Glucolipotoxicity diminishes cardiomyocyte TFEB and inhibits lysosomal autophagy during obesity and diabetes.

Impaired cardiac metabolism in the obese and diabetic heart leads to glucolipotoxicity and ensuing cardiomyopathy. Glucolipotoxicity causes cardiomyocyte injury by increasing energy insufficiency, impairing proteasomal-mediated protein degradation and inducing apoptosis. Proteasome-evading proteins are degraded by autophagy in the lysosome, whose metabolism and function are regulated by master regulator transcription factor EB (TFEB). Limited studies have examined the impact of glucolipotoxicity on intra-lysosomal signaling proteins and their regulators. By utilizing a mouse model of diet-induced obesity, type-1 diabetes (Akita) and ex-vivo model of glucolipotoxicity (H9C2 cells and NRCM, neonatal rat cardiomyocyte), we examined whether glucolipotoxicity negatively targets TFEB and lysosomal proteins to dysregulate autophagy and cause cardiac injury. Despite differential effects of obesity and diabetes on LC3B-II, expression of proteins facilitating autophagosomal clearance such as TFEB, LAMP-2A, Hsc70 and Hsp90 were decreased in the obese and diabetic heart. In-vivo data was recapitulated in H9C2 and NRCM cells, which exhibited impaired autophagic flux and reduced TFEB content when exposed to a glucolipotoxic milieu. Notably, overloading myocytes with a saturated fatty acid (palmitate) but not an unsaturated fatty acid (oleate) depleted cellular TFEB and suppressed autophagy, suggesting a fatty acid specific regulation of TFEB and autophagy in the cardiomyocyte. The effect of glucolipotoxicity to reduce TFEB content was also confirmed in heart tissue from patients with Class-I obesity. Therefore, during glucolipotoxicity, suppression of lysosomal autophagy was associated with reduced lysosomal content, decreased cathepsin-B activity and diminished cellular TFEB content likely rendering myocytes susceptible to cardiac injury.

Evaluation of the capability of the PCV2 genome to encode miRNAs: lack of viral miRNA expression in an experimental infection.

Porcine circovirus type 2 (PCV2) is a ssDNA virus causing PCV2-systemic disease (PCV2-SD), one of the most important diseases in swine. MicroRNAs (miRNAs) are a new class of small non-coding RNAs that regulate gene expression post-transcriptionally. Viral miRNAs have recently been described and the number of viral miRNAs has been increasing in the past few years. In this study, small RNA libraries were constructed from two tissues of subclinically PCV2 infected pigs to explore if PCV2 can encode viral miRNAs. The deep sequencing data revealed that PCV2 does not express miRNAs in an in vivo subclinical infection.

Identification of microRNAs in PCV2 subclinically infected pigs by high throughput sequencing.

Porcine circovirus type 2 (PCV2) is the essential etiological infectious agent of PCV2-systemic disease and has been associated with other swine diseases, all of them collectively known as porcine circovirus diseases. MicroRNAs (miRNAs) are a new class of small non-coding RNAs that regulate gene expression post-transcriptionally. miRNAs play an increasing role in many biological processes. The study of miRNA-mediated host-pathogen interactions has emerged in the last decade due to the important role that miRNAs play in antiviral defense. The objective of this study was to identify the miRNA expression pattern in PCV2 subclinically infected and non-infected pigs. For this purpose an experimental PCV2 infection was carried out and small-RNA libraries were constructed from tonsil and mediastinal lymph node (MLN) of infected and non-infected pigs. High throughput sequencing determined differences in miRNA expression in MLN between infected and non-infected while, in tonsil, a very conserved pattern was observed. In MLN, miRNA 126-3p, miRNA 126-5p, let-7d-3p, mir-129a and mir-let-7b-3p were up-regulated whereas mir-193a-5p, mir-574-5p and mir-34a down-regulated. Prediction of functional analysis showed that these miRNAs can be involved in pathways related to immune system and in processes related to the pathogenesis of PCV2, although functional assays are needed to support these predictions. This is the first study on miRNA gene expression in pigs infected with PCV2 using a high throughput sequencing approach in which several host miRNAs were differentially expressed in response to PCV2 infection.

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.

Intron retention is a mechanism of erythropoietin regulation in brain cell models.

Intron retention is a mechanism of post-transcriptional gene regulation, including genes involved in erythropoiesis. Erythropoietin (EPO) is a hormone without evidence of intracellular vesicle storage that regulates erythropoiesis. We hypothesize that EPO uses intron retention as a mechanism of post-transcriptional regulation in response to hypoxia and ischemia. Cell models of hypoxia and ischemia for kidney, liver, and brain cells were examined for intron retention by real time quantitative PCR. EPO expression increased in most cells except for blood brain barrier and liver cells. The intron retained transcript ratio decreased in brain cells, except for Astrocytes, but showed no change in kidney or liver after 24 h of ischemia. The shift in intron ratio was maintained when using poly (A) enriched cDNA, suggesting that intron retention is not due to immature transcripts. The expression of EPO was elevated at variable time points amongst cell models with the intron ratio also changing over a time course of 2 to 16 h after ischemia. We conclude that intron retention is a mechanism regulating EPO expression in response to ischemia in a tissue specific manner.

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.

Parallel evolution of picobirnaviruses from distinct ancestral origins.

IMPORTANCE: Picobirnaviruses (PBVs) are highly heterogeneous viruses encoding a capsid and RdRp. Detected in a wide variety of animals with and without disease, their association with gastrointestinal and respiratory infections, and consequently their public health importance, has rightly been questioned. Determining the "true" host of Picobirnavirus lies at the center of this debate, as evidence exists for them having both vertebrate and prokaryotic origins. Using integrated and time-stamped phylogenetic approaches, we show they are contemporaneous viruses descending from two different ancestors: avian Reovirus and fungal Partitivirus. The fungal PBV-R2 species emerged with a single segment (RdRp) until it acquired a capsid from vertebrate PBV-R1 and PBV-R3 species. Protein and RNA folding analyses revealed how the former came to resemble the latter over time. Thus, parallel evolution from disparate hosts has driven the adaptation and genetic diversification of the Picobirnaviridae family.

Temporal and coevolutionary analyses reveal the events driving the emergence and circulation of human mamastroviruses.

Characterized by high genetic diversity, broad host range, and resistance to adverse conditions, coupled with recent reports of neurotropic astroviruses circulating in humans, mamastroviruses pose a threat to public health. The current astrovirus classification system based on host source prevents determining whether strains with distinct tropism or virulence are emerging. By using integrated phylogeny, we propose a standardized demarcation of species and genotypes, with reproducible cut-off values that reconcile the pairwise sequence distribution, genetic distances between lineages, and the topological reconstruction of the Mamastrovirus genus. We further define the various links established by co-evolution and resolve the dynamics of transmission chains to identify host-jump events and the sources from which different mamastrovirus species circulating in humans have emerged. We observed that recombination is relatively infrequent and restricted to within genotypes. The well-known "human" astrovirus, defined here as mamastrovirus species 7, has co-speciated with humans, while there have been two additional host-jumps into humans from distinct hosts. Newly defined species 6 genotype 2, linked to severe gastroenteritis in children, resulted from a marmot to human jump taking place ∼200 years ago while species 6 genotype 7 (MastV-Sp6Gt7), linked to neurological disease in immunocompromised patients, jumped from bovines only ∼50 years ago. Through demographic reconstruction, we determined that the latter reached coalescent viral population growth only 20 years ago and is evolving at a much higher evolutionary rate than other genotypes infecting humans. This study constitutes mounting evidence of MastV-Sp6Gt7 active circulation and highlights the need for diagnostics capable of detecting it.

The Principles of SARS-CoV-2 Intervariant Competition Are Exemplified in the Pre-Omicron Era of the Colombian Epidemic.

The first 18 months of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections in Colombia were characterized by three epidemic waves. During the third wave, from March through August 2021, intervariant competition resulted in Mu replacing Alpha and Gamma. We employed Bayesian phylodynamic inference and epidemiological modeling to characterize the variants in the country during this period of competition. Phylogeographic analysis indicated that Mu did not emerge in Colombia but acquired increased fitness there through local transmission and diversification, contributing to its export to North America and Europe. Despite not having the highest transmissibility, Mu's genetic composition and ability to evade preexisting immunity facilitated its domination of the Colombian epidemic landscape. Our results support previous modeling studies demonstrating that both intrinsic factors (transmissibility and genetic diversity) and extrinsic factors (time of introduction and acquired immunity) influence the outcome of intervariant competition. This analysis will help set practical expectations about the inevitable emergences of new variants and their trajectories. IMPORTANCE Before the appearance of the Omicron variant in late 2021, numerous SARS-CoV-2 variants emerged, were established, and declined, often with different outcomes in different geographic areas. In this study, we considered the trajectory of the Mu variant, which only successfully dominated the epidemic landscape of a single country: Colombia. We demonstrate that Mu competed successfully there due to its early and opportune introduction time in late 2020, combined with its ability to evade immunity granted by prior infection or the first generation of vaccines. Mu likely did not effectively spread outside of Colombia because other immune-evading variants, such as Delta, had arrived in those locales and established themselves first. On the other hand, Mu's early spread within Colombia may have prevented the successful establishment of Delta there. Our analysis highlights the geographic heterogeneity of early SARS-CoV-2 variant spread and helps to reframe the expectations for the competition behaviors of future variants.

Purifying selection decreases the potential for Bangui orthobunyavirus outbreaks in humans.

Pathogens carried by insects, such as bunyaviruses, are frequently transmitted into human populations and cause diseases. Knowing which spillover events represent a public health threat remains a challenge. Metagenomic next-generation sequencing (mNGS) can support infectious disease diagnostics by enabling the detection of any pathogen from clinical specimens. mNGS was performed on blood samples to identify potential viral coinfections in human immunodeficiency virus (HIV)-positive individuals from Kinshasa, the Democratic Republic of the Congo (DRC), participating in an HIV diversity cohort study. Time-resolved phylogenetics and molecular assay development assisted in viral characterization. The nearly complete genome of a novel orthobunyavirus related to Nyangole virus, a virus previously identified in neighboring Uganda, was assembled from a hepatitis B virus-positive patient. A quantitative polymerase chain reaction assay was designed and used to screen >2,500 plasma samples from Cameroon, the DRC, and Uganda, failing to identify any additional cases. The recent sequencing of a US Center for Disease Control Arbovirus Reference Collection revealed that this same virus, now named Bangui virus, was first isolated in 1970 from an individual in the Central African Republic. Time-scaled phylogenetic analyses of Bangui with the related Anopheles and Tanga serogroup complexes indicate that this virus emerged nearly 10,000 years ago. Pervasive and episodic models further suggest that this virus is under purifying selection and that only distant common ancestors were subject to positive selection events. This study represents only the second identification of a Bangui virus infection in over 50 years. The presumed rarity of Bangui virus infections in humans can be explained by its constraint to an avian host and insect vector, precluding efficient transmission into the human population. Our results demonstrate that molecular phylogenetic analyses can provide insights into the threat posed by novel or re-emergent viruses identified by mNGS.

An SYBR Green-based real-time RT-PCR assay for the detection of H5 hemagglutinin subtype avian influenza virus.

Increasing diversity among H5 hemagglutinin (HA) subtype avian influenza (AI) viruses has resulted in the need of novel sensitive and specific molecular assays. In this study, an SYBR Green-based real-time reverse transcription-PCR (RRT-PCR) assay was developed for the detection of H5 subtype AI virus. Sequence analysis of the Mexican lineage H5N2 isolates (subgroup B) revealed several mismatches in the primer/hydrolysis probe set reported in the commonly used RRT-PCR assay for the detection of H5 North American lineage. The present assay was designed to circumvent the challenge that these viruses represent for the specific detection of H5 subtype AI viruses. This RRT-PCR assay successfully detected a range of different H5 subtype AI strains from both Eurasian and North American lineages representing different avian H5 HA clades from diverse geographical locations. The sensitivity of the present method was determined by using in vitro-transcribed RNA and 10-fold serial dilutions of titrated AI viruses. High sensitivity levels were obtained, with limits of detection of 10(0) 50% egg infectious dose (EID50)/mL and 4.2 gene copies/µl. The linear ranges of the assay span within 10(6)-10(0) EID50/mL and 10(6)-10(0) gene copies/µl. The results obtained from this method were directly compared with those of the H5 RRT-PCR assay recommended by the OIE. The comparison was performed with 110 tracheal and cloacal swabs from various bird species collected during field and laboratory investigations in Eurasia and Africa in 2006 and 2008 and showed 100% agreement. This assay is recommended as an alternative method, also allowing a 'double check' approach detection, to be use mainly in outbreak scenarios with higher risk of poultry infections by Central American/Caribbean H5 AI viruses.

Oropouche virus as an emerging cause of acute febrile illness in Colombia.

Arbovirus infections are frequent causes of acute febrile illness (AFI) in tropical countries. We conducted health facility-based AFI surveillance at four sites in Colombia (Cucuta, Cali, Villavicencio, Leticia) during 2019-2022. Demographic, clinical and risk factor data were collected from persons with AFI that consented to participate in the study (n = 2,967). Serologic specimens were obtained and tested for multiple pathogens by RT-PCR and rapid test (Antigen/IgM), with 20.7% identified as dengue positive from combined testing. Oropouche virus (OROV) was initially detected in serum by metagenomic next-generation sequencing (mNGS) and virus target capture in a patient from Cúcuta. Three additional infections from Leticia were confirmed by conventional PCR, sequenced, and isolated in tissue culture. Phylogenetic analysis determined there have been at least two independent OROV introductions into Colombia. To assess OROV spread, a RT-qPCR dual-target assay was developed which identified 87/791 (10.9%) viremic cases in AFI specimens from Cali (3/53), Cucuta (3/19), Villavicencio (38/566), and Leticia (43/153). In parallel, an automated anti-nucleocapsid antibody assay detected IgM in 27/503 (5.4%) and IgG in 92/568 (16.2%) patients screened, for which 24/68 (35.3%) of PCR positives had antibodies. Dengue was found primarily in people aged <18 years and linked to several clinical manifestations (weakness, skin rash and petechiae), whereas Oropouche cases were associated with the location, climate phase, and odynophagia symptom. Our results confirm OROV as an emerging pathogen and recommend increased surveillance to determine its burden as a cause of AFI in Colombia.

The early SARS-CoV-2 epidemic in Senegal was driven by the local emergence of B.1.416 and the introduction of B.1.1.420 from Europe.

Molecular surveillance of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is growing in west Africa, especially in the Republic of Senegal. Here, we present a molecular epidemiology study of the early waves of SARS-CoV-2 infections in this country based on Bayesian phylogeographic approaches. Whereas the first wave in mid-2020 was characterized by a significant diversification of lineages and predominance of B.1.416, the second wave in late 2020 was composed primarily of B.1.1.420. Our results indicate that B.1.416 originated in Senegal and was exported mainly to Europe. In contrast, B.1.1.420 was introduced from Italy, gained fitness in Senegal, and then spread worldwide. Since both B.1.416 and B.1.1.420 lineages carry several positive selected mutations in the spike and nucleocapsid genes, each of which may explain their local dominance, their mutation profiles should be carefully monitored. As the pandemic continues to evolve, molecular surveillance in all regions of Africa will play a key role in stemming its spread.

The Novel Genetic Background of Infectious Bursal Disease Virus Strains Emerging from the Action of Positive Selection.

The circulation in Europe of novel reassortant strains of infectious bursal disease virus (IBDV), containing a unique genetic background composition, represents a serious problem for animal health. Since the emergence of this novel IBDV mosaic was first described in Poland, this scenario has become particularly attractive to uncover the evolutionary forces driving the genetic diversity of IBDV populations. This study additionally addressed the phenotypic characterization of these emergent strains, as well as the main features affecting the viral fitness during the competition process of IBDV lineages in the field. Our results showed how different evolutionary mechanisms modulate the genetic diversity of co-existent IBDV lineages, leading to the error catastrophe effect, Muller ratchet effect, or prevalence, depending on their genetic compositions. We also determined that the action of the positive selection pressure, depending on the genomic segment on which it is acting, can drive two main phenotypes for IBDV: immune-escaping strains from the selection on segment A or strains with functional advantages from the selection on segment B. This last group seems to possess an increased fitness landscape in the viral quasispecies composition, presenting better adaptability to dissimilar environmental conditions and likely becoming the dominant population. The reassortant strains also exhibited a lower mortality rate compared with the well-known vvIBDV strains, which can facilitate their spreading.

Understanding the Genetic Diversity of Picobirnavirus: A Classification Update Based on Phylogenetic and Pairwise Sequence Comparison Approaches.

Picobirnaviruses (PBVs) are small, double stranded RNA viruses with an ability to infect a myriad of hosts and possessing a high degree of genetic diversity. PBVs are currently classified into two genogroups based upon classification of a 200 nt sequence of RdRp. We demonstrate here that this phylogenetic marker is saturated, affected by homoplasy, and has high phylogenetic noise, resulting in 34% unsolved topologies. By contrast, full-length RdRp sequences provide reliable topologies that allow ancestralism of members to be correctly inferred. MAFFT alignment and maximum likelihood trees were established as the optimal methods to determine phylogenetic relationships, providing complete resolution of PBV RdRp and capsid taxa, each into three monophyletic groupings. Pairwise distance calculations revealed these lineages represent three species. For RdRp, the application of cutoffs determined by theoretical taxonomic distributions indicates that there are five genotypes in species 1, eight genotypes in species 2, and three genotypes in species 3. Capsids were also divided into three species, but sequences did not segregate into statistically supported subdivisions, indicating that diversity is lower than RdRp. We thus propose the adoption of a new nomenclature to indicate the species of each segment (e.g., PBV-C1R2).

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.

Emergence of a Distinct Picobirnavirus Genotype Circulating in Patients Hospitalized with Acute Respiratory Illness.

Picobirnaviruses (PBV) are found in a wide range of hosts and typically associated with gastrointestinal infections in immunocompromised individuals. Here, a divergent PBV genome was assembled from a patient hospitalized for acute respiratory illness (ARI) in Colombia. The RdRp protein branched with sequences previously reported in patients with ARI from Cambodia and China. Sputa from hospitalized individuals (n = 130) were screened by RT-qPCR which enabled detection and subsequent metagenomic characterization of 25 additional PBV infections circulating in Colombia and the US. Phylogenetic analysis of RdRp highlighted the emergence of two dominant lineages linked to the index case and Asian strains, which together clustered as a distinct genotype. Bayesian inference further established capsid and RdRp sequences as both significantly associated with ARI. Various respiratory-tropic pathogens were detected in PBV+ patients, yet no specific bacteria was common among them and four individuals lacked co-infections, suggesting PBV may not be a prokaryotic virus nor exclusively opportunistic, respectively. Competing models for the origin and transmission of this PBV genotype are presented that attempt to reconcile vectoring by a bacterial host with human pathogenicity. A high prevalence in patients with ARI, an ability to reassort, and demonstrated global spread indicate PBV warrant greater public health concern.

Emergence and expansion of novel pathogenic reassortant strains of infectious bursal disease virus causing acute outbreaks of the disease in Europe.

Infectious bursal disease virus (IBDV) is the aetiological agent of a highly contagious chicken immunodeficiency disorder known as Gumboro disease, which cause severe economic loses to the poultry worldwide. The emergence of very virulent IBDV strains (vvIBDV) during the late 80s resulted in drastic changes to the epidemiology of IBDV with a dramatic increase in the mortality of the animals affected. Molecular studies determined that the emergence of the vvIBDV was a consequence of a genomic reorganization of IBDV known as reassortant event by which the virus combined two emergent genetic background vvIBDV for segment A and vvIBDV for segment B. In the current study, a retrospective analysis was conducted, and samples collected during acute outbreaks of Gumboro disease in Poland during 1992-2015 were submitted to sequencing and further molecular and phylogenetic analyses. The results obtained not only revealed a high genetic diversity for Polish IBDV strains but a new population of IBDV was identified. These novel reassortant strains with a unique genetic background contain the segment A from very virulent strains and segment B from an unidentified source, phylogenetically segregated and classified as 'transitional lineage'. The results obtained also showed the presence of this new lineage in Finland, evidencing the expansion of this new genomic reorganized viral strain in Europe representing an additional threat to the global situation of IBDV.

A Validated Multiplex Real-Time PCR Assay for the Diagnosis of Infectious Leptospira spp.: A Novel Assay for the Detection and Differentiation of Strains From Both Pathogenic Groups I and II.

Leptospirosis is recognized as the most globally widespread reemerging zoonosis and represents a serious threat for both human and animal health. Indeed, leptospirosis is linked to more than 60,000 human deaths per year and to incalculable economic burden as consequence of medical treatment costs and livestock loss. The increasing number of reports from species of pathogenic Leptospira spp. group II causing disease in both humans and animals constitutes an additional concern to the complex epidemiology of this zoonotic agent. Diagnostic methods based on qPCR have improved the diagnosis of Leptospira spp. in terms of cost, time, and reliability, but most of the validated assays fail to detect species from the pathogenic group II. Hence, the current study was aimed to develop and validate a novel multiplex qPCR to enable the specific and selective detection of the whole group of infectious Leptospira spp., including both pathogenic groups I and II and moreover, selectively discriminate between them. To fit the "fitness of purpose" for the specific detection of infectious Leptospira spp. and further discrimination between both pathogenic groups three target regions on the 16S RNA gene were selected. These targets facilitated a broad and selective spectrum for the detection of all infectious Leptospira spp. with the exclusion of all saprophytic groups and the novel clade of environmental Leptospira spp. The analytical sensitivity (ASe) showed by the new assay also enables a wide window of detection for the agent at different stages of infection since the assay was able to efficiently detect at 95% of confidence ∼5 leptospires/reaction. From the evaluation of the analytical specificity (ASp) by in silico and in vitro approaches, it was congruently revealed that the primers and probes selected only recognized the specific targets for which the assay was intended. Bayesian latent class analysis of performance of the new assay on 684 clinical samples showed values of diagnostic sensitivity of 99.8% and diagnostic specificity of 100%. Thus, from the evaluation of the analytical and diagnostic parameters, the new multiplex qPCR assay is a reliable method for the diagnosis of Leptospira spp.

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.