infected calves. Lungs samples from two naturally infected calves were tested and resulted positive for BRSV by the IHC test. These results suggest that this test may be used in the future for diagnosis as well as a useful tool to assess the distribution of BRSV infections in Brazilian herds.

Desenvolveu-se um teste de imunohistoquímica (IHQ) para detecção do vírus respiratório sincicial bovino (BRSV) multiplicado em cultivo celular e em tecidos de camundongos e bezerros infectados experimentalmente, utilizando um anticorpo monoclonal comercial contra o vírus respiratório sincicial humano (HRSV), como uma alternativa para eliminar os custos de produção de anticorpos monoclonais específicos para o BRSV. Amostras clínicas de bezerros com sintomatologia respiratória foram analisadas. A técnica mostrou-se eficiente na detecção de antígenos do BRSV em traquéias (3, 5 e 7 dias pós-infecção) e pulmões (5 e 7 dias pós-infecção) dos camundongos infectados e em uma das três amostras de pulmões dos bezerros infectados experimentalmente. Amostras de pulmões de dois animais com infecção natural foram positivas para BRSV. Conclui-se que o teste de IHQ pode ser usado no diagnóstico das infecções por BRSV e na avaliação da distribuição dessas infecções nos rebanhos bovinos brasileiros.

Characterization of bovine respiratory syncytial virus isolated in Brazil

This paper presents the first isolation of bovine respiratory syncytial virus in Brazil and its physicochemical, morphological and molecular characterization. The virus was isolated from 33 samples of nasotracheal secretions, successively inoculated into a Madin-Darby bovine kidney cell culture, which was characterized by physicochemical tests and morphological observation by electron microscopy. The Brazilian sample is an RNA pleomorphic, enveloped, thermolabile and non-hemagglutinating spicular virus. Reverse transcription, followed by nested polymerase chain reaction (nRT-PCR) assay was carried out using oligonucleotides B1, B2A, B3 and B4 for the fusion proteins (F) and B5A, B6A, B7A and B8 for the attachment protein (G). The nRT-PCR-F amplified a fragment of 481 bp corresponding to part of the gene that codes for protein F, whereas nRT-PCR-G amplified a fragment of 371 bp, in agreement with part of the G gene. The virus isolated from Brazilian samples in this study corresponded to the bovine respiratory syncytial virus, and RT-PCR proved to be useful for the diagnosis of bovine clinical samples