Rotavirus trends and distribution of genotypes before and during COVID-19 pandemic era: Bangladesh, 2017-2021.

Rotavirus gastroenteritis is accountable for an estimated 128 500 deaths among children younger than 5 years worldwide, and the majority occur in low-income countries. Although the clinical trials of rotavirus vaccines in Bangladesh revealed a significant reduction of severe rotavirus disease by around 50%, the vaccines are not yet included in the routine immunization program. The present study was designed to provide data on rotavirus diarrhea with clinical profiles and genotypes before (2017-2019) and during the COVID-19 pandemic period (2020-2021). Fecal samples were collected from 2% of the diarrheal patients at icddr,b Dhaka hospital of all ages between January 2017 and December 2021 and were tested for VP6 rotavirus antigen using ELISA. The clinical manifestations such as fever, duration of diarrhea and hospitalization, number of stools, and dehydration and so on were collected from the surveillance database (n = 3127). Of the positive samples, 10% were randomly selected for genotyping using Sanger sequencing method. A total of 12 705 fecal samples were screened for rotavirus A antigen by enzyme immunoassay. Overall, 3369 (27%) were rotavirus antigen-positive, of whom children <2 years had the highest prevalence (88.6%). The risk of rotavirus A infection was 4.2 times higher in winter than in summer. Overall, G3P[8] was the most prominent genotype (45.3%), followed by G1P[8] (32.1%), G9P[8] (6.8%), and G2P[4] (6.1%). The other unusual combinations, such as G1P[4], G1P[6], G2P[6], G3P[4], G3P[6], and G9P[6], were also present. Genetic analysis on Bangladeshi strains revealed that the selection pressure (dN/dS) was estimated as <1. The number of hospital visits showed a 37% drop during the COVID-19 pandemic relative to the years before the pandemic. Conversely, there was a notable increase in the rate of rotavirus positivity during the pandemic (34%, p < 0.00) compared to the period before COVID-19 (23%). Among the various clinical symptoms, only the occurrence of watery stool significantly increased during the pandemic. The G2P[4] strain showed a sudden rise (19%) in 2020, which then declined in 2021. In the same year, G1P[8] was more prevalent than G3P[8] (40% vs. 38%, respectively). The remaining genotypes were negligible and did not exhibit much fluctuation. This study reveals that the rotavirus burden remained high during the COVID-19 prepandemic and pandemic in Bangladesh. Considering the lack of antigenic variations between the circulating and vaccine-targeted strains, integrating the vaccine into the national immunization program could reduce the prevalence of the disease, the number of hospitalizations, and the severity of cases.

An essential vesicular-trafficking phospholipase mediates neutral lipid synthesis and contributes to hemozoin formation in Plasmodium falciparum.

BACKGROUND: Plasmodium falciparum is the pathogen responsible for the most devastating form of human malaria. As it replicates asexually in the erythrocytes of its human host, the parasite feeds on haemoglobin uptaken from these cells. Heme, a toxic by-product of haemoglobin utilization by the parasite, is neutralized into inert hemozoin in the food vacuole of the parasite. Lipid homeostasis and phospholipid metabolism are crucial for this process, as well as for the parasite's survival and propagation within the host. P. falciparum harbours a uniquely large family of phospholipases, which are suggested to play key roles in lipid metabolism and utilization. RESULTS: Here, we show that one of the parasite phospholipase (P. falciparum lysophospholipase, PfLPL1) plays an essential role in lipid homeostasis linked with the haemoglobin degradation and heme conversion pathway. Fluorescence tagging showed that the PfLPL1 in infected blood cells localizes to dynamic vesicular structures that traffic from the host-parasite interface at the parasite periphery, through the cytosol, to get incorporated into a large vesicular lipid rich body next to the food-vacuole. PfLPL1 is shown to harbour enzymatic activity to catabolize phospholipids, and its transient downregulation in the parasite caused a significant reduction of neutral lipids in the food vacuole-associated lipid bodies. This hindered the conversion of heme, originating from host haemoglobin, into the hemozoin, and disrupted the parasite development cycle and parasite growth. Detailed lipidomic analyses of inducible knock-down parasites deciphered the functional role of PfLPL1 in generation of neutral lipid through recycling of phospholipids. Further, exogenous fatty-acids were able to complement downregulation of PfLPL1 to rescue the parasite growth as well as restore hemozoin levels. CONCLUSIONS: We found that the transient downregulation of PfLPL1 in the parasite disrupted lipid homeostasis and caused a reduction in neutral lipids essentially required for heme to hemozoin conversion. Our study suggests a crucial link between phospholipid catabolism and generation of neutral lipids (TAGs) with the host haemoglobin degradation pathway.

Plasmodium falciparum OTU-like cysteine protease (PfOTU) is essential for apicoplast homeostasis and associates with noncanonical role of Atg8.

The metabolic pathways associated with the mitochondrion and the apicoplast in Plasmodium, 2 parasite organelles of prokaryotic origin, are considered as suitable drug targets. In the present study, we have identified functional role of a novel ovarian tumour unit (OTU) domain-containing cysteine protease of Plasmodium falciparum (PfOTU). A C-terminal regulatable fluorescent affinity tag on native protein was utilised for its localization and functional characterization. Detailed studies showed vesicular localization of PfOTU and its association with the apicoplast. Degradation-tag mediated knockdown of PfOTU resulted in abnormal apicoplast development and blocked development of parasites beyond early-schizont stages in subsequent cell cycle; downregulation of PfOTU hindered apicoplast protein import. Further, the isoprenoid precursor-mediated parasite growth-rescue experiments confirmed that PfOTU knockdown specifically effect development of functional apicoplast. We also provide evidence for a possible biological function of PfOTU in membrane deconjugation of Atg8, which may be linked with the apicoplast protein import. Overall, our results show that the PfOTU is involved in apicoplast homeostasis and associates with the noncanonical function of Atg8 in maintenance of parasite apicoplast.

Eps15 homology domain containing protein of Plasmodium falciparum (PfEHD) associates with endocytosis and vesicular trafficking towards neutral lipid storage site.

The human malaria parasite, Plasmodium falciparum, takes up numerous host cytosolic components and exogenous nutrients through endocytosis during the intra-erythrocytic stages. Eps15 homology domain-containing proteins (EHDs) are conserved NTPases, which are implicated in membrane remodeling and regulation of specific endocytic transport steps in eukaryotic cells. In the present study, we have characterized the dynamin-like C-terminal Eps15 homology domain containing protein of P. falciparum (PfEHD). Using a GFP-targeting approach, we studied localization and trafficking of PfEHD in the parasite. The PfEHD-GFP fusion protein was found to be a membrane bound protein that associates with vesicular network in the parasite. Time-lapse microscopy studies showed that these vesicles originate at parasite plasma membrane, migrate through the parasite cytosol and culminate into a large multi-vesicular like structure near the food-vacuole. Co-staining of food vacuole membrane showed that the multi-vesicular structure is juxtaposed but outside the food vacuole. Labeling of parasites with neutral lipid specific dye, Nile Red, showed that this large structure is neutral lipid storage site in the parasites. Proteomic analysis identified endocytosis modulators as PfEHD associated proteins in the parasites. Treatment of parasites with endocytosis inhibitors obstructed the development of PfEHD-labeled vesicles and blocked their targeting to the lipid storage site. Overall, our data suggests that the PfEHD is involved in endocytosis and plays a role in the generation of endocytic vesicles at the parasite plasma membrane, that are subsequently targeted to the neutral lipid generation/storage site localized near the food vacuole.

The prokaryotic ClpQ protease plays a key role in growth and development of mitochondria in Plasmodium falciparum.

The ATP-dependent ClpQY system is a prokaryotic proteasome-like multi-subunit machinery localized in the mitochondrion of malaria parasite. The ClpQY machinery consists of ClpQ threonine protease and ClpY ATPase. In the present study, we have assessed cellular effects of transient interference of PfClpQ protease activity in Plasmodium falciparum using a trans-dominant negative approach combined with FKBP degradation domain system. A proteolytically inactive mutant PfClpQ protein [PfClpQ(mut)] fused with FKBP degradation domain was expressed in parasites, which gets stabilized by Shield1 drug treatment. We show that the inactive PfClpQ(mut) interacts with wild-type PfClpQ and associates within multi-subunit complex in the parasite. Stabilization of the PfClpQ(mut) and its association in the protease machinery caused dominant negative effect in the transgenic parasites, which disrupted the growth cycle of asexual blood stage parasites. The mitochondria in these parasites showed abnormal morphology, these mitochondria were not able to grow and divide in the parasite. We further show that the dominant negative effect of PfClpQ(mut) disrupted transcription of mitochondrial genome encoded genes, which in turn blocked normal development and functioning of the mitochondria.

The cysteine-rich regions of Plasmodium falciparum RON2 bind with host erythrocyte and AMA1 during merozoite invasion.

Invasion of Plasmodium falciparum merozoites into host erythrocyte involves a series of highly specific and sequential interaction between merozoite and host erythrocyte surface protein. The key step in the invasion process is the formation of a tight protein-protein interaction between host and parasite called as moving junction. A number of parasite proteins secreted from two organelles, microneme and rhoptry, play a role in initial interaction and junction formation between merozoite with host red blood cells (RBCs) during the invasion process. In the present study, we investigated the role of different domains of a P. falciparum rhoptry neck protein PfRON2. Immunofluorescence assay revealed close association of PfAMA1 and PfRON2 in the merozoites during the invasion process. PfRON2 domains were expressed on COS-7 cell surface, and their interaction was analysed with host RBCs and PfAMA1 protein by rosetting assays. The rosetting assays suggest that the C-terminal cysteine-rich domain of PfRON2 plays a role in binding with host erythrocyte. The C-terminal as well as the central cysteine-rich domain of PfRON2 interact with PfAMA1; this binding can be inhibited by monoclonal antibody (mAb 4 G2) against PfAMA1, suggesting that the hydrophobic groove of PfAMA1 binds to PfRON2. These results suggest that PfRON2 plays a role in merozoite invasion and thus it can be an important vaccine candidate antigen.

Hospital-based Surveillance for Pediatric Norovirus Gastroenteritis in Bangladesh, 2012-2016.

BACKGROUND: Globally, noroviruses are recognized as an important cause of acute gastroenteritis (AGE), but data from low and middle-income countries are limited. AIMS: To examine the epidemiology and strain diversity of norovirus infections among children hospitalized for AGE in Bangladesh. METHODS: We implemented active surveillance of children <5 years of age hospitalized with AGE at 8 geographically dispersed tertiary care hospitals in Bangladesh from July 2012 to June 2016. We tested random samples of AGE cases stratified by site and age group for norovirus by real-time RT-PCR. Noro-positive specimens were genotyped. Coinfection with rotavirus was assessed based on prior EIA testing. RESULTS: We enrolled 5622 total AGE cases, of which 1008 were tested for norovirus. Total of 137 (14%) AGE cases tested positive for norovirus (range, 11%-17% by site). Most (94%) norovirus-associated hospitalizations were among children less than 2 years of age. Norovirus was detected year-round, with higher detection from March to June (20%-38%) and November to January (9%-18%). Genogroup II (GII) noroviruses were detected in 96% of cases, and the most frequent genotypes were GII.4 Sydney [P4 New Orleans] (33%), GII.3 [P16] (20%), and GII.4 Sydney [P16] (11%). The proportion of norovirus-positive specimens was significantly greater among rotavirus-negative AGE patients compared with rotavirus-positive AGE patients (27% vs. 5%, P < 0.001). As measured by the Vesikari severity score, a similar proportion of norovirus and rotavirus positive AGE patients were considered severe (68% vs. 70%, P = 0.86). CONCLUSIONS: Norovirus is an important cause of AGE hospitalization in Bangladeshi children with most infections caused by GII viruses.

Reference-Free Assessment of Speech Intelligibility Using Bispectrum of an Auditory Neurogram.

Sensorineural hearing loss occurs due to damage to the inner and outer hair cells of the peripheral auditory system. Hearing loss can cause decreases in audibility, dynamic range, frequency and temporal resolution of the auditory system, and all of these effects are known to affect speech intelligibility. In this study, a new reference-free speech intelligibility metric is proposed using 2-D neurograms constructed from the output of a computational model of the auditory periphery. The responses of the auditory-nerve fibers with a wide range of characteristic frequencies were simulated to construct neurograms. The features of the neurograms were extracted using third-order statistics referred to as bispectrum. The phase coupling of neurogram bispectrum provides a unique insight for the presence (or deficit) of supra-threshold nonlinearities beyond audibility for listeners with normal hearing (or hearing loss). The speech intelligibility scores predicted by the proposed method were compared to the behavioral scores for listeners with normal hearing and hearing loss both in quiet and under noisy background conditions. The results were also compared to the performance of some existing methods. The predicted results showed a good fit with a small error suggesting that the subjective scores can be estimated reliably using the proposed neural-response-based metric. The proposed metric also had a wide dynamic range, and the predicted scores were well-separated as a function of hearing loss. The proposed metric successfully captures the effects of hearing loss and supra-threshold nonlinearities on speech intelligibility. This metric could be applied to evaluate the performance of various speech-processing algorithms designed for hearing aids and cochlear implants.