Pathogenicity studies of the brazilian variant strain (BR) of infectious bronchitis virus

Orientadores: Clarice Weis Arns, Helena Lage FerreiraDissertação (mestrado) - Universidade Estadual de Campinas, Instituto de BiologiaResumo: Estudos recentes identificaram variantes do vírus da bronquite infecciosa das galinhas (Infectious Bronchitis Virus - IBV) exclusivamente brasileiras (BR), cujo genótipo não está relacionado com o grupo Massachusetts. As únicas vacinas vivas permitidas no Brasil são deste sorotipo, o que sugere uma proteção ineficiente contra os variantes presentes no país. Devido à carência de estudos de patogenicidade da variante BR do IBV, se faz necessário o conhecimento de seus efeitos na população avícola, elucidando seus sinais clínicos, lesões macroscópicas e microscópicas, além da produção de anticorpos induzida pela mesma. O presente estudo visa analisar a patogenicidade e o tropismo da variante BR do IBV para frangos de corte, através de um experimento in vivo utilizando aves SPF de um dia. Previamente, foram desenvolvidas 12 passagens em ovos embrionados com o objetivo de se aumentar a carga viral e realizar a titulação das amostras 801 e 810, obtendo os efeitos esperados nos embriões, de nanismo enrolamento e hemorragia. Quanto ao título viral, obteve-se um título de aproximadamente 105 EID, que quando inoculado nas aves, gerou sinais clínicos respiratórios e lesões macroscópicas esperadas nos tratos respiratório e renal, principalmente. Ao teste da inibição da atividade ciliar, ou ciliostase, fragmentos de traqueia das aves foram coletados e observados em microscópio. Verificou-se maior inibição da atividade ciliar entre o 7º e 9º d.p.i. Os resultados obtidos com a técnica de qRT-PCR mostram os órgãos do trato respiratório como sítio de replicação primária, concordando com os sinais clínicos nos dias iniciais. Os níveis de RNA detectados nos órgãos do trato digestório foram os mais altos e apresentaram a maior persistência entre todos os órgãos coletados. Entretanto, o exame histopatológico confirmou o achado de outros estudos, nos quais apesar do alto nível de RNA detectado nos órgãos do trato digestório, houve poucas alterações microscópicas nestes órgãos. No trato respiratório, porém, as lesões microscópicas foram compatíveis com os sinais clínicos e a detecção de RNA. Ambos os grupos geraram uma baixa resposta de produção de anticorpos. Com este estudo, foi possível concluir que estas variantes brasileiras apresentam patogenicidade muito similar à de amostras pertencentes ao genótipo Massachusetts, no qual estão incluídas as vacinas mais utilizadas, com tropismo pelo trato respiratório e também sendo capaz de se replicar em grandes quantidades no trato digestório e urinário, porém com poucas ou nenhuma lesãoAbstract: Recent studies identified variants of the Infectious bronchitis vírus (IBV) exclusives from Brazil (BR), which genotype is not related to the Massachussets serotype. In Brazil, alive vaccines for IBV are only allowed if belonging to this serotype, what suggests an inefficient protection against brazilian variants. Due to the lack of studies focusing the pathogenicity of the BR variant of IBV, the knowledge of its effects become necessary, elucidating clinical signs, macroscopic and microscopic lesions, besides the immunity against this virus. This study aims to analyse the pathogenicity and tropism of the BR variant of IBV for poultries through a in vivo experiment, using day old specific pathogen free (SPF) chicks. Previously, 12 serial passages in embryonated eggs were performed with the aim to increase the viral load and to perform the titration of strains 801 ans 810, reaching the expected effects on the embryos (curling, dwarfing and hemorrhage). The viral titre obtained was around 105 EID. When inoculated in the chicks, produced respiratory clinical signs and macroscopic lesions in kidneys and trachea. In the ciliary activity test, tracheal fragments were collected and examined. A ciliary activity inhibition was observed between 7 and 9 d.p.i. (days post inoculation). The results obtained using the qRT-PCR technique showed the respiratory organs as a primary replication site, in consonance with the clinical signs observed in the first days of experiment. The highest RNA levels were detected in the digestory system, where the persistence was also the longest. Nevertheless, the histopathological examination confirmed the findings of others studies, that showed only a few microscopic lesions, despite the high RNA level detected. In the respiratory system, however, microscopic lesions were consistent with clinical signs and RNA detection. Both groups presented a low antibody response. With the results of this study, it is possible to conclude that these brazilian varianst presented a pathogenicity very similar to the Massachusetts strains, with respiratoty tropism and are also able to replicate in large quantitites in the digestory and urinary system, but causing only a few or no lesionsMestradoMicrobiologiaMestre em Genética e Biologia Molecular2013/02058-6FAPES

Investigação do microbioma e ocorrência de patógenos zoonóticos em gambás "Didelphis" de fragmentos florestais da região de Campinas - SP

Orientador: Clarice Weis ArnsTese (doutorado) - Universidade Estadual de Campinas, Instituto de BiologiaResumo: A composição da diversidade bacteriana e viral de mamíferos é modelada por diversas características intrínsecas e extrínsecas que influenciam a relação patógeno-hospedeiro. Identificar espécies reservatórios naturais e quais características estão associadas à ocorrência de patógenos são essenciais para o controle de doenças infecciosas emergentes. A biodiversidade encontrada no Brasil representa um potencial para a emergência de novas zoonoses. Marsupiais do gênero "Didelphis" estão amplamente distribuídos pelo continente americano e as espécies "Didelphis" albiventris e "Didelphis" aurita ocorrem nas áreas mais populosas do Brasil e são comuns em toda sua área de distribuição. Adaptam-se à uma variedade de habitats, incluindo grandes centros urbanos e florestas secundárias modificadas pela ação humana. No entanto, seus potenciais como hospedeiros de patógenos zoonóticos ainda não foram profundamente estudados. O objetivo deste trabalho foi realizar um estudo metagenômico focando em amostras de duas espécies de gambás capturados em três fragmentos florestais da região de Campinas, através de sequenciamento de nova geração. A microbiota basal de suabes orais e anais obtidos de gambás saudáveis é descrita e a ocorrência de patógenos zoonóticos que podem ser alvos para pesquisas epidemiológicas futuras também é discutida. Foram observadas diferenças entre a composição do microbioma oral e anal, com microbiomas do mesmo tipo de amostras mais similares entre sí independentemente da espécie e local de coleta. Swabs orais apresentaram uma maior diversidade bacteriana. Também foram detectadas sequências relacionadas a patógenos zoonóticos, incluindo Campylobacter, Salmonella, Burkholderia, Chlamydia e Hepacivirus. Alguns patógenos foram detectados somente em uma espécie de gambá ou somente em um local de coleta, sugerindo que fatores extrínsecos podem contribuir para a ocorrência destes patógenos, mas fatores instrínsecos como dieta e filogenia também podem explicar os padrões observados. Hepacivirus C foi detectado em todas as amostras, sugerindo que gambás didelfídeos são possíveis hospedeiros deste vírus zoonótico. A detecção de patógenos zoonóticos em espécies comuns como gambás alerta para a possibilidade de emergência de doenças em outras espécies, incluindo humanos, especialmente quando seus nichos ecológicos se sobrepõem naturalmente ou devido à fragmentação. Investigações futuras podem abordar a relação entre os padrões de ocorrência de patógenos e fatores extrínsecos como as características ambientaisAbstract: Mammalian wildlife gut bacterial and viral diversity composition are shaped by numerous host traits and environmental factors influencing host - pathogen relationships. Identifying natural reservoir species and characterizing which traits are associated with pathogens occurrence are key to controlling emerging infectious diseases. The biodiversity found in Brazil is a potential for the emergence of new zoonoses. Marsupials of the "Didelphis" genus are widely distributed in the American Continent and the species "Didelphis" albiventris and "Didelphis" aurita occur in the most populated areas of Brazil and are common in all of their area of distribution. They adapt to a broad variety of habitats, including great urban centers and secundary forests modified by human action. However, their potential as zoonotic pathogens hosts has not been deeply studied yet. Here we describe a metagenomic study that focused on samples from opossums representing two species captured in three forest fragments in the region of Campinas, State of São Paulo, Brazil, using high throughput sequencing. The baseline microbiome of oral and anal swabs of healthy opossums is described in this article. We also discuss the occurrence of zoonotic pathogens that can be targets for future epidemiological surveys. Differences between oral and gut microbiomes composition were observed, with microbiomes from the same body site being more similar across especies and sampling sites. Oral swabs presented higher bacterial diversity than anal swabs. At species level, we also detected sequences related to zoonotic pathogens, including Campylobacter, Salmonella, Burkholderia, Chlamydia and Hepacivirus. Some pathogen species were assigned only to one opossum specie or only one sampling site, suggesting that extrinsic factors may contribute at some level to the occurrence of these pathogens but intrinsic factors such as diet and phylogeny can also explain the patterns observed. Hepacivirus C was detected in all of the samples, suggesting that didelphid opossums are possible hosts for this zoonotic virus and can also be competent hosts for other viruses from the Flaviviridae family. The detection of zoonotic pathogens in such abundant species warns for the possibility of emergence in other species including humans, specially when their habitats overlap naturally or due to the fragmentation. Further investigations could address the relation between these patterns and extrinsic factors such as environmental characteristicsDoutoradoMicrobiologiaDoutor em Genetica e Biologia Molecular154978/2016-7CNP

Rapid genotyping of porcine reproductive and respiratory syndrome virus (PRRSV) using MinION nanopore sequencing.

The global distribution and constant evolution are challenges for the control of porcine reproductive and respiratory syndrome virus (PRRSV), one of the most important viruses affecting swine worldwide. Effective control of PRRSV benefits from genotyping, which currently relies on Sanger sequencing. Here we developed and optimized procedures for real-time genotyping and whole genome sequencing of PRRSV directly from clinical samples based on targeted amplicon- and long amplicon tiling sequencing using the MinION Oxford Nanopore platform. Procedures were developed and tested on 154 clinical samples (including lung, serum, oral fluid and processing fluid) with RT-PCR Ct values ranging from 15 to 35. The targeted amplicon sequencing (TAS) approach was developed to obtain sequences of the complete ORF5 (main target gene for PRRSV genotyping) and partial ORF4 and ORF6 sequences of both PRRSV-1 and PRRSV-2 species. After only 5 min of sequencing, PRRSV consensus sequences with identities to reference sequences above 99% were obtained, enabling rapid identification and genotyping of clinical PRRSV samples into lineages 1, 5 and 8. The long amplicon tiling sequencing (LATS) approach targets type 2 PRRSV, the most prevalent viral species in the U.S. and China. Complete PRRSV genomes were obtained within the first hour of sequencing for samples with Ct values below 24.9. Ninety-two whole genome sequences were obtained using the LATS procedure. Fifty out of 60 sera (83.3%) and 18 out of 20 lung samples (90%) had at least 80% of genome covered at a minimum of 20X sequence depth per position. The procedures developed and optimized in this study here are valuable tools with potential for field application during PRRSV elimination programs

Infection and tissue distribution of highly pathogenic avian influenza A type H5N1 (clade 2.3.4.4b) in red fox kits (Vulpes vulpes)

ABSTRACTAvian influenza H5N1 is a highly pathogenic virus that primarily affects birds. However, it can also infect other animal species, including mammals. We report the infection of nine juvenile red foxes (Vulpes vulpes) with Highly Pathogenic Avian Influenza A type H5N1 (Clade 2.3.4.4b) in the spring of 2022 in the central, western, and northern regions of New York, USA. The foxes displayed neurologic signs, and examination of brain and lung tissue revealed lesions, with brain lesions ranging from moderate to severe meningoencephalitis. Analysis of tissue tropism using RT-PCR methods showed a comparatively lower Ct value in the brain, which was confirmed by in situ hybridization targeting Influenza A RNA. The viral RNA labelling was highly clustered and overlapped the brain lesions, observed in neurons, and grey matter. Whole viral genome sequences obtained from the affected foxes were subjected to phylogenetic and mutation analysis to determine influenza A clade, host specificity, and potential occurrence of viral reassortment. Infections in red foxes likely occurred due to preying on infected wild birds and are unlikely due to transmission between foxes or other mammals

Experimental infection of inbred BALB/c and A/J mice with Massachusetts and Brazilian strains of infectious bronchitis virus (IBV)

The ability of avian coronaviruses to replicate in mice was investigated to investigate interspecies transmission. Two inbred mouse strains (BALB/c and A/J) with different genetic backgrounds were inoculated with the avian coronavirus strains Mass and BR-I and monitored for at least 10 days. Analysis of viral RNA, histopathological examinations, immunohistochemistry and serology were performed. After virus inoculation, neither clinical signs nor evident gross lesions were observed. Viral RNA, histopathological changes, and viral nucleoprotein were observed in the lung, trachea and sinus of all inoculated mice. Our study demonstrates the importance of elucidating the epidemiology of coronaviruses, including in rodents that are pests in poultry production.Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq

Bat Coronavirus in Brazil Related to Appalachian Ridge and Porcine Epidemic Diarrhea Viruses

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq

Bat Coronavirus in Brazil related to appalachian ridge and porcine epidemic diarrhea viruses

Sem informação214729731CONSELHO NACIONAL DE DESENVOLVIMENTO CIENTÍFICO E TECNOLÓGICO - CNPQFUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULO - FAPESP307738/2011-62011/50919-