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The combination of ultra-high-performance liquid chromatography (UHPLC) with time-of-flight mass spectrometry (ToF-MS) is one of the most powerful analytical tools in terms of selectivity, sensitivity and speed. However, due to the gap on regulation and guidelines concerning the validation criteria for the use of ToF-MS methodology not only for screening but also for confirmatory and quantification purposes, this study presents, for the first time, an approach on how to use the referred methodology for confirmatory and quantification purposes. Thus, an UHPLC-ToF-MS method was developed and validated for the simultaneous determination of 43 antibiotic residues from 7 different families (tetracyclines, sulfonamides, quinolones, penicillins, macrolides, cephalosporins and trimethoprim) in piglet kidneys as a confirmatory method. A simple sample preparation consisting of extraction with a mixture of acetonitrile and ethylenediaminetetraacetic acid (EDTA) followed by solid-phase extraction (SPE) with a hydrophilic-lipophilic balanced (HLB) cartridge and finalized with a defatting step with n-hexane was used. The methodology was validated according the Commission Decision 2002/657/EC, by defining suitable criteria for antibiotic identification, as well as by evaluating required parameters like selectivity, specificity and ruggedness that fulfil the required criteria. The calculated values of decision limit (CCα) and of detection capacity (CCβ) for antibiotics with established maximum residue limits (MRLs) fully complied with required criteria. For antibiotics for which no MRL was set, values ranging from 0.2 to 0.5 μg kg−1 and from 0.5 to 0.8 μg kg−1 were achieved for CCα and for CCβ, respectively. Linearity (all R2 > 0.95), recovery (81.4–110.0%) (lowest values of 4.6% and 7.1% were obtained for repeatability and for reproducibility, respectively), LODs (0.04–13.0 μg kg−1), LOQs (0.09–32.0 μg kg−1) and matrix effects (35.5–118.1 %) were also evaluated. After validation, the method was applied to analyse piglet kidney samples. Antibiotic residues were determined in 10 of the 15 analysed samples. However, all the antibiotics concentrations were below the MRLs.

Research Areas: Cell Biology Commercial hare and rabbit immortalized cell lines are extremely limited regarding the many species within the lagomorpha order. To overcome this limitation, researchers and technicians must establish primary cell cultures derived from biopsies or embryos. Among all cell types, fibroblasts are plastic and resilient cells, highly convenient for clinical and fundamental research but also for diagnosis, particularly for viral isolation. Here, we describe a fast and cheap method to produce primary fibroblast cell cultures from leporid species, using dispase II, a protease that allows dermal–epidermal separation, followed by a simple enzymatic digestion with trypsin. This method allows for the establishment of an in vitro cell culture system with an excellent viability yield and purity level higher than 85% and enables the maintenance and even immortalization of leporid fibroblastic cells derived from tissues already differentiated. info:eu-repo/semantics/publishedVersion

Background West Nile virus (WNV) is a mosquito-borne pathogen with a globally expanding geographical range. Many European countries regularly report outbreaks affecting both human and equine populations, with an increase in epidemic activity in the past decade. Portugal, on the far west of Europe, has not yet experienced large outbreaks of WNV, despite its suitable Mediterranean climate. Objectives To provide a comprehensive, data-driven perspective on the past epidemiology, surveillance and transmission suitability of WNV in Portugal. Methods Literature and database reviews were performed, covering the period between 1966 and 2020 on existing evidence for WNV circulation. Climatic data was used to estimate the theoretical transmission potential of WNV, and to analyse long-term climate trends between 1981 and 2019. Results Existing data show that WNV-capable mosquito species are spread across the country, while WNV molecular and serological evidence is vastly concentrated in the south. Our analyses suggest that two different climate types, characteristic of the north and south of Portugal, contribute to quantitative differences in WNV theoretical transmission potential. This supports the north-south divide observed from previous molecular and serological evidence. Due to long-term changes in local climate, suitability for WNV transmission is shown to have been slowly increasing in the country. Discussion Data and theoretical analyses support the notion that the south of Portugal is more suitable for WNV transmission, while not rejecting the possibility of transmission in the north. Mosquito monitoring has been effective, but surveillance of key sentinel species remains passive, creating important knowledge gaps for epidemic control and public health. To date, evidence supports WNV circulation, but it remains unclear whether it is endemic or sporadically transmitted. This study supports a timely change towards WNV active surveillance, to avoid and mitigate potential public health emergencies as those recently observed in other European countries.

Aves polyomavirus 1 (APyV) is the causative agent of acute disease in birds and causes high mortality rates in nestlings. Infections have been reported worldwide in a significant number of caged bird species, such as parrots, caiques, budgerigars, lovebirds, and macaws. However, the number of complete viral sequences available in public databases is scarce, especially those with associated clinical histories. In this study, the clinical, pathological, epidemiological, and molecular characteristics of 2 APyV strains detected in Portugal are described. In the autumn of 2015, a 2-month-old female green-winged macaw ( Ara chloroptera ) from a small breeder died with signs of dehydration, weight loss, and depression, raising the suspicion of polyomavirus infection. Histopathological analysis revealed lesions compatible with APyV infection, and the presence of polyomavirus in several organs was confirmed by polymerase chain reaction. From the cohabitants tested ( n = 14), 1 eclectus parrot ( Eclectus roratus ), which was more than 1-year-old, was also APyV DNA-positive. The full genomic sequences of the 2 strains were obtained and found identical, suggesting a single introduction in the premises and the occurrence of subsequent infections by the same virus. When compared with sequences of other APyVs available in public databases, high nucleotide similarity percentages were obtained, confirming the close genetic relationship among polyomaviruses worldwide. Interestingly, strain APV7, detected in a white-bellied parrot in 2008 in Japan, was the closest strain to those isolated in this report. Attempts to isolate the virus in eggs and cell lines failed. Phylogenetic analysis was performed by the Bayesian method to determine the phylogenetic classification of the macaw and parrot strains. Both clustered into group V, together with other strains from different bird species with no host or spatial-temporal relationships being observed.

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.

The use of antibiotics in livestock production has become a common practice. In the EU, the use of antibiotics as growth promoters is forbidden. However, the (mis)use of antibiotics may lead to antimicrobial resistance, which adversely affects humans, animals and the environment. Thus, the present study, using a previously validated multiclass and multi-residue UHPLC-ToF-MS methodology for the determination of 43 antibiotics, presents an assessment of data obtained from 60 piglets, using their livers and kidneys as samples. Eleven antimicrobials from different families were detected, which included ciprofloxacin, danofloxacin, doxycycline, enrofloxacin, oxytetracycline, spiramycin, sulfisomidine, sulfachloropyridazine, sulfadiazine, tilmicosin, and trimethoprim. The obtained concentrations ranged from 0.3 to 438.7 μg kg−1, confirming that both kidney and liver matrices are relevant in monitoring drug residue control programs.

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.

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.

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.

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.

The circulation of West Nile virus in Portugal was assessed by serological surveys conducted during 2004-2010 in horses and birds. The detection of WNV antibodies in both species in all the years covered by the study as well as the presence of anti-WNV IgM in symptomatic horses that had not traveled outside the country, support the notion that WNV circulates in Portugal.

Beak and feather disease virus (BFDV), a member of the genus Circovirus, was detected in six dead African grey parrots (Psittacus erithacus) in Portugal. The complete nucleotide sequences of these six BFDVs (PT05, PT08, PT08-2, PT08-3, PT09, and PT09-2) were determined and analyzed. The seven open reading frames (ORFs) described for other BFDVs were detected in all strains, except for PT05 and PT08, in which ORFs 4 and 7 are absent. Bayesian inference of phylogeny based on complete genomes of BFDVs isolated in Portugal and 32 other BFDVs found in other parts of the world revealed that PT05 is included in lineage IV, whereas the others form a new proposed genotype lineage IX. The nucleotide diversity ranged from 2% to 12% between the BFDV strains isolated in Portugal and other BFDVs found worldwide.

Vaccination programs for the control of bluetongue (BT) in ruminants have limitations due to difficulties in differentiating infected from vaccinated animals (DIVA). To overcome this problem a DIVA test that looks at a differential immune response to bluetongue virus (BTV) non-structural protein 3 (NS3) was developed. The NS3 encoding gene of strain BTV4/22045/PT04 was inserted into expression vector pET-28a and expressed in Escherichia coli strain JM109. Recombinant NS3 protein was used as an antigen in an indirect ELISA (NS3-ELISA) to measure the serologic response to NS3 protein in cattle and sheep. Following a cattle vaccination/challenge experiment with a bivalent inactivated BTV 2-4 vaccine, NS3 antibodies were detected at approximately 15 days post-infection in control unvaccinated animal, while vaccinated animals did not develop detectable NS3 antibodies and, with exception of one, remained negative even after virus challenge. The inactivated vaccine induced antibodies against the major core structural protein (VP7) of BTV as well as neutralizing antibodies in all animals. To evaluate the applicability of NS3-ELISA in field scenario, a total of 562 serum samples collected from uninfected, BTV-infected and vaccinated animals were tested for NS3 antibodies. Taken together, the results confirm that NS3 antibodies were induced to the greatest levels in animals infected with BTV in comparison to the levels induced in animals immunized with inactivated BTV vaccines, implying that antibody response to NS3 allows the differentiation between infected and vaccinated animals.

A total of 300 Ixodes ricinus ticks were tested by polymerase chain reaction (PCR) for the presence of Borrelia spp., Rickettsia spp., and Anaplasma phagocytophilum. Sequence analysis demonstrated 8 (2.7%) ticks infected with B. lusitaniae, 60 (20%) with Rickettsia spp., and 1 (0.3%) with A. phagocytophilum. Seven (2.3%) ticks were coinfected with B. lusitaniae and Rickettsia spp., 2 (0.6%) with R. monacensis, and 5 (1.7%) with Rickettsia sp. IRS3. The results of this study suggest simultaneous transmission of multiple tick-borne agents on Madeira Island, Portugal.

After 44 years of epidemiological silence, bluetongue virus (BTV) was reintroduced in Portugal in the autumn of 2004. The first clinical cases of bluetongue disease (BT) were notified in sheep farms located in the South of Portugal, close to the Spanish border. A total of six BTV, five of serotype 4 and one of serotype 2 were isolated from sheep and cattle during the 2004-2006 epizootics. The nucleotide sequence of gene segments L2, S7 and S10 of BTV-4 prototype strain (BTV4/22045/PT04) obtained from the initial outbreak and of BTV-2 (BTV2/26629/PT05) was fully determined and compared with those from other parts of the world. The phylogenetic analysis revealed that BTV4/22045/PT04 is related to other BTV-4 strains that circulate in the Mediterranean basin since 1998, showing the highest identity (99%) with BTV-4 isolates of 2003 from Sardinia and Corsica, whereas BTV2/26629/PT05 is almost indistinguishable from the Onderstepoort BTV-2 live-attenuated vaccine strain and its related field strain isolated in Italy. Since live-attenuated BTV-2 vaccine was never used in Portugal, the isolation of this strain may represent a natural circulation of the vaccine virus used in other countries in Mediterranean Europe.

FOLLOWING on from our letter published earlier this year ( VR , January 31, 2015, vol 176, p 130), which reported the detection of rabbit haemorrhagic disease virus variant 2 (RHDV-2) in wild rabbits from four islands of the western and central group of the Azorean archipelago, we would like to communicate the diagnosis of RHDV-2 in domestic rabbits from Terceira Island. Two adult rabbits were diagnosed with RHDV-2 (Duarte and others 2015). They originated from the localities of Quatro-Ribeiras and Sao Bento, located on the north and south coasts of Terceira Island. The deaths …

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.

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.

RABBIT haemorrhagic disease virus (RHDV) causes a highly infectious and lethal disease in European rabbits ( Oryctolagus cuniculus ). In Portugal the disease was first reported on the island of Madeira in 1987 and, in the following year, in the Azores. The first report of the disease on the mainland occurred in 1989. Since then, the disease has become endemic throughout the country. The genetically distinct RHDV variant 2 (RHDV-2) was reported on the Portuguese mainland …

Bovine viral diarrhoea virus (BVDV) infections are an important cause of morbidity and mortality worldwide in dairy and beef cattle. To date, little is know about BVDV genotypes circulating in Portugal. For this purpose, a fragment within the 5′-untranslated region (5′-UTR) from 34 Portuguese field strains of BVDV was amplified by RT-PCR, cloned and sequenced. A maximum-likelihood phylogenetic analysis revealed that most of viruses, originated from cattle from different regions of the country, belong to BVDV type 1 (BVDV-1), genotypes 1b ( n = 19), 1a ( n = 6), 1d ( n = 3) and 1e ( n = 3); whereas three viruses clustered in BVDV type 2 (BVDV-2). The results from this study demonstrate that BVDV-lb is the most prevalent genotype and also shows the presence of BVDV-2 in Portugal.

The long terminal repeats (LTR) sequence divergence among Maedi Visna virus (MVV) isolates leads to LTRs with distinct transcriptional activities, which may result in distinct biological behaviours. The genetic heterogeneity, as well as basal and Tat-induced transcriptional activity of the LTRs from P1OLV and WLC-1 MVV viruses, slow/low and rapid/high isolates, respectively, have been examined and compared with LTRs from other strains of small ruminant lentiviruses (SRLV). Transfection assays using a reporter construct containing the LTR fused to a luciferase gene demonstrated that the LTR from P1OLV virus had the weakest promoter activity, suggesting a correlation between the level of promoter activity and the viral replication rate. To confirm this hypothesis, the promoter of P1OLV was cloned into infectious molecular clone KV1772kv72/67 and the resulting chimeric virus was tested for growth in various cell types. Compared to the parental KV1772, the LTR-chimeric virus KV1772/P1OLV exhibited a drastic reduction in replication rate in sheep choroid plexus (SCP) and lung cells, while in ovine macrophages and goat synovial membrane cells (GSM), chimeric virus showed a growth rate similar to that of parental virus. These observations suggest that the LTR is responsible for the slow/low in vitro phenotype presented by P1OLV in SCP and lung cells.

The complete genomic sequence of a sheep lentivirus isolate that presents a slow/low phenotype in vitro has been determined. The virus, designated P1OLV, was isolated from lung cells of a naturally infected sheep in Portugal. Three overlapping DNA fragments amplified by PCR, and encompassing the entire viral genome were cloned and sequenced. This isolate has an overall similarity of approximately 80% with the K1514 Maedi Visna virus (MVV) and approximately 70% with the caprine arthritis encephalitis virus (CAEV) Co strain. Phylogenetic analysis based on SU and RT nucleotide sequences grouped P1OLV with previously reported ovine MVV. To determine the virus replication rate, sheep choroid plexus (SCP) and lung cells, macrophages (MØ), and goat synovial membrane (GSM) cells were inoculated with either P1OLV or with the lytic North American strain WLC-1. Viral RNA in culture supernatants was measured by one-tube real time quantitative RT-PCR. Significant differences were observed between the replication rates of the two viruses, with WLC-1 growing rapidly and to high levels in all the cells tested, while P1OLV replicated more slowly and to lower levels inducing persistent infections in lung and SCP cells. The U3 region of the LTR of P1OLV lacks the sequence repeats that are present in the LTRs of WLC-1 and MVV prototype K1514 and that contain additional binding sites for the AML(vis) transcriptional factor. To evaluate the contribution of LTR in the virus replication rate in vitro, we measured the basal activity of the promoter from P1OLV and WLC-1 in a luciferase-driven gene expression assay and lower levels of expression were achieved for P1OLV. The genetic and biological properties of P1OLV will be useful for the study of virus transcriptional factors and genes that may be responsible for the slow/low phenotype.

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.

A monoclonal antibody (MAb) blocking ELISA (Blck-ELISA) was developed to detect antibodies against Maedi-Visna virus (MVV) in sheep sera. The assay employs a MAb directed against the envelope protein p90 of the virus in a sandwich blocking procedure. To determine whether the MAb was a potential antibody for developing a Blck-ELISA, a collection of three hundred sera obtained from several sheep flocks known to be infected with MVV were used to examine the sensitivity of the Blck-ELISA. A total of 50 serum samples originating from a flock free of MVV were tested to assess the specificity of the assay. The results were compared with a commercial indirect ELISA (I-ELISA) and samples giving a conflicting or doubtful result were tested by immunoblot. The Blck-ELISA proved to be specific, sensitive and it showed high reproducibility and low variability.

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.

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.

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). published

Porcine circovirus type 2 (PCV2) belongs to the genus Circovirus of the Circoviridae family and has been associated with a disease named postweaning multisystemic wasting syndrome (PMWS). In Portugal the disease caused severe problems mainly between 1995 and 2003, but is nowadays less evident probably as the result of vaccination programmes initiated in 2007. This study reports the molecular analysis of 22 PCV2 strains obtained from domestic pigs originated from different regions of Portugal. One hundred and seventy-seven samples from PCV2 suspected cases received between 2003 and 2010 were analysed, 79 (44.6%) of which tested PCV2 PCR-positive. The entire genomes of 22 viruses were amplified and sequenced. Two major groups were observed, according to the genome length and in single nucleotide polymorphisms. The nucleotide similarity found among the PCV2 strains ranged between 94% and 99%. The Bayesian inference of phylogeny based on the genome of the strains studied revealed the existence of two distinct genetic groups consistent with the nucleotide sequence observations. The majority of the strains branched in genotype PCV2b, while six strains with a common geographical origin obtained between 2007 and 2009, were included in genotype PCV2a. The analysis performed with cap and rep genes confirmed that cap is a reliable alternative to full-length genome based phylogenetic studies of PCV2.

Two cases of fatal infection caused by parvovirus in a white tiger (Panthera tigris) and an African lion (Panthera leo) at the Lisbon Zoo (Portugal) are described. Gross findings at necropsy were catharral enteritis in the tiger and severe hemorrhagic enteritis in the lion. Histopathologic examination revealed, in both animals, intestinal crypt necrosis and lymphocyte depletion in the germinal centers of the mesenteric lymph nodes. Bacteriologic examination was negative for common bacterial pathogens, including Salmonella. Amplification of the parvovirus VP2 complete gene was achieved in both cases and sequencing analysis identified these isolates as feline panleukopenia virus (FPLV). The nucleotide sequences obtained from these two viruses were genetically indistinguishable. The phylogenetic analysis of FPLV strains from domestic cats obtained in the Lisbon area revealed the circulation of FPLV strains highly similar to those isolated from the tiger and lion, which strongly suggests that stray cats may have been the source of infection.

This report describes the first establishment of five isolates of R. conorii Israeli tick typhus (ITT) strain from Rhipicephalus sanguineus ticks in different regions of Portugal. Ticks were collected on two separate expeditions that took place in Braganca, Montesinho Natural Park, and Portalegre City.

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.