Efficient protein tagging and cis-regulatory element engineering via precise and directional oligonucleotide-based targeted insertion in plants.

Efficient and precise targeted insertion holds great promise but remains challenging in plant genome editing. An efficient nonhomologous end-joining-mediated targeted insertion method was recently developed by combining clustered regularly interspaced short palindromic repeat (CRISPR)/Streptococcus pyogenes CRISPR-associated nuclease 9 (SpCas9) gene editing with phosphorothioate modified double-stranded oligodeoxynucleotides (dsODNs). Yet, this approach often leads to imprecise insertions with no control over the insertion direction. Here, we compared the influence of chemical protection of dsODNs on efficiency of targeted insertion. We observed that CRISPR/SpCas9 frequently induced staggered cleavages with 1-nucleotide 5' overhangs; we also evaluated the effect of donor end structures on the direction and precision of targeted insertions. We demonstrate that chemically protected dsODNs with 1-nucleotide 5' overhangs significantly improved the precision and direction control of target insertions in all tested CRISPR targeted sites. We applied this method to endogenous gene tagging in green foxtail (Setaria viridis) and engineering of cis-regulatory elements for disease resistance in rice (Oryza sativa). We directionally inserted 2 distinct transcription activator-like effector binding elements into the promoter region of a recessive rice bacterial blight resistance gene with up to 24.4% efficiency. The resulting rice lines harboring heritable insertions exhibited strong resistance to infection by the pathogen Xanthomonas oryzae pv. oryzae in an inducible and strain-specific manner.

Prime Editing in Plants: Prospects and Challenges.

Prime editors are reverse transcriptase (RT)-based genome editing tools utilizing double strand break (DSB) free mechanisms to decrease off-target editing in genomes and enhance the efficiency of targeted insertions. Multiple prime editors developed within a short span of time are a testament to the potential of this technique for targeted insertions. This is mainly because of the possibility of generation of all types of mutations including deletions, insertions, transitions and transversions. Prime editing reverses several bottlenecks of gene editing technologies that limit the biotechnological applicability to produce designer crops. The current mini review evaluates the status and evolution of prime editing technique in terms of the types of prime editors available till date up to prime editor 5 and twin prime editors, and the developments in plants in a systematic manner. The various factors affecting prime editing efficiency in plants including the effect of temperature, the pegRNA, and RT template amongst others are discussed in detail. We discuss the obstructions, key challenges, available resolutions, and provide future directions and further improvements feasible to elevate the efficiency in plants.

Metagenomics revealing molecular profiling of community structure and metabolic pathways in natural hot springs of the Sikkim Himalaya.

BACKGROUND: Himalaya is an ecologically pristine environment. The geo-tectonic activities have shaped various environmental niches with diverse microbial populations throughout the Himalayan biosphere region. Albeit, limited information is available in terms of molecular insights into the microbiome, including the uncultured microbes, of the Himalayan habitat. Hence, a vast majority of genomic resources are still under-explored from this region. Metagenome analysis has simplified the extensive in-depth exploration of diverse habitats. In the present study, the culture-independent whole metagenome sequencing methodology was employed for microbial diversity exploration and identification of genes involved in various metabolic pathways in two geothermal springs located at different altitudes in the Sikkim Himalaya. RESULTS: The two hot springs, Polok and Reshi, have distinct abiotic conditions. The average temperature of Polok and Reshi was recorded to be 62 °C and 43 °C, respectively. Both the aquatic habitats have alkaline geochemistry with pH in the range of 7-8. Community profile analysis revealed genomic evidence of plentiful bacteria, with a minute fraction of the archaeal population in hot water reservoirs of Polok and Reshi hot spring. Mesophilic microbes belonging to Proteobacteria and Firmicutes phyla were predominant at both the sites. Polok exhibited an extravagant representation of Chloroflexi, Deinococcus-Thermus, Aquificae, and Thermotogae. Metabolic potential analysis depicted orthologous genes associated with sulfur, nitrogen, and methane metabolism, contributed by the microflora in the hydrothermal system. The genomic information of many novel carbohydrate-transforming enzymes was deciphered in the metagenomic description. Further, the genomic capacity of antimicrobial biomolecules and antibiotic resistance were discerned. CONCLUSION: The study provided comprehensive molecular information about the microbial treasury as well as the metabolic features of the two geothermal sites. The thermal aquatic niches were found a potential bioresource of biocatalyst systems for biomass-processing. Overall, this study provides the whole metagenome based insights into the taxonomic and functional profiles of Polok and Reshi hot springs of the Sikkim Himalaya. The study generated a wealth of genomic data that can be explored for the discovery and characterization of novel genes encoding proteins of industrial importance.

Senna leaf curl virus: a novel begomovirus identified in Senna occidentalis.

Begomoviruses are whitefly-transmitted, single-stranded DNA viruses that infect a variety of cultivated (crop) and non-cultivated (weed) plants. The present study identified a novel begomovirus and satellites (alpha- and betasatellite) in Senna occidentalis (syn. Cassia occidentalis) showing leaf curl symptoms. The begomovirus shared a maximum sequence identity of 88.6 % with french bean leaf curl virus (JQ866297), whereas the alphasatellite and the betasatellite shared identities of 98 % and 90 % with ageratum yellow vein India alphasatellite (LK054802) and papaya leaf curl betasatellite (HM143906), respectively. No other begomovirus or satellites were detected in the suspected plants. We propose to name the virus "senna leaf curl virus" (SenLCuV).

In vitro regeneration and optimization of factors affecting Agrobacterium mediated transformation in Artemisia Pallens, an important medicinal plant.

Artemisia pallens is an important medicinal plant. In-vitro regeneration and multiplication of A. pallens have been established using attached cotyledons. Different growth regulators were considered for regeneration of multiple shoots. An average of 36 shoots per explants were obtained by culturing attached cotyledons on Murashige and Skoog's medium containing 2 mg/L BAP and 0.1 mg/L NAA, after 45 days. The shoots were rooted best on half Murashige and Skoog's medium with respect to media containing 1 mg/L IBA or 1 mg/L NAA. Different parameters such as type of bacterial strains, OD600 of bacterial culture, co-cultivation duration, concentration of acetosyringone and explants type were optimized for transient expression of the reporter gene. Agrobacterium tumefaciens harbouring pCambia1301 plasmid carrying ß-glucuronidase as a reporter gene and hygromycin phosphotransferase as plant selectable marker genes were used for genetic transformation of A. pallens. Hygromycin lethality test showed concentration of 15 mg/L were sufficient to inhibit the growth of attached cotyledons and multiple shoot buds of nontransgenics in selection media. Up to 83 % transient transformation was found when attached cotyledons were co-cultivated with Agrobacterium strain AGL1 for 2 days at 22 °C on shoot induction medium. The bacterial growth was eliminated by addition of cefotaxime (200 mg/L) in selection media. T0 transgenic plants were confirmed by GUS histochemical assay and further by polymerase chain reaction (PCR) using uidA and hpt gene specific primers. The study is useful in establishing technological improvement in A. pallens by genetic engineering.

Association of satellites with a mastrevirus in natural infection: complexity of Wheat dwarf India virus disease.

UNLABELLED: In contrast to begomoviruses, mastreviruses have not previously been shown to interact with satellites. This study reports the first identification of the association of satellites with a mastrevirus in field-grown plants. Two alphasatellite species were detected in different field samples of wheat infected with Wheat Dwarf India Virus (WDIV), a Cotton leaf curl Multan alphasatellite (CLCuMA) and a Guar leaf curl alphasatellite (GLCuA). In addition to the alphasatellites, a betasatellite, Ageratum yellow leaf curl betasatellite (AYLCB), was also identified in the wheat samples. No begomovirus was detected in the wheat samples, thus establishing association of the above-named satellites with WDIV. Agrobacterium-mediated inoculation of WDIV in wheat, in the presence of either of the alphasatellites or the betasatellite, resulted in infections inducing more severe symptoms. WDIV efficiently maintained each of the alphasatellites and the betasatellite in wheat. The satellites enhanced the level of WDIV DNA in wheat. Inoculation of the satellites isolated from wheat with various begomoviruses into Nicotiana tabacum demonstrated that these remain capable of interacting with the viruses with which they were first identified. Virus-specific small RNAs accumulated in wheat upon infection with WDIV but were lower in abundance in plants coinfected with the satellites, suggesting that both the alphasatellites and the betasatellite suppress RNA silencing. These results suggest that the selective advantage for the maintenance of the alphasatellites and the betasatellite by WDIV in the field is in overcoming RNA silencing-mediated host defense. IMPORTANCE: Wheat is the most widely cultivated cereal crop in the world. A number of viruses are important pathogens of wheat, including the viruses of the genus Mastrevirus, family Geminiviridae. This study reports the association of subgenomic components, called satellites (alpha- and betasatellites), with a mastrevirus, Wheat Dwarf India Virus (WDIV), isolated from two distant locations in India. This study reports the first identification of the satellites in a monocot plant. The satellites enhanced accumulation of WDIV and severity of disease symptoms. The satellites lowered the concentration of virus-specific small RNAs in wheat plants, indicating their silencing suppressor activity. The involvement of the satellites in symptom severity of the mastrevirus can have implications in the form of economic impact of the virus on crop yield. Understanding the role of the satellites in disease severity is important for developing disease management strategies.

ßC1 is a pathogenicity determinant: not only for begomoviruses but also for a mastrevirus.

ßC1 proteins, encoded by betasatellites, are known to be pathogenicity determinants, and they are responsible for symptom expression in many devastating diseases caused by begomoviruses. We report the involvement of ßC1 in pathogenicity determination of a mastrevirus. Analysis of field samples of wheat plants containing wheat dwarf India virus (WDIV) revealed the presence of a full-length and several defective betasatellite molecules. The detected betasatellite was identified as ageratum yellow leaf curl betasatellite (AYLCB). No begomovirus was detected in any of the samples. The full-length AYLCB contained an intact ßC1 gene, whereas the defective molecules contained complete or partial deletions of ßC1. Agroinoculation of wheat with the full-length AYLCB and WDIV or of tobacco with ageratum enation virus enhanced the pathogenicity and accumulation of the respective viruses, whereas the defective molecules could not. This study indicates that ßC1 is a pathogenicity determinant for WDIV and can interact functionally not only with begomoviruses but also with a mastrevirus.

Virus-induced gene silencing using a modified betasatellite: a potential candidate for functional genomics of crops.

Betasatellites are geminivirus-associated single-stranded DNA molecules that play an important role in symptom modulation. A VIGS vector was developed by modifying cotton leaf curl Multan betasatellite (CLCuMB). CLCuMB DNA was modified by replacing the ßC1 gene with a multiple cloning site. The silencing ability of the modified CLCuMB was investigated by cloning a fragment of a host gene (Su) or a reporter transgene (uidA) into the modified CLCuMB and co-agroinoculation with cotton leaf curl Multan virus, cotton leaf curl Kokhran virus, and ageratum enation virus, separately. The inoculated Nicotiana tabacum, N. benthamiana, Solanum lycopersicum, Arabidopsis thaliana and Gossypium hirsutum plants showed efficient silencing of the cognate genes.

Physiology, genomics, and evolutionary aspects of desert plants.

BACKGROUND: Despite the exposure to arid environmental conditions across the globe ultimately hampering the sustainability of the living organism, few plant species are equipped with several unique genotypic, biochemical, and physiological features to counter such harsh conditions. Physiologically, they have evolved with reduced leaf size, spines, waxy cuticles, thick leaves, succulent hydrenchyma, sclerophyll, chloroembryo, and photosynthesis in nonfoliar and other parts. At the biochemical level, they are evolved to perform efficient photosynthesis through Crassulacean acid metabolism (CAM) and C4 pathways with the formation of oxaloacetic acid (Hatch-Slack pathway) instead of the C3 pathway. Additionally, comparative genomics with existing data provides ample evidence of the xerophytic plants' positive selection to adapt to the arid environment. However, adding more high-throughput sequencing of xerophyte plant species is further required for a comparative genomic study toward trait discovery related to survival. Learning from the mechanism to survive in harsh conditions could pave the way to engineer crops for future sustainable agriculture. AIM OF THE REVIEW: The distinct physiology of desert plants allows them to survive in harsh environments. However, the genomic composition also contributes significantly to this and requires great attention. This review emphasizes the physiological and genomic adaptation of desert plants. Other important parameters, such as desert biodiversity and photosynthetic strategy, are also discussed with recent progress in the field. Overall, this review discusses the different features of desert plants, which prepares them for harsh conditions intending to translate knowledge to engineer plant species for sustainable agriculture. KEY SCIENTIFIC CONCEPTS OF REVIEW: This review comprehensively presents the physiology, molecular mechanism, and genomics of desert plants aimed towards engineering a sustainable crop.

Correction: Anwer et al. Molecular and Morphological Characterization of Exserohilum turcicum (Passerini) Leonard and Suggs Causing Northern Corn Leaf Blight of Maize in Bihar. Bioengineering 2022, 9, 403.

In the original publication [...].

Genome-resolved metagenomics inferred novel insights into the microbial community, metabolic pathways, and biomining potential of Malanjkhand acidic copper mine tailings.

Mine tailing sites provide profound opportunities to elucidate the microbial mechanisms involved in ecosystem functioning. In the present study, metagenomic analysis of dumping soil and adjacent pond around India's largest copper mine at Malanjkhand has been done. Taxonomic analysis deciphered the abundance of phyla Proteobacteria, Bacteroidetes, Acidobacteria, and Chloroflexi. Genomic signatures of viruses were predicted in the soil metagenome, whereas Archaea and Eukaryotes were noticed in water samples. Mesophilic chemolithotrophs, such as Acidobacteria bacterium, Chloroflexi bacterium, and Verrucomicrobia bacterium, were predominant in soil, whereas, in the water sample, the abundance of Methylobacterium mesophilicum, Pedobacter sp., and Thaumarchaeota archaeon was determined. The functional potential analysis highlighted the abundance of genes related to sulfur, nitrogen, methane, ferrous oxidation, carbon fixation, and carbohydrate metabolisms. The genes for copper, iron, arsenic, mercury, chromium, tellurium, hydrogen peroxide, and selenium resistance were found to be predominant in the metagenomes. Metagenome-assembled genomes (MAGs) were constructed from the sequencing data, indicating novel microbial species genetically related to the phylum predicted through whole genome metagenomics. Phylogenetic analysis, genome annotations, functional potential, and resistome analysis showed the resemblance of assembled novel MAGs with traditional organisms used in bioremediation and biomining applications. Microorganisms harboring adaptive mechanisms, such as detoxification, hydroxyl radical scavenging, and heavy metal resistance, could be the potent benefactions for their utility as bioleaching agents. The genetic information produced in the present investigation provides a foundation for pursuing and understanding the molecular aspects of bioleaching and bioremediation applications.

The curious case of genome packaging and assembly in RNA viruses infecting plants.

Genome packaging is the crucial step for maturation of plant viruses containing an RNA genome. Viruses exhibit a remarkable degree of packaging specificity, despite the probability of co-packaging cellular RNAs. Three different types of viral genome packaging systems are reported so far. The recently upgraded type I genome packaging system involves nucleation and encapsidation of RNA genomes in an energy-dependent manner, which have been observed in most of the plant RNA viruses with a smaller genome size, while type II and III packaging systems, majorly discovered in bacteriophages and large eukaryotic DNA viruses, involve genome translocation and packaging inside the prohead in an energy-dependent manner, i.e., utilizing ATP. Although ATP is essential for all three packaging systems, each machinery system employs a unique mode of ATP hydrolysis and genome packaging mechanism. Plant RNA viruses are serious threats to agricultural and horticultural crops and account for huge economic losses. Developing control strategies against plant RNA viruses requires a deep understanding of their genome assembly and packaging mechanism. On the basis of our previous studies and meticulously planned experiments, we have revealed their molecular mechanisms and proposed a hypothetical model for the type I packaging system with an emphasis on smaller plant RNA viruses. Here, in this review, we apprise researchers the technical breakthroughs that have facilitated the dissection of genome packaging and virion assembly processes in plant RNA viruses.

Innovative Approaches and Therapies to Enhance Neuroplasticity and Promote Recovery in Patients With Neurological Disorders: A Narrative Review.

Brain rehabilitation and recovery for people with neurological disorders, such as stroke, traumatic brain injury (TBI), and neurodegenerative diseases, depend mainly on neuroplasticity, the brain's capacity to restructure and adapt. This literature review aims to look into cutting-edge methods and treatments that support neuroplasticity and recovery in these groups. A thorough search of electronic databases revealed a wide range of research and papers investigating several neuroplasticity-targeting methods, such as cognitive training, physical activity, non-invasive brain stimulation, and pharmaceutical interventions. The results indicate that these therapies can control neuroplasticity and improve motor, mental, and sensory function. In addition, cutting-edge approaches, such as virtual reality (VR) and brain-computer interfaces (BCIs), promise to increase neuroplasticity and foster rehabilitation. However, many issues and restrictions still need to be resolved, including the demand for individualized treatments and the absence of defined standards. In conclusion, this review emphasizes the significance of neuroplasticity in brain rehabilitation. It identifies novel strategies and treatments that promise to enhance recovery in patients with neurological illnesses. Future studies should concentrate on improving these therapies and developing evidence-based standards to direct clinical practice and enhance outcomes for this vulnerable population.

An extensible vector toolkit and parts library for advanced engineering of plant genomes.

Plant biotechnology is rife with new advances in transformation and genome engineering techniques. A common requirement for delivery and coordinated expression in plant cells, however, places the design and assembly of transformation constructs at a crucial juncture as desired reagent suites grow more complex. Modular cloning principles have simplified some aspects of vector design, yet many important components remain unavailable or poorly adapted for rapid implementation in biotechnology research. Here, we describe a universal Golden Gate cloning toolkit for vector construction. The toolkit chassis is compatible with the widely accepted Phytobrick standard for genetic parts, and supports assembly of arbitrarily complex T-DNAs through improved capacity, positional flexibility, and extensibility in comparison to extant kits. We also provision a substantial library of newly adapted Phytobricks, including regulatory elements for monocot and dicot gene expression, and coding sequences for genes of interest such as reporters, developmental regulators, and site-specific recombinases. Finally, we use a series of dual-luciferase assays to measure contributions to expression from promoters, terminators, and from cross-cassette interactions attributable to enhancer elements in certain promoters. Taken together, these publicly available cloning resources can greatly accelerate the testing and deployment of new tools for plant engineering.

A novel mastrevirus infecting wheat in India.

A new mastrevirus (family Geminivridae) infecting wheat in India was detected by rolling-circle amplification (RCA). The complete nucleotide sequence of the virus was determined to be 2783 bp long. Analysis of the nucleotide sequence revealed identity and a genome organisation typical of a mastrevirus. An identical virus was detected in the candidate insect vector (leafhopper) collected from the field. Agroinoculation of young wheat plants with an infectious clone of the virus resulted in dwarfing of plants, identical to what was observed in the field, confirming that this novel virus was the causative agent of the disease. Considering the low degree of sequence identity to any known mastrevirus, the virus described here is suggested to be a member of a new species. Based on symptoms, we propose the name "wheat dwarf India virus".

Knockdown of capsid protein encoding novel ATPase domain inhibits genome packaging in potato leafroll virus.

Potato leafroll virus (PLRV) uses powerful molecular machines to package its genome into a viral capsid employing ATP as fuel. Although, recent bioinformatics and structural studies have revealed detailed mechanism of DNA packaging, little is known about the mechanochemistry of genome packaging in small plant viruses such as PLRV. We have identified a novel P-loop-containing ATPase domain with two Walker A-like motifs, two arginine fingers, and two sensor motifs distributed throughout the polypeptide chain of PLRV capsid protein (CP). The composition and arrangement of the ATP binding and hydrolysis domain of PLRV CP is unique and rarely reported. The discovery of the system sheds new light on the mechanism of viral genome packaging, regulation of viral assembly process, and evolution of plant viruses. Here, we used the RNAi approach to suppress CP gene expression, which in turn prevented PLRV genome packaging and assembly in Solanum tuberosum cv. Khufri Ashoka. Potato plants agroinfiltrated with siRNA constructs against the CP with ATPase domain exhibited no rolling symptoms upon PLRV infection, indicating that the silencing of CP gene expression is an efficient method for generating PLRV-resistant potato plants. In addition, molecular docking study reveals that the PLRV CP protein has ATP-binding pocket at the interface of each monomer. This further confirms that knockdown of the CP harboring ATP-binding domain could hamper the process of viral genome packaging and assembly. Moreover, our findings provide a robust approach to generate PLRV-resistant potato plants, which can be further extended to other species. Finally, we propose a new mechanism of genome packaging and assembly in plant viruses.

Molecular and Morphological Characterization of Exserohilum turcicum (Passerini) Leonard and Suggs Causing Northern Corn Leaf Blight of Maize in Bihar.

Maize is considered the third most important cereal crop in Asia after rice and wheat. Many diseases affect this crop due to the cultivation of various hybrids. This research aimed to characterize the causative agent of northern corn leaf blight disease in Bihar, India, caused by Exserohilum turcicum (Passerini) Leonard and Suggs. Leaf samples were collected from infected fields in five maize growing districts of Bihar in 2020-2022. A total of 45 fungal isolates from 135 samples were examined for cultural, morphological, and molecular characteristics and were identified as E. turcicum. The isolates were grouped into four groups based on colony color, i.e., olivaceous brown, blackish brown, whitish black, and grayish, and into two groups based on regular and irregular margins. The conidial shapes were observed to be elongated and spindle-shaped with protruding hilum, with conidial septa ranging from 2-12. Similarly, conidial length varied from 52.94 µm to 144.12 µm. ß-tubulin gene sequences analysis made it possible to verify the identities of fungal strains and the phylogenetic relationships of all isolates, which were clustered in the same clade. The ß-tubulin gene sequences of all the isolates showed a high level of similarity (100%) with reference isolates from GenBank accession numbers KU670342.1, KU670344.1, KU670343.1, KU670341.1, and KU670340.1. The findings of this study will serve as a baseline for future studies and will help to minimize yield losses.

Exserohilum turcicum (Passerini) Leonard and Suggs: Race Population Distribution in Bihar, India.

Northern corn leaf blight (NCLB) of maize, caused by Exserohilum turcicum (Pass.) Leonard and Suggs., is an important foliar disease common across maize-producing areas of the world, including Bihar, India. In this study, virulence and distribution of races were observed against Ht-resistant genes and also identified the E. turcicum race population distribution in Bihar. For that, 45 E. turcicum isolates were collected from maize fields in Bhagalpur, Begusarai, Khagaria, Katihar and Samastipur districts between 2020 and 2022. These isolates were screened on maize differential lines containing Ht1, Ht2, Ht3 and HtN1 resistance genes. Five different physiological races were observed based on the symptoms response of the differential maize lines. These races are race 0, race 1, race 3, race 23N and race 123N. E. turcicum race 3 was the most prevalent race having 26.6% frequency followed by race 0 (24.4%) and race 1 (22.2%) and the least prevalent races were race 23N and 123N having 13.3% each. Varied resistance response of different isolates was observed on differential lines having different resistant genes. Despite the fact that virulence was seen against all Ht resistance genes, NCLB control might be increased by combining qualitative Ht resistance genes with quantitative resistance.

Long-Term Neurological Impact of COVID-19.

INTRODUCTION: Recent research has observed the ability of coronavirus disease 2019 (COVID-19) to spread in the brain from the respiratory system. The associated neurological disorder includes encephalopathies, inflammatory syndromes, stroke, peripheral neuropathies, and various other central nervous system disorders. This study aims to highlight the long-term neurological sequelae in patients with COVID-19 disease. METHODS: This long-term study was carried out in the COVID-19 unit of a tertiary care hospital in Pakistan from July 2020 to July 2021. After obtaining informed consent, we enrolled 1000 patients who recovered from COVID-19 and were discharged. The participants were followed up after 30 and 90 days. RESULTS: At the time of enrollment, there were 602 (60.2%) males and 398 (39.8%) females. The most common neurological symptom on 30-day follow-up was headache (8.8%), followed by insomnia. The most common neurological symptom on day 90 follow-up was insomnia (5.07%), followed by an altered sense of smell (3.3%). CONCLUSION: COVID-19 tends to produce a wide range of neurological symptoms, ranging from headache to anosmia to increased risk of stroke, that complicates clinical management. Potential neurologic effects and drug interactions have been reported secondary to the medications used to treat COVID-19. In light of the aforementioned facts, COVID-19 could potentially have a long-term effect on the brain. Therefore, it is important that the clinicians must be aware of the potential neurologic complications. Lastly, proper follow-up is recommended that would aid in timely recognition and management of the neurological disorder.

Gastrointestinal Manifestation of COVID-19 in Hospitalized Patients.

INTRODUCTION: During the initial phase of the pandemic, gastrointestinal (GI) symptoms were less frequent but during the later stages, GI manifestations have become more frequent. This study aims to explore the prevalence of GI symptoms in COVID-19 patients, and also focuses on the frequency of these symptoms. METHODS: This longitudinal study was conducted in a COVID-19 unit of a tertiary care hospital, Pakistan. Data of patients hospitalized with COVID-19 infection between June 2021 and July 2021 was included in the study. A total of 412 participants were enrolled in the study via consecutive convenient non-probability sampling. Participants' symptoms and demographics were noted in a self-structured questionnaire. The collected data were analyzed using Statistical Package for Social Sciences (SPSS), version 23.0 (IBM Corp., Armonk, NY). RESULTS: A total of 261 (63.3%) participants had a minimum of one GI symptom. The most common symptom was anorexia (43.9%), followed by diarrhea (24.7%) and nausea/vomiting (17.9%). CONCLUSION:  Our study indicates high frequency of COVID-19 patients reporting GI symptoms. Anorexia, diarrhea, nausea, and vomiting were commonly reported symptoms. Therefore, COVID 19 testing should be considered with patients presenting with GI symptoms.