Olgotrelvir as a Single-Agent Treatment of Nonhospitalized Patients with Covid-19.

BACKGROUND: Olgotrelvir is an oral antiviral with dual mechanisms of action targeting severe acute respiratory syndrome coronavirus 2 main protease (i.e., Mpro) and human cathepsin L. It has potential to serve as a single-agent treatment of coronavirus disease 2019 (Covid-19). METHODS: We conducted a phase 3, double-blind, randomized, placebo-controlled trial to evaluate the efficacy and safety of olgotrelvir in 1212 nonhospitalized adult participants with mild to moderate Covid-19, irrespective of risk factors, who were randomly assigned to receive orally either 600 mg of olgotrelvir or placebo twice daily for 5 days. The primary and key secondary end points were time to sustained recovery of a panel of 11 Covid-19-related symptoms and the viral ribonucleic acid (RNA) load. The safety end point was incidence of treatment-emergent adverse events. RESULTS: The baseline characteristics of 1212 participants were similar in the two groups. In the modified intention-to-treat population (567 patients in the placebo group and 558 in the olgotrelvir group), the median time to symptom recovery was 205 hours in the olgotrelvir group versus 264 hours in the placebo group (hazard ratio, 1.29; 95% confidence interval [CI], 1.13 to 1.46; P<0.001). The least squares mean (95% CI) changes of viral RNA load from baseline were -2.20 (-2.59 to -1.81) log10 copies/ml in olgotrelvir-treated participants and -1.40 (-1.79 to -1.01) in participants receiving placebo at day 4. Skin rash (3.3%) and nausea (1.5%) were more frequent in the olgotrelvir group than in the placebo group; there were no treatment-related serious adverse events, and no deaths were reported. CONCLUSIONS: Olgotrelvir as a single-agent treatment significantly improved symptom recovery. Adverse effects were not dose limiting. (Funded by Sorrento Therapeutics, a parent company of ACEA Therapeutics; ClinicalTrials.gov number, NCT05716425.).

Olgotrelvir, a dual inhibitor of SARS-CoV-2 Mpro and cathepsin L, as a standalone antiviral oral intervention candidate for COVID-19.

BACKGROUND: Oral antiviral drugs with improved antiviral potency and safety are needed to address current challenges in clinical practice for treatment of COVID-19, including the risks of rebound, drug-drug interactions, and emerging resistance. METHODS: Olgotrelvir (STI-1558) is designed as a next-generation antiviral targeting the SARS-CoV-2 main protease (Mpro), an essential enzyme for SARS-CoV-2 replication, and human cathepsin L (CTSL), a key enzyme for SARS-CoV-2 entry into host cells. FINDINGS: Olgotrelvir is a highly bioavailable oral prodrug that is converted in plasma to its active form, AC1115. The dual mechanism of action of olgotrelvir and AC1115 was confirmed by enzyme activity inhibition assays and co-crystal structures of AC1115 with SARS-CoV-2 Mpro and human CTSL. AC1115 displayed antiviral activity by inhibiting replication of all tested SARS-CoV-2 variants in cell culture systems. Olgotrelvir also inhibited viral entry into cells using SARS-CoV-2 Spike-mediated pseudotypes by inhibition of host CTSL. In the K18-hACE2 transgenic mouse model of SARS-CoV-2-mediated disease, olgotrelvir significantly reduced the virus load in the lungs, prevented body weight loss, and reduced cytokine release and lung pathologies. Olgotrelvir demonstrated potent activity against the nirmatrelvir-resistant Mpro E166 mutants. Olgotrelvir showed enhanced oral bioavailability in animal models and in humans with significant plasma exposure without ritonavir. In phase I studies (ClinicalTrials.gov: NCT05364840 and NCT05523739), olgotrelvir demonstrated a favorable safety profile and antiviral activity. CONCLUSIONS: Olgotrelvir is an oral inhibitor targeting Mpro and CTSL with high antiviral activity and plasma exposure and is a standalone treatment candidate for COVID-19. FUNDING: Funded by Sorrento Therapeutics.

Influenza A(H5N1) Virus Infections in 2 Free-Ranging Black Bears (Ursus americanus), Quebec, Canada.

Wholly Eurasian highly pathogenic avian influenza H5N1 clade 2.3.4.4b virus was isolated from 2 free-ranging black bears with meningoencephalitis in Quebec, Canada. We found that isolates from both animals had the D701N mutation in the polymerase basic 2 gene, previously known to promote adaptation of H5N1 viruses to mammal hosts.

Characterization of neurotropic HPAI H5N1 viruses with novel genome constellations and mammalian adaptive mutations in free-living mesocarnivores in Canada.

The GsGd lineage (A/goose/Guangdong/1/1996) H5N1 virus was introduced to Canada in 2021/2022 through the Atlantic and East Asia-Australasia/Pacific flyways by migratory birds. This was followed by unprecedented outbreaks affecting domestic and wild birds, with spillover into other animals. Here, we report sporadic cases of H5N1 in 40 free-living mesocarnivore species such as red foxes, striped skunks, and mink in Canada. The clinical presentations of the disease in mesocarnivores were consistent with central nervous system infection. This was supported by the presence of microscopic lesions and the presence of abundant IAV antigen by immunohistochemistry. Some red foxes that survived clinical infection developed anti-H5N1 antibodies. Phylogenetically, the H5N1 viruses from the mesocarnivore species belonged to clade 2.3.4.4b and had four different genome constellation patterns. The first group of viruses had wholly Eurasian (EA) genome segments. The other three groups were reassortant viruses containing genome segments derived from both North American (NAm) and EA influenza A viruses. Almost 17 percent of the H5N1 viruses had mammalian adaptive mutations (E627 K, E627V and D701N) in the polymerase basic protein 2 (PB2) subunit of the RNA polymerase complex. Other mutations that may favour adaptation to mammalian hosts were also present in other internal gene segments. The detection of these critical mutations in a large number of mammals within short duration after virus introduction inevitably highlights the need for continually monitoring and assessing mammalian-origin H5N1 clade 2.3.4.4b viruses for adaptive mutations, which potentially can facilitate virus replication, horizontal transmission and posing pandemic risks for humans.

Phylogenetic Inference of the 2022 Highly Pathogenic H7N3 Avian Influenza Outbreak in Northern Mexico.

The Mexican lineage H7N3 highly pathogenic avian influenza virus (HPAIV) has persisted in Mexican poultry since its first isolation in 2012. To date, the detection of this virus has gradually expanded from the initial one state to 18 states in Mexico. Despite the HPAIV H7N3 outbreak occurring yearly, the transmission pathways have never been studied, disallowing the establishment of effective control measures. We used a phylogenetic approach to unravel the transmission pathways of 2022 H7N3 HPAIVs in the new outbreak areas in Northern Mexico. We present genetic data of H7N3 viruses produced from 18 poultry farms infected in the spring of 2022. Our results indicate that the virus responsible for the current outbreak in Northern Mexico evolved from the Mexican lineage H7N3 HPAIV discovered in 2012. In the current outbreak, we identified five clusters of infection with four noticeably different genetic backgrounds. It is a cluster IV-like virus that was transmitted into one northern state causing an outbreak, then spreading to another neighboring northern state, possibly via a human-mediated mechanical transmission mechanism. The long-distance transmission event highlights the necessity for the more rigorous enforcement of biosafety measures in outbreaks. Additionally, we examined the evolutionary processes shaping the viral genetic and antigenic diversities. It is imperative to enhance active surveillance to include birds, the environment, and humans to detect HPAI in domestic poultry at an earlier point and eliminate it.

A threat from both sides: Multiple introductions of genetically distinct H5 HPAI viruses into Canada via both East Asia-Australasia/Pacific and Atlantic flyways.

From 2016 to 2020, high pathogenicity avian influenza (HPAI) H5 viruses circulated in Asia, Europe, and Africa, causing waves of infections and the deaths of millions of wild and domestic birds and presenting a zoonotic risk. In late 2021, H5N1 HPAI viruses were isolated from poultry in Canada and also retrospectively from a great black-backed gull (Larus marinus), raising concerns that the spread of these viruses to North America was mediated by migratory wild bird populations. In February and April 2022, H5N1 HPAI viruses were isolated from a bald eagle (Haliaeetus leucocephalus) and broiler chickens in British Columbia, Canada. Phylogenetic analysis showed that the virus from bald eagle was genetically related to H5N1 HPAI virus isolated in Hokkaido, Japan, in January 2022. The virus identified from broiler chickens was a reassortant H5N1 HPAI virus with unique constellation genome segments containing PB2 and NP from North American lineage LPAI viruses, and the remaining gene segments were genetically related to the original Newfoundland-like H5N1 HPAI viruses detected in November and December 2021 in Canada. This is the first report of H5 HPAI viruses' introduction to North America from the Pacific and the North Atlantic-linked flyways and highlights the expanding risk of genetically distinct virus introductions from different geographical locations and the potential for local reassortment with both the American lineage LPAI viruses in wild birds and with both Asian-like and European-like H5 HPAI viruses. We also report the presence of some amino acid substitutions across each segment that might contribute to the replicative efficiency of these viruses in mammalian host, evade adaptive immunity, and pose a potential zoonotic risk.

Isolation and Characterization of Novel Reassortant Influenza A(H10N7) Virus in a Harbor Seal, British Columbia, Canada.

We isolated a novel reassortant influenza A(H10N7) virus from a harbor seal in British Columbia, Canada, that died from bronchointerstitial pneumonia. The virus had unique genome constellations involving lineages from North America and Eurasia and polymerase basic 2 segment D701N mutation, associated with adaptation to mammals.

Evolutionary Dynamics of Mexican Lineage H5N2 Avian Influenza Viruses.

We have demonstrated for the first time a comprehensive evolutionary analysis of the Mexican lineage H5N2 avian influenza virus (AIV) using complete genome sequences (n = 189), from its first isolation in 1993 until 2019. Our study showed that the Mexican lineage H5N2 AIV originated from the North American wild bird gene pool viruses around 1990 and is currently circulating in poultry populations of Mexico, the Dominican Republic, and Taiwan. Since the implementation of vaccination in 1995, the highly pathogenic AIV (HPAIV) H5N2 virus was eradicated from Mexican poultry in mid-1995. However, the low pathogenic AIV (LPAIV) H5N2 virus has continued to circulate in domestic poultry populations in Mexico, eventually evolving into five distinct clades. In the current study, we demonstrate that the evolution of Mexican lineage H5N2 AIVs involves gene reassortments and mutations gained over time. The current circulating Mexican lineage H5N2 AIVs are classified as LPAIV based on the amino acid sequences of the hemagglutinin (HA) protein cleavage site motif as well as the results of the intravenous pathogenicity index (IVPI). The immune pressure from vaccinations most likely has played a significant role in the positive selection of antigenic drift mutants within the Mexican H5N2 AIVs. Most of the identified substitutions in these viruses are located on the critical antigenic residues of the HA protein and as a result, might have contributed to vaccine failures. This study highlights and stresses the need for vaccine updates while emphasizing the importance of continued molecular monitoring of the HA protein for its antigenic changes compared to the vaccines used.

Validation of a competitive enzyme-linked immunosorbent assay to improve the serological diagnosis of swine vesicular disease.

Swine vesicular disease (SVD) is an infectious viral disease of pigs. The clinical symptoms of SVD are indistinguishable from other vesicular diseases. In countries free of vesicular diseases, rapid SVD diagnosis and differentiation from other vesicular diseases are essential. In this report, a competitive enzyme-linked immunosorbent assay (cELISA) was developed and validated to improve the current SVD serological diagnosis. In this cELISA, an anti-SVD monoclonal antibody (mAb) captures the recombinant SVD virus-like particle (SVD-VLP) antigen, and 5B7 mAb is used as a competitor to compete with polyclonal antibodies in SVD-positive sera. The cut-off value of the SVD-VLP based cELISA (SVD-VLP cELISA) is ≥ 65% inhibition (%). The determined diagnostic specificity was 99.2%. SVD-VLP cELISA successfully detected SVD antibodies in the sera of SVD-infected animals and produced a diagnostic sensitivity of 100%. A panel of SVD positive sere including outbreak samples (n = 11) and samples (n = 5) from experimentally inoculated pigs, were correctly identified as positive by the SVD-VLP cELISA. In terms of reducing false positives detected by the currently used cELISA (5B7 cELISA), the performance of SVD-VLP cELISA is comparable to the gold standard virus neutralization test.

La maladie vésiculeuse du porc (SVD) est une maladie virale infectieuse des porcs. Les symptômes cliniques de la SVD sont indiscernables des autres maladies vésiculaires. Dans les pays exempts de maladies vésiculaires, un diagnostic rapide de la SVD et une différenciation avec les autres maladies vésiculaires sont essentiels. Dans ce rapport, un test immuno-enzymatique compétitif (cELISA) a été développé et validé pour améliorer le diagnostic sérologique actuel de la SVD. Dans ce cELISA, un anticorps monoclonal anti-SVD (mAb) capture l'antigène recombinant de particules de type virus SVD (SVD-VLP), et le mAb 5B7 est utilisé comme compétiteur pour concurrencer les anticorps polyclonaux dans les sérums positifs pour la SVD. La valeur seuil du cELISA basé sur SVD-VLP (cELISA SVD-VLP) est ≥ 65 % d'inhibition (%). La spécificité diagnostique déterminée était de 99,2 %. SVD-VLP cELISA a détecté avec succès des anticorps SVD dans les sérums d'animaux infectés par SVD et a produit une sensibilité diagnostique de 100 %. Un panel de sérums positifs pour la SVD, comprenant des échantillons d'épidémie (n = 11) et des échantillons (n = 5) de porcs inoculés expérimentalement, a été correctement identifié comme positif par le cELISA SVD-VLP. En termes de réduction des faux positifs détectés par le cELISA actuellement utilisé (5B7 cELISA), les performances du cELISA SVD-VLP sont comparables au test de neutralisation du virus de référence.(Traduit par Docteur Serge Messier).

A Novel Third-generation EGFR Tyrosine Kinase Inhibitor Abivertinib for EGFR T790M-mutant Non-Small Cell Lung Cancer: a Multicenter Phase I/II Study.

PURPOSE: To establish recommended phase II dose (RP2D) in phase I and evaluate safety and efficacy of abivertinib in patients with EGFR Thr790Met point mutation (T790M)-positive(+) non-small cell lung cancer (NSCLC) with disease progression from prior EGFR inhibitors in phase II. PATIENTS AND METHODS: This multicenter, open-label study included 367 adult Chinese patients. Abivertinib at doses of 50 mg twice a day to 350 mg twice a day was evaluated in phase I in continual 28-day cycles, and the RP2D of 300 mg twice a day was used in phase II in continual 21-day cycles. Primary endpoints include RP2D in phase I and objective response rate (ORR) at RP2D in phase II. RESULTS: The RP2D of 300 mg twice a day for abivertinib was established based on pharmacokinetics, efficacy, and safety profiles across doses in phase I. In phase II, 227 patients received RP2D for a median treatment duration of 24.6 weeks (0.43-129). Among 209 response-evaluable patients, confirmed ORR was 52.2% [109/209; 95% confidence interval (CI): 45.2-59.1]. Disease control rate (DCR) was 88.0% (184/209; 95% CI: 82.9-92.1). The median duration of response (DoR) and progression-free survival (PFS) was 8.5 months (95% CI: 6.1-9.2) and 7.5 months (95% CI: 6.0-8.8), respectively. The median overall survival (OS) was 24.9 months [95% CI: 22.4-not reachable (NR)]. All (227/227) patients reported at least 1 adverse event (AE), with 96.9% (220/227) of treatment-related AEs. Treatment-related serious AEs were reported in 13.7% (31/227) of patients. Death was reported in 4.4% (10/227) of patients, and none was deemed as treatment-related. CONCLUSIONS: Abivertinib of 300 mg twice a day demonstrated favorable clinical efficacy with manageable side effects in patients with EGFR T790M+ NSCLC.

Genetic variation and evolution of foot-and-mouth disease virus serotype A in relation to vaccine matching.

Foot-and-mouth disease (FMD) is a severe, highly contagious viral disease that affects a wide variety of domestic and wild cloven-hoofed animals. FMD vaccines can play a vital role in disease control and are very widely used globally each year. However, due to the diversity of FMDV, the choice of FMD vaccine is still a huge challenge. In this study, 45 FMDV/A isolates were phylogenetically categorized into three topotypes: ASIA (n = 31), AFRICA (n = 10), and EURO-SA (n = 4). Three sera collected from vaccinated cattle with FMDV A22/IRQ/24/64, A/IRN/05, and A/ARG/01 were used to evaluate their antigenic relationship (r1) with the field isolates. The IRQ/24/64 serum demonstrated a 39% (17/44) match (r1 ≥ 0.3) to the field isolates, whereas IRN/05 serum and ARG/01serum showed an 18% (8/44) and a 2% (1/44) match (r1 ≥ 0.3) to the field isolates, respectively. The A22/IRQ/24/64 matched with isolates mainly from topotype ASIA, with limited cross-topotype match with isolates from topotypes AFRICA and EURO-SA. However, the A/IRN/05 did not show a cross-topotype match with topotype AFRICA isolates and A/ARG/01 failed to match any isolates from topotypes ASIA and AFRICA. After analyzing the amino acid variation of the known antigenic sites of 45 strains of FMDV/A, it was found that together antigenic sites 1 and 3 contributed about 71% of the amino acid changes to the vaccine evaluated. Based on the capsid sequences, the FMDV/A evolved unequally among topotypes. The topotypes of ASIA and AFRICA evolves faster than that of EURO-SA. The FMDV/A continues to show a high level of genetic diversity driven by a high substitution rate, purifying selection, and positive selection concentrated on antigenic sites or near antigenic sites. The current research shows the challenges of the FMDV/A vaccine selection and emphasizes the importance of continuous monitoring of antigenic evolution for the selection of effective vaccines.

Metabolic disposition of [14 C]-abivertinib, an epidermal growth factor receptor tyrosine kinase inhibitor: Role of glutathione conjugation.

AIMS: To determine the absorption, distribution, metabolism and excretion of abivertinib, a third-generation epidermal growth factor receptor tyrosine kinase inhibitor, in patients with advanced non-small cell lung cancer (NSCLC). METHODS: Seven patients with advanced NSCLC were given a single 200 mg/83 µCi oral suspension of [14 C]-abivertinib. Blood, urine and faeces were collected. Mass balance of radioactivity, the pharmacokinetics of abivertinib, and the total radioactivity were determined. Metabolite profiling and characterisation were performed. RESULTS: The mean recovery was 82.16%, with 2.38 and 79.78% of the radioactive dose excreted in urine and faeces, respectively. The unchanged abivertinib was the major radioactive component detected in plasma within the first 24 hours after dosing, accounting for 59.17% of the total drug-related radioactivity. Abivertinib in urine accounted for only 0.96% of the administered dose, whereas in faeces it accounted for 33.36%. Eight metabolites were detected and characterised in plasma, among which MII-7, a product of cysteine glycine conjugate, was the only circulating metabolite, accounting for approximate 10.6% of the total drug-related exposure. MII-2 (an abivertinib cysteine-glycine adduct) and M7 (a reduced product of abivertinib) were the 2 major metabolites in the excreta, accounting for 20.0 and 12.4%, respectively, of the drug-related radioactivity in faeces. CONCLUSION: Following a single oral administration, the unchanged abivertinib was the predominant drug-related material in plasma, urine and faeces. The drug-related materials were primarily eliminated via the faecal route. Direct glutathione conjugation of abivertinib played a significant role in the metabolic clearance and metabolite exposure of abivertinib.

Development of two rapid lateral flow test strips for detection of foot-and-mouth disease virus SAT 1 and SAT 3.

Foot-and-mouth disease (FMD) is a highly contagious disease that affects cattle, sheep, goats, pigs, and over 70 species of wildlife. FMD continues to be a major economic concern for livestock productivity in many countries. FMDV has seven serotypes O, A, Asia 1, C, and Southern Africa Territories (SAT) 1, 2, and 3. Although SAT 1, and SAT 3 outbreaks are not as common as serotypes O, A, Asia 1, and SAT 2, outbreaks have also been reported. The recent outbreaks of SAT 1 occurred in Cameroon, Zimbabwe, South Africa, and Uganda, while most recent SAT 3 occurred in Namibia in 2019. The development of rapid and easy-to-perform FMDV detection tests is critical to control the outbreak and spread of FMD. The current project has produced monoclonal antibodies (mAb) against FMDV serotypes SAT 1, and SAT 3. Using these mAbs, two lateral flow immunochromatographic (LFI) strip tests for the detection of FMDV SAT 1, and SAT 3 have been developed. SAT 1 strip test detected 14 out of 15 SAT 1 field isolates. The SAT 3 strip test detected all four SAT 3 isolates tested, but the signal is weak for UGA 10/97 and showed no cross-reactivity with other FMDV serotypes. The diagnostic specificities of the SAT 1 and the SAT 3 tests are 100 %, which are higher than double antibody sandwich (DAS) ELISA. The diagnostic sensitivity of the SAT 1 test strip is lower than that of DAS ELISA, while the diagnostic sensitivity of the SAT 3 test strip is similar to that of DAS ELISA. The first reported SAT 1 and SAT 3 strip test combined with the previously developed SAT 2 strip test can be used for quick diagnosis in endemic countries in Africa. Rapid identification of FMDV serotypes is critical for disease control and vaccine selection. Also, these strip tests can be used in the laboratory to quickly screen samples from the field.

Development of A recombinant nucleocapsid based indirect ELISA for the detection of antibodies to avian metapneumovirus subtypes, A, B, and C.

Nucleocapsid (N) protein is the most highly expressed of all avian metapneumovirus (aMPV) viral proteins and stimulates a substantial immune response in infected animals. Codon optimized recombinant N (rec-N) protein from aMPV subtypes A, B, and C were expressed using the baculoviral expression system in Trichoplusia ni (Tni) insect cells. A mixture of purified rec-N antigens from each subtype was used as a coating antigen and was evaluated in indirect ELISA (iELISA) to assess antibody response in serum samples collected from experimentally infected chickens and turkeys with different aMPV subtypes. Also, archived field serum samples that were collected from different poultry submissions were used. Receiver operating characteristic (ROC) analysis was performed using chicken and turkey serum samples that were confirmed by indirect fluorescent antibody (IFA) test for serostatus (positive n = 270, negative n = 610). The ROC analysis showed sensitivity and specificity of 97 % at a cut-off value of 0.25. The rec-N iELISA was compared with a commercial whole virus-based APV kit. The rec-N iELISA showed comparable results in detecting antibody response in aMPV infected chicken sera but was more sensitive in detecting early antibody response in aMPV infected turkey serum samples. Our results further confirm the presence of aMPV antibodies in Canadian domestic poultry populations. The developed aMPV-rec N iELISA offers a safe and valuable alternative to whole virus-based iELISA for serodiagnosis and seroepidemiological surveillance of the disease in domestic poultry.

Phylogenetic Inference of H3N2 Canine Influenza A Outbreak in Ontario, Canada in 2018.

The first Canadian H3N2 canine influenza A outbreak involving an Asian-origin H3N2 canine influenza virus (CIV) began in southwestern Ontario, Canada, in late December 2017. More H3N2 CIV cases were identified in central and eastern Ontario between March and October 2018. Based on epidemiological investigation, 5 clusters were identified (C1, C2, C3a, C3b, and C4); however, the origin of infection has only been revealed for epidemiological cluster C1. Here, we use phylogenetic analyses to unravel the links of virus transmission between the 5 epidemiological clusters and the origin of infection for all epidemiological clusters. Our results demonstrate that the Canadian H3N2 CIV sequences were grouped into four distinct phylogenetic clusters with minimal genetic diversity between these clusters. Large scale phylogenetic analysis of H3N2 CIV from around the globe showed that the Canadian CIVs formed a distinct new clade along with CIVs that have been circulating in the USA since 2017-2018 and in China since 2017. This clade shares a common ancestor of Asian origin. This study concludes that the H3N2 CIV outbreak in Ontario was driven by multiple introductions of South Korean/Chinese-origin H3N2 CIVs over 10 months.

Development and evaluation of swine vesicular disease isotype-specific antibody ELISAs based on recombinant virus-like particles.

Swine vesicular disease (SVD) is a contagious viral disease of pigs. The clinical signs of SVD are indistinguishable from other vesicular diseases, such as senecavirus A infection (SVA) and foot-and-mouth disease (FMD). Rapid and accurate diagnostic tests of SVD are considered essential in countries free of vesicular diseases. Competitive ELISA (cELISA) is the serological test used routinely. However, although cELISA is the standard test for SVD antibody testing, this test produces a small number of false-positive results, which caused problems in international trade. The current project developed a SVD isotype antibody ELISA using recombinant SVD virus-like particles (VLP) and an SVD-specific monoclonal antibody (mAb) to reduce the percentage of false positives. The diagnostic specificities of SVD-VLP isotype ELISAs were 98.7% and 99.6% for IgM and IgG. The SVD isotype ELISAs were SVD-specific, without cross-reactivity to other vesicular diseases. A panel of 16 SVD-positive reference sera was evaluated using the SVD-VLP isotype ELISAs. All sera were correctly identified as positive by the two combined SVD-VLP isotype ELISAs. Comparison of the test results showed a high level of correlation between the SVDV antigen isotype ELISAs and SVD-VLP isotype ELISAs. 303 sera from animals lacking clinical signs and history of SVDV exposure were identified positive using SVD cELISA. These samples were examined using SVD-VLP isotype ELISAs. Of the 303 serum samples, five were positive for IgM, and five of 303 were positive for IgG. Comparable to virus neutralization test results, SVD isotype ELISAs significantly reduced the false-positive samples. Based on above test results, the combined use of cELISA and isotype ELISAs can reduce the number of false-positive samples and the use of time-consuming virus neutralization tests, with benefit for international trade in swine and related products.

Overcoming Resistance to AC0010, a Third Generation of EGFR Inhibitor, by Targeting c-MET and BCL-2.

AC0010 is a pyrrolopyrimidine-based irreversible inhibitor of epidermal growth factor receptor (EGFR), structurally distinct from previously reported pyrimidine-based irreversible EGFR inhibitors such as osimertinib and rociletinib. AC0010 selectively inhibits EGFR T790M mutation in both preclinical and clinical studies. However, AC0010 treatment eventually triggers drug resistance with unknown mechanism. To this end, we established two H1975 NSCLC-derived lines resistant to AC0010 after a series of drug exposure and selection in either nude-mice xenograft tumor (H1975-P) or cell culture (H1975-AVR) settings. Both lines obtained 100-fold resistance to AC0010 as compared to the parental lines. To elucidate underlying mechanism, we performed unbiased RNAseq-based profiling analysis and found that H1975-P cells had c-MET overexpression, whereas H1975-AVR cells had BCL-2 overexpression. AC0010 resistance was partially abrogated by targeting c-MET or BCL-2 using either pharmacological (small molecule inhibitors) and/or genetic (siRNA-based knockdown) approach, respectively. Our study shows that drug resistance to AC0010 can be developed via the different mechanism in a cell context-dependent manner and provides the proof-of-concept evidence for rational drug combinations to overcome resistance for maximal therapeutic efficacy.

Synchronized electromechanical integration recording of cardiomyocytes.

Cardiac issues are always one of major health problems that attract wide attention by the public. It is urgent to explore a preclinical strategy to efficiently prevent the life-threatening arrhythmias by precisely assessing the cardiac excitation-contraction behavior. Conventional label-free asynchronous strategies are difficult to synchronously record and precisely match the excitation and contraction signals in vitro, while label-based strategies generally present pharmacological adverse effects and phototoxicity that significantly interfere the natural excitation and contraction signals. Both types of strategies preclude to exactly understand how cardiac excitation-contraction coupling changes in quantitative and coherent detail when dysfunctions occur. Here, we show a label-free synchronized electromechanical integration detection strategy that can synchronously monitor electrical and mechanical signals of cardiomyocytes over a long period of time by an integrated microelectrode-interdigitated electrode (ME-IDE). ME-IDE can detect subtle changes in electromechanical integration signals induced by drugs that target excitation-contraction coupling. Moreover, electromechanical integration delay is explored to specifically recognize the sodium channel inhibition. Furthermore, biomimetic electronic pacemaker function provides an alternative way to efficiently assess the drug-induced arrhythmia using refractory period of cardiomyocytes.

Genetic and antigenic relationship of foot-and-mouth disease virus serotype O isolates with the vaccine strain O1/BFS.

Foot-and-mouth disease serotype O viruses (FMDV/O) are responsible for the most outbreaks in FMD endemic countries. O1/BFS is one of the recommended FMD/O vaccine strains by World Reference Laboratory for FMD. In the current study, FMDV/O1 BFS vaccine strain and serotype O field isolates (45) were analyzed phylogenetically and antigenically to gain more insight into the genetic and antigenic characteristics of the vaccine strain and field isolates. O1/BFS showed similarity with 89% of the field isolates using a virus neutralization test (VNT). The P1 region encoding the FMDV capsid was sequenced and analysed for 46 strains of FMDV/O. Phylogenetic analysis showed these viruses originated from five continents and covered eight of 11 reported topotypes. Five isolates that demonstrated low antigenic similarities with O1/BFS were analyzed for their antigenic variation at the known neutralizing antigenic sites. Three of the five isolates demonstrated unique amino acid substitutions at various antigenic sites. No unique amino acid substitutions were observed for the other two unmatched isolates. Positively selected residues were identified on the surface of the FMD virus capsid supporting that it is important to continuously monitor field isolates for their antigenic and phenotypic changes. In conclusion, the vaccine strain O1/BFS is likely to confer protection against 89% of the 45 FMDV/O isolates based on VNT. Thus O1/BFS vaccine strain is still suitable for use in global FMD serotype O outbreak control. Combining data from phylogenetic, molecular and antigenic analysis can provide improvements in the process of vaccine selection.