Biology subjects

Manandhar, P.

Publications and source records attributed to Manandhar, P..

10 recordsLinked to original sources

bioRxiv2025

Tim-3 Promotes Early Differentiation of Tbet+ Effector T Cells During Acute Viral Infection

The transmembrane protein Tim-3 has received significant attention in recent years as a possible immunotherapy target. This is due to its robust expression on dysfunctional, exhausted, T cells found in the settings of cancer and chronic infection and biochemical evidence suggesting an inhibitory function of Tim-3. However, numerous clinical trials of putative Tim-3 blocking antibodies have yielded modest benefits, at best, in clinical trials for various cancers. Thus, there is a need to more fully understand the function of Tim-3 in vivo. Here we have studied the function of Tim-3 early during a T cell response to LCMV Armstrong, which causes an acute viral infection in mice. We show that Tim-3 is rapidly expressed after infection and that the expression of Tim-3 is associated with acquisition of a type I effector phenotype, including expression of T-bet and downregulation of Tcf-1, by both CD4+ and CD8+ T cells. In addition, we demonstrate that knockout or cytoplasmic truncation of Tim-3 attenuates the acquisition of the effector program by T cells after LCMV Armstrong infection. Together, these data help to clarify the role of Tim-3 during acute infection.

immunology↗

bioRxiv2023

One Health Assessment of an Urban Temporary Settlement Reveals Gut Microbiome Serving as Antimicrobial Resistance Gene Reservoir

Antimicrobial resistance (AMR) is an emerging and growing global health challenge that could result in 10.2 million deaths annually by 2050. The unrestricted and haphazard use of antibiotics is contributing to the rapid emergence and spread of AMR, and the problem is exacerbated by release of untreated waste water from high-risk sources like hospitals into rivers. Bacteria often develop resistance through horizontal gene transfer mechanism and gut flora can act as a source for new Antimicrobial Resistance Genes (ARG). Upcoming methods like metagenomics can identify the resistance profile (AMR) of gut microbiome, and detect bacterial infections that otherwise go unnoticed. Our study focused on understanding the presence of AMR mutations and gene transfer dynamics in human, animal and environmental samples collected in one of the temporary settlements of Kathmandu (Nepal) using One Health approach. Current AMR reporting based on clinical cases is limited and does not provide information on specific pathogen and associated AMR genes-our study is an effort to contribute information to fulfill this gap. Twenty-one samples were collected from a temporary settlement in Thapathali (Kathmandu), which included fecal samples from birds (n=3) and humans (n=14), and environmental samples (n=4). Microbiological assessment was carried out based on 16S sequence metagenomic analysis using MiSeq (Illumina, USA). Taxonomic classification on obtained 16S sequences were determined by using Metaphlan 2 and Qiime 2 bioinformatics tools. ShortBRED was used to classify ARG and virulence factors, and WAFFLE was used for horizontal gene transfer event prediction. The network analysis was carried out using Gephi v0.9 and the ResistoXplorer web tool to identify ARG in the collected samples. Prevotella spp. was the dominant gut microbiome in humans. We detected diverse phages and viruses, including Stx-2 converting phages. 72 virulence factors and 53 ARG subtypes were detected, with poultry samples having the highest number of subtypes. The cluster and network analysis showed a strong association between gut microbiome and ARG, which was also supported by Horizontal Gene Transfer (HGT) analysis. One-Health interface showed ARG dynamics and revealed gut microbiomes of humans and animals serving as a reservoir for the circulating ARG.

genomics↗

bioRxiv2023

Application of DNA forensic to identify a problem leopard and its implications for human-leopard conflict mitigation

Attacks on humans by leopards Panthera pardus often escalate human-leopard conflict, influence extreme negative tolerance and encourage retaliatory killings. In the rural hilly region of Arghakhanchi district, mid-western Nepal, a leopard killed a child in November 2018. Government authorities captured a leopard a week later which was immediately killed by the villagers. We collected the predators salivary DNA from the victims bite wound and compared its DNA fingerprint profile with the killed leopards profile to resolve the case using 13 microsatellite markers for leopard individualization. Our genetic analysis confirmed that the leopard persecuted by the villagers was the same leopard that had killed the victim. We urge the government to devise dedicated policy and guidelines for human-leopard conflict management and mitigation in Nepal, and to incorporate protocols, including leopard individualization microsatellite panel we have standardized, that mandate correct identification of captured leopard before any interventions such as persecutions and translocations are attempted. We also recommend steering community programs to proactively safeguard children, people and livestock to avoid conflicts and to influence positive tolerance towards leopards. This will benefit leopard conservation and save human lives and livelihoods leading to a healthy coexistence.

genetics↗

bioRxiv2023

Phylogenetic analysis shows canine distemper virus outbreak in stray dogs possibly occurs through spillover from wild carnivore reservoirs

Canine distemper is a highly contagious, often fatal disease caused by canine distemper virus (CDV) in domestic dogs and wild carnivores. The virus has caused mass epidemics in wild carnivores of high conservation value such as tigers, lions and leopards in both wild and captivity. Hence, understanding and managing CDV outbreaks is particularly important in Nepal, which is home to many species of threatened wild carnivores including tigers, leopards, snow leopards, dholes and wolves, as well as a large population of stray dogs. Previous studies have suggested that CDV may pose a threat to wild carnivores, but there has not been any studies characterizing the genetic strains of the virus circulating in Nepals carnivores. We collected invasive and non-invasive biological samples from stray dogs in Kathmandu Valley and genetically characterized the strains of CDV in the dogs to belong to Asia-5 lineage by using phylogenetic analysis. The same lineage also contained CDV strains isolated from dogs, civets, red panda and lions in India. Based on our phylogenetic analysis, we think it is likely that in Nepal CDV is maintained through sylvatic cycle among small carnivore guilds allowing the recurring spillovers and outbreaks among free-ranging stray dogs and possibly large carnivores. It is crucial to prevent the virus transmission from reservoir hosts to other species, especially threatened populations of large carnivores in Nepal. Hence, we recommend for regular surveillance of CDV targeting small wild carnivores as well as vaccination programmes to control the disease spillover in stray dogs.

genetics↗

bioRxiv2022

Chorography and conformational dynamism of the Soluble Human Fibrinogen in solution

Fibrinogen is a soluble, multi-subunit and multi-domain dimeric protein, which, upon its proteolytic cleavage by thrombin, is converted to insoluble fibrin initiating polymerization that substantially contributes to clot growth. The consentaneous structural view of the soluble form of fibrinogen is relatively straight and rigid-like. However, fibrinogen contains numerous, transiently-accessible "cryptic" epitopes for hemostatic and immunologic proteins, suggesting that fibrinogen exhibits conformational flexibility, which may play functional roles in its temporal and spatial interactions. Hitherto, there has been limited information on the solution structure and internal flexibility of soluble fibrinogen. Here, utilizing an integrative, biophysical approach involving temperature-dependent hydrogen-deuterium exchange mass spectrometry, small angle X-ray scattering, and negative stain electron microscopy, we present a holistic, conformationally dynamic model of human fibrinogen in solution. Our data reveal a high degree of internal flexibility, accommodated by four major and distinct flexible sites along the central axis of the protein. We propose that the fibrinogen structure in solution consists of a complex, conformational landscape with multiple local minima, rather than a single, rigid topology. This is further supported by the location of numerous point mutations that are linked to dysfibrinogenemia, and post-translational modifications, residing near fibrinogen flexions. This work provides a molecular basis for the structural "dynamism" of fibrinogen that is expected to influence the broad swath of functionally diverse macromolecular interactions and fine-tune the structural and mechanical properties of blood clots.

biochemistry↗

bioRxiv2022

Screening Avian Pathogens in Eggs from Commercial Hatcheries in Nepal- an Effective Poultry Disease Surveillance Tool

BackgroundCommercial hatcheries play an important role in the overall poultry value chain-providing small to large poultry farmers with day old chicks. Any outbreak in such hatcheries can spread diseases to other farms. Regular screening of major avian pathogens, along with strict bio-security measures, can prevent spread of diseases in hatcheries. Newcastle Disease Virus (NDV), Infectious Bronchitis Virus (IBV), Mycoplasma gallisepticum (MG), Mycoplasma synoviae (MS), Infectious Bursal Disease Virus (IBDV) and Influenza A Virus (IAV) are among the most prevalent poultry diseases which can be detected in egg albumin. MethodWe retrospectively (August 2020-August 2021, except October 2020) analyzed diagnostic results for six selected avian pathogens (NDV, IBV, MS, MG, IBDV and IAV) on eggs (n=4343) received from eleven major commercial poultry hatcheries located in the five adjoining districts of Kathmandu, Nepal. Albumin from 10% randomly selected eggs from each hatchery were tested for the six avian pathogens using multiplex PCR. ResultMajority (7/11, 64%) of the poultry hatcheries had at least one of the six pathogens present. We detected at least one avian pathogen in nine out of eleven months (82%) of screening. Except for IBDV, we found one or more of the other major avian pathogens-Influenza A (IAV) (n=4 times) and Mycoplasma gallisepticum (MG) (n=4 times) were detected the most, followed by Newcastle Virus (NDV) (n=3 times). Infectious bronchitis virus (IBV) were detected twice, and Mycoplasma synoviae (MS) was detected once. ConclusionIn a resource strapped country like Nepal, poultry disease outbreak investigation in particular and surveillance in general are challenging. Meanwhile, poultry production is highly impacted by disease outbreaks often triggered by poor bio-security and lack of pathogen screening practices. Our molecular screening tests have picked up major poultry pathogens present throughout the year in eggs collected from hatcheries. Influenza A was detected at 4 different incidences throughout the year, which is of concern to both human and animal health. Quick systematic screening of eggs at key distribution points (hatcheries) for major avian pathogens is an effective surveillance tool for early disease detection and containment of outbreaks.

microbiology↗

bioRxiv2022

Identification and Characterization of Probiotics Isolates from Indigenous Chicken (Gallus domesticus) of Nepal

BackgroundExcessive and irrational use of antibiotics as growth promoters in poultry has been one of key factors contributing to increased emergence of antibiotics resistant bacteria. Drug resistant infections are becoming major concerns in poultry production impacting both human and poultry health. Several alternatives for antibiotic growth promoters are being sought, and the search for effective probiotics to be used as feed additives is amongst the promising ones. Our study aimed to isolate and test potential probiotics bacteria from cloacal swabs of various indigenous chicken (Gallus domesticus) breeds from rural outskirts of the Kathmandu valley (Nepal). MethodsSelective isolation of probiotics was conducted by micro-aerophilic enrichment of sample in MRS Broth at 37{degrees}C, followed by culturing on MRS agar supplemented with 5 g/L of CaCO3. Isolated bacterial colonies producing transparent halo were selected as potential lactic acid bacteria (LAB), and tested for their antibacterial activity, phenotypic and biochemical characteristics, acidic yield, and tolerance to acid and bile. ResultsA total of 90 potential LAB were isolated from cloacal samples collected from 41 free-ranging chickens of indigenous breeds. Of these, 52 LAB isolates (57%) showed variable antibacterial activity to at least one bacterial pathogen. Of 52 LAB, 46 isolates fulfilled phenotypic and biochemical criteria of Lactobacillus spp. Of these, 37 isolates produced varying percentage yields of lactic acid, 27 isolates showed survival at pH 3.0, and 17 isolates showed survival tolerances in the presence of 0.3% and 0.5% bile salts for 24 hours. Phylogenetic analysis of 16SrDNA sequencing of LAB isolates fulfilling in vitro probiotics properties showed that 3 isolates had genetic identity of 99.38% with Lactobacillus plantarum, while one isolate was genetically similar (99.85%) with the clade of L. reuteri, L. antri and L. panis. ConclusionsOur study identified four Lactobacillus spp. strains having potential probiotics properties. Further investigations are needed to evaluate these isolates to be used as poultry probiotics feed supplement.

microbiology↗

bioRxiv2022

Newcastle disease burden in Nepal and efficacy of Tablet I-2 vaccine in commercial and backyard poultry production

Poultry (Gallus domesticus) farming plays an important role as an income generating enterprise in a developing country like Nepal, contributing more than 4% to the national GDP. It is also one of the major sources of protein for growing population. Newcastle Disease (ND) is a major poultry disease affecting both commercial and backyard poultry production worldwide. There were more than 90 reported cases of ND outbreaks in Nepal in 2018, with over 74,986 birds being affected. ND might be responsible for over 7% of total poultry mortality in the country. Recent outbreak of ND in 2021 affected many farms throughout Nepal, and caused massive poultry production loss. ND is caused by a single stranded RNA virus which presents very similar clinical symptoms as Influenza A (commonly known as Bird flu), adding much complexity to clinical disease identification and intervention. We conducted a nationwide ND and Influenza A prevalence study, collecting samples from commercial and backyard poultry farms from across the major poultry production hubs of Nepal, and conducted both serological and molecular assessments-giving us disease exposure history and identification of floating strains of ND Virus (NDV). Of 600 commercial chickens tested from various farms, both NDV (n=381, 64%) and IA (n=125, 21%) antibodies were detected in the majority of the samples. In backyard chicken (n=108, 39 farms), sero-prevalence was also relatively high for both NDV (n=38, 35%) and IA (n=17, 16%). Out of the 40 commercial farms, majority had detectable NDV (n=31, 78%) and IA (n=15, 38%) virus present. In backyard farms (n=36), we also detected NDV (n=6, 16%) and IA (n=1, 3%) virus. We Genotyped (strain) detected NDV, and found Genotype II to be present in most of the commercial farms (which might be coming from live vaccine usage) and Genotype I in some backyard poultry samples. The identified Genotype I strain is reported for the first time, and hence could be an endemic NDV strain found in Nepal. Our 2021 ND outbreak investigation identified Genotype VII c as the causative strain. Additionally, we have developed a thermostable I-2 NDV vaccine (Ranigoldunga) in tablet formulation and tested on various (mixed) breeds of chicken (G. domesticus). This vaccine seems to be highly effective against NDV, including a virulent 2021 outbreak strain (Genotype VII c). The I-2 Tablet ND vaccine showed more than 85% efficacy when administered either ocularly or in water, and has a stability of 30 days in room temperature.

microbiology↗

bioRxiv2022

Novel strains of Campylobacter cause diarrheal outbreak in Rhesus macaques (Macaca mulatta) of Kathmandu Valley

Campylobacter spp. is often underreported and underrated bacteria that present real health risks to both humans and animals, including non-human primates. It is a commensal microorganism of gastrointestinal tract known to cause gastroenteritis in humans. Commonly found in many wild animals including non-human primates (monkeys-Rhesus macaques) these pathogens are known to be a common cause of diarrhea in humans in many parts of developing and under developed countries. Rhesus macaques from the two holy sites in Kathmandu (Pashupati and Swoyambhu) were included in this cross-sectional study. Opportunistic diarrheal samples of monkeys were analyzed to detect and characterize the pathogen using 16S rRNA-based PCR screening, followed by DNA sequencing and phylogenetic analysis. Out of a total 67 collected diarrheal samples, Campylobacter spp. were detected in the majority of the samples (n=64; 96%). DNA sequences of the amplified PCR products were successfully obtained from 13 samples. Phylogenetic analysis identified Candidatus Campylobacter infans (n=10, Kimura-2 parameter (K2P) pairwise distance values of 0.002287). Remaining three sequences might potentially belong to a novel Campylobacter species/sub-species-closely relating to known species of C. helviticus (K2P pairwise distance of 0.0267). Both Candidatus Campylobacter infans and C. helvitucus are known to infect humans and animals. Additionally, we also detected the bacteria in water and soil samples from the sites. Campylobacter spp. caused the 2018 diarrhea outbreak in Rhesus macaques in the Kathmandu valley. Campylobacter might be one of the important contributing pathogens in diarrheal outbreaks-both in humans and animals (monkeys) in Nepal. Due to close interactions of these animals with humans and other animals, One Health approach might be the most effective way to prevent and mitigate the threat posed by this pathogen.

microbiology↗

bioRxiv2019

The co-stimulatory activity of Tim-3 requires Akt and MAPK signaling and immune synapse recruitment

Expression of the transmembrane protein Tim-3 is increased on dysregulated T cells undergoing chronic T cell activation, including in chronic infection and solid tumors. We and others previously reported that Tim-3 exerts apparently paradoxical co-stimulatory activity in T cells (and other cells), including enhancement of ribosomal S6 protein phosphorylation (pS6). Here we examined the upstream signaling pathways that control Tim3-mediated increases in pS6 in T cells. We have also defined the localization of Tim-3 relative to the T cell immune synapse and impacts on downstream signaling. Recruitment of Tim-3 to the immune synapse was mediated exclusively by the transmembrane domain, replacement of which impaired Tim-3 co-stimulation of pS6. Strikingly, enforced localization of the Tim-3 cytoplasmic domain to the immune synapse in the context of a chimeric antigen receptor still allowed for robust T cell activation. Our findings are consistent with a model whereby Tim-3 enhances TCR-proximal signaling under acute conditions. One Sentence SummaryHere we define elements of signaling and localization associated with Tim-3 co-stimulatory function in T cells.

immunology↗