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.

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.

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.

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.

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.

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.

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.

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).

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.

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.

Adverse Outcomes in Obese Cardiac Surgery Patients Correlates With Altered Branched-Chain Amino Acid Catabolism in Adipose Tissue and Heart.

Background: Predicting relapses of post-operative complications in obese patients who undergo cardiac surgery is significantly complicated by persistent metabolic maladaptation associated with obesity. Despite studies supporting the linkages of increased systemic branched-chain amino acids (BCAAs) driving the pathogenesis of obesity, metabolome wide studies have either supported or challenged association of circulating BCAAs with cardiovascular diseases (CVDs). Objective: We interrogated whether BCAA catabolic changes precipitated by obesity in the heart and adipose tissue can be reliable prognosticators of adverse outcomes following cardiac surgery. Our study specifically clarified the correlation between BCAA catabolizing enzymes, cellular BCAAs and branched-chain keto acids (BCKAs) with the severity of cardiometabolic outcomes in obese patients pre and post cardiac surgery. Methods: Male and female patients of ages between 44 and 75 were stratified across different body mass index (BMI) (non-obese = 17, pre-obese = 19, obese class I = 14, class II = 17, class III = 12) and blood, atrial appendage (AA), and subcutaneous adipose tissue (SAT) collected during cardiac surgery. Plasma and intracellular BCAAs and BC ketoacids (BCKAs), tissue mRNA and protein expression and activity of BCAA catabolizing enzymes were assessed and correlated with clinical parameters. Results: Intramyocellular, but not systemic, BCAAs increased with BMI in cardiac surgery patients. In SAT, from class III obese patients, mRNA and protein expression of BCAA catabolic enzymes and BCKA dehydrogenase (BCKDH) enzyme activity was decreased. Within AA, a concomitant increase in mRNA levels of BCAA metabolizing enzymes was observed, independent of changes in BCKDH protein expression or activity. BMI, indices of tissue dysfunction and duration of hospital stay following surgery correlated with BCAA metabolizing enzyme expression and metabolite levels in AA and SAT. Conclusion: This study proposes that in a setting of obesity, dysregulated BCAA catabolism could be an effective surrogate to determine cardiac surgery outcomes and plausibly predict premature re-hospitalization.

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.

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.

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.

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.