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Canine parvovirus-2(CPV-2) causes a highly contagious disease of dogs characterised by acute hemorrhagic gastroenteritis, lethargy, vomiting, fever and usually bloody or mucoid diarrhoea. In the present study, 41 faecal samples collected from dogs exhibiting the signs of fever, vomition, bloody or mucoid diarrhoea in Kolkata, India were screened by haemagglutination test and PCR for detection of capsid protein coding VP2 gene. The viral genotype was detected by multiplex PCR and analysis of partial VP2 gene nucleotide sequences of selected PCR products with bioinformatics tool. Thirteen (31.71%) samples were found positive with HA titre ≥ 32 whereas 28 (68.29%) samples were positive by PCR of VP2 gene indicating higher sensitivity of PCR. Highest occurrence of CPV-2 was observed in the age group of 1-6 months (80.65%) and non-descript breeds with no history of vaccination (85%). Three samples were antigenic type CPV-2a, rest were CPV-2b/CPV 2c. Six CPV sequences were found to be highly similar to published CPV 2c sequences in BLAST analysis revealing a maximum identity of 99-100% with other CPV-2c strains and clustered together with CPV-2c strains of India and other countries in phylogenetic analysis. The present study highlights the need for continuous monitoring of samples to detect gradual changes in circulating CPV-2 genotypes in India.

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Polygenic risk scores (PRSs) quantify the contribution of multiple genetic loci to an individual's likelihood of a complex trait or disease. However, existing PRSs estimate this likelihood with common genetic variants, excluding the impact of rare variants. Here, we report on a method to identify rare variants associated with outlier gene expression and integrate their impact into PRS predictions for body mass index (BMI), obesity, and bariatric surgery. Between the top and bottom 10%, we observed a 20.8% increase in risk for obesity (p = 3 × 10-14), 62.3% increase in risk for severe obesity (p = 1 × 10-6), and median 5.29 years earlier onset for bariatric surgery (p = 0.008), as a function of expression outlier-associated rare variant burden when controlling for common variant PRS. We show that these predictions were more significant than integrating the effects of rare protein-truncating variants (PTVs), observing a mean 19% increase in phenotypic variance explained with expression outlier-associated rare variants when compared with PTVs (p = 2 × 10-15). We replicated these findings by using data from the Million Veteran Program and demonstrated that PRSs across multiple traits and diseases can benefit from the inclusion of expression outlier-associated rare variants identified through population-scale transcriptome sequencing.

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There is limited data on host-specific genetic determinants of susceptibility to bacterial and viral infections. Genome-wide association studies using large population cohorts can be a first step towards identifying patients prone to infectious diseases and targets for new therapies. Genetic variants associated with clinically relevant entities of bacterial and viral infections (e.g., abdominal infections, respiratory infections, and sepsis) in 337,484 participants of the UK Biobank cohort were explored by genome-wide association analyses. Cases (n = 81,179) were identified based on ICD-10 diagnosis codes of hospital inpatient and death registries. Functional annotation was performed using gene expression (eQTL) data. Fifty-seven unique genome-wide significant loci were found, many of which are novel in the context of infectious diseases. Some of the detected genetic variants were previously reported associated with infectious, inflammatory, autoimmune, and malignant diseases or key components of the immune system (e.g., white blood cells, cytokines). Fine mapping of the HLA region revealed significant associations with HLA-DQA1, HLA-DRB1, and HLA-DRB4 locus alleles. PPP1R14A showed strong colocalization with abdominal infections and gene expression in sigmoid and transverse colon, suggesting causality. Shared significant loci across infections and non-infectious phenotypes in the UK Biobank cohort were found, suggesting associations for example between SNPs identified for abdominal infections and CRP, rheumatoid arthritis, and diabetes mellitus. We report multiple loci associated with bacterial and viral infections. A better understanding of the genetic determinants of bacterial and viral infections can be useful to identify patients at risk and in the development of new drugs.

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Long non-coding RNA (lncRNA) genes have well-established and important impacts on molecular and cellular functions. However, among the thousands of lncRNA genes, it is still a major challenge to identify the subset with disease or trait relevance. To systematically characterize these lncRNA genes, we used Genotype Tissue Expression (GTEx) project v8 genetic and multi-tissue transcriptomic data to profile the expression, genetic regulation, cellular contexts, and trait associations of 14,100 lncRNA genes across 49 tissues for 101 distinct complex genetic traits. Using these approaches, we identified 1,432 lncRNA gene-trait associations, 800 of which were not explained by stronger effects of neighboring protein-coding genes. This included associations between lncRNA quantitative trait loci and inflammatory bowel disease, type 1 and type 2 diabetes, and coronary artery disease, as well as rare variant associations to body mass index.

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BACKGROUND: Population structure among study subjects may confound genetic association studies, and lack of proper correction can lead to spurious findings. The Genotype-Tissue Expression (GTEx) project largely contains individuals of European ancestry, but the v8 release also includes up to 15% of individuals of non-European ancestry. Assessing ancestry-based adjustments in GTEx improves portability of this research across populations and further characterizes the impact of population structure on GWAS colocalization. RESULTS: Here, we identify a subset of 117 individuals in GTEx (v8) with a high degree of population admixture and estimate genome-wide local ancestry. We perform genome-wide cis-eQTL mapping using admixed samples in seven tissues, adjusted by either global or local ancestry. Consistent with previous work, we observe improved power with local ancestry adjustment. At loci where the two adjustments produce different lead variants, we observe 31 loci (0.02%) where a significant colocalization is called only with one eQTL ancestry adjustment method. Notably, both adjustments produce similar numbers of significant colocalizations within each of two different colocalization methods, COLOC and FINEMAP. Finally, we identify a small subset of eQTL-associated variants highly correlated with local ancestry, providing a resource to enhance functional follow-up. CONCLUSIONS: We provide a local ancestry map for admixed individuals in the GTEx v8 release and describe the impact of ancestry and admixture on gene expression, eQTLs, and GWAS colocalization. While the majority of the results are concordant between local and global ancestry-based adjustments, we identify distinct advantages and disadvantages to each approach.

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BACKGROUND: The prevalence of obesity and its comorbidities, including type 2 diabetes mellitus (T2DM), is dramatically increasing throughout the world; however, the underlying aetiology is incompletely understood. Genome-wide association studies (GWAS) have identified hundreds of genec susceptibility loci for obesity and T2DM, although the causal genes and mechanisms are largely unknown. SPRY2 is a candidate gene identified in GWAS of body fat percentage and T2DM, and has recently been linked to insulin production in pancreatic ß-cells. In the present study, we aimed to further understand SPRY2 via functional characterisation in HepG2 cells, an in vitro model of human hepatocytes widely used to investigate T2DM and insulin resistance. METHODS: CRISPR-Cas9 genome editing was used to target SPRY2 in HepG2 cells, and the functional consequences of SPRY2 knockout (KO) and overexpression subsequently assessed using glucose uptake and lipid droplet assays, measurement of protein kinase phosphorylation and RNA sequencing. RESULTS: The major functional consequence of SPRY2 KO was a significant increase in glucose uptake, along with elevated lipid droplet accumulation. These changes were attenuated, but not reversed, in cells overexpressing SPRY2. Phosphorylation of protein kinases across key signalling pathways (including Akt and mitogen activated protein kinases) was not altered after SPRY2 KO. Transcriptome profiling in SPRY2 KO and mock (control) cells revealed a number of differentially expressed genes related to cholesterol biosynthesis, cell cycle regulation and cellular signalling pathways. Phospholipase A2 group IIA (PLA2G2A) mRNA level was subsequently validated as significantly upregulated following SPRY2 KO, highlighting this as a potential mediator downstream of SPRY2. CONCLUSION: These findings suggest a role for SPRY2 in glucose and lipid metabolism in hepatocytes and contribute to clarifying the function of this gene in the context of metabolic diseases.

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PURPOSE OF REVIEW: Large genome-wide association studies (GWAS) have identified variants accounting for a substantial portion of the heritable risk for coronary artery disease (CAD). These studies have catalyzed drug discovery and generated the possibility of improved risk prediction and stratification. Here, we review the current state-of-the art in polygenic risk scores (PRSs) and look to the future, as these scores move towards clinical application. RECENT FINDINGS: Over the last decade, multilocus PRSs for CAD have expanded to include millions of variants and demonstrated strong association with CAD outcomes, even when adjusted for traditional risk factors. Recently, PRSs have shown better prediction of CAD outcomes than any single traditional risk factor alone. Advances in statistical methods used to generate PRSs have improved their predictive ability and transferability between populations with varied ancestries. Initial clinical studies have also demonstrated the potential of genetic information to impact shared decision-making between patients and providers, leading to improved outcomes. SUMMARY: PRSs can improve risk stratification for CAD especially in white/European populations and have the potential to alter routine clinical care. However, unlocking this potential will require additional research in PRSs in nonwhite populations and substantial investment in clinical implementation studies.

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BACKGROUND: PCSK9 inhibition is a potent new therapy for hypercholesterolemia and cardiovascular disease. Although short-term clinical trial results have not demonstrated major adverse effects, long-term data will not be available for some time. Genetic studies in large biobanks offer a unique opportunity to predict drug effects and provide context for the evaluation of future clinical trial outcomes. METHODS: We tested the association of the PCSK9 missense variant rs11591147 with predefined phenotypes and phenome-wide, in 337 536 individuals of British ancestry in the UK Biobank, with independent discovery and replication. Using a Bayesian statistical method, we leveraged phenotype correlations to evaluate the phenome-wide impact of PCSK9 inhibition with higher power at a finer resolution. RESULTS: The T allele of rs11591147 showed a protective effect on hyperlipidemia (odds ratio, 0.63±0.04; P=2.32×10-38), coronary heart disease (odds ratio, 0.73±0.09; P=1.05×10-6), and ischemic stroke (odds ratio, 0.61±0.18; P=2.40×10-3) and was associated with increased type 2 diabetes mellitus risk adjusted for lipid-lowering medication status (odds ratio, 1.24±0.10; P=1.98×10-7). We did not observe associations with cataracts, heart failure, atrial fibrillation, and cognitive dysfunction. Leveraging phenotype correlations, we observed evidence of a protective association with cerebral infarction and vascular occlusion. These results explore the effects of direct PCSK9 inhibition; off-target effects cannot be predicted using this approach. CONCLUSIONS: This result represents the first genetic evidence in a large cohort for the protective effect of PCSK9 inhibition on ischemic stroke and corroborates exploratory evidence from clinical trials. PCSK9 inhibition was not associated with variables other than those related to LDL (low-density lipoprotein) cholesterol, atherosclerosis, and type 2 diabetes mellitus, suggesting that other effects are either small or absent.

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BACKGROUND: Military personnel and civilians living in areas of armed conflict have increased risk of exposure to blast overpressures that can cause significant hearing loss and/or brain injury. The equipment used to simulate comparable blast overpressures in animal models within laboratory settings is typically very large and prohibitively expensive. NEW METHOD: To overcome the fiscal and space limitations introduced by previously reported blast wave generators, we developed a compact, low-cost blast wave generator to investigate the effects of blast exposures on the auditory system and brain. RESULTS: The blast wave generator was constructed largely from off the shelf components, and reliably produced blasts with peak sound pressures of up to 198dB SPL (159.3kPa) that were qualitatively similar to those produced from muzzle blasts or explosions. Exposure of adult rats to 3 blasts of 188dB peak SPL (50.4kPa) resulted in significant loss of cochlear hair cells, reduced outer hair cell function and a decrease in neurogenesis in the hippocampus. COMPARISON TO EXISTING METHODS: Existing blast wave generators are typically large, expensive, and are not commercially available. The blast wave generator reported here provides a low-cost method of generating blast waves in a typical laboratory setting. CONCLUSIONS: This compact blast wave generator provides scientists with a low cost device for investigating the biological mechanisms involved in blast wave injury to the rodent cochlea and brain that may model many of the damaging effects sustained by military personnel and civilians exposed to intense blasts.

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OBJECTIVES: The ideal vascular reconstruction strategy for anomalies associated with Tetralogy of Fallot (ToF) is often driven by observations made at the operating table. A method to conduct accurate studies to assess the virtues of a certain surgical technique to guide surgical decisions is found wanting. We hypothesize that patient-specific computed tomography (CT)-based morphometry followed by in silico reconstruction of viable surgical options with haemodynamic function assessment using computational fluid dynamics (CFD) can guide surgical decisions and help forecast functional outcomes without invasive measurements. METHODS: A ToF patient associated with additional left pulmonary artery (LPA) stenosis and a patent ductus arteriosus (PDA) who underwent a successful correction using a single pericardial patch (SPP) was selected as a reference for morphological characterization after 3D anatomical reconstruction from CT images. A second patient with morphological similarities established after scaled, co-registration with the reference patient was selected for virtual correction using the same strategy (i.e. SPP repair). CFD was employed for functional analysis of pulmonary artery (PA) pressure gradients in the baseline preoperative and virtually corrected models, using patient-specific cardiac output and Qp/Qs information. RESULTS: SPP repair was modelled in silico following surgical steps of PDA ligation, creation of an incision along the LPA and main PA (MPA) and finally suturing a rectangular SPP, effectively reducing MPA to LPA angle. Analysis of SPP repair revealed significant reduction in right ventricular outflow tract-LPA pressure gradient with improved left-right PA flow distribution in both patients. CONCLUSIONS: In silico surgery followed by CFD evaluation has the potential in augmenting morphology-guided decisions on surgical strategy and holds promise in preoperatively determining optimal intervention strategy. This is a paradigm-shifting concept to evaluate patient-specific anomalies in a manner more objective than mere visual inspection of anatomical traits from radiology images. Present studies are focused on an analysis with a larger patient cohort to establish a library of ToF patients' successful surgical outcomes to inform morphology-based selection of surgical strategy.

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Objective Gonioscopy provides limited quantitative information to compare the iridocorneal anatomy across different species. In addition, the anatomic relationships by histologic examination are altered during processing. As a result, the comparative anatomy of the iridocorneal angle across several mammalian species was evaluated by Optical Coherence Tomography (OCT). Methods Cats, beagle dogs, minipigs, owl monkeys, cynomolgus monkeys, and rhesus monkeys (n = 6 or 7 per species) were evaluated. Imaging was performed using the OCT. The anterior chamber angle (ACA), angle opening distance (AOD), and the angle recess area (ARA) were evaluated. Results AC angle: cat (63 ± 6°) > owl monkey (54 ± 4°) > beagle dog (42 ± 4°) > minipig (40 ± 3°) > rhesus monkey (36 ± 1°) > cynomolgus monkey (34 ± 2°). AOD: cat (3.3 ± 0.5 mm) > owl monkey (2.05 ± 0.2 mm) > beagle dog (1.08 ± 0.1 mm) > rhesus monkey (0.92 ± 0.06 mm) > minipig (0.64 ± 0.04 mm) > cynomolgus monkey (0.43 ± 0.03 mm). ARA: cat (3.5 ± 0.1 mm(2) ) > owl monkey (1.41 ± 0.2 mm(2) ) > dog (0.88 ± 0.1 mm(2) ) > rhesus monkey (0.62 ± 0.06 mm(2) ) > minipig (0.21 ± 0.05 mm(2) ) > cynomolgus monkey (0.15 ± 0.01 mm(2) ). Conclusions This study benchmarks the normative iridocorneal angle measurements across different mammalian species by OCT. These data can be useful to compare iridocorneal angle measurements in disease states as OCT evolves as a common diagnostic tool in veterinary ophthalmic research and practice.

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The lamina cribrosa has been postulated from in vitro studies as an early site of damage in glaucoma. Prior in vivo measures of laminar morphology have been confounded by ocular aberrations. In this study the lamina cribrosa was imaged after correcting for ocular aberrations using the adaptive optics scanning laser ophthalmoscope (AOSLO) in normal and glaucomatous eyes of rhesus monkeys. All measured laminar morphological parameters showed increased magnitudes in glaucomatous eyes relative to fellow control eyes, indicating altered structure. The AOSLO provides high-quality images of the lamina cribrosa and may have potential as a tool for early identification of glaucoma.

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PURPOSE: Losses of retinal ganglion cells (RGCs) in glaucoma are the cause of visual field defects and thinning of the retinal nerve fiber layer (RNFL), but methods of correlating these events have not been developed. The present study was conducted to investigate the relationship between standard automated perimetry (SAP) measures of RGCs and optical coherence tomography (OCT) measures of the ganglion cell axons entering the optic nerve from corresponding visual field locations. METHODS: SAP and OCT data from normal monkeys were used to develop methods for estimating neuron counts and mapping SAP visual field locations onto the optic nerve head (ONH). The procedures developed for normal eyes were applied to monkeys with experimental glaucoma. RESULTS: The number of neurons derived from SAP and OCT data for normal eyes were in close agreement. The estimates of the number of RGCs in retinal areas of the Humphrey Field Analyzer 24-2 (Carl Zeiss Meditec, Inc., Dublin, CA) visual field and the axons entering the ONH were both approximately 1.5 million. The neural losses derived from subjective and objective measurements in monkeys with early experimental glaucoma correlated highly, with a mean +/- SD difference of 0.6% +/- 22% between the two estimates in control eyes and 3% +/- 24% in laser-treated eyes. CONCLUSIONS: SAP measures of visual field defects and OCT measures of RNFL defects are correlated measures of glaucomatous neuropathy. The normal intersubject variability and the dynamic ranges of the measurements suggest that RNFL thickness may be a more sensitive measurement for early stages and perimetry a better measure for moderate to advanced stages of glaucoma.

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PURPOSE: Accommodation can be restored to presbyopic human eyes by refilling the capsular bag with a soft polymer. This study was conducted to test whether accommodation, measurable as changes in optical refraction, can be restored with a newly developed refilling polymer in a rhesus monkey model. A specific intra- and postoperative treatment protocol was used to minimize postoperative inflammation and to delay capsular opacification. METHODS: Nine adolescent rhesus monkeys underwent refilling of the lens capsular bag with a polymer. In the first four monkeys (group A) the surgical procedure was followed by two weekly subconjunctival injections of corticosteroids. In a second group of five monkeys (group B) a treatment intended to delay the development of capsular opacification was applied during the surgery, and, in the postoperative period, eye drops and two subconjunctival injections of corticosteroids were applied. Accommodation was stimulated with carbachol iontophoresis or pilocarpine and was measured with a Hartinger refractometer at regular times during a follow-up period of 37 weeks in five monkeys. In one monkey, lens thickness changes were measured with A-scan ultrasound. RESULTS: In group A, refraction measurement was possible in one monkey. In the three other animals in group A, postoperative inflammation and capsular opacification prevented refraction measurements. In group B, the maximum accommodative amplitude of the surgically treated eyes was 6.3 D. In three monkeys the accommodative amplitude decreased to almost 0 D after 37 weeks. In the two other monkeys, the accommodative amplitude remained stable at +/-4 D during the follow-up period. In group B, capsular opacification developed in the postoperative period, but refraction measurements could still be performed during the whole follow-up period of 37 weeks. CONCLUSIONS: A certain level of accommodation can be restored after lens refilling in adolescent rhesus monkeys. During the follow-up period refraction measurements were possible in all five monkeys that underwent the treatment designed to prevent inflammation and capsular opacification.

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Experiments were undertaken to understand the relationship between dynamic accommodative refractive and biometric (lens thickness (LT), anterior chamber depth (ACD) and anterior segment length (ASL=ACD+LT)) changes during Edinger-Westphal stimulated accommodation in rhesus monkeys. Experiments were conducted on three rhesus monkeys (aged 11.5, 4.75 and 4.75 years) which had undergone prior, bilateral, complete iridectomies and implantation of a stimulating electrode in the Edinger-Westphal (EW) nucleus. Accommodative refractive responses were first measured dynamically with video-based infrared photorefraction and then ocular biometric responses were measured dynamically with continuous ultrasound biometry (CUB) during EW stimulation. The same stimulus amplitudes were used for the refractive and biometric measurements to allow them to be compared. Main sequence relationships (ratio of peak velocity to amplitude) were calculated. Dynamic accommodative refractive changes are linearly correlated with the biometric changes and accommodative biometric changes in ACD, ASL and LT show systematic linear correlations with increasing accommodative amplitudes. The relationships are relatively similar for the eyes of the different monkeys. Dynamic analysis showed that main sequence relationships for both biometry and refraction are linear. Although accommodative refractive changes in the eye occur primarily due to changes in lens surface curvature, the refractive changes are well correlated with A-scan measured accommodative biometric changes. Accommodative changes in ACD, LT and ASL are all well correlated over the full extent of the accommodative response.

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Changes in accommodative dynamics with repeated accommodation were studied in three anesthetized rhesus monkeys and two conscious humans. Maximum accommodation was centrally stimulated via the Edinger-Westphal nucleus in monkeys with a 4 s on, 4 s off paradigm (4 x 4) for 17 min, 4 x 1.5 for 27 min and 2 x 1 for 16 min. Humans accommodated repeatedly to visual targets (5 x 5; 5D and 2 x 2; 6D) for 30 min. In all cases, accommodation was sustained throughout. The anesthetized monkeys showed inter-individual variability in the extent of changes in accommodative dynamics over time while no systematic changes were detected in the human accommodative responses. Little accommodative fatigue was found compared to previous studies which have reported a complete loss of accommodation after 5 min of repeated stimulation in monkeys.

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Wave aberrations were measured with a Shack-Hartmann wavefront sensor (SHWS) in the right eye of a large young adult population when accommodative demands of 0, 3, and 6 D were presented to the tested eye through a Badal system. Three SHWS images were recorded at each accommodative demand and wave aberrations were computed over a 5-mm pupil (through 6th order Zernike polynomials). The accommodative response was calculated from the Zernike defocus over the central 3-mm diameter zone. Among all individual Zernike terms, spherical aberration showed the greatest change with accommodation. The change of spherical aberration was always negative, and was proportional to the change in accommodative response. Coma and astigmatism also changed with accommodation, but the direction of the change was variable. Despite the large inter-subject variability, the population average of the root mean square for all aberrations (excluding defocus) remained constant for accommodative levels up to 3.0 D. Even though aberrations change with accommodation, the magnitude of the aberration change remains less than the magnitude of the uncorrected aberrations, even at high accommodative levels. Therefore, a typical eye will benefit over the entire accommodative range (0-6 D) if aberrations are corrected for distance viewing.

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This study investigated the changes in ocular aberrations that occur over the entire lens equatorial diameter during accommodation in iridectomized rhesus monkey eyes to understand the nature of accommodative lenticular deformation. Accommodation was centrally stimulated to a range of different response amplitudes (0 D to approximately 11 D), and ocular aberrations were measured with a Shack-Hartmann wavefront sensor in both eyes of one previously iridectomized 10-year-old rhesus monkey. At the highest amplitude in the two eyes, aberrations were analyzed over entrance pupil diameters ranging from 3 to 8 mm in steps of 1 mm. Root mean square error of the total measured aberrations, excluding defocus, increased systematically with increasing accommodation from about 1 to 3.5 microns. Spherical aberration became systematically more negative, and vertical coma increased significantly in magnitude with accommodation. There was a strong accommodative change in power near the center of the lens and little change in power at the periphery. At the highest accommodative state, decreasing the analyzed entrance pupil diameter from 8 to 3 mm considerably reduced the wavefront error. The greater increase in optical power near the central region of the lens, combined with an accommodative pupillary miosis, would serve to maximize accommodative refractive change while maintaining acceptable image quality.

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Dynamics of accommodation (far-to-near focus) and disaccommodation (near-to-far focus) are described as a function of response amplitude. Accommodative responses to step stimuli of various amplitudes presented in real space were measured in eight 20-30 year old subjects. Responses were fitted with exponential functions to determine amplitude, time constant and peak velocity. Despite the intersubject variability, the results show that time constants of accommodation and peak velocity of disaccommodation increase with amplitude in all subjects. The dynamics of accommodation and disaccommodation are dependent on amplitude, but have different properties in each case.

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