Safety and Efficacy upon Infection in Sheep with Rift Valley Fever Virus ZH548-rA2, a Triple Mutant Rescued Virus.

The introduction of three single nucleotide mutations into the genome of the virulent RVFV ZH548 strain allows for the rescue of a fully attenuated virus in mice (ZH548-rA2). These mutations are located in the viral genes encoding the RdRp and the non-structural protein NSs. This paper shows the results obtained after the subcutaneous inoculation of ZH548-rA2 in adult sheep and the subsequent challenge with the parental virus (ZH548-rC1). Inoculation with the ZH548-rA2 virus caused no detectable clinical or pathological effect in sheep, whereas inoculation of the parental rC1 virus caused lesions compatible with viral infection characterised by the presence of scattered hepatic necrosis. Viral infection was confirmed via immunohistochemistry, with hepatocytes within the necrotic foci appearing as the main cells immunolabelled against viral antigen. Furthermore, the inoculation of sheep with the rA2 virus prevented the liver damage expected after rC1 virus inoculation, suggesting a protective efficacy in sheep which correlated with the induction of both humoral and cell-mediated immune responses.

Genetic diversification patterns in swine influenza A virus (H1N2) in vaccinated and nonvaccinated animals.

Influenza A viruses (IAVs) are characterized by having a segmented genome, low proofreading polymerases, and a wide host range. Consequently, IAVs are constantly evolving in nature causing a threat to animal and human health. In 2009 a new human pandemic IAV strain arose in Mexico because of a reassortment between two strains previously circulating in pigs; Eurasian "avian-like" (EA) swine H1N1 and "human-like" H1N2, highlighting the importance of swine as adaptation host of avian to human IAVs. Nowadays, although of limited use, a trivalent vaccine, which include in its formulation H1N1, H3N2, and, H1N2 swine IAV (SIAV) subtypes, is one of the most applied strategies to reduce SIAV circulation in farms. Protection provided by vaccines is not complete, allowing virus circulation, potentially favoring viral evolution. The evolutionary dynamics of SIAV quasispecies were studied in samples collected at different times from 8 vaccinated and 8 nonvaccinated pigs, challenged with H1N2 SIAV. In total, 32 SIAV genomes were sequenced by next-generation sequencing, and subsequent variant-calling genomic analysis was carried out. Herein, a total of 364 de novo single nucleotide variants (SNV) were found along all genetic segments in both experimental groups. The nonsynonymous substitutions proportion found was greater in vaccinated animals suggesting that H1N2 SIAV was under positive selection in this scenario. The impact of each substitution with an allele frequency greater than 5% was hypothesized according to previous literature, particularly in the surface glycoproteins hemagglutinin and neuraminidase. The H1N2 SIAV quasispecies evolution capacity was evidenced, observing different evolutionary trends in vaccinated and nonvaccinated animals.

Lactate Dehydrogenase and Hemorrhagic Stroke During Extracorporeal Membrane Oxygenation for COVID-19.

PURPOSE: Hemorrhagic stroke (HS) is a devastating complication during extracorporeal membrane oxygenation (ECMO) but markers of risk stratification during COVID-19 are unknown. Lactate dehydrogenase (LDH) is a readily available biomarker of cell injury and permeability. We sought to determine whether an elevated LDH before ECMO placement is related to the occurrence of HS during ECMO for COVID-19. METHODS: Adult patients with COVID-19 requiring ECMO between March 2020 and February 2022 were included. LDH values prior to ECMO placement were captured. Patients were categorized into high (> 750 U/L) or low (≤ 750 U/L) LDH groups. Multivariable regression modeling was used to determine the association between LDH and HS during ECMO. RESULTS: There were 520 patients that underwent ECMO placement in 17 centers and 384 had an available LDH. Of whom, 122 (32%) had a high LDH. The overall incidence of HS was 10.9%, and patients with high LDH had a higher incidence of HS than those with low LDH level (17% vs 8%, p = 0.007). At 100 days, the probability of a HS was 40% in the high LDH group and 23% in those with a low LDH, p = 0.002. After adjustment for clinical covariates, high LDH remained associated with subsequent HS (aHR: 2.64, 95% CI 1.39-4.92). Findings were similar when restricting to patients supported by venovenous ECMO only. CONCLUSION: Elevated LDH prior to ECMO cannulation is associated with a higher incidence of HS during device support. LDH can risk stratify cases for impending cerebral bleeding during ECMO.

Outcomes With Peripheral Venoarterial Extracorporeal Membrane Oxygenation for Suspected Acute Myocarditis: 10-Year Experience From the Extracorporeal Life Support Organization Registry.

BACKGROUND: Acute myocarditis can result in severe hemodynamic compromise requiring venoarterial extracorporeal membrane oxygenation (VA-ECMO). Outcomes and factors associated with mortality among myocarditis patients are not well described in the modern ECMO era. METHODS: We queried the Extracorporeal Life Support Organization registry from 2011 to 2020 for adults with suspected acute myocarditis undergoing peripheral VA-ECMO support. The primary outcome was in-hospital mortality and was compared to all-comers receiving VA-ECMO in the registry over the same period. Secondary outcomes were rates of bridging to advanced therapies and ECMO complications. We used multivariable logistic regression to examine factors associated with in-hospital mortality. RESULTS: Among 850 patients with suspected acute myocarditis receiving peripheral VA-ECMO, the mean age was 41 years, 52% were men, 39% Asian race, and 14.8% underwent extracorporeal cardiopulmonary resuscitation. During the study period, in-hospital mortality steadily declined and was 58.3% for all all-comers receiving VA-ECMO compared with 34.9% for patients with myocarditis (P<0.001). After multivariable modeling, risk factors for mortality were earlier year of support, older age, higher weight, Asian race, need for extracorporeal cardiopulmonary resuscitation, sepsis, and lower mean arterial pressure and pH prior to ECMO initiation. ECMO complications including bleeding, limb ischemia, infections and ischemic stroke were more common among nonsurvivors and significantly declined during the study period. CONCLUSIONS: Compared with all-comers supported with VA-ECMO, in-hospital mortality for patients with acute myocarditis is significantly lower, with nearly two-thirds of patients surviving to discharge. Major modifiable risk factors for mortality were ongoing cardiopulmonary resuscitation requiring ECMO and markers of illness severity prior to ECMO.

Pulmonary Protection from Left Ventricular Distension During Venoarterial Extracorporeal Membrane Oxygenation: Review and Management Algorithm.

The use of venoarterial extracorporeal membrane oxygenation (VA-ECMO) in adults for refractory cardiogenic shock has risen exponentially during the prior decade. Although VA-ECMO provides cardiopulmonary support, it can alter left ventricular (LV) loading conditions leading to LV distension, which makes the lungs susceptible to congestion and promotes intracardiac thrombosis. These conditions can be alleviated by pharmacologic and mechanical unloading, but gaps in knowledge remain on optimal timing and methods of this approach. This review provides an overview of the epidemiology of VA-ECMO, describes pathophysiology and methods for monitoring and reducing LV loading and summarizes contemporary studies examining the association between LV unloading and adverse events. We offer a simple protocol for implementing LV unloading during VA-ECMO to provide pulmonary protection and improve outcomes.

Vaccination against swine influenza in pigs causes different drift evolutionary patterns upon swine influenza virus experimental infection and reduces the likelihood of genomic reassortments.

Influenza A viruses (IAVs) can infect a wide variety of bird and mammal species. Their genome is characterized by 8 RNA single stranded segments. The low proofreading activity of their polymerases and the genomic reassortment between different IAVs subtypes allow them to continuously evolve, constituting a constant threat to human and animal health. In 2009, a pandemic of an IAV highlighted the importance of the swine host in IAVs adaptation between humans and birds. The swine population and the incidence of swine IAV is constantly growing. In previous studies, despite vaccination, swine IAV growth and evolution were proven in vaccinated and challenged animals. However, how vaccination can drive the evolutionary dynamics of swine IAV after coinfection with two subtypes is poorly studied. In the present study, vaccinated and nonvaccinated pigs were challenged by direct contact with H1N1 and H3N2 independent swine IAVs seeder pigs. Nasal swab samples were daily recovered and broncho-alveolar lavage fluid (BALF) was also collected at necropsy day from each pig for swine IAV detection and whole genome sequencing. In total, 39 swine IAV whole genome sequences were obtained by next generation sequencing from samples collected from both experimental groups. Subsequently, genomic, and evolutionary analyses were carried out to detect both, genomic reassortments and single nucleotide variants (SNV). Regarding the segments found per sample, the simultaneous presence of segments from both subtypes was much lower in vaccinated animals, indicating that the vaccine reduced the likelihood of genomic reassortment events. In relation to swine IAV intra-host diversity, a total of 239 and 74 SNV were detected within H1N1 and H3N2 subtypes, respectively. Different proportions of synonymous and nonsynonymous substitutions were found, indicating that vaccine may be influencing the main mechanism that shape swine IAV evolution, detecting natural, neutral, and purifying selection in the different analyzed scenarios. SNV were detected along the whole swine IAV genome with important nonsynonymous substitutions on polymerases, surface glycoproteins and nonstructural proteins, which may have an impact on virus replication, immune system escaping and virulence of virus, respectively. The present study further emphasized the vast evolutionary capacity of swine IAV, under natural infection and vaccination pressure scenarios.

Clinical, Pathological and Virological Outcomes of Tissue-Homogenate-Derived and Cell-Adapted Strains of Porcine Epidemic Diarrhea Virus (PEDV) in a Neonatal Pig Model.

Porcine epidemic diarrhea virus (PEDV) is characterized by diarrhea, vomiting, dehydration, and high mortality rates in neonatal piglets. Two distinct genogroups, S-INDEL (G1a, G1b) and non-S INDEL (G2a, G2b, and G2c), circulate worldwide and are characterized by varying degrees of virulence. Here, we compared the early pathogenesis of a PEDV S-INDEL strain obtained from intestine homogenate (CALAF-HOMOG) or adapted to cell culture by 22 passages (CALAF-ADAP) and a virulent non-S INDEL strain (PEDV-USA) in newborn piglets. After orogastric inoculation of PEDV strains, body weight, temperature and clinical signs were monitored for 48 hpi. Pathological studies were performed at 48 hpi and RNA extracts from jejunal content (at 48 hpi) and rectal swabs (at 0 and 48 hpi) were tested for the presence of PEDV RNA as well as sequenced and compared to the inoculum. Piglets inoculated with PEDV-USA and CALAF-HOMOG isolates showed more severe weight loss, diarrhea, villi fusion and atrophy compared to CALAF-ADAP inoculated piglets. The viral load of rectal swabs was higher in the PEDV-USA inoculated group, followed by CALAF-HOMOG and CALAF-ADAP isolates. Similarly, viral RNA load in jejunal content was comparable among PEDV-USA and CALAF-HOMOG inoculated piglets and higher than that of CALAF-ADAP ones. The comparison of three full PEDV sequences of the inocula with the corresponding ones of pigs after 48 hpi yielded a nucleotide identity >99.9%. This study highlights variations in virulence among S-INDEL and non-S INDEL strains and between S-INDEL isolates obtained from homogenate and cell culture.

Evolution of Swine Influenza Virus H3N2 in Vaccinated and Nonvaccinated Pigs after Previous Natural H1N1 Infection.

Swine influenza viruses (SIV) produce a highly contagious and worldwide distributed disease that can cause important economic losses to the pig industry. Currently, this virus is endemic in farms and, although used limitedly, trivalent vaccine application is the most extended strategy to control SIV. The presence of pre-existing immunity against SIV may modulate the evolutionary dynamic of this virus. To better understand these dynamics, the viral variants generated in vaccinated and nonvaccinated H3N2 challenged pigs after recovery from a natural A(H1N1) pdm09 infection were determined and analyzed. In total, seventeen whole SIV genomes were determined, 6 from vaccinated, and 10 from nonvaccinated animals and their inoculum, by NGS. Herein, 214 de novo substitutions were found along all SIV segments, 44 of them being nonsynonymous ones with an allele frequency greater than 5%. Nonsynonymous substitutions were not found in NP; meanwhile, many of these were allocated in PB2, PB1, and NS1 proteins. Regarding HA and NA proteins, higher nucleotide diversity, proportionally more nonsynonymous substitutions with an allele frequency greater than 5%, and different domain allocations of mutants, were observed in vaccinated animals, indicating different evolutionary dynamics. This study highlights the rapid adaptability of SIV in different environments.

Mechanical Left Ventricular Unloading in Patients Undergoing Venoarterial Extracorporeal Membrane Oxygenation.

BACKGROUND: Venoarterial extracorporeal membrane oxygenation (VA-ECMO) increases left ventricular (LV) afterload, potentially provoking LV distention and impairing recovery. LV mechanical unloading (MU) with intra-aortic balloon pump (IABP) or percutaneous ventricular assist device (pVAD) can prevent LV distension, potentially at the risk of more complications, and net clinical benefit remains uncertain. OBJECTIVES: This study aims to determine the association between MU and outcomes for patients undergoing VA-ECMO. METHODS: The authors queried the Extracorporeal Life Support Organization registry for adults receiving peripheral VA-ECMO from 2010 to 2019 and stratified them by MU with IABP or pVAD. The primary outcome was in-hospital mortality; secondary outcomes included on-support mortality and complications during VA-ECMO. RESULTS: Among 12,734 VA-ECMO patients, 3,399 (26.7%) received MU: 2,782 (82.9%) IABP and 580 (17.1%) pVAD. MU patients were older (age 56.3 vs 52.7 years) and, before extracorporeal membrane oxygenation, more often required >2 vasopressors (41.7% vs 27.2%) and had respiratory (21.1% vs 15.9%), renal (24.6% vs 15.8%), and liver failure (4.4% vs 3.1%) (all P < 0.001). MU patients had lower in-hospital mortality (56.6% vs 59.3%, P = 0.006), which persisted in multivariable modeling (adjusted OR [aOR]: 0.84; 95% CI: 0.77-0.92; P < 0.001). MU was associated with more cannula site bleeding (aOR: 1.25; 95% CI: 1.11-1.40; P < 0.001) and hemolysis (aOR: 1.27; 95% CI: 1.03-1.57; P = 0.02). Compared to pVAD, MU patients with IABP had similar mortality (aOR: 0.80; 95% CI: 0.64-1.01; P = 0.06) and less medical bleeding (aOR: 0.45; 95% CI: 0.31-0.64; P < 0.001), cannula site bleeding (aOR: 0.72; 95% CI: 0.54-0.96; P = 0.03), and renal injury (aOR: 0.78; 95% CI: 0.62-0.98; P = 0.03). CONCLUSIONS: Among adults receiving VA-ECMO, MU was associated with lower in-hospital mortality despite increased complications including hemolysis and cannulation site bleeding. Compared to pVAD, MU with IABP was associated with similar mortality and lower complication rates.

Outcomes With Phosphodiesterase-5 Inhibitor Use After Left Ventricular Assist Device: An STS-INTERMACS Analysis.

BACKGROUND: Elevated right ventricular afterload following continuous-flow left ventricular assist device (CF-LVAD) may contribute to late right heart failure (LRHF). PDE5i (phosphodiesterase-5 inhibitors) are used to treat pulmonary hypertension and right heart dysfunction after CF-LVAD, but their impact on outcomes is uncertain. METHODS: We queried Interagency Registry for Mechanically Assisted Circulatory Support from 2012 to 2017 for adults receiving a primary CF-LVAD and surviving ≥30 days from index discharge. Patients receiving early PDE5i (ePDE5i) at 1 month were propensity-matched 1:1 with controls. The primary outcome was the cumulative incidence of LRHF, defined using prevailing Interagency Registry for Mechanically Assisted Circulatory Support criteria; secondary outcomes included all-cause mortality and major bleeding. RESULTS: Among 9627 CF-LVAD recipients analyzed, 2463 (25.6%) received ePDE5i and 1600 were propensity-matched 1:1 with controls. Before implant, ePDE5i patients had more severe RV dysfunction (13.1% versus 9.6%) and higher pulmonary vascular resistance (2.8±2.7 versus 2.2±2.4 WU), both P<0.001, but clinical factors were well-balanced after propensity-matching. In the unmatched cohort, ePDE5i patients had a higher 3-year cumulative incidence of LRHF, mortality, and major bleeding, but these differences were attenuated in the propensity-matched cohort: LRHF 40.8% versus 35.7% (hazard ratio, 1.14 [95% CI, 0.99-1.32]; P=0.07); mortality 38.6% versus 35.8% (hazard ratio, 0.99 [95% CI, 0.86-1.15]; P=0.93); major bleeding 51.2% versus 46.0% (hazard ratio, 1.12 [95% CI, 0.99-1.27]; P=0.06). CONCLUSIONS: Compared with propensity-matched controls, adult CF-LVAD patients receiving ePDE5i had similar rates of LRHF, mortality, and major bleeding. While intrinsic patient risk factors likely account for more adverse outcomes with ePDE5i in the unmatched cohort, there is no obvious benefit of ePDE5i in the LVAD population.

Bleeding and thrombotic events in adults supported with venovenous extracorporeal membrane oxygenation: an ELSO registry analysis.

PURPOSE: This study aimed at analyzing the prevalence, mortality association, and risk factors for bleeding and thrombosis events (BTEs) among adults supported with venovenous extracorporeal membrane oxygenation (VV-ECMO). METHODS: We queried the Extracorporeal Life Support Organization registry for adults supported with VV-ECMO from 2010 to 2017. Multivariable logistic regression modeling was used to assess the association between BTEs and in-hospital mortality and the predictors of BTEs. RESULTS: Among 7579 VV-ECMO patients meeting criteria, 40.2% experienced ≥ 1 BTE. Thrombotic events comprised 54.9% of all BTEs and were predominantly ECMO circuit thrombosis. BTE rates decreased significantly over the study period (p < 0.001). The inpatient mortality rate was 34.9%. Bleeding events (1.69 [1.49-1.93]) were more strongly associated with in-hospital mortality than thrombotic events (1.23 [1.08-1.41]) p < 0.01 for both. The BTEs most strongly associated with mortality were ischemic stroke (4.50 [2.55-7.97]) and medical bleeding, including intracranial (5.71 [4.02-8.09]), pulmonary (2.02 [1.54-2.67]), and gastrointestinal (1.54 [1.2-1.98]) hemorrhage, all p < 0.01. Risk factors for bleeding included acute kidney injury and pre-ECMO vasopressor support and for thrombosis were higher weight, multisite cannulation, pre-ECMO arrest, and higher PaCO2 at ECMO initiation. Longer time on ECMO, younger age, higher pH, and earlier year of support were associated with bleeding and thrombosis. CONCLUSIONS: Although decreasing over time, BTEs remain common during VV-ECMO and have a strong, cumulative association with in-hospital mortality. Thrombotic events are more frequent, but bleeding carries a higher risk of inpatient mortality. Differential risk factors for bleeding and thrombotic complications exist, raising the possibility of a tailored approach to VV-ECMO management.

Identification and Characterization of Swine Influenza Virus H1N1 Variants Generated in Vaccinated and Nonvaccinated, Challenged Pigs.

Influenza viruses represent a continuous threat to both animal and human health. The 2009 H1N1 A influenza pandemic highlighted the importance of a swine host in the adaptation of influenza viruses to humans. Nowadays, one of the most extended strategies used to control swine influenza viruses (SIVs) is the trivalent vaccine application, whose formulation contains the most frequently circulating SIV subtypes H1N1, H1N2, and H3N2. These vaccines do not provide full protection against the virus, allowing its replication, evolution, and adaptation. To better understand the main mechanisms that shape viral evolution, here, the SIV intra-host diversity was analyzed in samples collected from both vaccinated and nonvaccinated animals challenged with the H1N1 influenza A virus. Twenty-eight whole SIV genomes were obtained by next-generation sequencing, and differences in nucleotide variants between groups were established. Substitutions were allocated along all influenza genetic segments, while the most relevant nonsynonymous substitutions were allocated in the NS1 protein on samples collected from vaccinated animals, suggesting that SIV is continuously evolving despite vaccine application. Moreover, new viral variants were found in both vaccinated and nonvaccinated pigs, showing relevant substitutions in the HA, NA, and NP proteins, which may increase viral fitness under field conditions.

Hemocompatibility-Related Adverse Events and Survival on Venoarterial Extracorporeal Life Support: An ELSO Registry Analysis.

OBJECTIVES: This study sought to determine the frequency, incidence rates over time, association with mortality, and potential risk factors for hemocompatibility-related adverse events (HRAEs) occurring during venoarterial-extracorporeal life support (VA-ECLS). BACKGROUND: HRAEs are common complications of VA-ECLS. Studies examining relevant clinical predictors and the association of HRAEs with survival are limited by small sample size and single-center setting. METHODS: We queried adult patients supported with VA-ECLS from 2010 to 2017 in the Extracorporeal Life Support Organization database to assess the impact of HRAEs on in-hospital mortality. RESULTS: Among 11,984 adults meeting study inclusion, 8,457 HRAEs occurred; 62.1% were bleeding events. The HRAE rate decreased significantly over the study period (p trend <0.001), but rates of medical bleeding and ischemic stroke remained stable. HRAEs had a cumulative association with mortality in adjusted analysis: 1 event, odds ratio (OR) of 1.43; 2 events, OR of 1.86; ≥3 events, OR of 3.27 (p < 0.001 for all). HRAEs most strongly associated with mortality were medical bleeding, including intracranial (OR: 7.71), pulmonary (OR: 3.08), and gastrointestinal (OR: 1.95) hemorrhage and ischemic stroke (OR: 2.31); p < 0.001 for all. Risk factors included the following: for bleeding: older age, lower pH, and female sex; for thrombosis: younger age, male sex, Asian race, and non-polymethylpentene oxygenator; and for both: time on ECLS, central cannulation, and renal failure. CONCLUSIONS: Although decreasing, HRAEs remain common during VA-ECLS and have a cumulative association with survival. Bleeding events are twice as common as thrombotic events, with a hierarchy of HRAEs influencing survival. Differential risk factors for bleeding and thrombotic complications exist and raise the possibility of a tailored approach to ECLS management.

Detection of Porcine Circovirus 3 in Wildlife Species in Spain.

Porcine circovirus 3 (PCV-3) is the third member of the family Circoviridae, genus Circovirus, able to infect swine. A high prevalence of viral DNA has been recorded in wild boars. Recently, PCV-3 DNA was identified in Italian wild ruminants. Based on these previous results, this study assessed the frequency of PCV-3 DNA detection in free-ranging ruminants and Lagomorpha species in Spain. In addition, the genetic characterization of the PCV-3 PCR-positive samples was performed. A total of 801 serum samples, including red deer (Cervus elaphus, [CE]; n = 108), roe deer (Capreolus capreolus, [CC]; n = 87), Pyrenean chamois (Rupicapra pyrenaica, [RP]; n = 133), Iberian ibex (Capra pyrenaica, [CP]; n = 92), mouflon (Ovis aries, [OA]; n = 91), fallow deer (Dama dama, [DD]; n = 104), European rabbit (Oryctolagus cuniculus, [OC]; n = 101), and European hare (Lepus europaeus, [LE]; n = 85) from Catalonia (northeast Spain) were tested by conventional polymerase chain reaction (PCR) and, when positive, sequenced. Overall, PCV-3 DNA was found in three out of 801 analyzed sera (0.37%) corresponding to one red deer (1/108, 0.9%), one mouflon (1/91, 1.1%), and one fallow deer (1/104, 0.96%). None of the samples collected from Lagomorpha species resulted PCR positive. The partial genome sequences detected in positive samples displayed high identity with some PCV-3 sequences detected in wild boars and domestic pigs (99.7% and 100%, respectively). In conclusion, the present study indicated that free-ranging ruminant and Lagomorpha species are not relevant in the epidemiology of PCV-3 in Spain.

Similar frequency of Porcine circovirus 3 (PCV-3) detection in serum samples of pigs affected by digestive or respiratory disorders and age-matched clinically healthy pigs.

Porcine circovirus 3 (PCV-3) has been identified in pigs affected by different disease conditions, although its pathogenicity remains unclear. The objective of the present study was to assess the frequency of PCV-3 infection in serum samples from animals suffering from post-weaning respiratory or digestive disorders as well as in healthy animals. A total of 315 swine serum samples were analysed for PCV-3 DNA detection by conventional PCR; positive samples were further assayed with a quantitative PCR and partially sequenced. Sera were obtained from 4 week- to 4 month-old pigs clinically diagnosed with respiratory (n = 129) or digestive (n = 126) disorders. Serum samples of age-matched healthy animals (n = 60) served as negative control. Pigs with clinical respiratory signs had a wide variety of pulmonary lesions including suppurative bronchopneumonia, interstitial pneumonia, fibrinous-necrotizing pneumonia and/or pleuritis. Animals with enteric signs displayed histopathological findings like villus atrophy and fusion, catarrhal enteritis and/or catarrhal colitis. Overall, PCV-3 DNA was detected in 19 out of 315 analysed samples (6.0%). Among the diseased animals, PCV-3 was found in 6.2% (8 out of 129) and 5.6% (7 out of 126) of pigs with respiratory and digestive disorders, respectively. The frequency of PCV-3 PCR positive samples among healthy pigs was 6.7% (4 out of 60). No apparent association was observed between PCR positive cases and any type of histopathological lesion. The phylogenetic analysis of the partial genome sequences obtained showed high identity among viruses from the three groups of animals studied. In conclusion, PCV-3 was present in the serum of diseased and healthy pigs to similar percentages, suggesting that this virus does not seem to be causally associated with respiratory or enteric disorders.

African swine fever virus does not express viral microRNAs in experimentally infected pigs.

BACKGROUND: African swine fever virus (ASFV) is the etiological agent of African swine fever (ASF), a re-expanding devastating and highly lethal hemorrhagic viral disease. microRNAs (miRNAs) are a new class of small non-coding RNAs that regulate gene expression post-transcriptionally. The discovery of virus specific miRNAs has increased both in number and importance in the past few years. We have recently described the differential expression of several porcine miRNAs during in vivo infection with attenuated and virulent ASFV strains. Here, we have extended these studies trying to identify the presence of viral miRNAs encoded by ASFV in an in vivo infection in pigs. RESULTS: Sixteen small RNA libraries were analyzed from spleen and submandibular lymph nodes obtained from eight pigs, seven infected with either the virulent E75 ASFV strain or its attenuated counterpart E75CV1, or from pigs surviving E75CV1-infection and challenged with BA71 (heterologous challenge) and one non infected as negative control. Samples were recovered at different times post-infection. Libraries were analyzed by next-generation sequencing. Some viral miRNA candidates were initially identified, which did not correspond to porcine miRNAs. Further structural analyses were carried out in order to confirm if they met the conformational requirements to be considered a viral miRNA. CONCLUSIONS: The analysis of sixteen small RNA libraries prepared from two different tissues obtained from pigs experimentally infected with E75, E75CV1 or with E75CV1 plus BA71, revealed the presence of six potential miRNA sequences but none of them met the requirements to be considered as viral miRNAs. Thus, we can conclude that ASFV does not express miRNAs in vivo, at least under the experimental conditions described here.

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.