Miguel Fevereiro

Antigenic and biological characterisation of avian paramyxovirus type I isolates from pigeons--an international collaborative study.

During 1981-1983 a disease of pigeons (Columba livia), characterised predominantly by nervous signs, spread across Europe. In the present study 57 viruses isolated from pigeons from 15 countries (12 European, Japan, Israel and Sudan) were characterised. All were shown to be avian paramyxoviruses of the A/PMV-1 serotype. Monoclonal antibody binding tests showed 53 of the viruses to be identical. The virus from Sudan was similar to these viruses but showed distinguishable variation. One vaccinal virus from France and two virulent viruses from Czechoslovakia were unrelated to the other pigeon A/PMV-1 isolates.

Suppression of lymphocyte blastogenesis to mitogens in cats experimentally infected with feline immunodeficiency virus

Lymphocytes from normal cats or cats experimentally infected with feline immunodeficiency virus (FIV) were stimulated with phytohemagglutinin, pokeweed mitogen, or concanavalin A. Lymphocytes from infected cats had lower responses than those from uninfected cats. These results support the hypothesis that FIV induces immunosuppression.

Genomic Characterization of a Slow/Low Maedi Visna Virus

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.

Characterization of two monoclonal antibodies against feline immunodeficiency virus gag gene products and their application in an assay to evaluate neutralizing antibody activity

Monoclonal antibodies (MAbs) 3B7 and 1C11 were produced against the gag gene products of feline immunodeficiency virus (FIV). These MAbs reacted strongly with FIV p24 in Western blots (immunoblots) and recognized p50 with a lower intensity. They specifically bound antigens in the cytoplasm of FIV-infected cells as determined by indirect immunofluorescence and immunocytochemistry. Although neither MAb inhibited viral replication in vitro, they were useful in a simple assay for the detection and quantification of infectious virus and neutralizing antibody activity. The assay utilizes Crandell feline kidney cells and requires 4 days for completion. Neutralizing antibodies in cats were detected 3 to 4 weeks after experimental infection with FIV. Antibody titres progressively increased during the first year of infection reaching high titres which were maintained 2.5 years post-infection. The MAbs produced should be valuable reagents for the monitoring of viral replication in cells or tissues from FIV-infected cats and for other in vitro applications.

A DIVA system based on the detection of antibodies to non-structural protein 3 (NS3) of Bluetongue virus

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.

Cellular specificity and replication rate of Maedi Visna virus in vitro can be controlled by LTR sequences

Summary.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.

Phylogenetic analysis of five Portuguese strains of FIV

Summary Feline immunodeficiency virus (FIV) was isolated from five Portuguese cats. The five strains were named RP1, PP2, TLP3, FP4 and CP5. The LTR, the CA region of the gag gene, and the V3-V5 region of the env gene were amplified by PCR, cloned and sequenced. The phylogenetic relationships among these three regions and other previously published sequences revealed an independent clustering of the Portuguese isolates in the LTR and CA protein. Based on the V3-V5 region the Portuguese isolates were classified as Subtype B, although they appear to be a subcluster within Subtype B. The study of these new FIV isolates showed the presence of Subtype B in Portugal and could provide a contribution for the understanding of FIV’s genetic diversity.

Development of a monoclonal antibody blocking-ELISA for detection of antibodies against Maedi-Visna virus.

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.

Simultaneous detection of West Nile and Japanese encephalitis virus RNA by duplex TaqMan RT-PCR

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.

Serological evidence of West Nile virus circulation in Portugal

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.