Influenza A Virus Uses PSMA2 for Downregulation of the NRF2-Mediated Oxidative Stress Response.

Influenza A virus (IAV), an obligatory intracellular parasite, uses host cellular molecules to complete its replication cycle and suppress immune responses. Proteasome subunit alpha type 2 (PSMA2) is a cellular protein highly expressed in IAV-infected human lung epithelial A549 cells. PSMA2 is part of the 20S proteasome complex that degrades or recycles defective proteins and involves proteolytic modification of many cellular regulatory proteins. However, the role of PSMA2 in IAV replication is not well understood. In this study, PSMA2 knockdown (KD) in A549 cells caused a significant reduction in extracellular progeny IAV, but intracellular viral protein translation and viral RNA transcription were not affected. This indicates that PSMA2 is a critical host factor for IAV maturation. To better understand the interplay between PSMA2 KD and IAV infection at the proteomic level, we used the SomaScan 1.3K version, which measures 1,307 proteins to analyze alterations induced by these treatments. We found seven cellular signaling pathways, including phospholipase C signaling, Pak signaling, and nuclear factor erythroid 2p45-related factor 2 (NRF2)-mediated oxidative stress response signaling, that were inhibited by IAV infection but significantly activated by PSMA2 KD. Further analysis of NRF2-mediated oxidative stress response signaling indicated IAV inhibits accumulation of reactive oxygen species (ROS), but ROS levels significantly increased during IAV infection in PSMA2 KD cells. However, IAV infection caused significantly higher NFR2 nuclear translocation that was inhibited in PSMA2 KD cells. This indicates that PSMA2 is required for NRF2-mediated ROS neutralization and that IAV uses PSMA2 to escape viral clearance via the NRF2-mediated cellular oxidative response. IMPORTANCE Influenza A virus (IAV) remains one of the most significant infectious agents, responsible for 3 million to 5 million illnesses each year and more than 50 million deaths during the 20th century. The cellular processes that promote and inhibit IAV infection and pathogenesis remain only partially understood. PSMA2 is a critical component of the 20S proteasome and ubiquitin-proteasome system, which is important in the replication of numerous viruses. This study examined host protein responses to IAV infection alone, PSMA2 knockdown alone, and IAV infection in the presence of PSMA2 knockdown and determined that interfering with PSMA2 function affected IAV maturation. These results help us better understand the importance of PSMA2 in IAV replication and may pave the way for designing additional IAV antivirals targeting PSMA2 or the host proteasome for the treatment of seasonal flu.

Effects of a Water, Sanitation, and Hygiene Mobile Health Program on Diarrhea and Child Growth in Bangladesh: A Cluster-randomized Controlled Trial of the Cholera Hospital-based Intervention for 7 Days (CHoBI7) Mobile Health Program.

BACKGROUND: The Cholera Hospital-Based Intervention for 7 Days (CHoBI7) mobile health (mHealth) program was a cluster-randomized controlled trial of diarrhea patient households conducted in Dhaka, Bangladesh. METHODS: Patients were block-randomized to 3 arms: standard message on oral rehydration solution use; health facility delivery of CHoBI7 plus mHealth (no home visits); and health facility delivery of CHoBI7 plus 2 home visits and mHealth. The primary outcome was reported diarrhea in the past 2 weeks collected monthly for 12 months. The secondary outcomes were stunting, underweight, and wasting at a 12-month follow-up. Analysis was intention-to-treat. RESULTS: Between 4 December 2016 and 26 April 2018, 2626 participants in 769 households were randomly allocated to 3 arms: 849 participants to the standard message arm, 886 to mHealth with no home visits arm, and 891 to the mHealth with 2 home visits. Children <5 years had significantly lower 12-month diarrhea prevalence in both the mHealth with 2 home visits arm (prevalence ratio [PR]: 0.73 [95% confidence interval {CI}, .61-.87]) and the mHealth with no home visits arm (PR: 0.82 [95% CI, .69-.97]). Children <2 years were significantly less likely to be stunted in both the mHealth with 2 home visits arm (33% vs 45%; odds ratio [OR]: 0.55 [95% CI, .31-.97]) and the mHealth with no home visits arm (32% vs 45%; OR: 0.54 [95% CI, .31-.96]) compared with children in the standard message arm. CONCLUSIONS: The CHoBI7 mHealth program lowered pediatric diarrhea and stunting among diarrhea patient households. CLINICAL TRIALS REGISTRATION: NCT04008134.

Process evaluation for the delivery of a water, sanitation and hygiene mobile health program: findings from the randomised controlled trial of the CHoBI7 mobile health program.

OBJECTIVE: The Cholera-Hospital-Based Intervention for 7-days (CHoBI7) mobile health (mHealth) program delivers mobile messages to diarrhoea patient households promoting water treatment and handwashing with soap. The randomised controlled trial (RCT) of the CHoBI7 mHealth program demonstrated this intervention was effective in significantly reducing diarrhoea and stunting amoung young children. The objective of this study was to assess the implementation of the CHoBI7 mHealth program in delivering mHealth messages during this RCT. METHODS: 517 diarrhoea patient households with 1777 participants received weekly text, voice and interactive voice response (IVR) messages from the CHoBI7 mHealth program over the 12-month program period. The program process evaluation indicators were the following: the percentage of CHoBI7 mHealth messages received and fully listened to by program households (program fidelity and dose), and household members reporting receiving and sharing an mHealth message from the program in the past two weeks (program reach). RESULTS: Ninety two percent of text messages were received by program households. Eighty three percent of voice and 86% of IVR messages sent were fully listened to by at least one household member. Eighty one percent of IVR quiz responses from households were answered correctly. Program households reported receiving a CHoBI7 mHealth message in the past two weeks at 79% of monthly household visits during the 12-month program. Seventy seven percent of participants reported sharing a program message with a spouse, 55% with a neighbour and 49% with a child during the program period. CONCLUSION: There was high fidelity, dose and reach of mobile messages delivered for the CHoBI7 mHealth program. This study presents an approach for process evaluation that can be implemented to evaluate future mHealth programs.

OBJECTIF: Le programme CHoBI7 (Cholera-Hospital-Based-Intervention-for-7-days) de santé mobile (mHealth) délivre des messages mobiles aux ménages avec patients atteints de diarrhée pour promouvoir le traitement de l'eau et le lavage des mains au savon. L'essai contrôlé randomisé (ECR) du programme mHealth CHoBI7 a démontré que cette intervention était efficace pour réduire de manière significative la diarrhée et le retard de croissance chez les jeunes enfants. L'objectif de cette étude était d'évaluer la mise en œuvre du programme CHoBI7 mHealth dans la diffusion des messages mHealth au cours de cet ECR. MÉTHODES: 517 ménages avec des patients atteints de diarrhée ont reçu chaque semaine des messages SMS, vocaux et de réponse vocale interactive (RVI) du programme mHealth de CHoBI7 pendant les 12 mois du programme. Les indicateurs d'évaluation du processus du programme étaient les suivants: le pourcentage de messages mHealth du programme CHoBI7 reçus (fidélité au programme et dose) et entièrement écoutés par les ménages participant au programme (fidélité au programme et dose) et les bénéficiaires déclarant avoir reçu et partagé un message mHealth du programme (portée du programme) au cours des deux dernières semaines. RÉSULTATS: 92% des SMS ont été reçus par les ménages participant au programme. 83% des messages vocaux et 86% des messages RVI envoyés ont été entièrement écoutés par au moins un membre du ménage. 81% des réponses aux quiz RVI des ménages ont été correctement répondues. Les ménages du programme ont déclaré avoir reçu un message CHoBI7 mHealth au cours des deux dernières semaines dans 79% des visites mensuelles des ménages pendant les 12 mois du programme. 77% des participants ont déclaré avoir partagé un message du programme avec un conjoint, 55% avec un voisin et 49% avec un enfant pendant la durée du programme. CONCLUSION: La dose et les messages délivrés dans le cadre du programme mHealth de CHoBI7 l'ont été avec une fidélité élevée. Cette étude présente une approche d'évaluation des processus qui peut être mise en œuvre pour évaluer les futurs programmes mHealth.

Child mouthing of soil and presence of animals in child sleeping spaces are associated with growth faltering among young children in Dhaka, Bangladesh (CHoBI7 Program).

OBJECTIVE: The objective of the study was to investigate potential risk factors for growth faltering among children under 5 years of age. METHOD: We conducted a prospective cohort study of 553 children under 5 years from diarrhoea patient households in urban Dhaka, Bangladesh. Height and weight measurements were obtained at baseline and at a 12-month follow-up. Caregivers of young children were administered a monthly questionnaire on household sociodemographic characteristics and hygiene practices. RESULTS: Children with caregiver reports of mouthing soil at the majority of household visits had a significant reduction in their height-for-age z-scores (HAZ) from baseline to the 12-month follow-up (ΔHAZ: -0.28 (95% confidence interval (CI): -0.51, -0.05)). A significant reduction in HAZ was also observed for children in households with animals in their sleeping space (ΔHAZ: -0.37 (95% CI: -0.71, -0.04)). CONCLUSION: These findings provide further evidence to support the hypothesis that child mouthing of soil and the presence of animals in the child's sleeping space are potential risk factors for growth faltering among young children. Interventions are urgently needed to provide clean play and sleeping spaces for young children to reduce exposure to faecal pathogens through child mouthing.

OBJECTIF: L'objectif de l'étude était d'examiner les facteurs de risque potentiels de retard de croissance chez les enfants de moins de 5 ans. MÉTHODE: Nous avons mené une étude de cohorte prospective sur 553 enfants de moins de 5 ans provenant de ménages avec des patients diarrhéiques dans la ville de Dhaka, au Bangladesh. Les mesures de taille et de poids ont été obtenues au départ et à 12 mois de suivi. Les personnes s'occupant de jeunes enfants (les gardiens) ont reçu un questionnaire mensuel sur les caractéristiques sociodémographiques des ménages et les pratiques d'hygiène. RÉSULTATS: Les enfants pour lesquels les gardiens ont déclaré qu'ils mâchouillaient de la terre lors de la majorité des visites à domicile présentaient une diminution du score Z de taille pour l'âge (TAZ) de manière significative de l'âge de référence au 12 mois de suivi (ΔTAZ −0,28 (intervalle de confiance (IC) à 95%: −0,51, −0,05)). Une réduction significative des TAZ a également été observée pour les enfants des ménages ayant des animaux dans leur espace de couchage (ΔTAZ −0,37 (IC95%: −0,71, −0,04)). CONCLUSION: Ces résultats apportent des données supplémentaires pour appuyer l'hypothèse selon laquelle le fait que l'enfant mâchouille de la terre et la présence d'animaux dans son espace de couchage sont des facteurs de risque potentiels de retard de croissance chez les jeunes enfants. Des interventions sont urgemment nécessaires pour fournir des espaces de jeu et de sommeil sains aux jeunes enfants afin de réduire l'exposition aux agents pathogènes fécaux par le mâchouillement des enfants.

Serum-reduced media impacts on cell viability and protein expression in human lung epithelial cells.

Serum starvation is a widely used condition in molecular biology experiments. Opti-MEM is a serum-reduced media used during transfection of genetic molecules into mammalian cells. However, the impact of such media on cell viability and protein synthesis is unknown. A549 human lung epithelial cell viability and morphology were adversely affected by growing in Opti-MEM. The cellular protein levels of chloride intracellular channel protein 1, proteasome subunit alpha Type 2, and heat shock 70 kDa protein 5 were dysregulated in A549 cells after growing in serum-reduced media. Small interfering RNA transfection was done in Dulbecco's modified Eagle's medium (DMEM) with 10% fetal bovine serum, and knockdown efficacy was determined compared with Opti-MEM. Similar amounts of knockdown of the target proteins were achieved in DMEM, and cell viability was higher compared with Opti-MEM after transfection. Careful consideration of the impact of Opti-MEM media during the culture or transfection is important for experimental design and results interpretation.

Rapid dipstick detection of Vibrio cholerae in household stored and municipal water in Dhaka, Bangladesh: CHoBI7 trial.

OBJECTIVE: In urban Dhaka, Bangladesh, 30% of source water samples collected from the households of patients with cholera had detectable Vibrio cholerae. These findings indicate an urgent need for a public health intervention for this population. The Crystal VC® dipstick test is a rapid method for detecting V. cholerae in stool and water. However, to date no study has investigated the use of the rapid dipstick test for household surveillance of stored drinking water. METHODS: The efficacy of the Crystal VC® dipstick test for detecting V. cholerae in the Dhaka city municipal water supply and stored household drinking water sources after enrichment for 18 h in alkaline peptone water (APW) was compared to bacterial culture as the gold standard. RESULTS: A total of 1648 water samples (824 stored household drinking water samples and 824 municipal water supply samples) were collected from households of patients with cholera. The overall specificity and sensitivity of the dipstick test compared to bacterial culture was 99.6% (95% confidence interval (CI): 99.2%, 99.9%) and 65.6% (95% CI: 55.2%, 75%), respectively. The specificities for stored household drinking water and Dhaka city municipal supply water compared to bacterial culture were 99.8% (95% CI: 99.1%, 100%) and 99.5% (95% CI: 98.6%, 99.9%), respectively (P = 0.138), and the sensitivities were 66.7% (95% CI: 43.0%, 85.4%) and 65.3% (95% CI: 53.5%, 76.0%), respectively (P = 0.891). CONCLUSION: The Crystal VC® dipstick is a promising screening tool for cholera outbreak surveillance in resource-limited settings where elimination of false-positive results is critical. The lower than expected sensitivity should be further investigated in future studies.

Randomized Controlled Trial of Hospital-Based Hygiene and Water Treatment Intervention (CHoBI7) to Reduce Cholera.

The risk for cholera infection is >100 times higher for household contacts of cholera patients during the week after the index patient seeks hospital care than it is for the general population. To initiate a standard of care for this high-risk population, we developed Cholera-Hospital-Based-Intervention-for-7-Days (CHoBI7), which promotes hand washing with soap and treatment of water. To test CHoBI7, we conducted a randomized controlled trial among 219 intervention household contacts of 82 cholera patients and 220 control contacts of 83 cholera patients in Dhaka, Bangladesh, during 2013-2014. Intervention contacts had significantly fewer symptomatic Vibrio cholerae infections than did control contacts and 47% fewer overall V. cholerae infections. Intervention households had no stored drinking water with V. cholerae and 14 times higher odds of hand washing with soap at key events during structured observation on surveillance days 5, 6, or 7. CHoBI7 presents a promising approach for controlling cholera among highly susceptible household contacts of cholera patients.

Evaluation of enrichment method for the detection of Vibrio cholerae O1 using a rapid dipstick test in Bangladesh.

BACKGROUND: To evaluate the specificity of the Crystal VC dipstick test for detecting cholera. METHODS: We compared direct testing using the Crystal VC dipstick test and testing after enrichment for 6 h in alkaline peptone water (APW) to bacterial culture as the gold standard. Samples positive by dipstick but negative by culture were also tested using PCR. RESULTS: Stool was collected from 125 patients. The overall specificities of the direct testing and testing after 6-h enrichment in APW compared to bacterial culture were 91.8% and 98.4% (P = 0.125), respectively, and the sensitivities were 65.6% and 75.0% (P = 0.07), respectively. CONCLUSION: The increase in the sensitivity of the Crystal VC kit with the use of the 6-h enrichment step in APW compared to direct testing was marginally significant. The Crystal VC dipstick had a much higher specificity than previously reported (91-98%). Therefore, this method might be a promising screening tool for cholera outbreak surveillance in resource-limited settings where elimination of false-positive results is critical.

Cholera outbreaks (2012) in three districts of Nepal reveal clonal transmission of multi-drug resistant Vibrio cholerae O1.

BACKGROUND: Although endemic cholera causes significant morbidity and mortality each year in Nepal, lack of information about the causal bacterium often hinders cholera intervention and prevention. In 2012, diarrheal outbreaks affected three districts of Nepal with confirmed cases of mortality. This study was designed to understand the drug response patterns, source, and transmission of Vibrio cholerae associated with 2012 cholera outbreaks in Nepal. METHODS: V. cholerae (n = 28) isolated from 2012 diarrhea outbreaks {n = 22; Kathmandu (n = 12), Doti (n = 9), Bajhang (n = 1)}, and surface water (n = 6; Kathmandu) were tested for antimicrobial response. Virulence properties and DNA fingerprinting of the strains were determined by multi-locus genetic screening employing polymerase chain reaction, DNA sequencing, and pulsed-field gel electrophoresis (PFGE). RESULTS: All V. cholerae strains isolated from patients and surface water were confirmed to be toxigenic, belonging to serogroup O1, Ogawa serotype, biotype El Tor, and possessed classical biotype cholera toxin (CTX). Double-mismatch amplification mutation assay (DMAMA)-PCR revealed the V. cholerae strains to possess the B-7 allele of ctx subunit B. DNA sequencing of tcpA revealed a point mutation at amino acid position 64 (N → S) while the ctxAB promoter revealed four copies of the tandem heptamer repeat sequence 5'-TTTTGAT-3'. V. cholerae possessed all the ORFs of the Vibrio seventh pandemic island (VSP)-I but lacked the ORFs 498-511 of VSP-II. All strains were multidrug resistant with resistance to trimethoprim-sulfamethoxazole (SXT), nalidixic acid (NA), and streptomycin (S); all carried the SXT genetic element. DNA sequencing and deduced amino acid sequence of gyrA and parC of the NAR strains (n = 4) revealed point mutations at amino acid positions 83 (S → I), and 85 (S → L), respectively. Similar PFGE (NotI) pattern revealed the Nepalese V. cholerae to be clonal, and related closely with V. cholerae associated with cholera in Bangladesh and Haiti. CONCLUSIONS: In 2012, diarrhea outbreaks in three districts of Nepal were due to transmission of multidrug resistant V. cholerae El Tor possessing cholera toxin (ctx) B-7 allele, which is clonal and related closely with V. cholerae associated with cholera in Bangladesh and Haiti.

Chloride Intracellular Channel Protein 1 (CLIC1) Is a Critical Host Cellular Factor for Influenza A Virus Replication.

(1) Background: Influenza A Virus (IAV) uses host cellular proteins during replication in host cells. IAV infection causes elevated expression of chloride intracellular channel protein 1 (CLIC1) in lung epithelial cells, but the importance of this protein in IAV replication is unknown. (2) In this study, we determined the role of CLIC1 in IAV replication by investigating the effects of CLIC1 knockdown (KD) on IAV viral protein translation, genomic RNA transcription, and host cellular proteome dysregulation. (3) Results: CLIC1 KD in A549 human lung epithelial cells resulted in a significant decrease in progeny supernatant IAV, but virus protein expression was unaffected. However, a significantly larger number of viral RNAs accumulated in CLIC1 KD cells. Treatment with a CLIC1 inhibitor also caused a significant reduction in IAV replication, suggesting that CLIC1 is an important host factor in IAV replication. SomaScan®, which measures 1322 proteins, identified IAV-induced dysregulated proteins in wild-type cells and in CLIC1 KD cells. The expression of 116 and 149 proteins was significantly altered in wild-type and in CLIC1 KD cells, respectively. A large number of the dysregulated proteins in CLIC1 KD cells were associated with cellular transcription and predicted to be inhibited during IAV replication. (4) Conclusions: This study suggests that CLIC1 is involved in later stages of IAV replication. Further investigation should clarify mechanism(s) for the development of anti-IAV drugs targeting CLIC1 protein.