Thermostability study of virulent Newcastle disease viruses isolated in Southern Angola.

Newcastle disease (ND) is endemic in Angola. Several outbreaks of ND occurred in small backyard flocks and village chickens with high mortality in the southern provinces of the country, Cunene, Namibe and Huíla, in 2016 and 2018. In those years, 15 virulent ND virus (NDV) strains were isolated and grouped within subgenotype 2 of genotype VII (subgenotype VII.2). We now present a study on the thermostability of the isolates, aiming at the selection of the most thermostable strains that, after being genetically modified to reduce their virulence, can be adapted to the production of vaccines less dependent on cold chain and more adequate to protect native chickens against ND. Heat-inactivation kinetics of haemagglutinin (Ha) activity and infectivity (I) of the isolates were determined by incubating aliquots of virus at 56 °C for different time intervals. The two isolates from Namibe province showed a decrease in infectivity of 2 log10 in ≤ 10 min, therefore belonging to the I-phenotype, but while the NB1 isolate from 2016 maintained the Ha activity up to 30 min and was classified as thermostable virus (I-Ha+), the Ha activity of the 2018 NB2 isolate decreased by 2 log2 in 30 min, being classified as a thermolabile virus (I-Ha-). Of the 13 NDV isolates from Huíla province, 10 isolates were classified as thermostable, eight with phenotype I+Ha+ and 2 with phenotype I-Ha+. The other three isolates from this province were classified as thermolabile viruses (I-Ha-).Contribution: This study will contribute to the control and/or eradication of Newcastle disease virus in Angola. The thermostable viral strains isolated from chickens in the country can be genetically manipulated by reverse genetic technology in order to reduce their virulence and use them as a vaccine in the remote areas of Angola.

Molecular characterization and phylogenetic analysis of Newcastle disease viruses isolated in southern Angola, 2016-2018.

Newcastle disease (ND) is a highly contagious viral disease that affects many bird species worldwide. This study presents the results of the molecular characterization and phylogenetic analysis of 15 virulent ND viruses (NDV) isolated from chickens during outbreaks reported in 2016 and 2018, in the provinces of Namibe and Huíla, in southern Angola. A 561-nucleotide fragment of the F gene was amplified by RT-PCR and sequenced for molecular characterization. Results showed that in all isolates the amino acid sequence comprising the cleavage site of fusion protein is characteristic of virulent viruses (RRQKR/F). Blast analysis revealed high similarity (99.2%) between two isolates from Huíla province, HLA4 and HLA6, and strain 5620 (GenBank accession number KY747479) isolated from chickens in the neighboring country Namibia, in 2016. The other isolates investigated are more related (97.0%) with strain 6195 (GenBank accession number KY747480), also isolated in Namibia in 2016. Phylogenetic analysis performed by Maximum Likelihood, Neighbor-joining and Bayesian methods revealed that like the strains isolated in Namibia, the isolates from southern Angola also belong to subgenotype 2 of genotype VII (VII.2). The network analysis revealed that NBA1 isolate from Angola is closer to a common ancestor than the isolates from Namibia, suggesting that transmission of ND viruses occurred from Angola to Namibia.

A molecular test based on RT-LAMP for rapid, sensitive and inexpensive colorimetric detection of SARS-CoV-2 in clinical samples.

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.

First description of a fatal equine infection with Halicephalobus gingivalis in Portugal. Relevance for public health.

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.

Development and validation of a real-time PCR for the detection and quantification of porcine circovirus type 2.

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.

West Nile virus in horses during the summer and autumn seasons of 2015 and 2016, Portugal.

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.

Emergence of rabbit haemorrhagic disease virus 2 in the archipelago of Madeira, Portugal (2016-2017).

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.

Development and validation of a blocking ELISA test for the detection of avian influenza antibodies in poultry species.

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.

DNA Vaccines Against Maedi-Visna Virus.

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.

AVIAN POXVIRUS INFECTION IN A FLAMINGO (PHOENICOPTERUS RUBER) OF THE LISBON ZOO.

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.

Human case of West Nile neuroinvasive disease in Portugal, 2015.

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.

Rabbit haemorrhagic disease virus 2 (RHDV2) outbreak in Azores: Disclosure of common genetic markers and phylogenetic segregation within the European strains.

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 real time Taqman RT-PCR for the detection of rabbit hemorrhagic disease virus 2 (RHDV2).

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.

New insight into the epidemiology of rabbit hemorrhagic disease viruses in Portugal: retrospective study reveals the circulation of genogroup 5 (G5) in Azores and discloses the circulation of G1 and G6 strains in mainland until 2008.

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.

U exon variability as a discriminatory tool for Bayesian analysis of adenoviruses.

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.

Enhancement of DNA vaccine efficacy by intracellular targeting strategies.

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.

Development and validation of a real time PCR for the detection of myxoma virus based on the diploid gene M000.5L/R.

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.

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.