Diversity and Adaptation of Human Respiratory Syncytial Virus Genotypes Circulating in Two Distinct Communities: Public Hospital and Day Care Center

HRSV is one of the most important pathogens causing acute respiratory tract diseases as bronchiolitis and pneumonia among infants. HRSV was isolated from two distinct communities, a public day care center and a public hospital in Sao Jose do Rio Preto - SP, Brazil. We obtained partial sequences from G gene that were used on phylogenetic and selection pressure analysis. HRSV accounted for 29% of respiratory infections in hospitalized children and 7.7% in day care center children. On phylogenetic analysis of 60 HRSV strains, 48 (80%) clustered within or adjacent to the GA1 genotype; GA5, NA1, NA2, BA-IV and SAB1 were also observed. SJRP GA1 strains presented variations among deduced amino acids composition and lost the potential O-glycosilation site at amino acid position 295, nevertheless this resulted in an insertion of two potential O-glycosilation sites at positions 296 and 297. Furthermore, a potential O-glycosilation site insertion, at position 293, was only observed for hospital strains. Using SLAC and MEME methods, only amino acid 274 was identified to be under positive selection. This is the first report on HRSV circulation and genotypes classification derived from a day care center community in Brazil.FAPESP [2010/50444-4]FAPES

Aspectos da patogenia e análise da variabilidade da glicoproteína S do Coronavirus dos Perus isolado no Brasil

No últimos anos, a avicultura brasileira posicionou o país como o terceiro maior produtor mundial de perus, entretanto o setor permanece em acentuado crescimento e tem uma grande importância econômica, movimentando cerca de 10 milhões de dólares por ano. A indústria avícola brasileira obteve novos mercados, mostrando novas perspectivas no comércio internacional. O Coronavirus dos Perus (Turkey Coronavirus, ¨TCoV) é um vírus envelopado, com RNA de fita simples, não segmentado, com sentido positivo e pertence a ordem Nidovirales, família Coronavirus e gênero Coronavirus (grupo III). Trata-se de um agente causador de enterites altamente transmissíveis, gerando grandes perdas econômicas no Brasil e no mundo. O presente trabalho teve como objetivo avaliar o processo de apoptose celular e a variabilidade molecular em tecidos de embriões de perus experimentalmente infectados com TCoV (primeiro isolado brasileiro, 2007). A infecção experimental foi desenvolvida por meio da inoculação viral na cavidade amniótica de embriões de perus com 23-25 dias de incubação por nove vezes consecutivas. Foi empregada a técnica de imunoistoquímica no intuito de detectar os marcadores apoptóticos Anexina V, Caspase 2, Caspase 3, p53 e ainda a detecção de apoptose com o emprego do kit TUNEL. Foi realizado o sequenciamento do vírus original e suas passagens em ovos embrionados a fim de detectar mutações decorrentes das passagens virais. O TCoV propagou em ovos embrionados de perus (Melleagridis gallopavo) apresentando vacuolização dos enterócitos e congestão como lesões histopatológicas. A análise das lesões macroscópicas mostrou congestão moderada, acúmulo de líquidos e processo inflamatório. A apoptose foi identificada pelo uso do kit comercial TUNEL e mostrou a presença viral no intestino bem como a ocorrência de apoptose celular nos enterócitos. Os resultados...The Brazilian poultry industry represents third world producer of turkeys, in last years, whereas the sector is in growth and has a large economic importance moving around 10 million of dollars per year. Brazilian’s industry obtained new markets, showing new perspectives into the international trade. Turkey Coronavirus (TCoV) is an agent that causes high transmissible enteritis, causing great economic lost, in Brazil and worldwide. It is an enveloped virus, with single strand RNA, non segmented, positive sense, belongs to Nidovirales order, Coronaviridae family and Coronavirus group III. The present work evaluated the apoptosis process and the molecular variety in turkey embryo tissues experimentally infected by turkey Coronavirus (first isolated in Brazil, 2007). The experimental infection was performed trough viral inoculation into amniotic cavity in embryonated turkey eggs with 23-25 days of incubation for nine times consecutivelly. Immunohistochemistry was performed to detect the apoptotic factors Annexin V, Caspase 2, Caspase 3, p53 and apoptosis detection by TUNEL kit. The sequencing of original virus and the respective passages developed to detect mutations acquired through passages. TCoV propagated in turkey eggs (Melleagridis gallopavo) presenting enterocytes vacuolization and congestion as histopathologic lesions. Analysis of macroscopic lesions showed mild congestion, liquid accumulation and inflammation process. The apoptosis identification performed applying the commercial kit TUNEL shown the viral presence in intestinal tissue, as well, the cellular apoptosis occurrence. The results demonstrated positive signals to annexin V, caspase 2, caspase 3 and p53, exactly how described in others authors to others Coronavirus. Regarding to the sequences, it was detected the occurrence of three mutations never described before and 19 mutation of common occurrence... (Complete abstract click electronic access below)Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP

Estudo das interações entre proteína NS1 do hRSV e flavonóides utilizando métodos biofísicos

Human Respiratory Syncytial Virus (hRSV) is the major cause of acute respiratory infections in children, like bronchiolitis and pneumonia. This Paramyxovirus has a single strand RNA with 10 genes that codify 11 proteins. An important factor that contributes for the success hRSV replication is the immune system evasion, process wich Non-Structural Protein 1 (NS1) is involved. This protein can act by inhibiting or neutralizing steps of interferon type I pathway, as well as, by silencing the ribonucleoproteic complex of hRSV. The knowledge of NS1 protein interaction with ligands is important in the search for molecules that could interfere with protein function and hence result in a reduction of hRSV replication among them the flavonoids have been reported to be effective in suppressing viral replication. The aim of this study includes expression and purification of NS1 protein, characterization of its secondary structure and thermal stability through circular dicroism and perform fluorescence spectroscopy interaction study between NS1 and Quercetin. NS1 purification was performed using affinity resin coupled to a liquid cromatograph ÄKTA (GE HEALTHCARE LIFE SCIENCES). Circular dicroism spectra and thermal denaturation were carried out to investigate NS1 secondary structure and stability. Through fluorescence spectroscopy titration results it has been calculated the Stern Volmer constant (Ksv), the binding (Kb) constant and number of ligands per site (n). CD analysis shown that secondary structure composition of NS1 is 75% alpha-helix; 3% beta-sheet; 10% turn and 12% others and, melting temperature of NS1 is around 65°C. Fluorescence results shown that Ksv and Kb were in order of 104 and 105-106M-1, respectively. Ksv dependence with temperature indicates that the interaction between NS1 and quercetin is dynamic. The results suggest that this interaction may potentially contribute to block the xiii protein function and thus quercetin may ...O Vírus Respiratório Sincicial Humano (hRSV) é a principal causa de infecções respiratórias agudas em crianças, como bronquiolite e pneumonia. Este Paramyxovirus tem um RNA de fita simples, com 10 genes que codificam 11 proteínas. Um fator importante que contribui para o sucesso da replicação do hRSV é a evasão do sistema imune, processo no qual a proteína não estrutural 1 (NS1) está envolvida. Esta proteína pode atuar por meio da inibição ou neutralização das etapas da cascata do interferon do tipo I, bem como, pelo silenciamento do complexo ribonucleoproteico do hRSV. O conhecimento da interação da proteína NS1 com ligantes é importante na busca de moléculas que possam interferir na função da proteína e consequentemente resultar na redução da replicação do hRSV dentre os quais os flavonóides têm sido descritos como sendo supressores eficazes da replicação viral. Os objetivos deste estudo incluíram a expressão e purificação da proteína NS1, a caracterização da estrutura secundária e estabilidade térmica por dicroísmo circular e a realização do estudo de interação entre NS1 e quercetina por espectroscopia de fluorescência. Foi realizada a purificação da proteína NS1 em resina de afinidade utilizando cromatógrafo líquido ÄKTA (GE HEALTHCARE LIFE SCIENCES). As medidas de dicroísmo circular (CD) permitiram calcular a porcentagem de estruturas secundárias e a temperatura de melting da NS1. A partir das análises das titulações por fluorescência foram calculados a constante de Stern Volmer (Ksv), a constante de ligação (Kb) e o número de ligantes por sítio (n). Os resultados de CD mostraram que a composição da estrutura secundária de NS1 é de 75 % de alfa-hélice, 3% de folha-beta, 10% de voltas e 12% de outras estruturas e a temperatura de melting da NS1 é de cerca de 65°C. Os resultados de fluorescência mostraram que Ksv e Kb foram da ordem de 104 e 105-106M-1, ...Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES

Genetically Diverse Coronaviruses in Captive Bird Populations in a Brazilian Zoological Park

This study aimed to investigate the occurrence of coronaviruses (CoVs) in captive birds placed inside a zoological park in Brazil. The role of captive birds in the epidemiology of CoVs in the tropics is poorly understood. A total of 25 (n = 25) different species were tested for viral RNA using individual fecal samples collected from healthy birds. Reverse transcription-polymerase chain reaction targeting the 30 untranslated region was used to detect CoV RNA, and positive samples were submitted for sequence analysis. The phylogenetic search revealed nine mutations in the black shouldered peafowl (Pavus cristatus) CoV sequence, which clustered separately from samples previously described in England. This is the first report on the detection of the CoV genome in captive birds in Brazil.FAPESP (Fundacao Amparo a Pesquisa do Estado de Sao Paulo)CNP

Isolation and molecular characterization of Brazilian turkey reovirus from immunosuppressed young poults

In this study, we investigated turkey reovirus (TReoV) in tissue samples from young birds, aged 15 days. RT-PCR for TReoV detected 3.3 % positive samples and TReoV was successfully isolated in Vero cells. Histological analysis of positive bursa of Fabricius (BF) revealed atrophied follicles and lymphocyte depletion. The number of CD8+, CD4+ and IgM+ cells was lower in infected BF. Phylogenetic analysis based on S3 gene showed that the Brazilian TReoV isolates clustered in a single group with 98-100 % similarity to TReoV strains circulating in the United States. This is the first indication that TReoV infection may be a contributing factor to immunosuppression in young birds

Diversity and Adaptation of Human Respiratory Syncytial Virus Genotypes Circulating in Two Distinct Communities: Public Hospital and Day Care Center

HRSV is one of the most important pathogens causing acute respiratory tract diseases as bronchiolitis and pneumonia among infants. HRSV was isolated from two distinct communities, a public day care center and a public hospital in São José do Rio Preto – SP, Brazil. We obtained partial sequences from G gene that were used on phylogenetic and selection pressure analysis. HRSV accounted for 29% of respiratory infections in hospitalized children and 7.7% in day care center children. On phylogenetic analysis of 60 HRSV strains, 48 (80%) clustered within or adjacent to the GA1 genotype; GA5, NA1, NA2, BA-IV and SAB1 were also observed. SJRP GA1 strains presented variations among deduced amino acids composition and lost the potential O-glycosilation site at amino acid position 295, nevertheless this resulted in an insertion of two potential O-glycosilation sites at positions 296 and 297. Furthermore, a potential O-glycosilation site insertion, at position 293, was only observed for hospital strains. Using SLAC and MEME methods, only amino acid 274 was identified to be under positive selection. This is the first report on HRSV circulation and genotypes classification derived from a day care center community in Brazil

Evaluation of serum- and animal protein-free media for the production of infectious bronchitis virus (M41) strain in a continuous cell line

Infectious bronchitis virus (IBV) is produced in vitro using specific pathogen free chicken embryos, primary chicken kidney cells (CKC) and/or tracheal organ culture (TOC). Regulatory authorities in Europe (EMEA) and in the United States (FDA) have encouraged biological manufactures to reduce or eliminate the use of live animals and/or products of animal origin in biological manufacturing processes. In this paper, a stable chicken embryo-related (CER) cell line was adapted and maintained in serum free (SF) or animal protein free (APF) medium after a direct switch of the medium. The most suitable media were Ex Cell 520 and Ex Cell 302. CER monolayers adapted to SF or APF were infected with IBV (M41 strain) in agitated suspended culture and the IBV titre obtained 48 h post infection was 2.8 × 104 PFU/ml in both cases. Thus, propagation of CER cells and culture of IBV can be performed without the use of animal serum or animal protein.Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP

Pathological and molecular analysis of Turkey coronavirus replication in captive wild Turkey embryos (broad-breasted bronze breeder)

The degree of genetic and pathologic variation exhibited by a turkey Coronavirus (TCoV) strain was investigated after nine serial passages in 25-day-old turkey embryos obtained from wild broad-breasted bronze breeders. In spite of spleen, liver, kidneys, cloacal bursa and thymus have been collected and analysed, the main histopathological changes were only documented in the intestine sections. Microscopic lesions were characterized as mild enteritis, low degree of enterocyte vacuolization and detachment of the intestinal villous after five consecutive passages and were considered absent in the last passages. Genealogic analysis based on S1 and S2 DNA sequences suggested that Brazilian isolate might be considered as originated from TCoV strains circulating in the United States, as 100% identity with TCoV-Gl strain. Although S1 S2 sequences from each passage revealed no significant point mutations, and no correlation could be speculate between S2 nucleotide changes and pathologic features in infected embryos. This is the first demonstration of wild turkey embryos as a model for TCoV isolation and propagation

Poult intestinal organ culture for propagation of Turkey coronavirus and assessment of host-virus interactions

Infection of young poults with turkey coronavirus (TCoV) produces a syndrome characterized by acute enteritis, diarrhea, anorexia, ruffled feathers, decreased body weight gain and uneven flock growth. The objective of this study was to standardize an intestinal organ culture (IOC) in order to assess host-virus interaction related to apoptosis. For this purpose the Brazilian strain (TCoV/Brazil/2006 with GenBank accession number FJ188401), was used for infection. Infected IOC cells had mitochondrial dysfunction and initial nuclear activation with MTT value of 90.7 (± 2.4) and apoptotic factor 2.21 (± 2.1), considered statistically different from uninfected IOC cells (p > 0.05). The kinetics of TCoV antigens and viral RNA was directly correlated to annexin-V, caspases- 2 and -3, p53, BCl-2 antigens at 24, 72 and 96 h post-infection (p.i.). Morphological and biochemical features of apoptosis, such as in situ nuclear fragmentation (TUNEL and annexin-V) and DNA ladder formation were also detected in infected cells at all assayed p.i. intervals. Moreover, different from other coronaviruses, the expression of both effective caspase-2 and - 3 and p53 antigens were considered lower. However, at all p.i., the BCl-2 antigens were expressed quantitatively and qualitatively as viral antigen measured by immunofluorescence microscopy analysis. Because the diagnosis of TCoV infection is only performed by infecting embryonated poult eggs, the pathological characteri tics related to host-virus interaction remain unclear. This is the first report on apoptosis of TCoV infected IOC, and reveals that it may be useful immunological method to assess virus pathogenesis