Current status of virus-vectored vaccines against pathogens that affect poultry.

The most effective strategies for the control of disease in poultry are vaccination and biosecurity. Vaccines useful against pathogens affecting poultry must be safe, effective with a single dose, inexpensive, applicable by mass vaccination methods, and able to induce a protective immune response in the presence of maternal antibodies. Viral vector meet some of these characteristics and if the attenuated virus used as vector infects birds, the vaccine will have the advantage of being bivalent. Thus, viral vectors are currently a tool of choice for the development of new poultry vaccines. This review describes the main viruses used as vectors for the delivery and in vivo expression of antigens of poultry pathogens. It also presents the methodologies most frequently used to obtain recombinant viral vectors and summarizes the state-of-the-art related to vectored vaccines in poultry (some of them currently licensed), the pathogens targeted and their antigens, and the ability of these vaccines to induce an effective immune response. Finally, the review discusses the results of a few studies comparing recombinant viral vector vaccines and live-attenuated vaccines in vaccine matching challenges, and mentions strategies and future researches that can help to improve the efficacy of vectored vaccines in poultry birds.

Virus isolation and full-length genome sequencing of a representative canine distemper virus wild type strain of the South America 2 clade.

Canine Distemper Virus (CDV) is a highly contagious pathogen of dogs that causes severe respiratory, gastrointestinal and nervous signs. Although vaccines have been used to prevent infections, CDV has been reported worldwide, even in vaccinated animals. In the present study, a representative wild type CDV strain (Arg24) was isolated from a sick vaccinated dog and its genome was completely sequenced using Illumina technology. This strain produced a strong cytopathic effect in Vero SLAM (Signaling Lymphocyte Activation Molecule) cells with a higher titer of 1.1 × 105 Median Tissue Culture Infectious Dose (TCID50/mL) at 32 h post infection, in cell-associated virus. The Arg24 strain genome, showed values of 97.1, 90.3, 96.7, 90.6, 89.8 and 97.3 % of amino acid identity with respect to the Onderstepoort vaccine strain (Nucleoprotein, Phosphoprotein, Matrix, Fusion, Hemagglutinin and Large polymerase, respectively). Focusing on the Hemagglutinin gene, which is the target for genetic characterization, Arg24 showed four additional potential glycosylation sites, with respect to the Onderstepoort. The availability of Arg24 strain, which can be easily grown in Vero SLAM cells, is an important tool to perform immunological and antigenic comparative studies, between wild type and vaccine CDV strains.

RT-PCR and sequence analysis of the full-length fusion protein of Canine Distemper Virus from domestic dogs.

During 2007-2014, 84 out of 236 (35.6%) samples from domestic dogs submitted to our laboratory for diagnostic purposes were positive for Canine Distemper Virus (CDV), as analyzed by RT-PCR amplification of a fragment of the nucleoprotein gene. Fifty-nine of them (70.2%) were from dogs that had been vaccinated against CDV. The full-length gene encoding the Fusion (F) protein of fifteen isolates was sequenced and compared with that of those of other CDVs, including wild-type and vaccine strains. Phylogenetic analysis using the F gene full-length sequences grouped all the Argentinean CDV strains in the SA2 clade. Sequence identity with the Onderstepoort vaccine strain was 89.0-90.6%, and the highest divergence was found in the 135 amino acids corresponding to the F protein signal-peptide, Fsp (64.4-66.7% identity). In contrast, this region was highly conserved among the local strains (94.1-100% identity). One extra putative N-glycosylation site was identified in the F gene of CDV Argentinean strains with respect to the vaccine strain. The present report is the first to analyze full-length F protein sequences of CDV strains circulating in Argentina, and contributes to the knowledge of molecular epidemiology of CDV, which may help in understanding future disease outbreaks.

Resurgence of canine parvovirus 2a strain in the domestic dog population from Argentina.

Ninety-three rectal swab samples were taken, from dogs suspected of canine parvovirus (CPV) infection and analyzed by PCR. A fragment of the VP2 gene, was amplified in 41 (44%) of them, resulting CPV positive samples. Sequencing analysis of these PCR products showed that 37 samples (90.2%) belonged to the CPV2c type, whereas four samples (9.8%) were identified as CPV2a, which has not been found since 2008. It was also found that 24 out of 37 CPV2c samples (65%), carried the mutation Thr440Ala, whereas this mutation was absent in the four CPV2a strains reported herein. Using phylogenetic analysis of the full length VP2 gene, which was amplified by PCR in six local samples, it was seen that CPV2a Argentine strains reported in this study, were genetically closer to a previous local CPV2a isolate (year 2003) and to a South African CPV2a strain, than to any of the recently reported Uruguayan CPV2a strains. The results obtained in this work, together with those reported previously in Uruguay strongly suggest that, in spite of the geographical proximity, wild type CPV strains undergo different evolutive pathways in each country, resulting in the prevalence of different strains in related dog populations. Further extensive epidemiological studies are needed in order to improve the understanding of CPV evolution.

Study of canine parvovirus evolution: comparative analysis of full-length VP2 gene sequences from Argentina and international field strains.

The continuous emergence of new strains of canine parvovirus (CPV), poorly protected by current vaccination, is a concern among breeders, veterinarians, and dog owners around the world. Therefore, the understanding of the genetic variation in emerging CPV strains is crucial for the design of disease control strategies, including vaccines. In this paper, we obtained the sequences of the full-length gene encoding for the main capsid protein (VP2) of 11 canine parvovirus type 2 (CPV-2) Argentine representative field strains, selected from a total of 75 positive samples studied in our laboratory in the last 9 years. A comparative sequence analysis was performed on 9 CPV-2c, one CPV-2a, and one CPV-2b Argentine strains with respect to international strains reported in the GenBank database. In agreement with previous reports, a high degree of identity was found among CPV-2c Argentine strains (99.6-100% and 99.7-100% at nucleotide and amino acid levels, respectively). However, the appearance of a new substitution in the 440 position (T440A) in four CPV-2c Argentine strains obtained after the year 2009 gives support to the variability observed for this position located within the VP2, three-fold spike. This is the first report on the genetic characterization of the full-length VP2 gene of emerging CPV strains in South America and shows that all the Argentine CPV-2c isolates cluster together with European and North American CPV-2c strains.

Evolution of canine parvovirus in Argentina between years 2003 and 2010: CPV2c has become the predominant variant affecting the domestic dog population.

The current frequency of Canine Parvovirus variants (CPV2a, CPV2b and CPV2c) in the Argentine dog population was investigated by PCR amplification of a 583 bp fragment in the VP2 gene. From a total of 79 rectal swab samples that have been submitted to our laboratory since 2008, 55 (69.6%) resulted positive and were further analyzed by direct DNA sequencing. Fifty positives samples (91%) were characterized as CPV2c variant, which appeared in Argentina in the year 2003 and has been the prevalent type since 2008, whereas CPV2a and CPV2b, still found in Argentine dogs, were represented in 3.6% and 5.4% of the population, respectively. Considering that CPV2c is spreading worldwide, and that this variant is also affecting vaccinated dogs, efforts should be made towards the development of new matched CPV vaccines.

Effect of estradiol, progesterone and testosterone on apoptosis- and proliferation-induced MAPK signaling in human umbilical vein endothelial cells.

Sex hormones induce death or cell proliferation in various cell lines and in primary cultures. However, the signal transduction pathways involved in the regulation of proliferation and apoptosis in endothelial cells have not been fully elucidated. Here, we report that progesterone and testosterone induce apoptosis in HUVECs in a p38- and JNK-dependent manner, and that estradiol promotes proliferation via the activation of ERK2. We showed that, at physiological doses, progesterone and testosterone promoted p38, but not JNK, phosphorylation. Hormone inhibitors, on the other hand, prevented p38 phosphorylation. When supraphysiological doses were applied, both p38 and JNK were phosphorylated, causing apoptotic cell death. The addition of hormone inhibitors at an appropriate concentration did not prevent cell death or the phosphorylation of p38 and JNK. Estradiol, at physiological doses, promoted an increase in ERK2 phosphorylation that was blocked by fulvestrant. At physiological and supraphysiological doses, it promoted a proliferative effect. In conclusion, these findings suggest that JNK has an important pro-apoptotic function following progesterone and testosterone treatment in human endothelial cells, and that ERK2 has a proliferative effect following estradiol treatment.