Detection of rabbit Haemorrhagic disease virus 2 during the wild rabbit (Oryctolagus cuniculus) eradication from the Berlengas archipelago, Portugal.

BACKGROUND: In the regular wildlife monitoring action carried out in the summer of the past few years at the Berlenga Island, wild rabbits (Oryctolagus cuniculus) have been repeatedly found dead. However, the origin of those deaths was never investigated. Our aim was to investigate the cause of death of 11 rabbits collected between April and May 2016. RESULTS: While screening samples from rabbit carcasses for the major viral rabbit pathogens, five tested positive to RHDV2 but all were negative for RHDV and myxoma virus (MYXV). For six RHDV2-negative specimens, emaciation and parasitism were considered the most probable cause of death. Lesions identified in the RHDV2-positive rabbits included non-suppurative diffuse hepatic necrosis and pulmonary lesions varying from congestion and oedema of the lungs to interstitial pneumonia. Sequencing analysis of the vp60 gene obtained from two specimens showed identical vp60 sequences. Comparison with other known RHDV2 strains from public databases through BLAST analysis revealed a closer similarity with strains from Alentejo collected during 2013. Maximum Likelihood and Bayesian phylogenetic analysis showed that the 2016 strains from the archipelago have a higher resemblance with a group of strains mostly collected in the South of Portugal between 2013 and 2014. CONCLUSION: The results suggest that RHDV2 may have been introduced on the Berlenga Island a few years ago, having evolved separately from mainland strains due to insularity.

Challenges in the rabbit haemorrhagic disease 2 (RHDV2) molecular diagnosis of vaccinated rabbits.

Molecular methods are fundamental tools for the diagnosis of viral infections. While interpretation of results is straightforward for unvaccinated animals, where positivity represents ongoing or past infections, the presence of vaccine virus in the tissues of recently vaccinated animals may mislead diagnosis. In this study, we investigated the interference of RHDV2 vaccination in the results of a RT-qPCR for RHDV2 detection, and possible associations between mean Cq values of five animal groups differing in age, vaccination status and origin (domestic/wild). Viral sequences from vaccinated rabbits that died of RHDV2 infection (n=14) were compared with the sequences from the commercial vaccines used in those animals. Group Cq means were compared through Independent t-test and One-way ANOVA. We proved that RHDV2 vaccine-RNA is not detected by the RT-qPCR as early as 15days post-vaccination, an important fact in assisting results interpretation for diagnosis. Cq values of vaccinated and non-vaccinated infected domestic adults showed a statistically significant difference (p<0.05), demonstrating that vaccination-induced immunity reduces viral loads and delays disease progression. Contrarily, in vaccinated young rabbits higher viral loads were registered compared to non-vaccinated kittens. No significant variation (p=0.3824) was observed between viral loads of non-vaccinated domestic and wild RHDV2-victimised rabbits. Although the reduced number of vaccinated young animals analysed hampered a robust statistical analysis, this occurrence suggests that passively acquired maternal antibodies may inhibit the active immune response to vaccination, delaying protection and favouring disease progression. Our finding emphasises the importance of adapting kitten RHDV2 vaccination schedules to circumvent this interference phenomenon.

Detection of virulence-associated genes in pathogenic and commensal avian Escherichia coli isolates.

Poultry colibacillosis due to Avian Pathogenic Escherichia coli (APEC) is responsible for several extra-intestinal pathological conditions, leading to serious economic damage in poultry production. The most commonly associated pathologies are airsacculitis, colisepticemia, and cellulitis in broiler chickens, and salpingitis and peritonitis in broiler breeders. In this work a total of 66 strains isolated from dead broiler breeders affected with colibacillosis and 61 strains from healthy broilers were studied. Strains from broiler breeders were typified with serogroups O2, O18, and O78, which are mainly associated with disease. The serogroup O78 was the most prevalent (58%). All the strains were checked for the presence of 11 virulence genes: 1) arginine succinyltransferase A (astA); ii) E.coli hemeutilization protein A (chuA); iii) colicin V A/B (cvaA/B); iv) fimbriae mannose-binding type 1 (fimC); v) ferric yersiniabactin uptake A (fyuA); vi) iron-repressible high-molecular-weight proteins 2 (irp2); vii) increased serum survival (iss); viii) iron-uptake systems of E.coli D (iucD); ix) pielonefritis associated to pili C (papC); x) temperature sensitive haemaglutinin (tsh), and xi) vacuolating autotransporter toxin (vat), by Multiplex-PCR. The results showed that all genes are present in both commensal and pathogenic E. coli strains. The iron uptake-related genes and the serum survival gene were more prevalent among APEC. The adhesin genes, except tsh, and the toxin genes, except astA, were also more prevalent among APEC isolates. Except for astA and tsh, APEC strains harbored the majority of the virulence-associated genes studied and fimC was the most prevalent gene, detected in 96.97 and 88.52% of APEC and AFEC strains, respectively. Possession of more than one iron transport system seems to play an important role on APEC survival.

Phylogenetic analysis of six isolates of beak and feather disease virus from African grey parrots 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.

Effect of cationic liposomes/DNA charge ratio on gene expression and antibody response of a candidate DNA vaccine against Maedi Visna virus.

Maedi Visna virus (MVV) is an ovine lentivirus with high prevalence all over the world. Since conventional vaccines had failed in protecting animals against the infection, the development of a DNA vaccine can be an alternative. The candidate vaccine was constructed by cloning the sequence encoding MVV p25 protein and was tested both in vitro and in vivo experiments associated with cationic liposomes. The lipoplexes (plasmid DNA-liposome complexes) with charge ratios ranging from 0 to 18 were prepared in physiological saline solution and characterized at a physical-chemistry level. Agarose gel electrophoresis was used as a first approach to evaluate qualitatively the amount of unbounded DNA by the liposomes. Dynamic light scattering measurements revealed that under the studied conditions lipoplexes with theoretical charge ratios (+/-) from 3 to 6 are unstable and prone to aggregation displaying sizes higher than 1 microm. At lower and higher charge ratios lipoplex size range from 200 to 500 nm. Using a Foster Resonance Energy Transfer methodology previously reported by us, complexation efficiency of the same complexes was related to in vitro and in vivo results. Higher transfection efficiencies were obtained in vitro with lipoplexes with charge ratio (+/-)=10, where 97% of the DNA were protected by the liposomes. However, the subcutaneous immunization of mice induced higher antibody titers with lipoplexes at charge ratio (+/-)=1, in which only 23% DNA is protected by the liposomes. Moreover, use of cationic liposomes has shown an increased antibody response when compared with a naked DNA immunization.

Pacheco's parrot disease in macaws of the Lisbon's Zoological Garden. Description of an outbreak, diagnosis and management, including vaccination.

The Lisbon's Zoological Garden, Portugal, has maintained for many years a large collection of psittacine birds without any serious health problems. Unexpectedly, in April 1999, a total of nine macaws died after a short period of illness. Clinical signs consisted mainly of anorexia, ruffled feathers and yellowish droppings. A herpesvirus was isolated from brain, trachea, lung, liver, spleen, kidney and intestine of each of the examined dead birds, confirming that all animals succumbed during viraemia. Serotyping of the isolate in cross neutralization tests with reference sera prove that the outbreak was caused by serotype 3 of Pacheco's parrot disease herpesviruses. An autogenous, formalin-inactivated vaccine with adjuvant (aluminium hydroxid gel) was prepared from one of the isolates and injected intramuscularly 14 days and six weeks after the onset of mortality in an attempt to protect the remaining psittacine birds in the zoo from the disease. The autogenous vaccine was well tolerated and was able to rapidly stop virus spread and morbidity and mortality among the psittacine birds. Follow-up studies demonstrate that all nine blood samples from vaccinated birds obtained nine month' after the second vaccination contain neutralizing antibodies. Twenty five month' after vaccination two out of four serum samples were still antibody positive. No herpesvirus was isolated from faecal samples nine and twenty five months after the onset of the outbreak. These data prove that the autogenous vaccine played a major role in containing a severe outbreak of Pacheco's parrot disease in a large collection of psittacine birds.

Optimization of the primary recovery of human interferon alpha2b from Escherichia coli inclusion bodies.

The human interferon alpha2b (hu-IFNalpha2b) gene was cloned in Escherichia coli JM109(DE3) and the recombinant protein was expressed as cytoplasmic inclusion bodies (IB). The present work discusses the recovery of hu-IFNalpha2b IB from the E. coli cells. An optimized protocol is proposed based on the sequential evaluation of recovery steps and parameters: (i) cell disruption, (ii) IB recovery and separation from cell debris, (iii) IB washing, and (iv) IB solubilization. Parameters such as hu-IFNalpha2b purity and recovery yield were measured after each step. The optimized recovery protocol yielded 60% of hu-IFNalpha2b with a purity of up to 80%. The protein was renatured at high concentration after recovery and it was found to display biological activity.

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

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.

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.

Enhanced neutralization of feline immunodeficiency virus by complement viral lysis.

The ability of complement to inactivate feline immunodeficiency virus (FIV) was examined. Treatment of virus with complement plus sub-neutralizing titers of antiserum resulted in a significant reduction in the virus titer compared with treatment of the virus with complement or antibody alone. One of the mechanisms by which cat complement inactivates FIV was shown to be by viral lysis as determined by a reverse transcriptase release assay. Kinetic studies revealed that viral lysis is initiated soon after the addition of complement to a mixture of virus and antiserum. Treatment of FIV with normal non-complement-inactivated human serum resulted in virus inactivation and release of viral RT in the absence of specific antiserum. It appears that FIV activates complement directly through the classical pathway and that integrity of the membrane attack components is a requirement for FIV lysis by human serum. The vulnerability of two distinct isolates of FIV to complement lysis was compared using complement from different species. Oradell isolate was more sensitive to complement lysis than the Petaluma isolate as assessed by reverse transcriptase release. It appears that factors intrinsic to the virus isolate may influence the amplitude of complement-dependent viral lysis.

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.

Antibody response to reverse transcriptase in cats infected with feline immunodeficiency virus.

The antibody response in cats to feline immunodeficiency virus (FIV) reverse transcriptase (RT) was followed for 3 years. Eight of the nine cats used in this study produced reverse transcriptase-inhibiting (RTI) antibodies. Relative inhibitory means of 2.9%, 18.4%, 33%, and 47% were found 6, 12, 24, and 36 months, respectively, after infection with FIV. The enzyme activity was suppressed by greater than or equal to 78% with the use of 100 micrograms of FIV-associated IgG. The RTI antibodies were FIV-specific, as they did not inhibit other mammalian retroviral polymerases, including feline leukemia virus RT. An RT-inhibition assay with sera in the presence of protein A and immunoblot analysis showed that antibody binding to FIV RT protein p62 is independent of antibody ability to block enzyme activity. Viral RT released by detergent-treated virus was stable for more than 6 weeks at 4 degrees C, whereas its activity was reduced by 50% after 2 weeks at 37 degrees C. Because significant concentrations of RTI antibodies are detected only at 1 to 2 years after infection, they can be used to determine the approximate time of virus infection and as a marker for disease progression.

Anti-idiotypic antibodies to feline leukemia virus: an approach for retroviral immunization strategies.

The present study describes an approach to the development and use of anti-idiotypic antibodies as a possible immunization strategy to prevent retroviral infection. The rationale for using anti-idiotypes (anti-Ids) to try to elicit an antigenic-specific immune response is examined, and the production and characterization of polyclonal and monoclonal anti-Ids are described. Several techniques were used to determine antigenic mimicry and anti-Id subtypes. The potential use of anti-Ids in feline leukemia virus (FeLV) receptor studies and vaccine trials in vivo were investigated. Results from these studies suggest that the anti-Id strategy is feasible for the FeLV model. Polyclonal Ab2 reagents were developed that blocked virus-receptor binding and thus inhibited viral infection in vitro and induced humoral immune responses in 6- to 8-week old kittens characterized by production of Ab3 with the ability to bind the original FeLV envelope protein gp70 as assessed by Western blot analysis.

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.

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.