Application of in-situ casted ZnO-starch nanocomposite for packaging of strawberries (Fragaria x ananassa).

Nanomaterials have recently been used in packaging as antibacterial coatings because of their desirable antibacterial and antifungal properties. In this study, we describe the synthesis of zinc oxide nanoparticles (ZnO NPs) and evaluate potato peel starch based ZnO NPs nanocomposite paper (ZnO-starch NC paper) on microbiological activity, storage, and properties of strawberries (Fragaria x ananassa). The ZnO-starch NC paper was used to package the strawberries and then stored at 4 °C and 27 °C for 10 days, respectively, controls were incubated without the ZnO-starch NC paper. The ZnO NPs were synthesized and characterized, the formation of sizes ranging from 35 to 40 nm were confirmed by SEM and XRD. The SEM showed that the ZnO NPs were successfully embedded in the starch forming the ZnO-starch NC. The antimicrobial and antifungal analysis showed that the ZnO-starch NC paper was effective against the fungi, B. cinerea, gram positive bacteria, B. subtilis and S. aureus but not against the gram negative bacteria, E. coli and P. aeruginosa. Total phenolic compounds and Vitamin C content of both the ZnO-starch NC packaged and controls were found to be in the normal range of recommended quality parameters of strawberries as there was no change in intrinsic factors of the fruits during incubation. Application of the ZnO-starch NC in packaged strawberries resulted in the reduction of weight loss and microbial growth compared to the controls. The overall weight loss showed that the loss of moisture and nutrients was not statistically significant (p > 0.05). We conclude that the ZnO-starch NC packaging is a promising safe alternative to extend storage period and increase the shelf-life of strawberries.

Proteomic evaluation of nanotoxicity in aquatic organisms: A review.

The alteration of organisms protein functions by engineered nanoparticles (ENPs) is dependent on the complex interplay between their inherent physicochemical properties (e.g., size, surface coating, shape) and environmental conditions (e.g., pH, organic matter). To date, there is increasing interest on the use of 'omics' approaches, such as proteomics, genomics, and others, to study ENPs-biomolecules interactions in aquatic organisms. However, although proteomics has recently been applied to investigate effects of ENPs and associated mechanisms in aquatic organisms, its use remain limited. Herein, proteomics techniques widely applied to investigate ENPs-protein interactions in aquatic organisms are reviewed. Data demonstrates that 2DE and mass spectrometry and/or their combination, thereof, are the most suitable techniques to elucidate ENPs-protein interactions. Furthermore, current status on ENPs and protein interactions, and possible mechanisms of nanotoxicity with emphasis on those that exert influence at protein expression levels, and key influencing factors on ENPs-proteins interactions are outlined. Most reported studies were done using synthetic media and essay protocols and had wide variability (not standardized); this may consequently limit data application in actual environmental systems. Therefore, there is a need for studies using realistic environmental concentrations of ENPs, and actual environmental matrixes (e.g., surface water) to aid better model development of ENPs-proteins interactions in aquatic systems.

Detection of Enteric Viruses from Wastewater and River Water in Botswana.

Waterborne diseases remain a public health concern in developing countries where many lack access to safe water. Water testing mainly uses bacterial indicators to assess water quality, which may not fully indicate the threat from other non-bacterial pathogens like enteric viruses. This study was done to ascertain and establish the viral load, the temporal and spatial distribution of rotavirus A and norovirus (GI and GII) in sewage and river water samples. A total of 45 samples of raw and treated sewage, and surface water, were collected from a sludge activated wastewater treatment plant in Gaborone, and after treatment from the Notwane River, Botswana, over a period of 9 months (February 2016 to October 2016). Viruses were concentrated using polyethylene glycol/NaCl precipitation. Virus detection was performed using real-time polymerase chain reaction (RT-PCR). Rotavirus A was the most prevalent (84.4% positive samples), followed by Norovirus GI (48.9% positive samples), and Norovirus GII 46.7% positive samples). Detected viral loads went up to 104 genome copies per liter (copies/L) for all the viruses. The enteric viruses were detected in all the study sites with highest detection from site S1 (inlet). There was no significant association between physicochemical parameters and viral loads, except for pH which showed significant relationship with rotavirus and norovirus GII (p ≤ 0.05). This is the first study in Botswana to highlight the occurrence and quantification of the enteric viruses in treated and untreated wastewater, as well as surface water.

Occurrence, Fate, Effects, and Risks of Dexamethasone: Ecological Implications Post-COVID-19.

The recent outbreak of respiratory syndrome-coronavirus-2 (SARS-CoV-2), which causes coronavirus disease (COVID-19), has led to the widespread use of therapeutics, including dexamethasone (DEXA). DEXA, a synthetic glucocorticoid, is among the widely administered drugs used to treat hospitalized COVID-19 patients. The global COVID-19 surge in infections, consequent increasing hospitalizations, and other DEXA applications have raised concerns on eminent adverse ecological implications to aquatic ecosystems. Here, we aim to summarize published studies on DEXA occurrence, fate, and effects on organisms in natural and engineered systems as, pre-COVID, the drug has been identified as an emerging environmental contaminant. The results demonstrated a significant reduction of DEXA in wastewater treatment plants, with a small portion, including its transformation products (TPs), being released into downstream waters. Fish and crustaceans are the most susceptible species to DEXA exposure in the parts-per-billion range, suggesting potential deleterious ecological effects. However, there are data deficits on the implications of DEXA to marine and estuarine systems and wildlife. To improve DEXA management, toxicological outcomes of DEXA and formed TPs should entail long-term studies from whole organisms to molecular effects in actual environmental matrices and at realistic exposure concentrations. This can aid in striking a fine balance of saving human lives and protecting ecological integrity.

Next generation sequencing of near-full length genome of norovirus GII.4 from Botswana.

Noroviruses are highly diverse, with genotype GII.4 causing most epidemics. This study aimed to investigate the evolutionary dynamics of norovirus genogroup GII strains among acutely infected children under 5 years in Botswana, between 2016 and 2018. Reverse transcriptase polymerase chain reaction (RT-PCR) was used to amplify the whole norovirus genome, followed by next-generation sequencing using Oxford Nanopore technology. Twelve samples were successfully analyzed, with 11 identified as norovirus GII.4 Sydney [P31] and one as GII.4 Sydney [P13]. This study generated the first near-full length norovirus sequences in Botswana (93-95% coverage). Our results show that the norovirus GII.4 strains circulating in Botswana are under evolution through recombination and antigenic drift. Recombination in the GII.4 Sydney [P31] and GII.4 Sydney [P13] strains occurred in the ORF1/ORF2 junction and within ORF1, respectively. This study provides the first description of the GII.4 Sydney [P13] recombinant. Amino acid variation in the immunogenic sites was analyzed. Mutations in epitope A correlate with the emergence of novel norovirus GII.4 strains with altered antigenicity. In this study, we identified 43 unique amino acid substitutions in the VP1 region, with six occurring in epitopes, A (G295N, and E368Q) and E (S40T, N412D, N412K and T413H). The shell subdomain of the GII.4 Sydney [P13] variant was closely related to norovirus GII.17. Lastly, we also observed several mutations in the T cell restricted epitopes of both strains. Our study has made a novel contribution to understanding the evolution of norovirus GII.4 in Botswana.

Distribution and Genetic Variability of Sapoviruses in Africa.

In this review, we describe the distribution and genetic diversity of sapoviruses detected among humans, animals and the environment in African countries. Databases were searched for studies conducted in African countries and published between Jan 2005 and Mar 2019. Only studies where RT- PCR was used for initial detection were included in the systematic review. We identified 27 studies from 14 African countries with 18 focused on human sapoviruses, two on animal sapoviruses and seven on sapoviruses observed in the environment. Samples. The overall estimated pooled prevalence of human sapovirus infections among symptomatic and asymptomatic individuals was similar at 5.0% (95% Confidence Interval (CI): 3.0-7.0) and 2.0% (95% CI: 1.0-3.0), respectively. In environmental samples sapovirus detection rates ranged from 0% to 90% while in animal studies it was 1.7% to 34.8%. Multiple causes of gastroenteritis, sensitivity of detection method used, diversity of sapovirus strains and rotavirus vaccine coverage rate are some of the factors that could have contributed to the wide range of sapovirus detection rates that were reported. The studies reported human genogroups GI, GII, and GIV, with genogroup GI being the most prevalent. Some potential novel strains were detected from animal samples. Most studies genotyped a small portion of either the capsid and/or polymerase region. However, this is a limitation as it does not allow for detection of recombinants that occur frequently in sapoviruses. More studies with harmonized genotyping protocols that cover longer ranges of the sapovirus genome are needed to provide more information on the genomic characterization of sapoviruses circulating in African countries. Further investigations on animal to human transmission for sapoviruses are needed as inter-species transmissions have been documented for other viruses.

Genetic and epidemiological analysis of norovirus from children with gastroenteritis in Botswana, 2013-2015.

BACKGROUND: Norovirus is a leading cause of viral gastroenteritis worldwide with a peak of disease seen in children. The epidemiological analysis regarding the virus strains in Africa is limited. The first report of norovirus in Botswana was in 2010 and currently, the prevalence and circulating genotypes of norovirus are unknown, as the country has no systems to report the norovirus cases. This study investigated the prevalence, patterns and molecular characteristics of norovirus infections among children ≤5 years of age admitted with acute gastroenteritis at four hospitals in Botswana. METHODS: A total of 484 faecal samples were collected from children who were admitted with acute gastroenteritis during the rotavirus vaccine impact survey between July 2013 and December 2015. Norovirus was detected using real-time RT-PCR. Positive samples were genotyped using conventional RT-PCR followed by partial sequencing of the capsid and RdRp genes. Norovirus strains were determined by nucleotide sequence analysis using the online Norovirus Genotyping Tool Version 1.0, and confirmed using maximum likelihood tree construction as implemented in MEGA 6.0. RESULTS: The prevalence of norovirus was 9.3% (95% CI 6.7-11.9). The genotype diversity was dominated by the GII.4 strain at 69.7%. This was followed by GII.2, GII.12 each at 9.1%, GI.9 at 6.6% and GII.6, GII.10 each at 3.0%. The most common combined RdRp/Capsid genotype was the GII.Pe/GII.4 Sydney 2012. Norovirus was detected during most part of the year; however, there was a preponderance of cases in the wet season (December to March). CONCLUSION: The study showed a possible decline of norovirus infections in the last 10 years since the first report. An upward trend seen between 2013 and 2015 may be attributable to the success of rotavirus vaccine introductions in 2012. Knowledge of circulating genotypes, seasonal trends and overall prevalence is critical for prevention programming and possible future vaccine design implications.

Extraction of nanocellulose and in-situ casting of ZnO/cellulose nanocomposite with enhanced photocatalytic and antibacterial activity.

Polymer hosted metallic nanostructures with diverse applications have become a prominent area of materials science, engineering and technology. In this study nanocellulose (NC) was synthesized from oil palm empty fruit bunches biomass via alkaline treatment and acid hydrolysis and characterized. The obtained NC was used as a host polymer for the synthesis of zinc oxide (ZnO) nanostructures through in-situ solution casting method. Alkaline treatment and acid hydrolysis increased the percentage crystalline index from 35.7% to 43.3% and 53.3% respectively. X-ray diffraction studies pointed to cellulose I, with a monoclinic structure. Zinc oxide/cellulose nanocomposite displayed more photocatalytic activity than pure ZnO nanostructures upon degradation of methylene blue, and also improved antibacterial activity against Gram-positive Staphylococcus aureus and Gram-negative Escherichia coli.

Proteomic analysis reveals down-regulation of surfactant protein B in murine type II pneumocytes infected with influenza A virus.

Infection of type II alveolar epithelial (ATII) cells by influenza A viruses (IAV) correlates with severe respiratory disease in humans and mice. To understand pathogenic mechanisms during IAV infection of ATII cells, murine ATII cells were cultured to maintain a differentiated phenotype, infected with IAV-PR8, which causes severe lung pathology in mice, and proteomics analyses were performed using liquid chromatography-mass spectrometry. PR8 infection increased levels of proteins involved in interferon signaling, antigen presentation, and cytoskeleton regulation. Proteins involved in mitochondrial membrane permeability, energy metabolism, and chromatin formation had reduced levels in PR8-infected cells. Phenotypic markers of ATII cells in vivo were identified, confirming the differentiation status of the cultures. Surfactant protein B had decreased levels in PR8-infected cells, which was confirmed by immunoblotting and immunofluorescence assays. Analysis of ATII cell protein profiles will elucidate cellular processes in IAV pathogenesis, which may provide insight into potential therapies to modulate disease severity.

Differentiated phenotypes of primary murine alveolar epithelial cells and their susceptibility to infection by respiratory viruses.

Severe respiratory viral infections are associated with spread to the alveoli of the lungs. There are multiple murine models of severe respiratory viral infections that have been used to identify viral and host factors that contribute to disease severity. Primary cultures of murine alveolar epithelial cells provide a robust in vitro model to perform mechanistic studies that can be correlated with in vivo studies to identify cell type-specific factors that contribute to pathology within the alveoli of the lung during viral infection. In this study, we established an in vitro model to compare the responses of type I (ATI) and type II (ATII) alveolar epithelial cells to infection by respiratory viruses used in murine models: mouse-adapted severe acute respiratory syndrome-associated coronavirus (SARS-CoV, v2163), murine coronavirus MHV-1, and influenza A (H1N1) virus, strain PR8. Murine alveolar cells cultured to maintain an ATII cell phenotype, determined by expression of LBP180, were susceptible to infection by all three viruses. In contrast, ATII cells that were cultured to trans-differentiate into an ATI-like cell phenotype were susceptible to MHV-1 and PR8, but not mouse-adapted SARS-CoV. Epithelial cells produce cytokines in response to viral infections, thereby activating immune responses. Thus, virus-induced cytokine expression was quantified in ATI and ATII cells. Both cell types had increased expression of IL-1ß mRNA upon viral infection, though at different levels. While MHV-1 and PR8 induced expression of a number of shared cytokines in ATI cells, there were several cytokines whose expression was induced uniquely by MHV-1 infection. In summary, ATI and ATII cells exhibited differential susceptibilities and cytokine responses to infection by respiratory viruses. This in vitro model will be critical for future studies to determine the roles of these specialized cell types in the pathogenesis of respiratory viral infection.

Timing constraints of in vivo gag mutations during primary HIV-1 subtype C infection.

BACKGROUND: Aiming to answer the broad question "When does mutation occur?" this study examined the time of appearance, dominance, and completeness of in vivo Gag mutations in primary HIV-1 subtype C infection. METHODS: A primary HIV-1C infection cohort comprised of 8 acutely and 34 recently infected subjects were followed frequently up to 500 days post-seroconversion (p/s). Gag mutations were analyzed by employing single-genome amplification and direct sequencing. Gag mutations were determined in relation to the estimated time of seroconversion. Time of appearance, dominance, and completeness was compared for different types of in vivo Gag mutations. RESULTS: Reverse mutations to the wild type appeared at a median (IQR) of 62 (44;139) days p/s, while escape mutations from the wild type appeared at 234 (169;326) days p/s (p<0.001). Within the subset of mutations that became dominant, reverse and escape mutations appeared at 54 (30;78) days p/s and 104 (47;198) days p/s, respectively (p<0.001). Among the mutations that reached completeness, reverse and escape mutations appeared at 54 (30;78) days p/s and 90 (44;196) days p/s, respectively (p=0.006). Time of dominance for reverse mutations to and escape mutations from the wild type was 58 (44;105) days p/s and 219 (90;326) days p/s, respectively (p<0.001). Time of completeness for reverse and escape mutations was 152 (100;176) days p/s and 243 (101;370) days p/s, respectively (p=0.001). Fitting a Cox proportional hazards model with frailties confirmed a significantly earlier time of appearance (hazard ratio (HR): 2.6; 95% CI: 2.3-3.0), dominance (4.8 (3.4-6.8)), and completeness (3.6 (2.3-5.5)) of reverse mutations to the wild type Gag than escape mutations from the wild type. Some complex mutational pathways in Gag included sequential series of reversions and escapes. CONCLUSIONS: The study identified the timing of different types of in vivo Gag mutations in primary HIV-1 subtype C infection in relation to the estimated time of seroconversion. Overall, the in vivo reverse mutations to the wild type occurred significantly earlier than escape mutations from the wild type. This shorter time to incidence of reverse mutations remained in the subsets of in vivo Gag mutations that reached dominance or completeness.

Better control of early viral replication is associated with slower rate of elicited antiviral antibodies in the detuned enzyme immunoassay during primary HIV-1C infection.

BACKGROUND: Estimation of HIV incidence rates is important for timing interventions, planning prevention studies, and monitoring the epidemic. This requires accurate estimation of the "recency period" (also known as the "window period") between seroconversion and achievement of specific detectable levels of anti-HIV antibody titers, such as the standardized optical density (SOD) in the early phase of HIV-1 infection. METHODS: To obtain a better understanding of interpatient variation of the recency period, prospective measurements of antiviral antibody titers in the early phase of HIV-1 subtype C infection were quantified by Vironostika-LS. Time of seroconversion was estimated by Fiebig staging. RESULTS: The profiles of SOD values during the first year of infection commonly showed slow initial increase followed by a more rapid increase, although in some patients, SOD values increased rapidly soon after seroconversion. Using an SOD cutoff of 1.0, the average duration of the recency period in subtype C infection in the local epidemic in Botswana was estimated to be 151 days (95% confidence interval: 130 to 172 days) post seroconversion. The recency period was significantly associated (P = 0.007) with the level of viral replication during the first 2-3 months post seroconversion. Reduction of SOD values after initiation of antiretroviral therapy (ART) was a dominant pattern in antiretroviral drug (ARV)-treated subjects. CONCLUSIONS: Our data suggest that HIV incidence estimation based on sensitive/less sensitive enzyme immunoassay cross-sectional testing could be potentially improved by incorporation of viral load levels at the time of detection of a recent infection.

Viral load and CD4+ T-cell dynamics in primary HIV-1 subtype C infection.

BACKGROUND: Most knowledge of primary HIV-1 infection is based on subtype B studies, whereas the evolution of viral parameters in the early phase of HIV-1 subtype C infection is not well characterized. METHODS: The kinetics of viral RNA, proviral DNA, CD4+ T-cell count, and subsets of CD4+ T cells expressing CCR5 or CXCR4 were characterized in 8 acute and 62 recent subtype C infections over the first year postseroconversion. RESULTS: The viral RNA peak was 6.25 +/- 0.92 log10 copies per milliliter. After seroconversion, heterogeneity among acute cases was evident by patterns of change in viral load and CD4+ T-cell count over time. The patterns were supported by the rate of viral RNA decline from peak (P = 0.022), viral RNA means (P = 0.005), CD4 levels (P < 0.001), and CD4 decline to 350 (P = 0.011) or 200 (P = 0.046). Proviral DNA had no apparent peak and its mean was 2.59 +/- 0.69 log10 per 106 peripheral blood mononuclear cell. In recent infections, viral RNA set point was 4.00 +/- 0.97 log10 and viral RNA correlated inversely with CD4+ T cells (P < 0.001) and directly with proviral DNA (P < 0.001). CONCLUSIONS: Distinct patterns of viral RNA evolution may exist shortly after seroconversion in HIV-1 subtype C infection. The study provides better understanding of the early phase of subtype C infection.

Evolution of proviral gp120 over the first year of HIV-1 subtype C infection.

The evolution of proviral gp120 during the first year after seroconversion in HIV-1 subtype C infection was addressed in a case series of eight subjects. Multiple viral variants were found in two out of eight cases. Slow rate of viral RNA decline and high early viral RNA set point were associated with a higher level of proviral diversity from 0 to 200 days after seroconversion. Proviral divergence from MRCA over the same period also differed between subjects with slow and fast decline of viral RNA, suggesting that evolution of proviral gp120 early in infection may be linked to the level of viral RNA replication. Changes in the length of variable loops were minimal, and length reduction was more common than length increase. Potential N-linked glycosylation sites ranged +/-one site, showing common fluctuations in the V4 and V5 loops. These results highlight the role of proviral gp120 diversity and diversification in the pathogenesis of acute HIV-1 subtype C infection.