A Critical Review about Different Vaccines against Classical Swine Fever Virus and Their Repercussions in Endemic Regions.

Classical swine fever (CSF) is, without any doubt, one of the most devasting viral infectious diseases affecting the members of Suidae family, which causes a severe impact on the global economy. The reemergence of CSF virus (CSFV) in several countries in America, Asia, and sporadic outbreaks in Europe, sheds light about the serious concern that a potential global reemergence of this disease represents. The negative aspects related with the application of mass stamping out policies, including elevated costs and ethical issues, point out vaccination as the main control measure against future outbreaks. Hence, it is imperative for the scientific community to continue with the active investigations for more effective vaccines against CSFV. The current review pursues to gather all the available information about the vaccines in use or under developing stages against CSFV. From the perspective concerning the evolutionary viral process, this review also discusses the current problematic in CSF-endemic countries.

Unraveling the Global Phylodynamic and Phylogeographic Expansion of Mycoplasma gallisepticum: Understanding the Origin and Expansion of This Pathogen in Ecuador.

Mycoplasma gallisepticum (MG) is among the most significant problems in the poultry industry worldwide, representing a serious threat to international trade. Despite the fact that the mgc2 gene has been widely used for diagnostic and molecular characterization purposes, there is a lack of evidence supporting the reliability of this gene as a marker for molecular epidemiology approaches. Therefore, the current study aimed to assess the accuracy of the mgc2 gene for phylogenetic, phylodynamic, and phylogeographic evaluations. Furthermore, the global phylodynamic expansion of MG is described, and the origin and extension of the outbreak caused by MG in Ecuador were tracked and characterized. The results obtained strongly supported the use of the mgc2 gene as a reliable phylogenetic marker and accurate estimator for the temporal and phylogeographic structure reconstruction of MG. The phylodynamic analysis denoted the failures in the current policies to control MG and highlighted the imperative need to implement more sensitive methodologies of diagnosis and more efficient vaccines. Framed in Ecuador, the present study provides the first piece of evidence of the circulation of virulent field MG strains in Ecuadorian commercial poultry. The findings derived from the current study provide novel and significant insights into the origin, diversification, and evolutionary process of MG globally.

Positive selection pressure on E2 protein of classical swine fever virus drives variations in virulence, pathogenesis and antigenicity: Implication for epidemiological surveillance in endemic areas.

Classical swine fever (CSF), caused by CSF virus (CSFV), is considered one of the most important infectious diseases with devasting consequences for the pig industry. Recent reports describe the emergence of new CSFV strains resulting from the action of positive selection pressure, due mainly to the bottleneck effect generated by ineffective vaccination. Even though a decrease in the genetic diversity of the positively selected CSFV strains has been observed by several research groups, there is little information about the effect of this selective force on the virulence degree, antigenicity and pathogenicity of this type of strains. Hence, the aim of the current study was to determine the effect of the positive selection pressure on these three parameters of CSFV strains, emerged as result of the bottleneck effects induced by improper vaccination in a CSF-endemic area. Moreover, the effect of the positively selected strains on the epidemiological surveillance system was assessed. By the combination of in vitro, in vivo and immunoinformatic approaches, we revealed that the action of the positive selection pressure induces a decrease in virulence and alteration in pathogenicity and antigenicity. However, we also noted that the evolutionary process of CSFV, especially in segregated microenvironments, could contribute to the gain-fitness event, restoring the highly virulent pattern of the circulating strains. Besides, we denoted that the presence of low virulent strains selected by bottleneck effect after inefficient vaccination can lead to a relevant challenge for the epidemiological surveillance of CSF, contributing to under-reports of the disease, favouring the perpetuation of the virus in the field. In this study, B-cell and CTL epitopes on the E2 3D-structure model were also identified. Thus, the current study provides novel and significant insights into variation in virulence, pathogenesis and antigenicity experienced by CSFV strains after the positive selection pressure effect.

Multi-Target Strategy for Pan/Foot-and-Mouth Disease Virus (FMDV) Detection: A Combination of Sequences Analysis, in Silico Predictions and Laboratory Diagnostic Evaluation.

Foot-and-mouth disease (FMD) is a highly contagious viral disease affecting cloven-hoofed animals that causes severe economic losses. The disease is characterized by a vesicular condition and it cannot be differentiated from other vesicular diseases. Therefore, laboratory confirmation of any suspected FMD case is compulsory. Despite viral isolation in cell cultures has been considered for many years as the gold standard for FMD diagnosis, the advantages of real-time reverse transcription polymerase chain reaction (rRT-PCR) technology have motivated its use directly in clinical specimens for FMD diagnosis. The current work was aimed to develop and validate a molecular multi-check strategy using rRT-PCR (mMulti-rRT-PCR) based on SYBR-Green I for pan/foot-and-mouth disease virus (pan/FMDV) diagnosis. From in silico approaches, different primer pairs previously reported were selected and modified to reduce the likelihood of viral escape as well as potential failures in the pan/FMDV detection. The analytical parameters were evaluated using a high number of representative viral strains. The repeatability of the assay and its performance on field samples were also assessed. The mMulti-rRT-PCR was able to detect emergent FMDV strains that circulated in South America between the years 2006-2010 and on which the single rRT-PCRs failed when they were applied independently. The results obtained here showed that the proposed system is an accurate and rapid diagnosis method for sensitive and specific detection of FMDV. Thus, a validated mMulti-rRT-PCR assay based on SYBR-Green I detection coupled to melting curves resolution for pan/FMDV diagnosis on clinical samples is proposed. This study also highlights the need to incorporate the multi-target detection principle in the diagnosis of highly variable agents, specially, of those listed by OIE like FMDV.

Revisiting the genetic diversity of classical swine fever virus: A proposal for new genotyping and subgenotyping schemes of classification.

Classical swine fever (CSF) is a highly contagious febrile viral disease caused by CSF virus (CSFV), and it is considered one of the most important infectious diseases that affect domestic pigs and wild boar. Previous molecular epidemiology studies have revealed that the diversity of CSFV comprises three main genotypes and different subgenotypes defined using a reliable cut-off to accurately classify CSFV at genotype and subgenotype levels. However, a growing number of CSFV both complete genome and full E2 gene sequences have been submitted to GenBank (more than 500 sequences are currently available, revised on December 1, 2017). Therefore, the aim of this study was to revisit the taxonomy of CSFV at genotype and subgenotype levels, to unify nomenclature and to provide an update to the classification of CSFV. We propose here a new genotyping scheme with five well-defined CSFV genotypes (CSFV Genotypes 1-5) and 14 subgenotypes (seven for each of the CSFV Genotype 1 and CSFV Genotype 2). The findings showed in this study are relevant for molecular epidemiology approaches and will help to better understand the genetic diversity and spreading of CSFV at a global scale. The update in the classification of CSFV will allow the scientific community to establish more accurately the links among different outbreaks of the disease.

Impact of RNA Degradation on Viral Diagnosis: An Understated but Essential Step for the Successful Establishment of a Diagnosis Network.

The current global conditions, which include intensive globalization, climate changes, and viral evolution among other factors, have led to an increased emergence of viruses and new viral diseases; RNA viruses are key drivers of this evolution. Laboratory networks that are linked to central reference laboratories are required to conduct both active and passive environmental surveillance of this complicated global viral environment. These tasks require a continuous exchange of strains or field samples between different diagnostic laboratories. The shipment of these samples on dry ice represents both a biological hazard and a general health risk. Moreover, the requirement to ship on dry ice could be hampered by high costs, particularly in underdeveloped countries or regions located far from each other. To solve these issues, the shipment of RNA isolated from viral suspensions or directly from field samples could be a useful way to share viral genetic material. However, extracted RNA stored in aqueous solutions, even at -70 °C, is highly prone to degradation. The current study evaluated different RNA storage conditions for safety and feasibility for future use in molecular diagnostics. The in vitro RNA-transcripts obtained from an inactivated highly pathogenic avian influenza (HPAI) H5N1 virus was used as a model. The role of secondary structures in the protection of the RNA was also explored. Of the conditions evaluated, the dry pellet matrix was best able to protect viral RNA under extreme storage conditions. This method is safe, cost-effective and assures the integrity of RNA samples for reliable molecular diagnosis. This study aligns with the globally significant "Global One Health" paradigm, especially with respect to the diagnosis of emerging diseases that require confirmation by reference laboratories.

Deciphering the emergence, genetic diversity and evolution of classical swine fever virus.

Classical swine fever (CSF) is one of the most important infectious diseases causing significant economic losses. Its causal agent, CSF virus (CSFV), is a member of the Pestivirus genus included into the Flaviviridae family. Previous molecular epidemiology studies have revealed the CSFV diversity is divided into three main genotypes and different subgenotypes. However, the classification system for CSFV has not yet been harmonized internationally. Similarly, the phylogeny and evolutionary dynamics of CSFV remain unclear. The current study provides novel and significant insights into the origin, diversification and evolutionary process of CSFV. In addition, the best phylogenetic marker for CSFV capable of reproducing the same phylogenetic and evolutionary information as the complete viral genome is characterized. Also, a reliable cut-off to accurately classify CSFV at genotype and subgenotype levels is established. Based on the time for the most recent common ancestor (tMRCA) reconstruction and cophylogenetic analysis, it was determined that CSFV emerged around 225 years ago when the Tunisian Sheep Virus jumped from its natural host to swine. CSFV emergence was followed by a genetic expansion in three main lineages, driven by the action of positive selection pressure and functional divergence, as main natural forces.

Validation of optimal reference genes for quantitative real time PCR in muscle and adipose tissue for obesity and diabetes research.

The global incidence of obesity has led to an increasing need for understanding the molecular mechanisms that drive this epidemic and its comorbidities. Quantitative real-time RT-PCR (RT-qPCR) is the most reliable and widely used method for gene expression analysis. The selection of suitable reference genes (RGs) is critical for obtaining accurate gene expression information. The current study aimed to identify optimal RGs to perform quantitative transcriptomic analysis based on RT-qPCR for obesity and diabetes research, employing in vitro and mouse models, and human tissue samples. Using the ReFinder program we evaluated the stability of a total of 15 RGs. The impact of choosing the most suitable RGs versus less suitable RGs on RT-qPCR results was assessed. Optimal RGs differed between tissue and cell type, species, and experimental conditions. By employing different sets of RGs to normalize the mRNA expression of peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC1α), we show that sub-optimal RGs can markedly alter the PGC1α gene expression profile. Our study demonstrates the importance of validating RGs prior to normalizing transcriptional expression levels of target genes and identifies optimal RG pairs for reliable RT-qPCR normalization in cells and in human and murine muscle and adipose tissue for obesity/diabetes research.

Evaluation of a Phylogenetic Marker Based on Genomic Segment B of Infectious Bursal Disease Virus: Facilitating a Feasible Incorporation of this Segment to the Molecular Epidemiology Studies for this Viral Agent.

BACKGROUND: Infectious bursal disease (IBD) is a highly contagious and acute viral disease, which has caused high mortality rates in birds and considerable economic losses in different parts of the world for more than two decades and it still represents a considerable threat to poultry. The current study was designed to rigorously measure the reliability of a phylogenetic marker included into segment B. This marker can facilitate molecular epidemiology studies, incorporating this segment of the viral genome, to better explain the links between emergence, spreading and maintenance of the very virulent IBD virus (vvIBDV) strains worldwide. METHODOLOGY/PRINCIPAL FINDINGS: Sequences of the segment B gene from IBDV strains isolated from diverse geographic locations were obtained from the GenBank Database; Cuban sequences were obtained in the current work. A phylogenetic marker named B-marker was assessed by different phylogenetic principles such as saturation of substitution, phylogenetic noise and high consistency. This last parameter is based on the ability of B-marker to reconstruct the same topology as the complete segment B of the viral genome. From the results obtained from B-marker, demographic history for both main lineages of IBDV regarding segment B was performed by Bayesian skyline plot analysis. Phylogenetic analysis for both segments of IBDV genome was also performed, revealing the presence of a natural reassortant strain with segment A from vvIBDV strains and segment B from non-vvIBDV strains within Cuban IBDV population. CONCLUSIONS/SIGNIFICANCE: This study contributes to a better understanding of the emergence of vvIBDV strains, describing molecular epidemiology of IBDV using the state-of-the-art methodology concerning phylogenetic reconstruction. This study also revealed the presence of a novel natural reassorted strain as possible manifest of change in the genetic structure and stability of the vvIBDV strains. Therefore, it highlights the need to obtain information about both genome segments of IBDV for molecular epidemiology studies.

Development of a new LAMP assay for the detection of CSFV strains from Cuba: a proof-of-concept study.

Classical swine fever (CSF) is a devastating animal disease of great economic impact worldwide. In many countries, CSF has been endemic for decades, and vaccination of domestic pigs is one of the measures to control the disease. Consequently, differentiating infected from vaccinated animals by antibody ELISA screening is not applicable. In some countries, such as Cuba, lack of molecular techniques for sensitive, rapid and reliable detection of virus genomes is a critical point. To overcome this problem, an easy-to-use one-tube assay based on the loop-mediated isothermal amplification (LAMP) principle has been developed for detection of the genome of CSF virus (CSFV) of endemic Cuban genotype 1.4 isolates. The assay reliably detected recent isolates from three different regions of Cuba with an analytical sensitivity 10-100 times lower than that of quantitative reverse transcription RT-qPCR. Diagnostic test sensitivity was examined using reference sera from two groups of pigs experimentally infected with Cuban virulent strain CSF0705 "Margarita" and the recent field isolate CSF1058 "Pinar del Rio". Differences in pathogenicity of the two viruses were reflected in the clinical course of disease as well as in virus loads of blood samples. Low viral RNA loads in samples from pigs infected with the field isolate caused serious detection problems in RT-LAMP as well as in RT-qPCR. Thus, it will be necessary in future research to focus on targeted sampling of diseased animals and to restrict diagnosis to the herd level in order to establish LAMP as an efficient tool for diagnosing CSF under field conditions.

Molecular epidemiology study of swine influenza virus revealing a reassorted virus H1N1 in swine farms in Cuba.

In this report, we describe the emergence of reassorted H1N1 swine influenza virus, originated from a reassortment event between the H1N1 pandemic influenza virus (H1N1p/2009) and endemic swine influenza virus in Cuban swine population. In November 2010, a clinical respiratory outbreak was reported on a pig fattening farm in Cuba. Phylogenetic analysis showed that all the genes of one of the isolate obtained, with the exception of neuraminidase, belonged to the H1N1p/2009 cluster. This finding suggests that H1N1pdm has been established in swine and has become a reservoir of reassortment that may produce new viruses with both animal and public health risks.

A duplex SYBR Green I-based real-time RT-PCR assay for the simultaneous detection and differentiation of Massachusetts and non-Massachusetts serotypes of infectious bronchitis virus.

Infectious bronchitis is a highly contagious viral disease of poultry caused by infectious bronchitis virus (IBV) and is considered one of the most economically important viral diseases of chickens. Control of IBV has been attempted using live attenuated and inactivated vaccines. Live attenuated vaccines of the Massachusetts (Mass.) serotype are the most commonly used for this purpose. Due to the continuous emergence of new variants of the infectious bronchitis virus, the identification of the type of IBV causing an outbreak in commercial poultry is important in the selection of the appropriate vaccine(s) capable of inducing a protective immune response. The present work was aimed at developing and evaluating a duplex SYBR Green I-based real-time RT-PCR (rRT-PCR) assay for the simultaneous detection and differentiation of Mass. and non-Mass. serotypes of IBV. The duplex rRT-PCR yielded curves of amplification with two specific melting curves (Tm1 = 83 °C ± 0.5 °C and Tm2 = 87 °C ± 0.5 °C) and only one specific melting peak (Tm = 87 °C ± 0.5 °C) when the IBV Mass. serotype and IBV non-Mass. serotype strains were evaluated, respectively. The detection limit of the assay was 8.2 gene copies/µL based on in vitro transcribed RNA and 0.1 EID50/mL. The assay was able to detect all the IBV strains assessed and discriminated well among the IBV Mass. and the IBV non-Mass. serotypes strains. In addition, amplification curves were not obtained with any of the other viruses tested. From the 300 field samples tested, the duplex rRT-PCR yielded a total of 80 samples that were positive for IBV (26.67%), 73 samples identified as the IBV Mass. serotype and seven samples as identified as the IBV non-Mass. serotype. A comparison of the performance of test as assessed with field samples revealed that the duplex rRT-PCR detected a higher number of IBV-positive samples than when conventional RT-PCR or virus isolation tests were used. The duplex rRT-PCR presented here is a useful tool for the rapid identification of outbreaks and for surveillance programmes during IB-suspected cases, particularly in countries with a vaccination control programme.