Analysis of ORF5 and Full-Length Genome Sequences of Porcine Reproductive and Respiratory Syndrome Virus Isolates of Genotypes 1 and 2 Retrieved Worldwide Provides Evidence that Recombination Is a Common Phenomenon and May Produce Mosaic Isolates

Recombination is currently recognized as a factor for high genetic diversity, but the frequency of such recombination events and the genome segments involved are not well known. In the present study, we initially focused on the detection of recombinant porcine reproductive and respiratory syndrome virus (PRRSV) isolates by examining previously published data sets of ORF5 sequences (genotypes 1 and 2) obtained worldwide. We then examined full-length genome sequences in order to determine potential recombination breakpoints along the viral genome. For ORF5, 11 sets of genotype 1 sequences from different geographical areas, including 2 Asian, 1 American, and 7 European regions, and three sets of genotype 2, including sets from China, Mexico, and the United States, were analyzed separately. Potential recombination breakpoints were detected in 10/11 genotype 1 sets, including 9 cases in which the clustering of at least one isolate was different before and after the breakpoints. In genotype 2, potential breakpoints and different tree clustering of at least one strain before and after the breakpoint were observed in 2 out of 3 sets. The results indicated that most of the ORF5 data sets contained at least one recombinant sequence. When the full-length genome sequences were examined, both genotype 1 and 2 sets presented breakpoints (10 and 9, respectively), resulting in significantly different topologies before and after the breakpoints. Mosaic genomes were detected in genotype 1 sequences. These results may have significant implications for the understanding of the molecular epidemiology of PRRSV. IMPORTANCE PRRSV is one of the most important viruses affecting swine production worldwide, causing big economic losses and sanitary problems. One of the key questions on PRRSV arises from its genetic diversity, which is thought to have a direct impact on immunobiology, epidemiology, diagnosis, and vaccine efficacy. One of the causes of this genetic diversity is recombination among strains. This study provides evidence that recombinant PRRSV isolates are common in most of the countries with significant swine production, especially PRRSV genotype 1. This observation has implications in the proper characterization of PRRSV strains, in the future development of phylogenetic studies, and in the development of new PRRSV control strategies. Moreover, the present paper emphasizes the need for a deeper understanding of the mechanisms and circumstances involved in the generation of genetic diversity of PRRSV

Development and Validation of an Epitope Prediction Tool for Swine (PigMatrix) Based on the Pocket Profile Method

Background: T cell epitope prediction tools and associated vaccine design algorithms have accelerated the development of vaccines for humans. Predictive tools for swine and other food animals are not as well developed, primarily because the data required to develop the tools are lacking. Here, we overcome a lack of T cell epitope data to construct swine epitope predictors by systematically leveraging available human information. Applying the “pocket profile method”, we use sequence and structural similarities in the binding pockets of human and swine major histocompatibility complex proteins to infer Swine Leukocyte Antigen (SLA) peptide binding preferences. We developed epitope-prediction matrices (PigMatrices), for three SLA class I alleles (SLA-1*0401, 2*0401 and 3*0401) and one class II allele (SLA-DRB1*0201), based on the binding preferences of the best-matched Human Leukocyte Antigen (HLA) pocket for each SLA pocket. The contact residues involved in the binding pockets were defined for class I based on crystal structures of either SLA (SLA-specific contacts, Ssc) or HLA supertype alleles (HLA contacts, Hc); for class II, only Hc was possible. Different substitution matrices were evaluated (PAM and BLOSUM) for scoring pocket similarity and identifying the best human match. The accuracy of the PigMatrices was compared to available online swine epitope prediction tools such as PickPocket and NetMHCpan. Results: PigMatrices that used Ssc to define the pocket sequences and PAM30 to score pocket similarity demonstrated the best predictive performance and were able to accurately separate binders from random peptides. For SLA-1*0401 and 2*0401, PigMatrix achieved area under the receiver operating characteristic curves (AUC) of 0.78 and 0.73, respectively, which were equivalent or better than PickPocket (0.76 and 0.54) and NetMHCpan version 2.4 (0.41 and 0.51) and version 2.8 (0.72 and 0.71). In addition, we developed the first predictive SLA class II matrix, obtaining an AUC of 0.73 for existing SLA-DRB1*0201 epitopes. Notably, PigMatrix achieved this level of predictive power without training on SLA binding data. Conclusions: Overall, the pocket profile method combined with binding preferences from HLA binding data shows significant promise for developing T cell epitope prediction tools for pigs. When combined with existing vaccine design algorithms, PigMatrix will be useful for developing genome-derived vaccines for a range of pig pathogens for which no effective vaccines currently exist (e.g. porcine reproductive and respiratory syndrome, influenza and porcine epidemic diarrhea)

Development of an Electrochemical Sensor Conjugated with Molecularly Imprinted Polymers for the Detection of Enrofloxacin

An electrochemical sensor was fabricated for the rapid and simple detection of enrofloxacin (EF). Modification of screen-printed gold electrodes (SPE) with molecularly imprinted polymers (MIPs) allowed the detection of enrofloxacin by square wave voltammetry (SWV), measuring the oxidation peak at +0.9 V. The detection principle of molecularly imprinted polymers (MIPs) is based on the formation of binding sites with affinities and specificities comparable with those of natural antibodies. The detection of enrofloxacin showed a linear range of 0.01–0.1 mM with a detection limit LOD of 0.02 mM. The development of a non-imprinted polymer (NIP) control sensor allowed for better and more efficient detection. In addition, the sensor is portable, having the advantage of analyzing and detecting molecules of interest without the need to take the sample to a laboratory

Development of an Electrochemical Sensor Conjugated with Molecularly Imprinted Polymers for the Detection of Enrofloxacin

An electrochemical sensor was fabricated for the rapid and simple detection of enrofloxacin (EF). Modification of screen-printed gold electrodes (SPE) with molecularly imprinted polymers (MIPs) allowed the detection of enrofloxacin by square wave voltammetry (SWV), measuring the oxidation peak at +0.9 V. The detection principle of molecularly imprinted polymers (MIPs) is based on the formation of binding sites with affinities and specificities comparable with those of natural antibodies. The detection of enrofloxacin showed a linear range of 0.01–0.1 mM with a detection limit LOD of 0.02 mM. The development of a non-imprinted polymer (NIP) control sensor allowed for better and more efficient detection. In addition, the sensor is portable, having the advantage of analyzing and detecting molecules of interest without the need to take the sample to a laboratory

Temperature stimuli-responsive nanoparticles from chitosan-graft-poly(N-vinylcaprolactam) as a drug delivery system

This work describes the preparation of thermosensitive chitosan-graft-poly(N-vinylcaprolactam) nanoparticles by ionic gelation and their potential use as a controlled drug delivery system, using doxorubicin as a model drug. A systematic study of the effect of the main processing parameters on both the size and thermoresponsive behavior of nanoparticles was investigated. The size of the particles is strongly dependent on the length of the poly(N-vinylcaprolactam) grafted chains and the concentration of the copolymer and crosslinking agent solutions. The molecular structure of the copolymer plays an essential role in the phase transition temperature of the particles, which decreases with the length of PVCL grafted chain. The system displayed proper drug-association parameters, and the drug-loaded nanoparticles exhibited dose-dependent cytotoxicity. A significant increase in the doxorubicin delivery rate was observed above the phase transition temperature (40 °C). These features indicate that these nanoparticles are suitable for the development of a new thermally controlled anti-cancer drug delivery system. © 2019 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 2019, 136, 47831.The authors acknowledge the Mexican Council of Science and Technology (CONACYT) for their financial support through the project PDCAPN-2014/248982. D.F.Q. acknowledges a grant from CONACYT (AIIVFPN-2017/291229). The authors are grateful for the technical support of Dr. Alvaro González-Gómez from ICTP-CSIC and M.C. Karla Martínez-Robinson, M.C. Luisa L. Silva, Q.B. Alma Campa-Mada, and Prof.Miguel A.Martínez-Tellez fromCIAD.Peer Reviewe

Bottlenecks in the transmission of porcine reproductive and respiratory syndrome virus (PRRSV1) to naïve pigs and the quasi-species variation of the virus during infection in vaccinated pigs

Abstract This paper describes the results of two experiments regarding porcine reproductive and respiratory syndrome virus (PRRSV1): the first one studied the existence of bottlenecks in an experimental one-to-one model of transmission in pigs; while the second analysed the differences between viral quasi-species in vaccinated pigs that developed shorter or longer viraemias after natural challenge. Serum samples, as well as the initial inoculum, were deep-sequenced and a viral quasi-species was constructed per sample. For the first experiment, the results consistently reported a reduction in the quasi-species diversity after a transmission event, pointing to the existence of bottlenecks during PRRSV1 transmission. However, despite the identified preferred and un-preferred transmitted variants not being randomly distributed along the virus genome, it was not possible to identify any variant producing a structural change in any viral protein. In contrast, the mutations identified in GP2, nsp9 and M of the second experiment pointed to changes in the amino acid charges and the viral RNA-dependent RNA polymerase structure. The fact that the affected proteins are known targets of the immunity against PRRSV, plus the differential level of neutralizing antibodies present in pigs developing short or long viraemias, suggests that the immune response selected those changes