Subtype 1a Research Articles

Genetic characterization of HIV-1 viruses among cases with antiretroviral therapy failure in Suzhou City, China

BackgroundThis retrospective study aimed to characterize the distribution of HIV-1 genotypes and the prevalence of drug resistance mutations in people with antiretroviral treatment (ART) failure in Suzhou City, China.MethodsPol gene of HIV-1 viruses in blood samples of EDTA anticoagulants from 398 patients with failed antiviral treatment was successfully amplified by using an in-house assay. Drug resistance mutations were analyzed by using the Stanford HIV Drug Resistance Database system (https://hivdb.stanford.edu/hivdb/by-mutations/). HIV-1 genotypes were determined by the REGA HIV subtyping tool (version 3.46, https://www.genomedetective.com/app/typingtool/hiv). Near full-length genomes (NFLG) of HIV-1 viruses were obtained by next generation sequencing method.ResultsSequences analysis of the pol gene revealed that CRF 01_AE (57.29%, 228/398) was the dominant subtype circulating in Suzhou City, followed by CRF 07_BC (17.34%, 69/398), subtype B (7.54%, 30/398), CRF 08_BC (6.53%, 26/398), CRF 67_01B (3.02%, 12/398) and CRF55_01B (2.51%, 10/398). The overall prevalence of drug-resistant mutations in cases with ART failure was 64.57% (257/398), including 45.48% (181/398) for nucleotide reverse transcriptase inhibitors (NRTIs) mutations, 63.32% (252/398) for non-nucleoside reverse transcriptase inhibitors (NNRTIs) mutations, and 3.02% (12/398) for protease inhibitors (PIs) mutations. Ten near full-length genomes (NFLG) of HIV-1 viruses were identified, including six recombinants of CRF 01_AE and subtype B, two recombinants of CRF 01_AE, subtype B and subtype C sequences, one recombinant of CRF 01_AE and subtype C and one recombinant of CRF 01_AE, subtype A1 and subtype C.ConclusionsThe high prevalence of drug-resistant HIV-1 viruses was a serious challenge for HIV prevention and treatment of people with HIV infection. Treatment regimens for ART failure patients should be adjusted over time based on the outcome of drug resistance tests. NFLG sequencing facilitates the identification of new recombinants of HIV-1.

Roles of Adenosine Receptor (subtypes A1 and A2A) in Cuprizone-Induced Hippocampal Demyelination.

Hippocampal demyelination in multiple sclerosis (MS) has been linked with cognitive deficits, however, patients could benefit from treatment that induces oligodendroglial cell function and promotes remyelination. We investigated the role of A1 and A2A adenosine receptors (AR) in regulating oligodendrocyte precursor cells (OPCs) and myelinating oligodendrocyte (OL) in the demyelinated hippocampus using the cuprizone model of MS. Spatial learning and memory were assessed in wild type C57BL/6 mice (WT) or C57BL/6 mice with global deletion of A1 (A1AR-/-) or A2A AR (A2AAR-/-) fed standard or cuprizone diet (CD) for four weeks. Histology, immunofluorescence, Western blot and TUNEL assays were performed to evaluate the extent of demyelination and apoptosis in the hippocampus. Deletion of A1 and A2A AR alters spatial learning and memory. In A1AR-/- mice, cuprizone feeding led to severe hippocampal demyelination, A2AAR-/- mice had a significant increase in myelin whereas WT mice had intermediate demyelination. The A1AR-/- CD-fed mice displayed significant astrocytosis and decreased expression of NeuN and MBP, whereas these proteins were increased in the A2AAR-/- CD mice. Furthermore, Olig2 was upregulated in A1AR-/- CD-fed mice compared to WT mice fed the standard diet. TUNEL staining of brain sections revealed a fivefold increase in the hippocampus of A1AR-/- CD-fed mice. Also, WT mice fed CD showed a significant decrease expression of A1 AR. A1 and A2A AR are involved in OPC/OL functions with opposing roles in myelin regulation in the hippocampus. Thus, the neuropathological findings seen in MS may be connected to the depletion of A1 AR.

Thermocycling Stain Challenge and Colour Stability of two Current Zirconium Silicate-filled Indirect Composite Restorative Materials

Aim: This study compares the colour stability of two zirconium silicate-filled indirect composite materials CM and its improved formulation CMD using an in vitro thermocycling stain challenge model with 2 immersion solutions. The rationale of the thermocycling stain challenge model is to replicate the intraoral conditions that the indirect composite materials may be exposed to during a clinical service, water or saliva absorption and cyclical exposure to staining food colourant at varying temperature extremes. Materials and Methods: Ten discs each from shade A1 subtypes of CM and CMD indirect composites (Dentine Body A1B, Incisal 58, and Translucent HVT) were subjected to a thermocycling stain challenge following final polishing, which consisted of static immersion in 37°C of distilled water, followed by 500 thermocycles in 5°C of tea and 55°C of soy sauce and finally a 10-minute cleaning using an ultrasonic device. L*, a* and b* colour coordinates were measured, and the mean colour difference expressed as Delta E (ΔE) at different time points of the experiment were calculated and compared. Results: CM and CMD indirect composites had a significant color difference at all experimental time points (F=37.818, p<0.001). ΔE for CM subtypes and CMD subtypes in descending order, respectively, were Translucent HVT>Incisal 58>Dentin Body A1B and Incisal 58>Translucent HVT>Dentin Body A1B. Conclusion: CMD indirect composite showed improved colour stability compared to CM indirect composite. Clinical Significance: Limiting the usage of translucent shades and polishing according to the manufacturer’s recommendation may improve the overall colour of prostheses made using indirect zirconium silicate-filled composite materials.

High-Level Drug-Resistant Mutations among HIV-1 Subtype A6 and CRF02_AG in Kazakhstan.

HIV incidence in Kazakhstan increased by 73% between 2010 and 2020, with an estimated 35,000 people living with HIV (PLHIV) in 2020. The development of antiretroviral drug resistance is a major threat to effective antiretroviral therapy (ART), yet studies on the prevalence of drug resistance in Kazakhstan are sparse. In this study on the molecular epidemiology of HIV in Kazakhstan, we analyzed 968 partial HIV-1 pol sequences that were collected between 2017 and 2020 from PLHIV across all regions of Kazakhstan, covering almost 3% of PLHIV in 2020. Sequences predominantly represented subtypes A6 (57%) and CRF02_AG (41%), with 32% of sequences exhibiting high-level drug resistance. We further identified distinct drug-resistant mutations (DRMs) in the two subtypes: subtype A6 showed a propensity for DRMs A62V, G190S, K101E, and D67N, while CRF02_AG showed a propensity for K103N and V179E. Codon usage analysis revealed that different mutational pathways for the two subtypes may explain the difference in G190S and V179E frequencies. Phylogenetic analysis highlighted differences in the timing and geographic spread of both subtypes within the country, with A62V-harboring subtype A6 sequences clustering on the phylogeny, indicative of sustained transmission of the mutation. Our findings suggest an HIV epidemic characterized by high levels of drug resistance and differential DRM frequencies between subtypes. This emphasizes the importance of drug resistance monitoring within Kazakhstan, together with DRM and subtype screening at diagnosis, to tailor drug regimens and provide effective, virally suppressive ART.

Transmitted Drug Resistance Against Integrase Strand Transfer Inhibitors in Iranian HIV-Infected Naïve Patients.

Human Immunodeficiency Virus (HIV) has claimed the lives of millions of people during the past decades. While several antiretroviral drugs like Integrase Strand Transfer Inhibitors (INSTIs) have been introduced to control HIV, Transmitted Drug Resistance (TDR) in HIV genome caused failure in treatment. This study aimed to investigate TDR and natural occurring mutations (NOPs) in HIV integrase gene in Iranian HIV patients. In this cross-sectional study, blood samples of 30 HIV-positive patients who had never taken integrase inhibitors were considered for CD4 T cell count, RT real-time PCR, and, Nested PCR. The sequencing results were analyzed by CLC sequence viewer software and Stanford University HIV Drug Resistance Database. In all samples, nine NOPs with a high prevalence were found; however, we did not find any drug resistance mutations, except for a mutation in one sample, which showed a low resistance level. Subtype A1 was dominant in all samples. Based on the findings and compared to our previous study, all patients were sustainable to main integrase inhibitors, including bictegravir, raltegravir, bictegravir, elvitegravir and dolutegravir. It seems the resistant mutation pattern attributed to integrase inhibitors was not diffent among studied patients; hence, the prescription of such inhibitors helps physicians to control HIV infection in Iranian HIV-infected patients.

Age and CD4+ T cell counts are inversely associated with HIV drug resistance mutations in treatment naive female sex workers.

The increasing prevalence of human immunodeficiency virus (HIV) drug resistance mutations (HIVDRM) in untreated seropositive persons has consequences for future treatment options. This is extremely important in key populations such as female sex workers (FSWs), where the prevalence of pretreatment drug resistance (PDR) and associated risk factors are unknown. In this study, we analyzed PDR and associated risk factors in recently diagnosed and treatment-naive FSWs in Nairobi, Kenya. In this cross-sectional study, we used 64 HIV-seropositive plasma samples collected from FSWs between November 2020 and April 2021. To identify HIVDRM, the pol gene was amplified and genotyped using sanger sequencing. The effects of age, tropism, CD4+ T cell count, subtype, and location on HIVDRM counts were examined using Poisson regression. Overall, the prevalence of PDR was 35.9% (95% CI: 24.3-48.9), which was strongly influenced by K103N and M184V mutations, which confer resistance to non-nucleoside reverse transcriptase inhibitors (NNRTIs) and nucleoside reverse transcriptase inhibitors (NRTI), respectively. Subtype A1 was predominant followed by subtype D with a notable increase in inter-subtype recombinants. We found statistically significant evidence that age was inversely related to HIVDRM. A FSW who is 1 year older had 12% less HIVDRM (incidence rate ratios [IRR]: 0.88; 95% CI: 0.82-0.95; P < .001), after adjusting for CD4+ T cell count, subtype, location, and tropism. Similarly, an increase in CD4+ T cell count by 1 unit, was associated with 0.4% fewer HIVDRM (IRR: 0.996; 95% CI: 0.994-0.998; P = .001), while controlling for the other variables. HIV-1 tropism was not associated with HIVDRM counts. In conclusion, our findings show a high prevalence of NNRTIs. Lower CD4+ T cell counts and younger age were significant risk factors that influenced HIVDRM loads. This finding underscores the relevance of targeted interventions and the importance of continuing to focus on FSWs as a way of addressing the HIV epidemic.

#3822 POLYOMAVIRUS INFECTION MONITORING BY QUANTITATIVE PCR IN KIDNEY RECIPIENTS AS AN INSTRUMENT FOR PREVENTING POSTTRANSPLANT COMPLICATIONS

Abstract Background and Aims Polyomaviruses (PyV) are ubiquitous human viral pathogens. BKV and JCV representing this viral family are common causative agents of viral complications among kidney recipients. Viral load higher than 1 × 107 copies/ml in urine (viruria) or 1 × 104 copies/ml in serum (viremia) in posttransplantation period may lead to polyomavirus-associated nephropathy (PVAN), hemorrhagic cystitis (HC) or even kidney transplant failure. The aim of the study was to assess PyV reactivation frequency in patients during 12 months after renal transplantation (RT) and to identify molecular subtypes of BKV and JCV. Method We examined 3207 samples of biological material (serum and urine) of 763 adult (&amp;gt;18 years) patients who underwent renal transplantation (RT) at the State Institution “Minsk Scientific and Practical Center for Surgery, Transplantology and Hematology”, Healthcare institutions “Brest Regional Clinical Hospital”, “Vitebsk Regional Clinical Hospital Belarus”, “Mogilev Regional Clinical Hospital”. These patients were divided into 2 groups: group 1 included 394 patients examined only for BKV infection, group 2 – 356 recipients examined for both BKV and JCV infection. Serum and urine samples for regular monitoring were collected from patients before RT, every 2 weeks first 3 months, then at 6, 9, 12 months after RT. In the case of complication development samples from patients were collected later then 1-year monitoring period. PyV DNA was detected by real-time PCR. Viral DNAs from 17 BKV-positive and 11 JCV-positive patients were molecular typed by partial sequencing of VP1 genome region. Confidence intervals for the proportions were calculated using Wald's method. Results Results showed that BKV detection total frequency in the group 1 was 14.47% [11.32%; 18.3%], almost all patients developed viruria, only 2.54% [1.32%; 4.67%] had viremia. In the group 2 PyV DNA was detected in 46.07% [40.96%; 51.26%] of recipients: 19.10% [15.34%; 23.52%] had BKV infection, 19.94% [16.11%; 24.42%] – JCV, 7.02% [4.76%; 10.2%] – BKV+JCV mixed infection. Frequency of viremia was 6.74% [4.53%; 9.87%] in this group. Maximal BKV viral load levels reached 1.2 × 1012 copies/ml in urine and 5.9 × 107 copies/ml in serum. JCV loads were up to 3 × 109 copies/ml in urine and 1.2 × 108 copies/ml in serum. Then we analyzed frequency of PyV detection before RT and during the first year after RT among the 102 recipients. Results displayed on the Fig. 1 showed that the peak of PyV infection registration and the higher risk for patient had a place on the 1.5-2.5 months after RT. Quantitative monitoring of viral load in posttransplant period was the basis for the correction of the applied immunosuppressive therapy regimens in relation to the recipients with a high viral load (higher than 1 × 107 copies/ml in urine or 1 × 104 copies/ml in serum). The results of molecular typing showed that 17 BKV isolates belonged to subgroups Ib-2 and IVc-2 (12 and 5 isolates, respectively). Within subgroups Ib-2 isolates formed 3 clusters corresponding 3 separate genovariants. JCV isolates belonged to subtype 1A, 1B and 2A (7, 3 and 1 of isolates, respectively). The last one had 99% nucleotide sequence similarity with Greece and South Korea isolates. Conclusion Our data demonstrated an importance of PyV DNA monitoring of kidney recipients in the posttransplant period starting from the first days after RT to predict development of PyV complications as PVAN, HC or others by correcting the immunosuppressive therapy.

#3822 POLYOMAVIRUS INFECTION MONITORING BY QUANTITATIVE PCR IN KIDNEY RECIPIENTS AS AN INSTRUMENT FOR PREVENTING POSTTRANSPLANT COMPLICATIONS

Abstract Background and Aims Polyomaviruses (PyV) are ubiquitous human viral pathogens. BKV and JCV representing this viral family are common causative agents of viral complications among kidney recipients. Viral load higher than 1×107 copies/ml in urine (viruria) or 1×104 copies/ml in serum (viremia) in posttransplantation period may lead to polyomavirus-associated nephropathy (PVAN), hemorrhagic cystitis (HC) or even kidney transplant failure. The aim of the study was to assess PyV reactivation frequency in patients during 12 months after renal transplantation (RT) and to identify molecular subtypes of BKV and JCV. Method We examined 3207 samples of biological material (serum and urine) of 763 adult (&amp;gt;18 years) patients who underwent renal transplantation (RT) at the State Institution "Minsk Scientific and Practical Center for Surgery, Transplantology and Hematology", Healthcare institutions "Brest Regional Clinical Hospital", "Vitebsk Regional Clinical Hospital Belarus", "Mogilev Regional Clinical Hospital". These patients were divided into 2 groups: group 1 included 394 patients examined only for BKV infection, group 2 – 356 recipients examined for both BKV and JCV infection. Serum and urine samples for regular monitoring were collected from patients before RT, every 2 weeks first 3 months, then at 6, 9, 12 months after RT. In the case of complication development samples from patients were collected later then 1-year monitoring period. PyV DNA was detected by real-time PCR. Viral DNAs from 17 BKV-positive and 11 JCV-positive patients were molecular typed by partial sequencing of VP1 genome region. Confidence intervals for the proportions were calculated using Wald's method. Results Results showed that BKV detection total frequency in the group 1 was 14.47% [11.32%; 18.3%], almost all patients developed viruria, only 2.54% [1.32%; 4.67%] had viremia. In the group 2 PyV DNA was detected in 46.07% [40.96%; 51.26%] of recipients: 19.10% [15.34%; 23.52%] had BKV infection, 19.94% [16.11%; 24.42%] – JCV, 7.02% [4.76%; 10.2%] – BKV+JCV mixed infection. Frequency of viremia was 6.74% [4.53%; 9.87%] in this group. Maximal BKV viral load levels reached 1.2×1012 copies/ml in urine and 5.9×107 copies/ml in serum. JCV loads were up to 3×109 copies/ml in urine and 1.2×108 copies/ml in serum. Then we analyzed frequency of PyV detection before RT and during the first year after RT among the 102 recipients. Results displayed on the fig.1 showed that the peak of PyV infection registration and the higher risk for patient had a place on the 1.5-2.5 months after RT. Quantitative monitoring of viral load in posttransplant period was the basis for the correction of the applied immunosuppressive therapy regimens in relation to the recipients with a high viral load (higher than 1×107 copies/ml in urine or 1×104 copies/ml in serum). The results of molecular typing showed that 17 BKV isolates belonged to subgroups Ib-2 and IVc-2 (12 and 5 isolates, respectively). Within subgroups Ib-2 isolates formed 3 clusters corresponding 3 separate genovariants. JCV isolates belonged to subtype 1A, 1B and 2A (7, 3 and 1 of isolates, respectively). The last one had 99% nucleotide sequence similarity with Greece and South Korea isolates. Conclusion Our data demonstrated an importance of PyV DNA monitoring of kidney recipients in the posttransplant period starting from the first days after RT to predict development of PyV complications as PVAN, HC or others by correcting the immunosuppressive therapy.

Growth history of hepatitis C virus among HIV/HCV co-infected patients in Guizhou Province.

Background: The evolutionary and epidemiological history and the regional differences of various hepatitis C virus (HCV) genotypes are complex. Our aim was to better understand the molecular epidemiology and evolutionary dynamics of HCV among HIV/HCV co-infected individuals in Guizhou Province. This information could contribute to improve HCV prevention and control strategies in Guizhou and surrounding provinces. Methods: The HCV RNA was extracted from the serum of HIV/HCV co-infected patients, and reverse transcription/nested PCR was performed to amplify nucleotide sequences of the C-E1 region. Then, the successfully amplified sequences were selected for phylogenetic analysis. The available C-E1 region reference sequences from the surrounding provinces of Guizhou (Guangxi, Yunnan, Hunan, and Sichuan) were retrieved in GenBank, and the evolutionary analysis by Bayesian Markov chain Monte Carlo (MCMC) algorithm was performed using BEAST software to reconstruct a phylogeographic tree in order to explore their migration patterns. Finally, the epidemiological history of HCV in the Guizhou region was retraced by reconstructing Bayesian skyline plots (BSPs) after excluding sequences from surrounding provinces. Results: Among 186 HIV/HCV co-infected patients, the C-E1 region sequence was successfully amplified in 177 cases. Phylogenetic analysis classified these sequences into six subtypes: 1a, 1b, 3a, 3b, 6a, and 6n. Among them, subtype 6a was the most dominant strain (n = 70), followed by 3b (n = 55), 1b (n = 31), 3a (n = 11), 1a (n = 8), and 6n (n = 2). By reconstructing the phylogeographic tree, we estimated that the 6a strain in Guizhou mainly originated from Yunnan and Guangxi, while the 3b strain emerged due to transmission from the IDU network in Yunnan. Subtypes 1b, 3a, 3b, and 6a, as the major subtypes of HCV in HIV/HCV co-infected individuals in Guizhou, emerged and later grew more rapidly than the national average. Notably, BSPs of the currently prevalent HCV predominant strain subtype 6a in Guizhou have shown a rapid population growth since 2004. Although the growth rate slowed down around 2010, this growth has continued to date. Conclusion: Overall, despite the improvement and implementation of a series of HCV prevention and control policies and measures, a delayed growth pattern may indicate a unique history of the spread of 6a in Guizhou. Its trend as the dominant strain in Guizhou in recent years may continue to increase slowly over subsequent years.

Site-Specific Differences in Bone Mineral Density of Proximal Femur Correlate with the Type of Hip Fracture.

The aim of this study was to investigate whether site-specific differences in bone mineral density (BMD) of proximal femur correlate with the type of hip fracture using quantitative computed tomography. Femoral neck (FN) fractures were classified as nondisplaced or displaced subtypes. Intertrochanteric (IT) fractures were classified as A1, A2, or A3. The severe hip fractures were identified as displaced FN fractures or unstable IT fractures (A2 and A3). In total, 404 FN fractures (89 nondisplaced and 317 displaced) and 189 IT fractures (76 A1, 90 A2, and 23 A3) were enrolled. Areal BMD (aBMD) and volumetric BMD (vBMD) were measured in the regions of total hip (TH), trochanter (TR), FN, and IT of the contralateral unfractured femur. IT fractures exhibited lower BMD than FN fractures (all p ≤ 0.01). However, unstable IT fractures had higher BMD compared with stable ones (p < 0.01). After adjusting for covariates, higher BMD in TH and IT were associated with IT A2 (A1 vs. A2: odds ratios (ORs) from 1.47 to 1.69, all p < 0.01). Low bone measurements were risk factors for stable IT fractures (IT A1 vs. FN fracture subtypes: ORs from 0.40 to 0.65, all p < 0.01). There are substantial site-specific differences in BMD between IT fractures A1 and displaced FN fractures. Higher bone density was associated with unstable IT fracture when compared with stable ones. The understanding of biomechanics of various fracture types could help to improve the clinical management of these patients.

Radiovirotherapy at twenty

Radiovirotherapy at twenty

Looking at the Molecular Target of NS5A Inhibitors throughout a Population Highly Affected with Hepatitis C Virus.

Hepatitis C virus (HCV) is associated with liver damage and an increased progression rate to cirrhosis and hepatocellular carcinoma. In Portugal, it is prevalent in vulnerable populations such as injection drug users (IDU). HCV is characterized by a high intra-host variability, and the selecting driving forces could select variants containing resistance-associated substitutions (RAS) that reduce treatment effectiveness. The main goal of this study was to analyze the sequence variation of NS5A in treatment-naïve IDU. The epidemiological and clinical status of hepatitis C were analyzed, and samples were sequenced by Sanger and Next-Generation sequencing (NGS) to assess RAS and confirm HCV subtypes. Phylogenetic classification was concordant: 1a, 52.4%; 1b, 10.7%; 3a, 20.2%; 4a, 8.3%; 4d, 7.1%; and one 2k/1b recombinant. A 1a/3a mixed infection was detected by NGS. RAS were found in 34.5% (29/84) of samples using Sanger sequencing, while in 42.9% (36/84) using NGS. In sequences from subtypes 1a and 1b, RAS K24R, M28V, Q30H/R, H58D/P/Q/R, and RAS L31M and P58S were detected, respectively. In subtype 3a, RAS A30S/T, Y93H and polymorphisms in position 62 were identified. Additionally, RAS P58L was detected in genotype 4. The strategy used for the molecular survey of baseline HCV resistance is of particular importance to achieve treatment effectiveness and contribute to the elimination of hepatitis C.

Brain-Derived Estrogen Regulates Neurogenesis, Learning and Memory with Aging in Female Rats.

Although 17β-estradiol (E2) can be locally synthesized in the brain, whether and how brain-derived E2 (BDE2) impacts neurogenesis with aging is largely unclear. In this study, we examined the hippocampal neural stem cells, neurogenesis, and gliogenesis of 1, 3, 6, 14, and 18-month (Mon) female rats. Female forebrain neuronal aromatase knockout (FBN-ARO-KO) rats and letrozole-treated rats were also employed. We demonstraed that (1) the number of neural stem cells declined over 14-Mon age, and the differentiation of astrocytes and microglia markedly elevated and exhibited excessive activation. KO rats showed declines in astrocyte A2 subtype and elevation in A1 subtype at 18 Mon; (2) neurogenesis sharply dropped from 1-Mon age; (3) KO suppressed dentate gyrus (DG) neurogenesis at 1, 6 and 18 Mon. Additionally, KO and letrozole treatment led to declined neurogenesis at 1-Mon age, compared to age-matched WT controls; (4) FBN-ARO-KO inhibited CREB-BDNF activation, and decreased protein levels of neurofilament, spinophilin and PSD95. Notably, hippocampal-dependent spatial learning and memory was impaired in juvenile (1 Mon) and adulthood (6 Mon) KO rats. Taken together, we demonstrated that BDE2 plays a pivotal role for hippocampal neurogenesis, as well as learning and memory during female aging, especially in juvenile and middle age.

An outbreak of mucosal disease in cattle caused by &lt;i&gt;Pestivirus H&lt;/i&gt;

An outbreak of infection caused by Pestivirus H (virus of bovine viral diarrhea – mucosal disease of the third kind, BVDV-3) in a dairy farm with high morbidity and mortality in animals of different ages is described. In some sick animals a full complex of pronounced symptoms characteristic of "classical" bovine mucosal disease was registered: erosions and ulcers on the nasal mirror and tongue, foaming from the mouth, serous discharge from the nose, hemorrhagic inflammation and pronounced longitudinal erosions on the mucosa of the esophagus, rennet stomach and intestine. Cows miscarried at different stages of pregnancy. The coefficient of effective insemination decreased to 20%. The course of the disease was complicated by the involvement of the bovine herpes virus type 4, bacteria of the family Pasteurellaceae and Clostridium spp. in the infectious process. The BVDV-3 genome was found in a wide range of internal organs of aborted fetuses, calves, and adult animals. According to sequencing data, the pathogen was classified as subtype 3a. Phylogenetic analysis of the 5'-untranslated region of the virus genome (5'-UTR) showed its close relationship to the strains isolated in Italy and Brazil, most of which were previously identified as contaminants of fetal bovine serum and live vaccines against viral infections of cattle. No specific prophylaxis against BVDV-3 infection has been developed at this time, therefore, it is necessary to update and improve diagnostic methods, optimize control measures to prevent the spread of virulent strains of the pathogen, and control the safety of the vaccines used.

Retreatment of patients with chronic hepatitis C, subtype 3a, and cirrhosis, who previously failed a regimen containing NS5A inhibitors with sofosbuvir/velpatasvir plus ribavirin for 24 weeks

Background & Aims The use of direct acting antiviral agents (DAAs) in patients with chronic HCV genotype (GT) 3a infection results in sustained virologic response (SVR) rates of 93–98%, but 5–8% of patients experience virologic failure. Methods. We observed 48 patients infected with HCV subtype 3a who failed previous treatment with DAAs, including 43 subjects (89.6%) with liver cirrhosis. Thirty-seven (77%) subjects previously received NS5A inhibitors of the first generation (daclatasvir) and 11 subjects (23%) – of the second generation (velpatasvir). All patients received retreatment with sofosbuvir (SOF)/ velpatasvir (VEL) and ribavirin (RBV) for 24 weeks. We compared SVR12 rates depending on fibrosis stage, presence of NS5A mutation (L31M or A30K and Y93H), and on the generation of previously used NS5A inhibitors. Results. Observed SVR12 rate were: 83,3% (40/48 patients) overall; 100% in patients without cirrhosis (n=5) vs 81,4% (n=35) in those with cirrhosis (n=43) (P=0,01); 86,7% (n=13) with single L31M or A30K(n=15) vs 77,8% (n=21) with Y93H mutation (n=27) (P=0,46), 86,5% (n=32) in patients who previously failed first generation (n=37) vs 72,7% (n=8) in those who failed second generation NS5A inhibitors (n=11) (P=0,35). Conclusions. Retreatment with SOF/VEL+RBV was highly effective and safe in patients with chronic HCV GT3a infection, including those with liver cirrhosis, who previously failed DAA containing NS5A inhibitors. Presence of NS5A RASs L31M, A30K, and Y93H at the baseline, as well as the generation of previously used NS5A inhibitors did not impact SVR12 rates.

Hyperglycemia Induces Tear Reduction and Dry Eye in Diabetic Mice through the Norepinephrine–α1 Adrenergic Receptor–Mitochondrial Impairment Axis of Lacrimal Gland

Dry eye syndrome is a common complication in diabetic patients with a prevalence of up to 54.3%. However, the pathogenic mechanisms underlying hyperglycemia-induced tear reduction and dry eye remain less understood. In the present study, we found that both norepinephrine (NE) and tyrosine hydroxylase levels were elevated in the lacrimal gland of diabetic mice, accompanied by increased c-FOS+ cells in the superior cervical ganglion. However, the elimination of NE accumulation by surgical and chemical sympathectomy significantly ameliorated the reduction in tear production, suppressed abnormal inflammation of the lacrimal gland, and improved the severity of dry eye symptoms in diabetic mice. Among various adrenoceptors (ARs), the α1 subtype played the predominant role in the regulation of tear production, as treatments of α1AR antagonists improved tear secretion in diabetic mice compared with βAR antagonist propranolol. Moreover, the α1AR antagonist alfuzosin treatment also alleviated functional impairments of the meibomian gland and goblet cells in diabetic mice. Mechanically, the α1AR antagonist rescued the mitochondrial bioenergetic deficit, increased the mitochondrial DNA copy numbers, and elevated the glutathione levels of the diabetic lacrimal gland. Overall, these results deciphered a previously unrecognized involvement of the NE-α1AR-mitochondrial bioenergetics axis in the regulation of tear production in the lacrimal gland, which may provide a potential strategy to counteract diabetic dry eye by interfering with the α1AR activity.

Impact of Direct-Acting Antiviral Therapy on Liver Fibrosis Regression among People with Chronic HCV Infection: Results from a Real-Life Cohort in Patients Who Achieved Sustained Virological Response.

Background and Objectives: The global prevalence of chronic hepatitis C virus (HCV) infection is 0.8%, affecting around 58 million people worldwide. Treatment with DAAs reduces all-cause HCV mortality by 49-68%. This work aims to determine whether there is liver fibrosis regression (LFR) in patients who achieved Sustained Virological Response (SVR) after treatment with DAAs. Materials and Methods: An analytical, observational, single-center, and cohort study was carried out. The final sample consisted of 248 HCV-infected patients. All started treatment with DAAs between January 2015 and December 2017. Five measurements were performed to determine the fibrotic stage in patients (measured in kilopascals (kPa)) using transient elastography (FibroScan®, Echosens, The Netherlands). Results: Taking the baseline fibrotic stage as a reference, the distribution in subgroups was as follows: 77 F4 patients (31.0%); 55 F3 patients (22.2%); 53 F2 patients (21.4%); and 63 F0/F1 patients (25.4%). There were 40 patients (16.1%) with at least one HCV complication and 13 (5.2%) who developed hepatocellular carcinoma. The overall LFR rate was 77.8% (144 of 185 F2/F3/F4 patients, p = 0.01) at the end of the follow-up period. The highest mean FibroScan® values were observed in patients with: "male gender"; "metabolic syndrome"; "subtype 1a"; "NRP DAA"; "at least one HCV complication"; "death from HCV complications"; and "liver transplantation requirement". Conclusions: Treatment with DAAs achieved high rates of LFR and a decrease in mean FibroScan® values in all subgroups.

Hydrogen sulfide inhibits lipopolysaccharide-based neuroinflammation-induced astrocyte polarization after cerebral ischemia/reperfusion injury

The effect of lipopolysaccharide (LPS)-based neuroinflammation following cerebral ischemia/reperfusion (I/R) on the genotypic transformation of reactive astrocytes and its relationship with endogenous hydrogen sulfide (H2S) were investigated in present study. We found that LPS promoted the cerebral I/R-induced A1 astrocytes proliferation in mouse hippocampal tissues and deteriorated the reduction of hydrogen sulfide (H2S) content in mouse sera, H2S donor NaHS could inhibit A1 astrocytes proliferation. Similarly, knockout of cystathionine γ-lyase (CSE), one of endogenous H2S synthases, likewise up-regulated the cerebral I/R-induced A1 astrocytes proliferation, which could also be blocked by NaHS. Besides, supplement with H2S promoted the A2 astrocytes proliferation in hippocampal tissues of CSE knockout (CSE KO) mice or LPS-treated mice following cerebral I/R. In the oxygen glucose deprivation/reoxygenation (OGD/R) model of astrocytes, H2S also promoted the transformation of astrocytes into A2 subtype. Moreover, we found that H2S could up-regulate the expression of α-subunit of large-conductance Ca2+-activated K+ (BKCa) channels in astrocytes, and the channel opener BMS-191011 likewise promoted the transformation of astrocyte into A2 subtype. In conclusion, H2S inhibits the proliferation of A1 astrocytes induced by LPS-based neuroinflammation following cerebral I/R and promotes the transformation of astrocytes into A2 subtype, which may be related to up-regulation of BKCa channels.

SARS-CoV-2 spike ectodomain targets α7 nicotinic acetylcholine receptors

Virus entry into animal cells is initiated by attachment to target macromolecules located on host cells. The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) trimeric spike glycoprotein targets host angiotensin converting enzyme 2 to gain cellular access. The SARS-CoV-2 glycoprotein contains a neurotoxin-like region that has sequence similarities to the rabies virus and the HIV glycoproteins, as well as to snake neurotoxins, which interact with nicotinic acetylcholine receptor (nAChR) subtypes via this region. Using a peptide of the neurotoxin-like region of SARS-CoV-2 (SARS-CoV-2 glycoprotein peptide [SCoV2P]), we identified that this area moderately inhibits α3β2, α3β4, and α4β2 subtypes, while potentiating and inhibiting α7 nAChRs. These nAChR subtypes are found in target tissues including the nose, lung, central nervous system, and immune cells. Importantly, SCoV2P potentiates and inhibits ACh-induced α7 nAChR responses by an allosteric mechanism, with nicotine enhancing these effects. Live-cell confocal microscopy was used to confirm that SCoV2P interacts with α7 nAChRs in transfected neuronal-like N2a and human embryonic kidney 293cells. The SARS-CoV-2 ectodomain functionally potentiates and inhibits the α7 subtype with nanomolar potency. Our functional findings identify that the α7 nAChR is a target for the SARS-CoV-2 glycoprotein, providing a new aspect to our understanding of SARS-CoV-2 and host cell interactions, in addition to disease pathogenesis.

Abstract 5308: Analysis of extracellular vesicle surface markers as small cell lung cancer biomarkers

Abstract Small Cell Lung Cancer (SCLC) is an aggressive tumor type, usually metastatic at diagnosis, with a median survival of less than a year. SCLC tumors are heterogeneous and are composed of neuroendocrine (A, A2, N) and non-neuroendocrine (Y) subtypes, with the cooperation by heterogeneous cells being key to drive tumor survival. Unfortunately, the current SCLC treatment approach does not take tumor heterogeneity into consideration. Importantly, there are no consensus markers that can identify SCLC subtypes or early tumors. Extracellular vesicles (EVs), small vesicles known to promote cell to cell communication, have been investigated as non-invasive biomarkers for cancer diagnostics, staging, and treatment monitoring. EVs are attractive candidates for biomarker research due to the selective packaging of RNA, DNA, and proteins that are then secreted in the blood. Therefore, our aim was to identify new biomarkers based on EV surface proteins with potential for early detection and tumor characterization. We used differential ultracentrifugation to isolate EVs from four SCLC subtypes A, A2, N, and Y, which were submitted to label-free mass spectrometry (EV-proteomics). We identified a total of 3011 proteins, with the two most distinct subtypes being A2 and N, with 786 differentially expressed EV-proteins. Meanwhile, the 2 most similar subtypes were N and A, with only 75 differentially expressed EV-proteins. Gene set enrichment analysis of EVs derived from the A2 subtype showed an association with pathways in cancer, chemokine signaling, and neuronal systems. In addition, PANTHER analysis identified enrichment in nicotine pharmacodynamic pathways on these EVs, which corroborate the association of A2 cell lines with drug resistance. EVs derived from the N and A subtypes were enriched in spliceosome, RNA processing, and metabolism, while EVs from Y cell lines were enriched in extracellular matrix organization and metabolic pathways, similar to pathways from cell line RNAseq data. 13 proteins were differentially expressed in all group comparisons, including proteins involved in PI3K and nicotinic acetylcholine receptor signaling pathways. Further comparison of EV-proteomics with top genes from CIBERSORT analysis using cell line RNAseq data showed an overlap of 9 surface proteins, with 3 possible SCLC pan markers. In conclusion, EV-proteins isolated from SCLC cell lines presented a unique signature for each subtype, especially EVs derived from A2 subtype, which has a signature associated with drug resistance. We identified surface proteins on SCLC EVs with potential as SCLC pan and tumor characterization, which will be validated on EVs derived from cell lines and patient plasma by Western blotting and single-EV microflow cytometry. Citation Format: Patricia M. Ozawa, Sarah M. Groves, Vito Quaranta, Alissa M. Weaver. Analysis of extracellular vesicle surface markers as small cell lung cancer biomarkers. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5308.