Biology subjects

Manandhar, K. D.

Publications and source records attributed to Manandhar, K. D..

5 recordsLinked to original sources

bioRxiv2026

Dengue serotype-1 virus like particles induce antibody responses following HeLa cell expression

Dengue disease remains a significant global health threat, with current vaccines exhibiting variable efficacy and safety concerns. Virus-like particles (VLPs) offer a promising alternative by mimicking native virus structures without infectious genomes. We engineered a mammalian expression plasmid encoding Dengue-1 prM and E proteins, optimized for secretion using Japanese Encephalitis virus signal sequences, and transiently expressed it in HeLa cells. Purified VLPs exhibited spherical morphology ([~]39 nm diameter) consistent with native virions, as confirmed by transmission electron microscopy. Immunization of mice with these VLPs elicited robust Dengue-1 specific IgG antibody responses. Our study demonstrates production of immunogenic Dengue-1 VLPs in HeLa cells, highlighting their potential as a vaccine candidate and a tool for serodiagnosis. Further characterization of VLP epitopes and protective efficacy is warranted to advance vaccine development. ImportanceDengue remains a significant global health challenge, with serotype 1 being one of the dominant strains causing recurrent outbreaks in Nepal. Existing vaccines demonstrate limited efficacy and pose significant safety concerns, particularly in seronegative populations. To address these limitations, this study explores virus-like particles (VLPs) as a safer alternative vaccine platform. VLPs elicit robust immunogenicity by mimicking the structure of native virus while completely lacking genetic components. This study combines DENV1 structural proteins with optimized expression systems to enhance immunogenicity. This work is particularly significant as the first dengue vaccine research conducted in Nepal, directly addressing antigenic mismatches between existing commercial vaccines and locally circulating viral strains. Furthermore, the study provides scalable platform for developing region-specific dengue vaccines for other serotypes and flaviviruses.

immunology↗

bioRxiv2025

Harnessing Lytic Phages for Biofilm Control in Carbapenem-Resistant Klebsiella pneumoniae Causing Urinary Tract Infection

BackgroundKlebsiella pneumoniae is a major opportunistic pathogen with rising multidrug resistance and biofilm-related infections. Molecular and phage characterization is crucial to understand resistance mechanisms and explore alternative therapies such as phage therapy. MethodsWe performed whole-genome sequencing and antibiotic susceptibility testing of hospital-isolated Klebsiella pneumoniae (KP6697). MLST, plasmid replicon analysis, and resistance gene identification were conducted using bioinformatics. Phage isolation, electron microscopy-based morphological and biofilm analysis, and evaluation of lytic activity, stability, and host range were performed. Phage genome sequencing and annotation identified functional genes. ResultsThe host strain Klebsiella pneumoniae (KP6697) was multidrug-resistant, exhibiting resistance to 18 of 22 tested antibiotics, and genome analysis identified ST16 with eight plasmid replicons and 23 resistance genes, including blaCTX-M-15, blaNDM-5, and blaOXA-181. Functional annotations revealed extensive metabolic versatility and a rich repertoire of genes for biofilm formation, quorum sensing, secretion systems, and stress response. A lytic phage, Phage_KP6697_Omshanti, was isolated and classified as a Caudoviricetes member with a 45.3kb genome encoding lysis, replication, and structural genes. It demonstrated short latency, high burst size, thermal and pH stability, and broad host range against CRKP and other MDR strains. Importantly, microscopy confirmed its ability to inhibit and degrade biofilms at multiple stages, highlighting strong therapeutic potential. ConclusionComprehensive analysis of carbapenem-resistant K. pneumoniae (KP6697) revealed multidrug resistance and strong biofilm formation. The lytic phage Phage_KP6697_Omshanti, with depolymerase and endolysin activity, disrupted biofilms, and its stability, high burst size, and genomic traits suggest potential as an anti-CRKP agent, especially with antibiotics IMPORTANCEKlebsiella pneumoniae is increasing multidrug resistance and robust biofilm formation pose severe clinical challenges, limiting treatment options. Understanding the molecular basis of its resistance and exploiting bacteriophages with strong biofilm-disrupting properties provide promising alternative therapeutic strategies. This study highlights the isolation and genomic characterization of a lytic phage with potent anti-biofilm activity against carbapenem-resistant K. pneumoniae, underscoring its potential in combating resistant infections.

microbiology↗

bioRxiv2025

PCR-Based Detection and Phylogenetic Analysis of Candidatus Liberibacter asiaticus in Citrus Orchards Across Nepal

Citrus greening disease, also known as huanglongbing (HLB), is caused by the gram-negative -proteobacteria Candidatus Liberibacter species. This disease poses a significant threat to citrus production worldwide, including in Nepal. This study aimed to conduct the diagnosis and phylogenetic analysis of the citrus greening pathogen in Nepal using both conventional PCR and computational methods. A total of 1,026 samples were collected from thirteen districts across six provinces in the country. PCR-based diagnosis was performed using the primer set Las606/LSS, which targets the 16S rRNA gene of Candidatus Liberibacter asiaticus. Additionally, 16S rRNA gene sequencing was performed using sanger sequencing for five samples collected from different geographical regions. The obtained sequences were deposited in GenBank, and a phylogenetic tree was constructed based on these sequences. Among the 1,026 samples tested, 255 were positive, indicating the widespread distribution of HLB across Nepal. All consensus sequences from Nepal showed strong evolutionary relatedness within the C. L. asiaticus cluster, displayed over 99% genetic similarity with reference sequences from various parts of the world. Phylogenetic analysis revealed that the Nepalese sequences were closely related to C. L. asiaticus sequences from India, such as OM522080.1 (Punjab) and MH473394.1 (Meerut). Additionally, sequences obtained from different regions of Nepal clustered closely together. The findings of this research provide valuable insights into the genetic diversity and prevalence of citrus greening disease, which will aid the development of better management strategies to combat this devastating disease. Furthermore, the sequence data deposited in GenBank will serve as a key reference for future molecular studies on citrus greening disease.

molecular biology↗

bioRxiv2024

Genomic sequencing and neutralizing serological profiles during acute dengue infection: A 2017 cohort study in Nepal

Dengue virus (DENV) is a mosquito-borne flavivirus that poses a threat to nearly 50% of the global population. DENV has been endemic in Nepal since 2006; however, little is known about how DENV is evolving or the prevalence of anti-DENV immunity within the Nepalese population. To begin to address these gaps, we performed a serologic and genetic study of 49 patients from across Nepal who presented at central hospitals during the 2017 dengue season with suspected DENV infection. Of the 49 subjects assessed, 21 (43%) were positive for DENV NS1 antigen; of these; 5 were also anti-DENV IgM+ IgG+; 7 were DENV IgM+ IgG-, 2 were IgM- IgG+, and 7 were IgM- IgG- by specific ELISAs. Seven of the 21 NS1+ sera were RNA+ by RT-PCR (six DENV2, one DENV3), suggesting that DENV2 was the dominant serotype in our cohort. Whole-genome sequencing of two DENV2 isolates showed similarity with strains circulating in Singapore in 2016, and the envelope genes were also similar to strains circulating in India in 2017. DENV-neutralizing antibodies (nAbs) were present in 31 of 47 sera tested (66%); among these, 20, 24, 26, and 12 sera contained nAbs against DENV1, 2, 3, and 4 serotypes, respectively. Serology analysis suggested that 12 (26%) and 19 (40%) of the 49 subjects were experiencing primary and secondary DENV infections, respectively. Collectively, our results provide evidence for current and/or past exposure to multiple DENV serotypes in our cohort, and the RNA analyses further indicate that DENV2 was the likely dominant serotype circulating in Nepal in 2017. These data suggest that expanded local surveillance of circulating DENV genotypes and population immunity will be important to effectively manage and mitigate future dengue outbreaks in Nepal.

genomics↗

bioRxiv2024

Dengue Virus Surveillance in Nepal Yields the First On-Site Whole Genome Sequences of Isolates from the 2022 Outbreak

BackgroundThe 4 serotypes of dengue virus (DENV1-4) can each cause potentially deadly dengue disease, and are spreading globally from tropical and subtropical areas to more temperate ones. Nepal provides a microcosm of this global phenomenon, having met each of these grim benchmarks. To better understand DENV transmission dynamics and spread into new areas, we chose to study dengue in Nepal and, in so doing, to build the onsite infrastructure needed to manage future, larger studies. Methods and ResultsDuring the 2022 dengue season, we enrolled 384 patients presenting at a hospital in Kathmandu with dengue-like symptoms; 79% of the study participants had active or recent DENV infection (NS1 antigen and IgM). To identify circulating serotypes, we screened serum from 50 of the NS1+ participants by RT-PCR and identified DENV1, 2, and 3 - with DENV1 and 3 codominant. We also performed whole-genome sequencing of DENV, for the first time in Nepal, using our new on-site capacity. Sequencing analysis demonstrated the DENV1 and 3 genomes clustered with sequences reported from India in 2019, and the DENV2 genome clustered with a sequence reported from China in 2018. ConclusionThese findings highlight DENVs geographic expansion from neighboring countries, identify China and India as the likely origin of the 2022 DENV cases in Nepal, and demonstrate the feasibility of building onsite capacity for more rapid genomic surveillance of circulating DENV. These ongoing efforts promise to protect populations in Nepal and beyond by informing the development and deployment of DENV drugs and vaccines in real time.

genomics↗