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1.
Sci Rep ; 11(1): 16430, 2021 08 12.
Artigo em Inglês | MEDLINE | ID: mdl-34385527

RESUMO

Until there is an effective implementation of COVID-19 vaccination program, a robust testing strategy, along with prevention measures, will continue to be the most viable way to control disease spread. Such a strategy should rely on disparate diagnostic tests to prevent a slowdown in testing due to lack of materials and reagents imposed by supply chain problems, which happened at the beginning of the pandemic. In this study, we have established a single-tube test based on RT-LAMP that enables the visual detection of less than 100 viral genome copies of SARS-CoV-2 within 30 min. We benchmarked the assay against the gold standard test for COVID-19 diagnosis, RT-PCR, using 177 nasopharyngeal RNA samples. For viral loads above 100 copies, the RT-LAMP assay had a sensitivity of 100% and a specificity of 96.1%. Additionally, we set up a RNA extraction-free RT-LAMP test capable of detecting SARS-CoV-2 directly from saliva samples, albeit with lower sensitivity. The saliva was self-collected and the collection tube remained closed until inactivation, thereby ensuring the protection of the testing personnel. As expected, RNA extraction from saliva samples increased the sensitivity of the test. To lower the costs associated with RNA extraction, we performed this step using an alternative protocol that uses plasmid DNA extraction columns. We also produced the enzymes needed for the assay and established an in-house-made RT-LAMP test independent of specific distribution channels. Finally, we developed a new colorimetric method that allowed the detection of LAMP products by the visualization of an evident color shift, regardless of the reaction pH.


Assuntos
Teste para COVID-19/métodos , COVID-19/virologia , Colorimetria/métodos , Técnicas de Diagnóstico Molecular/métodos , Técnicas de Amplificação de Ácido Nucleico/métodos , SARS-CoV-2/isolamento & purificação , COVID-19/diagnóstico , Humanos , Pandemias , Portugal/epidemiologia , RNA Viral/genética , SARS-CoV-2/genética , Saliva/química , Saliva/virologia , Sensibilidade e Especificidade
2.
Vet Med Sci ; 5(2): 222-229, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-30672155

RESUMO

Halicephalobus gingivalis is a small saprophytic rhabditid nematode, represented only by females with a typical rhabditoid oesophagus and one egg in the uterus, capable of infecting vertebrates. This opportunistic parasite present in the soil, manure and decaying humus, is thought to penetrate through previous injuries to the mouth, eyes and skin of horses and migrate to various organs. The brain is one such organ, where the females lay their eggs, leading to malacia and causing a sudden onset of neurological signs, such as anorexia, ataxia, urinary incontinence, blindness, decreased menace and tonal reflexes, tremors and aggressiveness. The disease is invariably fatal whenever brain lesions are present, and the diagnosis usually achieved only post-mortem. The present work aims to describe the first case of infection by H. gingivalis ever reported in Portugal. An 8-year old warmblood horse presented with an 8-day history of progressive blindness involving the left eye, initially with normal pupillary reflexes, advancing to bilateral blindness and increasing deterioration in clinical condition. After euthanasia, the animal was submitted for necropsy. Organ samples were collected and fixed in 10% neutral buffered formalin for routine histopathology. A large mass was found in the left kidney corresponding to fibrous tissue heavily infiltrated with inflammatory cells and numerous nematodes. In the brain, multiple, bilateral and asymmetrical foci of malacia containing several rhabditoid nematodes, larvae and zygotes, and high numbers of inflammatory cells were found. The nematodes were identified as H. gingivalis. The clinical history, necropsy and histological findings presented constitute a typical case of H. gingivalis infection in a horse, never previously described in Portugal to the authors' best knowledge. Humans can be infected by contact with contaminated manure, which makes this nematode a public health concern, especially for people living and/or working in close proximity to horses.


Assuntos
Doenças dos Cavalos/parasitologia , Infecções por Rhabditida/veterinária , Rabditídios/isolamento & purificação , Animais , Doenças dos Cavalos/patologia , Doenças dos Cavalos/fisiopatologia , Cavalos , Larva/crescimento & desenvolvimento , Portugal , Saúde Pública , Rabditídios/crescimento & desenvolvimento , Infecções por Rhabditida/parasitologia , Infecções por Rhabditida/patologia , Infecções por Rhabditida/fisiopatologia
3.
Virusdisease ; 29(3): 355-361, 2018 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-30159371

RESUMO

Porcine circovirus type 2 (PCV2) is a spherical and non-enveloped virus belonging to the genus Circovirus of the Circoviridae family with a single stranded circular DNA genome. This virus, already detected worldwide, has been associated to several diseases and was implicated as the etiological agent of a disease named postweaning multisystemic wasting syndrome. Several methods have been described for the detection of PCV2, being real-time PCR the most simple and reliable. As far as we know, all the real-time PCR systems described until now are based on ORF2 gene, that exhibit the highest variability. This paper reports the development and validation of a real-time PCR targeted to ORF1 and based on a TaqMan probe for the detection of porcine circovirus type 2 DNA in swine samples. Due to the lack of PCV1 samples, the ability of the test to discriminate between PCV1 and PCV2 positive samples was evaluated in silico. Estimations of 100% specificity and 100% sensitivity were obtained based on the qPCR results with panel of 81 swine samples (known PCV2-positive (n = 50); known PCV2-negative (n = 17); samples positive to other common swine viral pathogens (n = 13) and one sample from a BFDV-positive parrot (n = 1)). Intra- and inter-assay coefficients of variation obtained with three positive samples of different viral charges in five replicates or in five independent assays were below the acceptance threshold. The limit of detection determined with a recombinant plasmid containing the amplicon, led to conclude that this assay can detect at least three plasmid copies.

4.
Vet Microbiol ; 212: 75-79, 2017 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-29173592

RESUMO

West Nile fever (WNF) is an emergent disease in Europe, under surveillance in the European Union. Following a 5-year period of apparent silence (autumn 2010 to summer 2015), West Nile virus (WNV) reemerged in the South of Portugal, in July 2015. Here we present data from the onset, geographic location within mainland Portugal, and outcome of clinical cases of WNV infection in horses in 2015 and 2016. During the transmission seasons of 2015 and 2016, twenty-seven horses, most symptomatic (n=20) were found positive to IgM, pr-E immunoglobulins and VNT, leading to the subsequent report to Animal Disease Notification System of the European Commission (ADNS) by the Portuguese National Authority for Animal Health. Outbreaks occurred in the middle summer (August) and early/mid autumn (October/November) of 2015 and 2016, in the southern regions of the country (Alentejo and Algarve). Compared with the previous WNV transmission seasons of 2004 and 2010, a higher number of cases were reported in 2015 and 2016. The results of our study contribute to increase information concerning the geographic areas affected and time period for WNV transmission risk in Portugal.


Assuntos
Surtos de Doenças/veterinária , Doenças dos Cavalos/epidemiologia , Febre do Nilo Ocidental/veterinária , Vírus do Nilo Ocidental/imunologia , Animais , Monitoramento Epidemiológico , Feminino , Geografia , Doenças dos Cavalos/virologia , Cavalos , Masculino , Portugal/epidemiologia , Febre do Nilo Ocidental/epidemiologia , Febre do Nilo Ocidental/virologia , Vírus do Nilo Ocidental/isolamento & purificação
5.
Virus Genes ; 53(6): 922-926, 2017 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-28639220

RESUMO

We report the detection of rabbit haemorrhagic disease virus 2 (RHDV2) in the Madeira archipelago, Portugal. Viral circulation was confirmed by RT-qPCR and vp60 sequencing. Epidemiological data revealed the outbreak initiated in October 2016 in Porto Santo affecting wild and domestic rabbits. It was then detected three months later on the island of Madeira. Five haplotypes were identified and a genetic overall similarity of 99.54 to 99.89% was observed between the two viral populations. Unique single nucleotide polymorphisms were recognised in the Madeira archipelago strains, two of which resulting in amino acid substitutions at positions 480 and 570 in the VP60 protein. Phylogenetic investigation by Maximum Likelihood showed all the vp60 sequences from the Madeira archipelago group together with high bootstraps. The analysis also showed that the Madeira archipelago strains are closely related to the strains detected in the south of mainland Portugal in 2016, suggesting a possible introduction from the mainland. The epidemiological data and high genetic similarity indicate a common source for the Porto Santo and Madeira RHDV2 outbreaks. Human activity related to hunting was most probably at the origin of the Madeira outbreak.


Assuntos
Infecções por Caliciviridae/virologia , Vírus da Doença Hemorrágica de Coelhos/genética , Substituição de Aminoácidos/genética , Animais , Surtos de Doenças , Haplótipos , Filogenia , Polimorfismo de Nucleotídeo Único/genética , Portugal , Coelhos
6.
J Virol Methods ; 236: 47-53, 2016 10.
Artigo em Inglês | MEDLINE | ID: mdl-27421625

RESUMO

Avian influenza (AI) is an infectious viral disease usually asymptomatic in wild birds that can spread to domestic poultry and cause large-scale outbreaks. Some of the AI viruses (AIV) can cross the species barrier and induce fatal disease to humans, a matter of great concern worldwide. Early detection of AIV is of major importance for disease control, since prompt implementation of adequate measures can prevent spread of the virus and therefore further outbreaks. This paper reports the development and validation of a blocking ELISA using a monoclonal antibody against a conserved structural protein for the serologic diagnosis of all AI virus subtypes from domestic bird species, allowing the quick, easily automated and low-cost screening of a high number of farms. The test will be of great value not only for surveillance, but also for monitoring the efficiency of vaccination programs. Cut-off values were established in 20% of inhibition for turkey sera and in 50% of inhibition for chicken and duck sera. Estimations of 100% specificity and 100% sensitivity were obtained based on the results of known AI positive (n=130) and negative (n=208) sera, including serum samples from birds infected with other common avian viral pathogens (n=7). ROC analysis showed an area under curve (AUC) of 1.0 for chicken, duck and turkey sera, indicating that the blocking ELISA was able to perfectly discriminate between negative and positive samples of any of the poultry species tested. Inter- and intra-assay coefficients of variation were above the acceptance threshold. Furthermore, the ELISA titers were similar to the known hemagglutination inhibition titers of three positive reference sera indicating sensitivity comparable with the golden standard HI method. The method here described is an economically attractive alternative to the commercial ELISAs currently available.


Assuntos
Anticorpos Antivirais/sangue , Ensaio de Imunoadsorção Enzimática/métodos , Influenza Aviária/diagnóstico , Orthomyxoviridae/imunologia , Doenças das Aves Domésticas/diagnóstico , Animais , Galinhas , Patos , Influenza Aviária/virologia , Programas de Rastreamento/métodos , Doenças das Aves Domésticas/virologia , Curva ROC , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Turquia
7.
Methods Mol Biol ; 1404: 59-76, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27076290

RESUMO

Maedi-visna virus (MVV) is an ovine retrovirus of the Lentivirus genus, responsible for a chronic and progressive disease of sheep with a high prevalence all over the world. Therefore, measures aiming at the control of MVV infection are necessary, and the development of DNA vaccines may be the ideal approach. A DNA vaccine is an antigen-encoding bacterial plasmid designed to mimic infections safely, with ability to generate both humoral and cellular long-lasting immune responses once it is delivered to the host.Here, we describe the development and evaluation of DNA vaccines against ovine maedi-visna virus. The first step is the design of the vaccines, including the choice of the backbone vector and the nucleotide sequences to use as antigen-encoding sequences. Once constructed, the vaccines may be produced with high quality for use in in vitro and in vivo tests. In vitro assays are performed through transfection of animal cells to confirm the expression of the protein, while in vivo tests are carried out by mouse and/or sheep immunization in order to check humoral and cellular responses to the vaccines and conclude about their efficiency. Several approaches may be later performed in order to enhance the effectiveness of the vaccines, such as the introduction of targeting sequences, the use of a prime-boost strategy, the administration of a combined vaccine, and the use of liposomes as delivery vehicle.


Assuntos
Vacinas de DNA/imunologia , Vacinas Virais/imunologia , Vírus Visna-Maedi/imunologia , Animais , Técnicas de Cultura de Células , Clonagem Molecular , Feminino , Citometria de Fluxo , Imunidade Humoral , Imunização , Camundongos , Transfecção , Vacinas de DNA/genética , Vacinas Virais/genética
8.
J Zoo Wildl Med ; 47(1): 161-74, 2016 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-27010277

RESUMO

Avian poxviruses (APV) are very large viruses spread worldwide in a variety of hosts. They are responsible for a disease usually referred to as pox, mainly characterized by nodular lesions on feather-free regions of the body. On May 2010, a young American flamingo (Phoenicopterus ruber) of the Lisbon Zoo (Portugal) developed a nodular lesion suggestive of poxvirus infection on its right foot. Avipoxvirus was isolated from the lesion and a fragment of the P4b-encoding gene was amplified by polymerase chain reaction. The nucleotide sequence of the amplicon was determined and analyzed. A close relationship (100% identity) was observed between the flamingo poxvirus and isolates from great bustard (Hungary 2005), house sparrow (Morocco 2009), MacQueen's bustard (Morocco 2011), and Houbara bustard (Morocco 2010 and 2011), suggesting interspecies transmission as a possible source of infection. To strengthen the investigation, the 5' and 3' ends of genes cnpv186 and cnpv 187, respectively, were also analyzed. The cnpv186-187 fragment exhibited 100% identity with MacQueen's bustard and Houbara bustard isolates, both from Morocco 2011. Phylogenetic analyses based in both fragments grouped the flamingo isolate consistently within clade B2 of canarypox. However, the phylogenetic relationships among the different representatives of avian poxviruses were more comprehensive in the tree based on the concatenated coding sequences of the cnpv186-187 fragment, rather than on the P4b-coding gene. The clearer displacement and distribution of the isolates regarding their host species in this last tree suggests the potential usefulness of this genomic region to refine avian poxvirus classification.


Assuntos
Doenças das Aves/virologia , Infecções por Poxviridae/veterinária , Poxviridae/isolamento & purificação , Animais , Animais de Zoológico , Doenças das Aves/epidemiologia , Doenças das Aves/patologia , Aves , Filogenia , Portugal/epidemiologia , Poxviridae/classificação , Poxviridae/genética , Infecções por Poxviridae/epidemiologia , Infecções por Poxviridae/patologia
9.
Euro Surveill ; 20(38)2015.
Artigo em Inglês | MEDLINE | ID: mdl-26535915

RESUMO

A case of West Nile virus (WNV) infection was reported in the Algarve region, Portugal, in the first week of September 2015. WNV is known to circulate in Portugal, with occasional reports in horses and birds (2004 to 2011) and very sporadically human cases (in 2004 and in 2010). Here we present the clinical and laboratory aspects related to the first human case of West Nile neuroinvasive disease reported in Portugal.


Assuntos
Anticorpos Antivirais/sangue , Febre do Nilo Ocidental/diagnóstico , Vírus do Nilo Ocidental/isolamento & purificação , Idoso , Humanos , Masculino , Testes de Neutralização , Portugal , Febre do Nilo Ocidental/sangue , Febre do Nilo Ocidental/virologia , Vírus do Nilo Ocidental/imunologia
10.
Infect Genet Evol ; 35: 163-71, 2015 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-26247721

RESUMO

Rabbit haemorrhagic disease virus 2 (RHDV2) is widespread in several countries of Western Europe, but it has not been introduced to other continents. However, between late 2014 and early 2015, the presence of RHDV2 was confirmed outside of the European continent, in the Azores, initially in the islands of Graciosa, Flores, S. Jorge and Terceira. In this study we report the subsequent detection of RHDV2 in wild rabbits from the islands of Faial, St. Maria and S. Miguel, and display the necropsy and microscopic examination data obtained, which showed lesions similar to those induced by classical strains of RHDV, with severe affection of lungs and liver. We also disclose the result of a genetic investigation carried out with RHDV2 positive samples from wild rabbits found dead in the seven islands. Partial vp60 sequences were amplified from 27 tissue samples. Nucleotide analysis showed that the Azorean strains are closely related to each other, sharing a high genetic identity (>99.15%). None of the obtained sequences were identical to any RHDV2 sequence publically known, hampering a clue for the source of the outbreaks. However, Bayesian and maximum likelihood phylogenetic analyses disclosed that Azorean strains are more closely related to a few strains from Southern Portugal than with any others presently known. In the analysed region comprising the terminal 942 nucleotides of the vp60 gene, four new single nucleotide polymorphisms (SNP) were identified. Based on the present data, these four SNPs, which are unique in the strains from Azores, may constitute putative molecular geographic markers for Azorean RHDV2 strains, if they persist in the future. One of these variations is a non-synonymous substitution that involves the replacement of one amino acid in a hypervariable region of the capsid protein.


Assuntos
Infecções por Caliciviridae/epidemiologia , Surtos de Doenças/veterinária , Vírus da Doença Hemorrágica de Coelhos/classificação , Vírus da Doença Hemorrágica de Coelhos/genética , Polimorfismo de Nucleotídeo Único , Proteínas Estruturais Virais/genética , Animais , Açores/epidemiologia , Infecções por Caliciviridae/patologia , Infecções por Caliciviridae/virologia , Europa (Continente)/epidemiologia , Marcadores Genéticos/genética , Vírus da Doença Hemorrágica de Coelhos/isolamento & purificação , Fígado/patologia , Fígado/virologia , Pulmão/patologia , Pulmão/virologia , Filogenia , Filogeografia , Coelhos , Análise de Sequência de RNA , Proteínas Estruturais Virais/análise
12.
J Virol Methods ; 219: 90-95, 2015 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-25823548

RESUMO

A specific real time RT-PCR for the detection of RHDV2 was developed and validated using RHDV and RHDV2 RNA preparations from positive field samples. The system was designed to amplify a 127 nucleotide-long RNA region located within the vp60 gene, based on the alignment of six sequences originated in Portugal, obtained in our laboratory, and 11 sequences from France and Italy. The primers and probe target sequences are highly conserved in the vast majority of the RHDV2 sequences presently known. In the sequences showing variability, only one mismatch is found per strain, usually outlying the 3' end of the primer or probe hybridization sequences. The specificity of the method was demonstrated in vitro with a panel of common rabbit pathogens. Standardization was performed with RNA transcripts obtained from a recombinant plasmid harboring the target sequence. The method was able to detected nine RNA molecules with an efficiency of 99.4% and a R(2) value of 1. Repeatability and reproducibility of the method were very high, with coefficients of variation lower than 2.40%. The assay was proven a valuable tool to diagnose most of RDVH2 circulating strains, and may be also useful to monitor viral loads, and consequently, disease progression and vaccination efficacy.


Assuntos
Infecções por Caliciviridae/diagnóstico , Infecções por Caliciviridae/virologia , Vírus da Doença Hemorrágica de Coelhos/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Animais , Vírus da Doença Hemorrágica de Coelhos/classificação , Coelhos , Reprodutibilidade dos Testes , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Sensibilidade e Especificidade
14.
Infect Genet Evol ; 27: 149-55, 2014 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-25062641

RESUMO

The genetic relationships between 10 rabbit hemorrhagic disease strains collected in Portugal between 2006 and 2013, originated in the mainland and Azorean islands, were investigated based on the vp60 gene variability. A genetic diversity ranging from 2% to 13% was determined among the 10-vp60 complete sequences revealing a significant level of genetic heterogeneity between same strains. Phylogenetic Bayesian analysis showed that the Portuguese RHDV strains fell within different genogroups, namely G1, G5 and G6. Interestingly, all strains obtained from Azores, where RHDV was first detected in 1988, belong to G5 genogroup. G5 strains, that were not identified in the continent so far, seem to be the dominant group in these Atlantic islands. G1-related strains belonging to the Iberian group 3 (n=3) and G6 (RHDVa) strains (n=2) were identified among the samples originated in mainland which were collected between 2006 and 2008. Although the presence of G1 and G6 in Portugal had been shown before, our data refines the time of circulation of these strains until at least 2008. In summary, this study revises the epidemiological information of RHDV in Portugal since it reports for the first time the presence of G5 strains in Azores and demonstrates the circulation of G1 and G6 strains in mainland Portugal until the late 2000s.


Assuntos
Infecções por Caliciviridae/epidemiologia , Infecções por Caliciviridae/virologia , Vírus da Doença Hemorrágica de Coelhos/classificação , Vírus da Doença Hemorrágica de Coelhos/genética , Animais , Açores/epidemiologia , Genes Virais , Genótipo , Geografia , Dados de Sequência Molecular , Filogenia , Polimorfismo Genético , Portugal/epidemiologia , Coelhos , Estudos Retrospectivos
15.
Infect Genet Evol ; 25: 117-21, 2014 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-24788000

RESUMO

Members of the family Adenoviridae are divided into five genera and infect a wide variety of vertebrates with a narrow host range, usually restricted to one species. Due to the high genetic diversity and distinct genomic organization, classification of adenoviruses is difficult to achieve and often performed by phylogenetic analysis. The most commonly used region for phylogenetic inference of adenoviruses is the DNA polymerase (AdPol) gene carried out at amino acid level. In this paper we investigated the suitability of the U exon to discriminate adenoviruses. The tree based on this genus-common feature, obtained with 23 short amino acid sequences, offered a clearest discrimination of the members of the adenovirus family (Adenoviridae) than the trees generated with the complete or partial polymerase protein sequences. Therefore, our results demonstrate that the U exon is an effective tool for a refined phylogenetic inference and genus classification of the Adenoviridae family.


Assuntos
Adenoviridae/classificação , DNA Viral/genética , DNA Polimerase Dirigida por DNA/genética , Proteínas Virais/genética , Adenoviridae/enzimologia , Adenoviridae/genética , Teorema de Bayes , Evolução Molecular , Éxons , Variação Genética , Filogenia
16.
Methods Mol Biol ; 1143: 33-59, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-24715281

RESUMO

Immune response against an encoded antigenic protein can be elicited by including targeting sequences to DNA vaccines that promote protein sorting to processing pathways, related with antigen presentation by major histocompatibility complexes (MHC). Candidate DNA vaccines coding for neuraminidase 3 of the avian influenza virus were designed to encode different sequences that direct the protein to specific cellular compartments such as endoplasmic reticulum (i.e., adenovirus E1A), lysosomes (i.e., LAMP), and the combination of protein targeting to the endoplasmic reticulum and lysosome (i.e., E1A-LAMP). The DNA vaccine prototypes were engineered by biomolecular techniques and subsequently produced in E. coli cells. The biological activity of the vaccines was tested firstly in vitro, in Chinese hamster ovary cells, through flow cytometry and real-time polymerase chain reaction analysis. Then, an essential in vivo study was performed in chickens, in order to evaluate the efficacy of DNA prototype vaccines, by measuring the antibody production by enzyme-linked immunosorbent assay.


Assuntos
Vacinas de DNA/genética , Vacinas de DNA/imunologia , Animais , Antígenos/genética , Antígenos/imunologia , Células CHO , Galinhas , Cricetinae , Cricetulus , Regulação da Expressão Gênica , Plasmídeos/genética , Plasmídeos/imunologia
17.
J Virol Methods ; 196: 219-24, 2014 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-24300832

RESUMO

The myxoma virus (MYXV) causes severe infections in European rabbits that may reach mortality rates up to 100% depending on the viral strain. The typical symptoms and lesions induced by the virus are usually enough to permit the correct clinical diagnosis. However, in peracute forms the infection may be accompanied by unspecific symptoms. Sudden death may also occur without evident clinical signs of myxomatosis. Likewise, a clinical diagnosis of atypical forms of myxomatosis (amyxomatous) is often complicated and delayed due to the scarceness of skin lesions. As the disease control often depends on an early and unequivocal diagnosis of MYXV, laboratorial methods play a relevant role in the confirmation of MYXV infection. This study describes the development and validation of a novel, high accurate real time polymerase chain reaction assay (rtPCR) for the detection of MYXV. Primers were designed to amplify a 125-bp within the gene M000.5L/R, which is duplicated in the termini of the genome and is unique among Leporipoxvirus. The assay was negative for SFV and other poxviruses and was able to detect 2.6 copies of MYXV DNA proving the effectiveness, specificity and sensitivity of this diagnosis tool. The rtPCR has been applied successfully in INIAV laboratory for routine diagnosis of myxomatosis since 2005.


Assuntos
Técnicas de Diagnóstico Molecular/métodos , Myxoma virus/isolamento & purificação , Mixomatose Infecciosa/diagnóstico , Reação em Cadeia da Polimerase em Tempo Real/métodos , Medicina Veterinária/métodos , Animais , Primers do DNA/genética , DNA Viral/química , DNA Viral/genética , Genes Virais , Dados de Sequência Molecular , Coelhos , Sensibilidade e Especificidade , Análise de Sequência de DNA
18.
J Virol Methods ; 193(2): 554-7, 2013 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-23892127

RESUMO

West Nile virus (WNV) and Japanese encephalitis virus (JEV) are important mosquito-borne viruses of the Flaviviridae family, associated with encephalitis, mainly in humans and horses. WNV is also pathogen for many bird species. The incidence of human and animal WNV infections in Europe has risen, mostly in recent years, and JEV was detected in 2011 in mosquitoes collected in Italy and may emerge in Europe in the same way as other flaviviruses had emerged recently (USUTU and Bagaza virus) and should be regarded as a potential threat to public health. Prompt identification and discrimination between WNV and JEV provides critical epidemiological data for prevalence studies and public and animal health management policies. Here we describe a quantitative one-step duplex TaqMan RT-PCR, targeting non-structural protein 2A gene (NS2A-qRT-PCR), based on only one primer pair and two probes for differential diagnosis of WNV and JEV. Also this assay enables the detection of both WNV lineages (WNV-1 and WNV-2). To access the specificity of NS2A-qRT-PCR a panel of different arboviruses were used. The assay was shown to be specific for both WNV lineages (WNV-1 and WNV-2), WNV related Kunjin virus and JEV, since no cross-reactions were observed with other tested arboviruses. Sensitivity of the assay was determined using serial dilutions of in vitro-transcribed RNA from WNV and JEV. The duplex NS2A-qRT-PCR assay was shown to be very sensitive, being able to detect 10 copies of WNV and JEV RNA. This assay is a suitable tool for the diagnosis of WNV and JEV, and provides a valuable addition to the methods currently available for routine diagnosis of these zoonoses and for surveillance studies.


Assuntos
Vírus da Encefalite Japonesa (Espécie)/isolamento & purificação , Encefalite Japonesa/diagnóstico , Reação em Cadeia da Polimerase Multiplex/métodos , RNA Viral/genética , Reação em Cadeia da Polimerase em Tempo Real/métodos , Febre do Nilo Ocidental/diagnóstico , Vírus do Nilo Ocidental/isolamento & purificação , Vírus da Encefalite Japonesa (Espécie)/genética , Encefalite Japonesa/virologia , Humanos , Técnicas de Diagnóstico Molecular/métodos , RNA Viral/isolamento & purificação , Sensibilidade e Especificidade , Febre do Nilo Ocidental/virologia , Vírus do Nilo Ocidental/genética
19.
PLoS One ; 8(3): e59399, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23527182

RESUMO

The exposure of wild carnivores to viral pathogens, with emphasis on parvovirus (CPV/FPLV), was assessed based on the molecular screening of tissue samples from 128 hunted or accidentally road-killed animals collected in Portugal from 2008 to 2011, including Egyptian mongoose (Herpestes ichneumon, n = 99), red fox (Vulpes vulpes, n = 19), stone marten (Martes foina, n = 3), common genet (Genetta genetta, n = 3) and Eurasian badger (Meles meles, n = 4). A high prevalence of parvovirus DNA (63%) was detected among all surveyed species, particularly in mongooses (58%) and red foxes (79%), along with the presence of CPV/FPLV circulating antibodies that were identified in 90% of a subset of parvovirus-DNA positive samples. Most specimens were extensively autolysed, restricting macro and microscopic investigations for lesion evaluation. Whenever possible to examine, signs of active disease were not present, supporting the hypothesis that the parvovirus vp2 gene fragments detected by real-time PCR possibly correspond to viral DNA reminiscent from previous infections. The molecular characterization of viruses, based on the analysis of the complete or partial sequence of the vp2 gene, allowed typifying three viral strains of mongoose and four red fox's as feline panleukopenia virus (FPLV) and one stone marten's as newCPV-2b type. The genetic similarity found between the FPLV viruses from free-ranging and captive wild species originated in Portugal and publicly available comparable sequences, suggests a closer genetic relatedness among FPLV circulating in Portugal. Although the clinical and epidemiological significance of infection could not be established, this study evidences that exposure of sympatric wild carnivores to parvovirus is common and geographically widespread, potentially carrying a risk to susceptible populations at the wildlife-domestic interface and to threatened species, such as the wildcat (Felis silvestris) and the critically endangered Iberian lynx (Lynx pardinus).


Assuntos
Distribuição Animal , Carnívoros/virologia , Suscetibilidade a Doenças/veterinária , Vírus da Panleucopenia Felina/genética , Herpestidae/virologia , Infecções por Parvoviridae/epidemiologia , Filogenia , Sequência de Aminoácidos , Animais , Anticorpos Antivirais/sangue , Área Sob a Curva , Sequência de Bases , Teorema de Bayes , Proteínas do Capsídeo/genética , Modelos Genéticos , Dados de Sequência Molecular , Infecções por Parvoviridae/patologia , Portugal/epidemiologia , Reação em Cadeia da Polimerase em Tempo Real/veterinária , Alinhamento de Sequência , Especificidade da Espécie
20.
Avian Pathol ; 40(6): 597-602, 2011 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-22107094

RESUMO

This report presents the results of a multiyear (2005 to 2009) study of avian influenza virus (AIV) occurrence in wild birds in Portugal. A total of 5691 samples from wild birds belonging to 13 different orders were examined. Ninety-three samples tested positive for AIV by matrix reverse transcriptase-polymerase chain reaction, giving a total prevalence of 1.63%. Twenty-one viruses were successfully cultured in embryonated chicken eggs, which represent a rate of viral infectivity of 22.6% in the samples. Nine subtypes of haemagglutinin (H1, H3 to H7, H9 to H11) and eight subtypes of neuraminidase (N1 to N4, N6 to N9) were identified in 20 different combinations. The most prevalent subtypes of haemagglutinin detected were H5, H1 and H4, while for neuraminidase subtypes N2 and N6 were the most common. The subtype combinations H4N6 and H1N1 were predominant (15.1%). All H5 and H7 viruses detected in the present study were low pathogenic for poultry as determined by the sequence of amino acids at the cleavage site of haemagglutinin. The full-length nucleotide sequences of five H5, one H7 and five N3 genes were analysed phylogenetically. The Bayesian analysis revealed that all but one of the strains analysed were closely related to isolates detected in the same period in North and Central European countries. Three H5N3 isolates, all from 2007, formed a separate cluster in both H5 and N3 phylogenetic trees. This study provides evidence that various subtypes of AIV, including subtypes H5 and H7, circulate in Portugal, which may pose a risk to industrial poultry.


Assuntos
Doenças das Aves/epidemiologia , Doenças das Aves/virologia , Vírus da Influenza A/genética , Influenza Aviária/epidemiologia , Filogenia , Animais , Sequência de Bases , Teorema de Bayes , Aves , Vírus da Influenza A/patogenicidade , Modelos Genéticos , Dados de Sequência Molecular , Vigilância da População , Portugal/epidemiologia , Prevalência , Reação em Cadeia da Polimerase Via Transcriptase Reversa/veterinária , Análise de Sequência de DNA , Sorotipagem/veterinária
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