Serological and molecular, detection and characterization of avian Paramyxovirus type 1 (class I and class II) in wild and synanthropic birds

Orientadores: Clarice Weis Arns, Helena Lage FerreiraDissertação (mestrado) - Universidade Estadual de Campinas, Instituto de BiologiaResumo: Os paramixovírus aviários do tipo 1 (APMV-1) pertencem à família Paramyxoviridae, subfamília Paramyxovirinae, gênero Avulavirus. O APMV-1 causador da doença de Newcastle (DN) é considerada uma das maiores causas de perdas econômicas para a avicultura mundial, devido a alta mortalidade e os embargos econômicos envolvidos. A compreensão da epidemiologia de infecções causadas por APMV-1 é dificultada pelo fato de que estes vírus não produzem sinais clínicos e frequentemente não são detectados por métodos de diagnóstico rápidos. As aves silvestres parecem ser um importante reservatório desses vírus. Recentemente, a análise dos tamanhos de genoma e sequências de genes revelou dois clados distintos dentro APMV-1: classe I e II. O presente estudo teve como objetivo investigar a presença de APMV-1 em aves selvagens e sinantrópicas através de testes moleculares e sorológicos. Para este fim, 387 amostras de aves (194 amostras de orofaringe e 193 esfregaços de cloaca) pertencentes a 37 espécies de 12 ordens foram testados. Um ensaio em tempo real de RT-PCR (RRT-PCR), para a detecção simultânea de vírus de classe I e classe II foi utilizado para a detecção molecular de genes L e M, respectivamente. Outros sete testes RT-PCR convencional direcionados ao gene de fusão (F) e ao gene de hemaglutinina-neuraminidase (HN) foram testados em comparação com a sensibilidade analítica do ensaio de RRT-PCR. Estes RT-PCR foram também utilizados para sequenciamentos das amostras positivas. Assim, das 387 amostras testadas 10 foram detectadas como positivas pelos RRT-PCR. Entre as amostras positivas, encontra-se, uma proveniente de Sporophila frontalis ave ameaçada de extinção, na qual ainda não se sabe totalmente o efeito do AMPV-1. Foi possível sequenciar três amostras com o gene HN e onze com gene F, estas sequências foram agrupadas dentro do genótipo II, classe II de APMV-1. No entanto, este grupo de amostras brasileiras parece representar um novo cluster dentro deste genótipo. Além disso, em um ELISA competitivo testado, obteve-se uma sensibilidade analítica e especificidade perto de 100% e 10% reprodutibilidade. No total, 123 soros foram testados por ELISA e pelo teste de Inibição da hemaglutinação (HI). A frequência de ocorrência, utilizando HI foi de 22,73% e 19,51% no ELISA competitivo. Essas duas técnicas tiveram uma correlação de 0,62. Portanto, nosso estudo reitera a importância de continuar a vigilância em aves selvagens para aumentar o conhecimento sobre a epidemiologia da AMPV -1 no BrasilAbstract: The avian paramyxovirus type 1 (APMV-1) belongs to family Paramyxoviridae, subfamily Paramyxovirinae, genus Avulavirus. APMV-1 that causes Newcastle disease (ND) is considered a major cause of economic losses to the global poultry industry, because of high mortality and economic embargoes. The Understanding of epidemiology of infections caused by APMV-1 is hampered by the fact that these viruses do not produce clinical signs and often are not detected by rapid diagnostic methods.Wild birds seem to be an important reservoir of these viruses. Recently, analysis of the genome sizes and sequences of genes has revealed two distinct clades within APMV-1: class I and II. The present study aimed to investigate the presence of APMV-1 in wild and feral birds through molecular and serological tests. To this end, 387 samples (194 oropharyngeal swabs and 193 cloacal swabs) of 37 species belonging to 12 orders of birds were tested. A test of real time RT-PCR (RRT-PCR), for the simultaneous detection of viruses of class I and class II was used for molecular detection of genes L and M, respectively. Seven others conventional RT-PCR tests targeting fusion (F) and hemagglutinin-neuraminidase (HN) genes were tested and analytical sensitivity compared with RRT-PCR assay. Those RT-PCR were used for DNA sequencing. Ten out of 387 tested samples were positive by RRT-PCR. Among the positive samples were found, one from Sporophila frontalis an endangered birds species, for which is not yet fully know about AMPV-1 effect. Three samples sequences with the HN gene and eleven with gene F were analyzed phylogenetically and grouped within the genotype II, class II of APMV-1. However, this brazilian group seems to represent a new cluster within this genotype. A competitive ELISA test was tested that performed analytical sensitivity and specificity close to 100% and 10% reproducibility using reference sera. In total, 123 sera were tested by ELISA and also tested by HI test. The occurrence frequency in birds, Anseriforms, especially was 22,73% and 19,51% using HI and ELISA tests, respectively. Those two techniques had a correlation of 0.62. Therefore, our study reiterates the importance of continuing surveillance in wild birds to increase knowledge about epidemiology of AMPV -1 in BrazilMestradoMicrobiologiaMestre em Genética e Biologia Molecular2013/02059-2CAPESFAPES

ArfGAP with dual pleckstrin domains 2 protein in the chicken innate immune system

Aves e mamíferos possuem algumas semelhanças em seu sistema imune inato, porém também apresentam diferenças pela sua divergência evolutiva. Alguns componentes da via do interferon (IFN) estão ausentes em células de galinha, como o gene RIG-I, IRF 3 e IRF 9. Outros componentes são únicos na resposta imune inata das galinhas, como o gene do receptor do tipo Toll 15 (TLR15). A via do IFN tipo I, ativa os genes estimulados por IFN (ISGs), os quais possuem um importante papel contra os vírus, estabelecendo o estado antiviral no hospedeiro. No entanto, os mecanismos moleculares dessa indução em aves são pouco compreendidos e podem ser evolutivamente distintos dos mamíferos. Em mamíferos, uma das proteínas responsivas ao vírus e reguladas por IFN é codificada pelo gene Arf-GAP com dois domínios plaquistrinos 2 (ADAP2, também conhecido como CENTA2 e Centaurina-β). A caracterização molecular e biológica da proteína ADAP2 de galinha (chADAP2) foi realizada neste estudo. Análises de bioinformática foram realizadas para identificar características da sequência de aminoácidos desta proteína e compará-las com as sequências obtidas de mamíferos. A expressão do gene chADAP2 foi calculada após a infecção em células de galinha (DF-1) com o vírus da influenza aviária (AIV) de dois subtipos (H2N1 e H5N1) em diferentes tempos. A localização da proteína chADAP2 e sua interação com a proteína MAVS de galinha (chMAVS) e grânulos de estresse (G3BP1) em células de galinhas foi acessada pela microscopia confocal e pela co- imunoprecipitação. A atividade antiviral foi avaliada após a transfecção de plasmídeos expressando as proteínas de chADAP2 em células infectadas com o subtipo H9N2 do AIV. Nossos resultados mostraram que as sequências de aminoácidos da proteína ADAP2 deram origem a dois clusters (mamíferos e aves) na análise filogenética. Após a infecção com os isolados AIV houve um aumento de expressão do gene chADAP2, entre o período de 0 a 48 horas em células aviárias. A co-localização entre as proteínas chADAP2, MAVS e grânulos de estresse (G3BP1) na região citoplasmática celular foi confirmada pela microscopia confocal. A interação entre chADAP2 e chMAVS foi também comprovada pela co-imunoprecipitação. Finalmente, a atividade antiviral foi confirmada pela diminuição de infecção de AIV em células transfectadas com o plasmídeo expressando a proteína chADAP2 em células de galinha. O presente estudo identificou que a proteína chADAP2 possui um papel semelhante ao descrito pela ADAP2 em células de mamíferos, apesar da distância genética das sequências de nucleotídeos e organização dos domínios proteicos entre eles. Nossos dados confirmam a interação entre os grânulos de estresse e as proteínas da resposta antiviral, com as proteínas chADAP2 e chMAVS envolvidas na cascata para expressão de IFN tipo I. Este é o primeiro relato sobre o papel chADAP2 em células aviárias. Este novo conhecimento sobre a resposta imune inata permitirá o desenvolvimento de ferramentas para o controle de infecções virais em aves.Avian and mammals have some similarities in their innate immune system, but they also show differences due to their evolutionary divergence. A few components of the interferon (IFN) pathway are absent in chicken cells, such as the RIG-I gene, IRF 3, and IRF 9. Other components are unique in their innate immune response, like the toll- like receptor 15 (TLR15) gene. The type I IFN pathway, activates the IFN-stimulated genes (ISGs), which plays a crucial role against viruses by establishing an antiviral state in the host. However, molecular mechanisms of this induction in avian are poorly understood and can be evolutionarily distinct from mammalian. In mammalian, one of the virus-responsive and IFN-regulated protein is encoded by Arf-GAP with dual PH Domain-Containing Protein 2 gene (ADAP2, also known as CENTA2 and Centaurin -β). Molecular and biological characterization of chicken ADAP2 protein (chADAP2) was performed in this study. Bioinformatics analyses were performed to identify molecular features in the amino acid sequence and compare them to sequences obtained from mammals. chADAP2 gene expression was calculated after infection in chicken cells (DF-1) with avian influenza viruses (AIV) from two subtypes (H2N1 and H5N1) at different time points. The co-localization of chADAP2 protein with the chicken MAVS (chMAVS) and stress granules (G3BP1) in chicken cells was evaluated by confocal microscopy and co-immunoprecipitation. The antiviral activity was evaluated after the transfection of plasmids expressing chADAP2 protein in cells infected with AIV from H9N2 subtype. Our results showed that the ADAP2 amino acid sequences had two clusters (mammals and avian) by phylogenetic analysis. After infection with AIV isolates, an upregulation of the ADAP2 gene was detected from 0 to 48 hours in chicken cells. Confocal microscopy assays confirmed the co-localization of chADAP2, MAVS, and G3BP1 proteins in the cells’ cytoplasmic region. The interaction between chADAP2 and chMAVS has been also proven by co-immunoprecipitation. Finally, the antiviral activity of the chADAP2 was confirmed by a decrease in AIV expression in cells transfected with the plasmid expressing the chADAP2 protein in chicken cells. The present study identified that chADAP2 has a similar role, as described in mammals’ cells, despite their high genetic distances of nucleotide sequences and protein domains organization. Our results confirm the interaction among stress granules and antiviral response proteins, such as ADAP2 and chMAVS proteins involved in the cascade for IFN type I. This is the first report of the chADAP2 role in avian cells. This new knowledge about the innate immune response will allow the development of tools for controlling virus infections in birds

Oncolytic effect of Newcastle disease virus is attributed to interferon regulation in canine mammary cancer cell lines

Canine mammary carcinoma (CMC) is one of the major health threats in dogs. The oncolytic virotherapy is a promising strategy to treat canine as well as human cancer patients with non‐pathogenic replicating viruses. Here, we evaluated the antitumor activity of one lentogenic, non‐lytic Newcastle disease virus (NDV) LaSota strain expressing GFP (NDV‐GFP) on five different CMCs and one non‐tumorigenic cell line, regarding cell viability, cell death, selectivity index, morphology, global and target gene expression analysis. As evidenced by the selectivity index, all CMC cell lines were more susceptible to NDV‐GFP in comparison with the non‐tumorigenic cells (~3.1× to ~78.7×). In addition, the oncolytic effect of NDV‐GFP was more evident in more malignant CMC cells. Also, we observed an inverse association of the IFN pathway expression and the susceptibility to NDV. The downregulated genes in NDV‐GFP‐sensitive cells were functionally enriched for antiviral mechanisms by interferon and immune system pathways, demonstrating that these mechanisms are the most prominent for oncolysis by NDV. To our knowledge, this is the first description of oncolysis by an NDV strain in canine mammary cancer cells. We also demonstrated specific molecular pathways related to NDV susceptibility in these cancer cells, opening the possibility to use NDV as a therapeutic‐targeted option for more malignant CMCs. Therefore, these results urge for more studies using oncolytic NDVs, especially considering genetic editing to improve efficacy in dogs

Draft genome sequence of Kocuria sp. SM24M-10 isolated from coral mucus

Here, we describe the genomic features of the Actinobacteria Kocuria sp. SM24M-10 isolated from mucus of the Brazilian endemic coral Mussismilia hispida. The sequences are available under accession number LDNX01000000 (http://www.ncbi.nlm.nih.gov/nuccore/LDNX00000000). The genomic analysis revealed interesting information about the adaptation of bacteria to the marine environment (such as genes involved in osmotic and oxidative stress) and to the nutrient-rich environment provided by the coral mucus. Keywords: Kocuria sp. SM24M-10, Coral mucus, Osmotic stress, Oxidative stres

Assessment of the Interferon-Lambda-3 Polymorphism in the Antibody Response to COVID-19 in Older Adults Seropositive for CMV

Background: Here, we investigated the impact of IFN-lambda-3 polymorphism on specific IgG responses for COVID-19 in older adults seropositive for CMV. Methods: Blood samples of 25 older adults of both sexes were obtained at three different times: during a micro-outbreak (MO) of SARS-CoV-2 in 2020; eight months after (CURE); and 30 days after the administration of the second dose of ChadOx-1 vaccine (VAC). The specific IgG for both SARS-CoV-2 and CMV antigens, neutralizing antibodies against SARS-CoV-2, and also the polymorphism profile for IFN-lambda-3 (rs12979860 C > T) were assessed. Results: Higher levels of specific IgG for SARS-CoV-2 antigens were found in the MO and VAC than in the CURE time-point. Volunteers with specific neutralizing antibodies against SARS-CoV-2 showed better specific IgG responses for SARS-CoV-2 and lower specific IgG levels for CMV than volunteers without specific neutralizing antibodies. Significant negative correlations between the specific IgG levels for SARS-CoV-2 and CMV were found at the MO time-point, as well as in the group of individuals homozygous for allele 1 (C/C) in the MO time-point and heterozygotes (C/T) in the CURE time-point. Conclusion: Our results suggested that both CMV seropositivity and the homozygosis for allele 1 (C/C) in IFN-lambda-3 gene can negatively impact the antibody response to COVID-19 infection and vaccination in older adults