Evaluation of the Viral Interference between Lentogenic Newcastle Disease Virus (Lasota) and Avian Influenza Virus (H9N2) using Real-Time Reverse Transcription Polymerase Chain Reaction in SPF Chicken

ABSTRACT Lentogenic Newcastle disease virus (lNDV) such as Lasota strain and low pathogenicity avian influenza such as H9N2 virus are two of the most economically important viruses affecting poultry worldwide, and little attention in recent years has been paid to simultaneous infections in chickens with these two viruses for the reason that co-infection do occur but are not easily detected. In the present study, chickens were inoculated with lNDV (Lasota) and LPAIV (A/chicken/Tehran/ZMT-173/99(H9N2)) simultaneously or sequentially three days apart. Oropharyngeal and cloacal swabs were collected from chickens from 1 to 14 days after inoculation. RRT-PCR for AIV and NDV detection was performed. The rate of viral shedding was measured within 14 days. No clinical symptoms were observed during the experiment however the pattern of virus shed was different with co-infection, thus comparing the results obtained from viral shedding showed that AIV is a much stronger agent than NDV in the occurrence of viral interference. This is due to the fact that in simultaneous inoculation, AIV replication delayed and reduced NDV replication, while replication of Lasota in simultaneous or pre-inoculated inoculation could not significantly disrupt H9N2 virus replication. These findings indicate that the infection with one virus can interfere with the replication of another, modifying the pathogenesis of the viruses. So, infection of the host with both viral agents simultaneously causes higher shedding of LPAIV than lNDV in OP and CL areas. In conclusion, co-infection with LPAVI in chickens did not impact clinical signs but affected the replication dynamics of these viruses.

Evaluation of the Viral Interference between Lentogenic Newcastle Disease Virus (Lasota) and Avian Influenza Virus (H9N2) using Real-Time Reverse Transcription Polymerase Chain Reaction in SPF Chicken

Lentogenic Newcastle disease virus (lNDV) such as Lasota strain and low pathogenicity avian influenza such as H9N2 virus are two of the most economically important viruses affecting poultry worldwide, and little attention in recent years has been paid to simultaneous infections in chickens with these two viruses for the reason that co-infection do occur but are not easily detected. In the present study, chickens were inoculated with lNDV (Lasota) and LPAIV (A/chicken/Tehran/ZMT-173/99(H9N2)) simultaneously or sequentially three days apart. Oropharyngeal and cloacal swabs were collected from chickens from 1 to 14 days after inoculation. RRT-PCR for AIV and NDV detection was performed. The rate of viral shedding was measured within 14 days. No clinical symptoms were observed during the experiment however the pattern of virus shed was different with co-infection, thus comparing the results obtained from viral shedding showed that AIV is a much stronger agent than NDV in the occurrence of viral interference. This is due to the fact that in simultaneous inoculation, AIV replication delayed and reduced NDV replication, while replication of Lasota in simultaneous or pre-inoculated inoculation could not significantly disrupt H9N2 virus replication. These findings indicate that the infection with one virus can interfere with the replication of another, modifying the pathogenesis of the viruses. So, infection of the host with both viral agents simultaneously causes higher shedding of LPAIV than lNDV in OP and CL areas. In conclusion, co-infection with LPAVI in chickens did not impact clinical signs but affected the replication dynamics of these viruses.(AU)

Evaluation of the Viral Interference between Lentogenic Newcastle Disease Virus (Lasota) and Avian Influenza Virus (H9N2) using Real-Time Reverse Transcription Polymerase Chain Reaction in SPF Chicken

Lentogenic Newcastle disease virus (lNDV) such as Lasota strain and low pathogenicity avian influenza such as H9N2 virus are two of the most economically important viruses affecting poultry worldwide, and little attention in recent years has been paid to simultaneous infections in chickens with these two viruses for the reason that co-infection do occur but are not easily detected. In the present study, chickens were inoculated with lNDV (Lasota) and LPAIV (A/chicken/Tehran/ZMT-173/99(H9N2)) simultaneously or sequentially three days apart. Oropharyngeal and cloacal swabs were collected from chickens from 1 to 14 days after inoculation. RRT-PCR for AIV and NDV detection was performed. The rate of viral shedding was measured within 14 days. No clinical symptoms were observed during the experiment however the pattern of virus shed was different with co-infection, thus comparing the results obtained from viral shedding showed that AIV is a much stronger agent than NDV in the occurrence of viral interference. This is due to the fact that in simultaneous inoculation, AIV replication delayed and reduced NDV replication, while replication of Lasota in simultaneous or pre-inoculated inoculation could not significantly disrupt H9N2 virus replication. These findings indicate that the infection with one virus can interfere with the replication of another, modifying the pathogenesis of the viruses. So, infection of the host with both viral agents simultaneously causes higher shedding of LPAIV than lNDV in OP and CL areas. In conclusion, co-infection with LPAVI in chickens did not impact clinical signs but affected the replication dynamics of these viruses.

Mumps virus genotypes identified during disease outbreaks in the state of São Paulo, Brazil: 2011 – 2016 / Genótipos dos vírus da caxumba identificados durante surtos da doença no estado de São Paulo, Brasil: 2011 - 2016

In São Paulo the mumps virus (MuV) outbreaks have been increasing from 2011 to nowadays. MuV epidemiological surveillance has been improving by using the polymerase chain reaction in real time (rRT-PCR) in addition to the specific IgM antibody (IgM-Ab) detection; in some cases, genome sequencing studies were performed. Increased virus transmission and recent outbreaks have raised interest on MuV genotyping, as a means to understand the transmission pathways and to identify the vaccine-associated cases. From January 2011 to August 2016, MuV infection was analyzed at Institute Adolfo Lutz. A total of 232 (77.33 %) throat wash samples showed positivity to mumps genome, and 68 (22.66 %) were negative when analyzed by rRT-PCR. Among 15 samples for molecular analysis, 10 serum samples from respective patients were also available for detecting anti-MuV IgM-Ab; and from these, four (40%) samples were seropositive. Vaccination status was available only for patients from Cedral and Araraquara. Phylogenetic analysis revealed the circulation of the following mumps virus genotypes in the investigated periods: 2011(M), 2012, and 2013 (K); 2014 (N); 2015 (G, K, and N); 2016 (G). Knowledge on MuV molecular epidemiology in São Paulo-Brazil could contribute to the surveillance and epidemiological program in Brazil, and globally as well.

No estado de São Paulo têm ocorrido surtos de caxumba desde 2011. O diagnóstico laboratorial tem sido realizado no Instituto Adolfo Lutz utilizando-se a técnica de identificação de material genético viral por meio de reação de cadeia de polimerase-em tempo real (rRT-PCR) e pela detecção de anticorpos IgM (Ac-IgM) específicos circulantes. Os recentes surtos de caxumba têm aumentado o interesse em investigar os genótipos dos vírus prevalentes para identificar os casos associadas à vacina. De janeiro de 2011 a agosto de 2016, 300 amostras de lavados da orofaringe coletadas de pacientes suspeitos de infecção foram analisadas. O material genético viral específico foi detectado em 232 (77,33 %) amostras e 68 (22,66 %) foram negativas. Das 10 amostras analisadas pelo teste sorológico, quatro (40 %) demonstraram positividade para Ac-IgM específicos anti-vírus da caxumba e seis foram negativas. Somente os municípios Cedral e Araraquara forneceram os dados referentes à vacinação. Análise filogenética mostrou a circulação dos seguintes genótipos do vírus da caxumba no período investigado: 2011 (M), 2012 e 2013 (K); 2014 (N); 2015 (GKN); 2016 (G). A vigilância virológica é mundialmente imprescindível, para identificar a diversidade e a distribuição dos diferentes genótipos, com vistas à composição de vacinas específicas.

Mumps virus genotypes identified during disease outbreaks in the state of São Paulo, Brazil: 2011 2016 / Genótipos dos vírus da caxumba identificados durante surtos da doença no estado de São Paulo, Brasil: 2011 - 2016

In São Paulo the mumps virus (MuV) outbreaks have been increasing from 2011 to nowadays. MuV epidemiological surveillance has been improving by using the polymerase chain reaction in real time (rRT-PCR) in addition to the specific IgM antibody (IgM-Ab) detection; in some cases, genome sequencing studies were performed. Increased virus transmission and recent outbreaks have raised interest on MuV genotyping, as a means to understand the transmission pathways and to identify the vaccine-associated cases. From January 2011 to August 2016, MuV infection was analyzed at Institute Adolfo Lutz. A total of 232 (77.33 %) throat wash samples showed positivity to mumps genome, and 68 (22.66 %) were negative when analyzed by rRT-PCR. Among 15 samples for molecular analysis, 10 serum samples from respective patients were also available for detecting anti-MuV IgM-Ab; and from these, four (40%) samples were seropositive. Vaccination status was available only for patients from Cedral and Araraquara. Phylogenetic analysis revealed the circulation of the following mumps virus genotypes in the investigated periods: 2011(M), 2012, and 2013 (K); 2014 (N); 2015 (G, K, and N); 2016 (G). Knowledge on MuV molecular epidemiology in São Paulo-Brazil could contribute to the surveillance and epidemiological program in Brazil, and globally as well.(AU)

No estado de São Paulo têm ocorrido surtos de caxumba desde 2011. O diagnóstico laboratorial tem sido realizado no Instituto Adolfo Lutz utilizando-se a técnica de identificação de material genético viral por meio de reação de cadeia de polimerase-em tempo real (rRT-PCR) e pela detecção de anticorpos IgM (Ac-IgM) específicos circulantes. Os recentes surtos de caxumba têm aumentado o interesse em investigar os genótipos dos vírus prevalentes para identificar os casos associadas à vacina. De janeiro de 2011 a agosto de 2016, 300 amostras de lavados da orofaringe coletadas de pacientes suspeitos de infecção foram analisadas. O material genético viral específico foi detectado em 232 (77,33 %) amostras e 68 (22,66 %) foram negativas. Das 10 amostras analisadas pelo teste sorológico, quatro (40 %) demonstraram positividade para Ac-IgM específicos anti-vírus da caxumba e seis foram negativas. Somente os municípios Cedral e Araraquara forneceram os dados referentes à vacinação. Análise filogenética mostrou a circulação dos seguintes genótipos do vírus da caxumba no período investigado: 2011 (M), 2012 e 2013 (K); 2014 (N); 2015 (GKN); 2016 (G). A vigilância virológica é mundialmente imprescindível, para identificar a diversidade e a distribuição dos diferentes genótipos, com vistas à composição de vacinas específicas.(AU)

Vigilância epidemiológica dos vírus da influenza aviária, da doença de Newcastle e da laringotraqueíte infecciosa das galinhas em propriedades avícolas

A sanidade avícola é necessária para garantir a posição de destaque mundial da avicultura brasileira. O objetivo deste trabalho foi realizar a vigilância epidemiológica do vírus da IA (VIA) e do vírus da DNC (VDNC) em 2017 e 2018, além de avaliar a circulação de estirpes do vírus de laringotraqueíte infecciosa das galinhas (VLTI) nas granjas de postura comercial em duas regiões quarentemadas (Bastos e Guatapará) entre 2010 e 2018. Diferentes programas vacinais contra a LTI foram implementados na região, com a substituição de vacinas vivas atenuadas por recombinantes em 2012 (Bastos) e 2013 (Guatapará). Para a vigilância de VIA e VDNC foram coletados pools de suabes (traqueais e cloacais, n=220) de aves de subsistências localizadas no entorno de propriedades avícolas de reprodutoras. Essas amostras foram testadas pela reação de transcrição reversa-PCR em tempo real (RRT-PCR). O estudo longitudinal de VLTI coletou amostras de pools de suabes orofaríngeos de aves localizadas em Bastos (n=364) e Guatapará (n=214) para a detecção de VLTI por PCR. O sequenciamento de DNA dos genes ICP4, TK e genomas completos das amostras positivas foram realizados usando sequenciamento Sanger e sequenciamento de última geração (NGS). As bibliotecas de DNA foram preparadas com o kit Nextera DNA Flex Library Prep e sequenciadas com o sequenciador MiSeq. Análise filogenética e as identidades genéticas foram inferidas com as sequências obtidas. Nosso estudo não detectou VIA e do VDNC em nenhuma das amostras no período avaliado. O VLTI foi detectado em 11,85% e 12,6% das amostras testadas de Bastos em 2013 e 2018. A região de Guatapará apresentou a maior taxa de detecção (60,5%) em 2010. A taxa de detecção de amostras testadas da região de Guatapará variou de 12,2% e 21,7% após 2013. Análise filogenética do gene ICP4 agrupou as sequências das amostras de Bastos com vacinas de CEO e outras sequências de Bastos detectadas em anos anteriores, com 99% da identidade genética entre estas sequências. As análises filogenéticas dos genes ICP4, TK e do genoma completo agruparam as amostras de Guatapará separadamente das amostras vacinais. A sequência completa do genoma de uma amostra obtida de aves sintomáticas sem vacinação na região de Guatapará em 2010 (IB8098) teve 152.985 nucleotídeos, com 4755 leituras e 98,2% de cobertura. A análise filogenética agrupou a amostra IB8098 com outras estirpes virulentas, como da Rússia (MF405079). As identidades genéticas apresentaram 99,9%, quando comparadas à estirpe russa e 99,6% em relação às vacinais (CEO e TCO). Nosso estudo mostrou o VLTI ainda está presente nas regiões de Bastos e Guatapará, apesar das medidas de controle e vacinação vigentes. Os programas de vacinação nas regiões quarentenadas diminuíram a circulação do vírus com um leve aumento da detecção do VLTI com a utilização apenas das vacinas recombinantes. Esse é o primeiro relato da obtenção do genoma completo de VLTI no Brasil e sua caracterização genômica desde o início da circulação do vírus na região há pelo menos dez anos. O monitoramento contínuo da área é essencial para auxiliar as ações da defesa agropecuária, avaliar as medidas de vacinação implementadas contribuindo para a melhoria da sanidade avícola.

Poultry health is important to ensure the world's leading position in Brazilian poultry production. The present study performed the epidemiological surveillance of avian influenza virus (AIV) and Newcastle disease virus (NDV) in 2017 and 2018; the circulation of virulent avian infection laryngotracheitis virus (ILTV) strains was also evaluated in commercial layer farms from two quarantined regions (Bastos and Guatapara) between 2010 and 2018. Different vaccine programs against ILT were established in the area, and a replacement of live vaccines by recombinant vaccines was done in 2012 (Bastos) and 2013 (Guatapara). For the AIV and NDV surveillance, swab pool samples (trachea and cloacal, n=220) were collected from backyard birds located closed to breeder farms. Real-time reverse-transcription polymerase chain reaction (RRT-PCR) tested these samples. The longitudinal study collected oropharyngeal swab pool samples from commercial layer chickens located in Bastos (n=364) and Guatapara (n=214) for virus ILTV detection by PCR. DNA sequencing from the ICP4, TK genes and complete genomes was performed in positive samples using Sanger sequencing and next-generation sequencing (NGS). DNA libraries were prepared with the Nextera DNA Flex Library Prep kit, sequenced with the MiSeq sequencer for NGS. Phylogenetic analysis and genetic identities were inferred using the obtained sequences. AIV and NDV were not detected in any sample during the study. ILTV was detected in 11.85% and 12.6% of tested samples from Bastos in 2013 and 2018. Guatapara region had the highest detection rate (60.5%) in 2010. The detection rate from tested samples of the Guatapara region ranged from 12.2% and 21.7% after 2013. Phylogenetic analysis based on ICP4 gene grouped sequences from Bastos samples with CEO vaccines and other sequences detected previously in Bastos, with 99% of nucleotide identity among them. Phylogenetic analysis based on ICP4, TK, and complete genomes grouped sequences from Guatapara separately from vaccine strains. The complete genome sequence of a sample (IB8098), collected from layer chickens displaying clinical signs without vaccination in the Guatapara region in 2010, had 152,985 nucleotides, with 4,755 reads and 98,2% coverage. The phylogenetic analysis grouped the IB8098 sample with other virulent ILTV strains, such as from Russia (MF405079). Nucleotide identities were 99.9% compared to the Russian, and 99.6% to vaccine strains (CEO and TCO). Our study showed the ILTV is still present in the Bastos and Guatapara regions, despite the control and vaccination measures. The vaccination programs in the quarantine regions decreased the virus circulation, slightly increasing when only recombinant vaccines were applied. That is the first report of complete genome ILTV sequences in Brazil and its genomic characterization, after its circulation in the region for at least ten years. Continuous monitoring in poultry flocks is essential to help the poultry health measures, evaluate the vaccination programs placed in the area contributing to the Brazilian poultry health plans improvement.

Metapneumovírus aviários em aves silvestres

Metapneumovírus Aviário (aMPV), família Pneumoviridae, gênero Metapneumovírus, é o agente etiológico responsável pela rinotraqueíte dos perus e também está associada à síndrome da cabeça inchada em galinhas, duas importantes doenças respiratórias que acometem aves comerciais e levam a grandes perdas econômicas. O objetivo deste estudo foi detectar a presença de aMPV em amostras de aves silvestres e realizar a caracterização moleculardos isolados encontrados, com o intuito maior de contribuir para o entendimento da epidemiologia desse vírus. Para isso foram coletados no total, 448 suabes orofaringeanos (OP) e cloacais (C) oriundos de 234 aves silvestres em quatro locais do estado de São Paulo. Três tipos de amostras foram processadas e testadas: 1) 266 suabes agrupados de um ou até cinco animais que estavam no mesmo recinto e respeitando o mesmo tipo de suabe (OP ou C); 2) 188 suabes foram agrupados na forma de pools de até dois animais, contendo os suabes OP e C de cada animal, formando então 48 polls; 3) amostras de tecido traqueal e pulmonar de três Egretta thula também foram coletados e testados. A purificação de RNA viral foi realizada utilizado o QIAmp RNA Mini Kit Kit (Qiagen). Para o teste de RT-PCR foi utilizado o lit OneStep RT-PCR (Qiagen) com primers baseados no gene N, previamente descritos, com fragmento esperado de 115 bp. As amostras foram testadas por RT-PCR em tempo real (RRT-PCR) com primers específicos previamente descritos, para os subtipos A e B, com base no gene G com fragmentos de 116 e 135 bp, respectivamente. Os fragmentos das amostras positivas foram purificados e sequencias pelo sequenciamento tipo Sanger para caracterização por análises filogenéticas. Das 126 amostras testadas pelo teste de RT-PCR baseado no gene N, quatorze foram positivas: oito amostras de Anseriformes (Aix sponsa, Aix galericulata, Dendrocygna viduata), três de Columbiformes (Columba livia), um de Falciformes (Falco sparverius), um de Psittaciformes (Psittacara leucophthalma) e um de Pelecaniformes (Egretta thula). Das quatorze amostraspositivas, treze eram provenientes de suabes, e a décima quarta era oriunda de tecido traqueal de Egretta thula. Pelo teste de RRT-PCR baseado no gene G nenhuma das 184 amostras testadas foi positiva. As análises filogenéticas realizadas com fragmentos de 44 e 54 bp do gene N de duas amostras positivas, que se agruparam com isolados pertencentes ao aMPV de subtipo A. Estes vírus apresentaram alta identidade com as estirpes derivadas de vacina e com estirpes vacinais, o que mostra uma possível ocorrência de escapers de aves de produção para as aves silvestres.

Avian Metapneumovirus (aMPV), family Pneumoviridae, genus Metapneumovirus, it is the etiologic agent responsible for turkey rhinotracheitis and is also associated with swollen head syndrome in chickens, two important respiratory diseases in poultry which leads to large economic losses. The aim of this study was detect the presence of aMPV wild birds samples, to perform the phylogenetic analysus of the isolates found with the major objetive of contributing to the understanding of the epidemiology of this virus in poultry farms. In total, 448 oropharyngeal (OP) and cloacal (C) swabs from 234 wild birds collected in four different locations within the state of São Paulo. The samples were processed and tested in three different ways: 1) 266 swabs were in the form of pools of one up five animals that were in the same enclosure and respecting the same type of swab (OP or C); 2) 188 swabs were grouped into pools of up to two animals, containing the oropharyngeal and cloacal swabs of each animal,; 3) tracheal and pulmonary tissue samples were also collected and tested. Purification was performed using the QIAmp RNA Mini Kit Kit (Qiagen). Viral detection was performed by conventional RT-PCR technique using the OneStep RT-PCR kit (Qiagen) with primers based on the N-gene, previously described with expected fragment of 115 bp. The samples were also tested by a real time RT-PCR (RRT-PCR) with specific primers previously described, for subtypes A and B, based on the G gene with fragments of 116 and 135 bp, respectively. Of the 126 samples tested by the N gene-based RT-PCR, fourteen were positive: eight samples of Anseriformes (Aix sponsa, Aix galericulata, Dendrocygna viduata), three Columbiformes (Columba livia), one Falcoformes (Falco sparverius), one Psittaciformes (Psittacara leucophthalma) and one of Pelecaniformes (Egretta thula). Of the swab samples, five were derived from oropharyngeal swabs and four from cloacal swabs, the other four samples were detected in the samples processed in pools of up to two animals, which contained the oropharyngeal and cloacal swabs of each bird. The positive Egretta thula sample was from a tracheal tissue sample. Based on the RRT-PCR G gene based, none of the 184samples tested were detected. Phylogenetic analyzes were performed on two positive samples that proved to belong to aMPV subtype A, showing high similarity with the strains derived from the vaccine and with vaccine strains.

Detecção e caracterização moleculares dos paramixovírus aviários tipo 1 em materiais provenientes de aves silvestres utilizando testes para a detecção dos vírus da família viral Paramyxoviridae

As aves silvestres são importantes reservatórios de vírus que podem acometer as aves domésticas. O monitoramento da circulação viral em aves silvestres é de extrema importância para garantir a sanidade dos plantéis avícolas. O presente estudo teve como objetivo 1) comparar dois testes moleculares de RT-PCR para a detecção dos vírus da família Paramyxoviridae em aves silvestres e sinantrópicas; 2) caracterizar os vírus detectados nestas amostras. A comparação da sensibilidade e especificidade analíticas foi realizada entre dois testes de RT-PCR e testes específicos de RT-PCR em tempo real (RRT-PCR) para o vírus da doença de Newcastle (NDV) e o metapneumovirus aviário (aMPV). A sensibilidade diagnóstica entre os dois testes de RT-PCR foi comparada. Um pequeno fragmento da região do sítio de clivagem do gene F das amostras positivas foi sequenciado. Os testes de RT-PCR foram validados com sucesso, mas apresentaram diferenças entre a sensibilidade analítica quando comparados aos testes específicos de RRT-PCR utilizando diferentes vírus. No total, 100 amostras de aves (suabes) foram testados pelo teste RT-PCR que apresentou uma sensibilidade analítica similar entre os diferentes agentes virais. O teste selecionado foi capaz de detectar duas amostras de aves silvestres que foram também detectadas pelo testes específico para NDV e. relacionadas às amostras de NDV vacinais do genótipo II da classe II referentes aos vírus de NDV lentogênico (113RQGR L117). Nosso estudo demonstra a deficiência na biosseguridade adotada pelos sistemas avícolas por permitir a saída dos vírus vacinais para as aves silvestres.

Wild birds are important reservoirs of viruses can affect poultry. Surveillance of circulating viruses in wild birds is of the most importance tool to ensure the poultry health. The present study aimed to 1) compare two molecular tests for detection of family Paramyxoviridae viruses in wild and feral birds; 2) characterize the detected viruses in those samples. Analytical and diagnostic sensitivity were compared among two RT-PCR techniques and specific real time RT-PCR (RRT-PCR) for detection of Newcastle disease virus (NDV) and avian metapneumovirus (aMPV). Diagnostic sensitivity was also compared between two RT-PCR tests. A small fragment of cleavage site region in F gene from positive samples was sequenced. The RT-PCR were successfully validated, however they had differences in the analytical sensitivity when compared to specific RRT-PCR assays using different viruses. In total, 100 wild bird samples (swabs) were tested by the selected RT-PCR with similar analytical sensitivity between tested viruses. Two samples were positive by this test and they were also detected by the specific RRT-PCR for NDV. These samples were closed related to vaccinal NDV strains belonging to genotype II class II. Deduced amino acid sequences of cleavage site region from detected samples were characterized as lentogenic NDV strains (113RQGR L117). Our study demonstrates the poor biosafety used by poultry industry allowing the vaccinal escape to wild bird species.

CARACTERIZAÇÃO GENÉTICA DE VÍRUS INFLUENZA ISOLADOS EM SUÍNOS NO RIO GRANDE DO SUL

O vírus influenza A (IAV) é um agente zoonótico de grande relevância tanto para saúde humana como animal. A influenza suína teve seu primeiro reconhecimento clínico em 1918, em suínos do Meio Oeste dos EUA, coincidindo com a pandemia de influenza em humanos. Desde então, o IAV permanece como um importante patógeno para a indústria suinícola em todo o mundo. A grande variabilidade genética destes vírus é causada por dois principais mecanismos genéticos: mutações pontuais e recombinações genéticas. A influenza é endêmica em muitos países e a emergências de recombinantes tem desafiado o controle e o diagnóstico desta enfermidade. No Brasil, a infecção pelo IAV em suínos (swIAV) não está bem caracterizada; poucos relatos evidenciam a prevalência deste agente antes do ano de 2009, especialmente no Estado do Rio Grande do Sul, que alberga um dos maiores rebanhos de suínos do Brasil. Em vista disso, este trabalho teve como objetivo investigar ocorrência de swIAV em alguns rebanhos suínos comerciais do Estado do Rio Grande do Sul, Brasil, no período de 2013-2014, e determinar os tipos e subtipos de vírus circulantesnaquelas propriedades. O primeiro capítulo deste estudo reporta os aspectos clínicos,patológicos e virológicos da ocorrência de influenza suína e co-infecções identificadas em seis propriedades suinícolas selecionadas na região do Vale do Taquari. Neste estudo foram analisados suabes nasais coletados de 66 animais e 6 amostras de tecido pulmonar de suínos com sinais de infecção respiratória. A detecção viral foi feita através de uma PCR de triagem e confirmada através do isolamento viral em célulasMDCK. A identificação dos subtipos virais foi feita através de uma PCR em Tempo Real (rRT-PCR) para o subtipo A(H1N1)pdm09 ou através de uma PCR multiplex(RT-PCR) para outros subtipos de swIAV. A detecção de agentes bacterianos foi realizada apenas nas amostras de tecido pulmonar, através da pesquisa de genomas bacterianos por PCR. O subtipo A(H1N1)pdm09 foi identificado em 4/6 granjas e o subtipo H1N2 em 2/6 granjas. Além disso, agentes envolvidos no complexo respiratório dos suínos foram identificados em todas as granjas; Pasteurella multocida foi identificada em 5/6 granjas e Mycoplasma hyopneumoniae em 3/6 granjas. Actinobacillus pleuropneumoniae (1/6), Haemophilus parasuis (1/6) e PCV2 (1/6) também foram detectados. O segundo capítulo deste estudo teve como objetivo o sequenciamento do genoma completo de um novo recombinante H1N2 de origem humana, detectado em suínos. O genoma completo foi gerado através de uma RT-PCR. Os produtos forampurificados e submetidos ao sequenciamento utilizando a plataforma MiSeq(illumina). A análise filogenética revelou que as sequencias dos genes HAe NA correspondem a genes de IAV de origem humana, enquanto que as sequencias dos genes que codificam as proteínas internas do vírus (PB1, PB2, PA, NP, M e NS) correspondem a genes de amostras do vírus A(H1N1)pdm09. O terceiro capítulo reporta o sequenciamento completo dos genomas de 8 amostras de vírus influenza identificados nas populações de suínos amostradas. Foram identificados dois subtipos virais de origem humana (H1N2 e H3N2), além do vírus A(H1N1)pdm09. Os subtipos de origem humana possuem os genes HAe NA similares a vírus sazonais de humanos e os genes internos são estreitamente relacionados com o vírus A(H1N1)pdm09.

Influenza A virus (IAV) is a zoonotic agent of great relevance to human and animal health. Swine influenza was first recognized clinically in pigs in the Midwestern U.S., in 1918, coinciding with the human influenza pandemic.Since that time swine influenza has remained of importance to the swine industry throughout the world. The great genetic variability of influenza viruses is caused by two main genetic mechanisms: point mutations (antigenic drift) and gene reassortment (antigenic shift). Influenza is endemic in pigs in many countries and the emergence of new viruses has been challenging its control and diagnostics. Influenza virus (swIAV) infection in Brazilian swine population is not well characterized, and little evidence existed of swIAV circulation before 2009, especially in Rio Grande do Sul State, which hosts one of the largest swine populations in Brazil. Thus, this study aimed to investigate the occurrence of IAV in commercial swine herds in the state of Rio Grande do Sul, Brazil, between 2013-2014 and to know the types and subtypes of swine influenza viruses that are circulating in these herd. The first chapter of this study reports the clinical, pathological and virological aspects of the occurrence of swine influenza and related co-infections in six pig properties of the Taquari Valley region. In this study were analyzed nasal swabs collected from 66 animals and six lung tissue samples from pigs showing clinical signs of respiratory disease. IAV detection was performed by PCR screening and confirmed by virus isolation in MDCK cells and hemagglutination (HA). Influenza A subtyping was performed by real-time reverse transcription PCR (rRT-PCR) to detect the 2009 H1N1pandemic A(H1N1)pdm09; other swIAV subtypes were identifieded by multiplex RT-PCR. Bacterial infections were identified through detection of bacterial genomes by PCR, only in lung samples. Influenza A was detected by screening PCR in 46/66 swab samples and from 5/6 lungs. Virus was recovered from pigs of the six herds. Subtype A(H1N1)pdm09 was detected in 4/6 herds and H1N2 in the other 2/6 herds. In lung tissues, further agents involved in porcine respiratory disease complex were detected in all cases; Pasteurella multocida was identified in 5/6 samples and Mycoplasma hyopneumoniae in 3/6. Actinobacillus pleuropneumoniae (1/6), Haemophilus parasuis (1/6) and PCV2 (1/6) were also detected. The aim ofthe second chapter was to sequence the whole-genome of a novel human-like H1N2 swine influenza virus. Whole-genome sequences were generated by RT-PCR. Amplicons were purified followed by sequencing in the MiSeq sequencing platform (Illumina). Phylogenetic analyses revealed that the HA and NA genes clustered with influenza viruses of human lineage, whereas the internal genes (PB1, PB2, PA, NP, M andNS) clustered with the A(H1N1)pdm09. The third chapter reports the geneticsequencing of the full genomes of eight swine influenza viruses circulating in the sampled pig population. Two swine human-like subtypes (H1N2 and H3N2) and the A(H1N1)pdm09 virus were identified. The human-like subtypes have the HA and NA genes similar to the human seasonal strains and the internal genes are closely related to the virus A(H1N1)pdm09.

Comparative evaluation of conventional RT-PCR and real-time RT-PCR (RRT-PCR) for detection of avian metapneumovirus subtype A

Avian metapneumovirus (AMPV) belongs to Metapneumovirus genus of Paramyxoviridae family. Virus isolation, serology, and detection of genomic RNA are used as diagnostic methods for AMPV. The aim of the present study was to compare the detection of six subgroup A AMPV isolates (AMPV/A) viral RNA by using different conventional and real time RT-PCR methods. Two new RT-PCR tests and two real time RT-PCR tests, both detecting fusion (F) gene and nucleocapsid (N) gene were compared with an established test for the attachment (G) gene. All the RT-PCR tested assays were able to detect the AMPV/A. The lower detection limits were observed using the N-, F- based RRT-PCR and F-based conventional RT-PCR (10(0.3) to 10¹ TCID50 mL-1). The present study suggests that the conventional F-based RT-PCR presented similar detection limit when compared to N- and F-based RRT-PCR and they can be successfully used for AMPV/A detection.

O metapneumovírus aviário (AMPV) pertence ao gênero Metapneumovirus, família Paramyxoviridae. Isolamento viral, sorologia e detecção do RNA genômico são atualmente as técnicas utilizadas para o diagnóstico desse agente. O objetivo do presente estudo foi comparar a detecção de RNA viral de seis isolados de AMPV, subtipo A (AMPV/A), utilizando diferentes métodos de RT-PCR convencional e real time RT-PCR (RRT-PCR). Duas novas técnicas de RT-PCR convencional e duas técnicas de RRT-PCR, ambas para a detecção dos genes da nucleoproteína (N) e da proteína de fusão (F), foram comparadas com um RT-PCR previamente estabelecido para a detecção do AMPV (gene da glicoproteína -G). Todos esses métodos foram capazes de detectar os isolados AMPV/A. As técnicas RRT-PCR (genes F e N) mostraram os menores limites de detecção (10(0.3) to 10¹ TCID50 mL-1). Os resultados sugerem que as técnicas RT-PCR convencional (gene F) e as técnicas de RRT-PCR (gene F e N) desenvolvidas no presente estudo podem ser utilizadas com sucesso para a detecção do AMPV/A. Além disso, o RRT-PCR gera resultados rápidos e sensíveis, o que o torna uma ferramenta alternativa para o isolamento viral.

Comparative evaluation of conventional RT-PCR and real-time RT-PCR (RRT-PCR) for detection of avian metapneumovirus subtype A

Avian metapneumovirus (AMPV) belongs to Metapneumovirus genus of Paramyxoviridae family. Virus isolation, serology, and detection of genomic RNA are used as diagnostic methods for AMPV. The aim of the present study was to compare the detection of six subgroup A AMPV isolates (AMPV/A) viral RNA by using different conventional and real time RT-PCR methods. Two new RT-PCR tests and two real time RT-PCR tests, both detecting fusion (F) gene and nucleocapsid (N) gene were compared with an established test for the attachment (G) gene. All the RT-PCR tested assays were able to detect the AMPV/A. The lower detection limits were observed using the N-, F- based RRT-PCR and F-based conventional RT-PCR (10(0.3) to 10¹ TCID50 mL-1). The present study suggests that the conventional F-based RT-PCR presented similar detection limit when compared to N- and F-based RRT-PCR and they can be successfully used for AMPV/A detection.

O metapneumovírus aviário (AMPV) pertence ao gênero Metapneumovirus, família Paramyxoviridae. Isolamento viral, sorologia e detecção do RNA genômico são atualmente as técnicas utilizadas para o diagnóstico desse agente. O objetivo do presente estudo foi comparar a detecção de RNA viral de seis isolados de AMPV, subtipo A (AMPV/A), utilizando diferentes métodos de RT-PCR convencional e real time RT-PCR (RRT-PCR). Duas novas técnicas de RT-PCR convencional e duas técnicas de RRT-PCR, ambas para a detecção dos genes da nucleoproteína (N) e da proteína de fusão (F), foram comparadas com um RT-PCR previamente estabelecido para a detecção do AMPV (gene da glicoproteína -G). Todos esses métodos foram capazes de detectar os isolados AMPV/A. As técnicas RRT-PCR (genes F e N) mostraram os menores limites de detecção (10(0.3) to 10¹ TCID50 mL-1). Os resultados sugerem que as técnicas RT-PCR convencional (gene F) e as técnicas de RRT-PCR (gene F e N) desenvolvidas no presente estudo podem ser utilizadas com sucesso para a detecção do AMPV/A. Além disso, o RRT-PCR gera resultados rápidos e sensíveis, o que o torna uma ferramenta alternativa para o isolamento viral.

Comparative evaluation of conventional RT-PCR and real-time RT-PCR (RRT-PCR) for detection of avian metapneumovirus subtype A

Avian metapneumovirus (AMPV) belongs to Metapneumovirus genus of Paramyxoviridae family. Virus isolation, serology, and detection of genomic RNA are used as diagnostic methods for AMPV. The aim of the present study was to compare the detection of six subgroup A AMPV isolates (AMPV/A) viral RNA by using different conventional and real time RT-PCR methods. Two new RT-PCR tests and two real time RT-PCR tests, both detecting fusion (F) gene and nucleocapsid (N) gene were compared with an established test for the attachment (G) gene. All the RT-PCR tested assays were able to detect the AMPV/A. The lower detection limits were observed using the N-, F- based RRT-PCR and F-based conventional RT-PCR (10(0.3) to 10¹ TCID50 mL-1). The present study suggests that the conventional F-based RT-PCR presented similar detection limit when compared to N- and F-based RRT-PCR and they can be successfully used for AMPV/A detection.

O metapneumovírus aviário (AMPV) pertence ao gênero Metapneumovirus, família Paramyxoviridae. Isolamento viral, sorologia e detecção do RNA genômico são atualmente as técnicas utilizadas para o diagnóstico desse agente. O objetivo do presente estudo foi comparar a detecção de RNA viral de seis isolados de AMPV, subtipo A (AMPV/A), utilizando diferentes métodos de RT-PCR convencional e real time RT-PCR (RRT-PCR). Duas novas técnicas de RT-PCR convencional e duas técnicas de RRT-PCR, ambas para a detecção dos genes da nucleoproteína (N) e da proteína de fusão (F), foram comparadas com um RT-PCR previamente estabelecido para a detecção do AMPV (gene da glicoproteína -G). Todos esses métodos foram capazes de detectar os isolados AMPV/A. As técnicas RRT-PCR (genes F e N) mostraram os menores limites de detecção (10(0.3) to 10¹ TCID50 mL-1). Os resultados sugerem que as técnicas RT-PCR convencional (gene F) e as técnicas de RRT-PCR (gene F e N) desenvolvidas no presente estudo podem ser utilizadas com sucesso para a detecção do AMPV/A. Além disso, o RRT-PCR gera resultados rápidos e sensíveis, o que o torna uma ferramenta alternativa para o isolamento viral.

Implementation and optimization of real time PCR for diagnosis of pandemic influenza A (H1N1) at Instituto Adolfo Lutz and perspectives for 2010 / Implantação e otimização da PCR em tempo real para o diagnóstico da influenza A (H1N1) pandêmica no Instituto Adolfo Lutz e perspectivas para 2010

The authors present their experience with the molecular diagnosis of Influenza A (H1N1) with 37.240 clinical samples obtained from individuals suspected of flu, sent to Instituto Adolfo Lutz for analysis. They show the used algorithms, compare their efficiency in terms of sensitivity, specificity and cost, and suggest a new algorithm to be employed in case of an outbreak of Influenza A (H1N1) in 2010.

Os autores apresentam sua experiência no diagnóstico laboratorial de Influenza A (H1N1) em 37.240 amostras clínicas obtidas de pacientes com suspeita de gripe, encaminhadas ao Instituto Adolfo Lutz para análise. Eles apresentam os algoritmos de testes moleculares empregados, comparam a eficiência dos mesmos quanto à sensibilidade, especificidade e custo e, finalmente, sugerem um novo algoritmo para ser usado em caso de nova epidemia de Influenza A (H1N1) em 2010.