An overview of influenza A virus genes, protein functions, and replication cycle highlighting important updates.

The recent research findings on influenza A virus (IAV) genome biology prompted us to present a comprehensive overview of IAV genes, protein functions, and replication cycle. The eight gene segments of the IAV genome encode 17 proteins, each having unique functions contributing to virus fitness in the host. The polymerase genes are essential determinants of IAV pathogenicity and virulence; however, other viral components also play crucial roles in the IAV replication, transmission, and adaptation. Specific adaptive mutations within polymerase (PB2, PB1, and PA) and glycoprotein-hemagglutinin (HA) and neuraminidase (NA) genes, may facilitate interspecies transmission and adaptation of IAV. The HA-NA interplay is essential for establishing the IAV infection; the low pH triggers the inactivation of HA-receptor binding, leading to significantly lower NA activities, indicating that the enzymatic function of NA is dependent on HA binding. While the HA and NA glycoproteins are required to initiate infection, M1, M2, NS1, and NEP proteins are essential for cytoplasmic trafficking of viral ribonucleoproteins (vRNPs) and the assembly of the IAV virions. The mechanisms that enable IAV to exploit the host cell resources to advance the infection are discussed. A comprehensive understanding of IAV genome biology is essential for developing antivirals to combat the IAV disease burden.

The impact of the COVID-19 pandemic on children with medical complexity.

BACKGROUND: Descriptions of the COVID-19 pandemic's indirect consequences on children are emerging. We aimed to describe the impacts of the pandemic on children with medical complexity (CMC) and their families. METHODS: A one-time survey of Canadian paediatricians using the Canadian Paediatric Surveillance Program (CPSP) was conducted in Spring 2021. RESULTS: A total of 784 paediatricians responded to the survey, with 70% (n = 540) providing care to CMC. Sixty-seven (12.4%) reported an adverse health outcome due to a COVID-19 pandemic-related disruption in healthcare delivery. Disruption of the supply of medication and equipment was reported by 11.9% of respondents (n = 64). Respondents reported an interruption in family caregiving (47.5%, n = 252) and homecare delivery (40.8%, n = 218). Almost 47% of respondents (n = 253) observed a benefit to CMC due to COVID-19 related changes in healthcare delivery, including increased availability of virtual care and reduction in respiratory illness. Some (14.4%) reported that CMC were excluded from in-person learning when their peers without medical complexity were not. CONCLUSION: Canadian paediatricians reported that CMC experienced adverse health outcomes during the COVID-19 pandemic, including disruptions to family caregiving and community supports. They also describe benefits related to the pandemic including the expansion of virtual care. These results highlight the need for healthcare, community and education policymakers to collaborate with families to optimize their health.

The HIV-1 Gag Protein Displays Extensive Functional and Structural Roles in Virus Replication and Infectivity.

Once merely thought of as the protein responsible for the overall physical nature of the human immunodeficiency virus type 1 (HIV-1), the Gag polyprotein has since been elucidated to have several roles in viral replication and functionality. Over the years, extensive research into the polyproteins' structure has revealed that Gag can mediate its own trafficking to the plasma membrane, it can interact with several host factors and can even aid in viral genome packaging. Not surprisingly, Gag has also been associated with HIV-1 drug resistance and even treatment failure. Therefore, this review provides an extensive overview of the structural and functional roles of the HIV-1 Gag domains in virion integrity, functionality and infectivity.

Deciphering transmission dynamics and spillover of avian influenza viruses from avian species to swine populations globally.

Genome sequences of eleven avian influenza virus (AIV) subtypes have been reported in swine populations from seven countries until August 2020. To unravel the transmission dynamics and spillover events of AIVs from avian reservoirs to swine, full-length hemagglutinin (HA) sequences of AIV subtypes (n = 11) reported from various avian species and swine were retrieved from the 'Influenza Research Database'. Phylogenetic analysis identified closely related avian and swine AIV sequences suggesting potential spillover events from multiple domestic and wild avian species, including chicken, duck, pigeon, goose, quail, and aquatic birds to swine. Furthermore, N-linked glycosylation analysis of these closely related AIV sequences supported the possibility of multiple spillover events of highly pathogenic H5N1 and low pathogenic H9N2 viruses from various avian species to swine. The principal coordinate analysis further validated these findings for H5N1 and H9N2 viruses; however, spillover events of the other nine AIV subtypes were limited. Interestingly, the presence of potential mammalian adaptation markers, particularly in some of the swine H5N1, H7N9, and H9N2 viruses, suggested that these viruses may have already adapted in swine. The occurrence and circulation of these AIVs in swine, especially the H5N1 and H9N2 viruses with numerous spillover events from the avian reservoirs to swine, pose a significant threat in terms of their reassortment with endemic swine viruses or circulating human influenza viruses within the swine which may facilitate the emergence of a novel influenza virus strain with pandemic potential.

Modulatory influences of antiviral bioactive compounds on cell viability, mRNA and protein expression of cytochrome P450 3A4 and P-glycoprotein in HepG2 and HEK293 cells.

The induction of cytochrome P450 3A4 (CYP3A4) and P-glycoprotein (ABCB1) influence drug plasma, and eventually decreases the drugs' therapeutic effects. The effects of Plant-derived compounds (PCs) on drug-metabolising proteins are largely unknown. This study investigated the cytotoxicity, cell viability profiles and regulatory influences of four PCs (epigallocatechin gallate (EGCG), kaempferol-7-glucoside (K7G), luteolin (LUT) and ellagic acid (EGA)) on the mRNA and protein expressions of CYP3A4 and ABCB1 in HepG2 and HEK293 cells. After treatment with the PCs (0-400 µM) for 24 h, 80% (IC20) and 50% (IC50) cell viability were determined. The PCs were not toxic to HepG2 (ATP levels increased at IC20, insignificant change in LDH (lactate dehydrogenase) with the exception of LUT, and ABCB1 protein expressions decreased. The PCs decreased CYP3A4 at IC20 (except LUT), EGCG and K7G at IC20 decreased mRNA expression. For HEK293 cells, no significant change in ATP, except for EGCG IC20 and K7G IC50 which decreased and increased, respectively. LDH decreased at IC20, but LUT IC50 significant increase LDH. ABCB1 protein expression increased at both IC20 and IC50, but LUT and EGA at IC50 decreased mRNA expression. The PCs at IC20, and IC50 of LUT, K7G and of EGCG may enhance drug bioavailability.

Candidate gene polymorphisms related to lipid metabolism in Asian Indians living in Durban, South Africa.

BACKGROUND & OBJECTIVES: Asian Indians have been shown to have a high prevalence of metabolic syndrome (MetS), related to insulin resistance and possibly genetic factors. The aim of this study was to determine the genetic patterns associated with MetS in Asian Indians living in Durban, South Africa. METHODS: Nine hundred and ninety nine participants from the Phoenix Lifestyle Project underwent clinical, biochemical and genetic assessment. MetS was diagnosed according to the harmonized definition. The apolipoprotein A5 Q139X, lipoprotein lipase (LPL) Hinf I, human paraoxonase 1 (PON1) 192Arg/Gln, cholesteryl ester transfer protein (CETP) Taq1B, adiponectin 45T>G and leptin (LEP) 25CAG were genotyped by real-time polymerase chain reaction in participants with and without MetS. Univariate-unadjusted and multivariate-adjusted relations were conducted for all analyses. RESULTS: The prevalence of MetS was high (49.0%). More females had MetS than males (51.0 vs 42.8%). There was no significant difference in the distribution of genotypes between participants with MetS and those without. Males with the MetS who had the adiponectin TG genotype and human paraoxonase 1 AA genotype were more likely to have reduced high-density lipoprotein cholesterol (HDL-C) (P=0.001) and higher systolic blood pressure (P=0.018), respectively. INTERPRETATION & CONCLUSIONS: About half of the Asian Indians living in Phoenix had MetS. No association between the polymorphisms studied and the risk for MetS was observed. The adiponectin TG genotype may be associated with reduced HDL-C and the human paraoxonase 1 AA genotype with hypertension in males. This suggested that lifestyle factors were the major determinant for MetS in this ethnic group and the genetic risk might be related to its component risk factors than to MetS as an entity.

Audit of pulse oximetry screening for critical congenital heart disease (CCHD) in newborns.

OBJECTIVES: To assess the efficacy of a new screening protocol for critical congenital heart disease (CCHD). BACKGROUND: In March 2014, the Ontario Provincial Council for Maternal Child Health (PCMCH) recommended screening for CCHD, utilizing pulse oximetry to measure oxygen saturation as part of the newborn examination. However, this is yet to be implemented in all hospitals. METHOD: An audit of consecutive healthy normal newborn patients in a secondary level centre in Ontario with early adoption of the screening recommendation over a 1-year period was undertaken. RESULTS: The median age of screening was 25 hours (6 to 80 hours). Compliance was 88% (95% if one excludes deliveries by a midwife as they did not agree to comply). Four patients screened positive and were seen by a paediatrician in consultation but did not have CCHD (specificity 99.4%). CONCLUSIONS: The current study shows that screening was successfully implemented in a Canadian hospital, with high specificity (99.4%) and good compliance (88%). Reasons for non-acceptance of screening by midwives need to be addressed.

Essential role of eIF5-mimic protein in animal development is linked to control of ATF4 expression.

Translational control of transcription factor ATF4 through paired upstream ORFs (uORFs) plays an important role in eukaryotic gene regulation. While it is typically induced by phosphorylation of eIF2α, ATF4 translation can be also induced by expression of a translational inhibitor protein, eIF5-mimic protein 1 (5MP1, also known as BZW2) in mammals. Here we show that the 5MP gene is maintained in eukaryotes under strong purifying selection, but is uniquely missing in two major phyla, nematoda and ascomycota. The common function of 5MP from protozoa, plants, fungi and insects is to control translation by inhibiting eIF2. The affinity of human 5MP1 to eIF2ß was measured as being equivalent to the published value of human eIF5 to eIF2ß, in agreement with effective competition of 5MP with eIF5 for the main substrate, eIF2. In the red flour beetle, Tribolium castaneum, RNA interference studies indicate that 5MP facilitates expression of GADD34, a downstream target of ATF4. Furthermore, both 5MP and ATF4 are essential for larval development. Finally, 5MP and the paired uORFs allowing ATF4 control are conserved in the entire metazoa except nematoda. Based on these findings, we discuss the phylogenetic and functional linkage between ATF4 regulation and 5MP expression in this group of eukaryotes.

A post-partum single-dose TDF/FTC tail does not prevent the selection of NNRTI resistance in women receiving pre-partum ZDV and intrapartum single-dose nevirapine to prevent mother-to- child HIV-1 transmission.

Although the rates of vertical transmission of HIV in the developing world have improved to around 3% in countries like South Africa, resistance to antiretrovirals (ARV) used in Prevention of Mother-to-Child transmission (pMTCT) strategies may thwart such outcomes and affect the efficacy of future ARV regimens in mothers and children. This study conducted in Durban, South Africa, between 2010 and 2013 found a high rate of nevirapine (NVP) resistance among women receiving Zidovudine (AZT) from 14 weeks gestation, single dose nevirapine (sd NVP) at the onset of labor and a single dose of coformulated Tenofovir/Emtricitabine (TDF/FTC) postpartum. Using Sanger sequencing, high and intermediate levels of nevirapine (NVP) resistance were detected in 15/44 (34%) and in 1/44 (2%) of women tested, respectively. Most subjects selected the K103N mutation (22% (10/45) of all patients and 66% (10/15) of those with high-level NVP resistance). Such rate of NVP resistance is comparable to studies where only sd NVP was used. In conclusion, a post-partum single-dose TDF/FTC tail does not prevent the selection of NNRTI resistance in women receiving pre-partum ZDV and intrapartum sd NVP to prevent mother-to-child HIV-1 transmission.

Early Results of an Initiative to Assess Exposure to Firearm Violence in Ambulatory Care: Descriptive Analysis of Electronic Health Record Data.

BACKGROUND:  Current research on firearm violence is largely limited to patients who received care in emergency departments or inpatient acute care settings or who died. This is because standardized disease classification codes for firearm injury only represent bodily trauma. As a result, research on pathways and health impacts of firearm violence is largely limited to people who experienced acute bodily trauma and does not include the estimated millions of individuals who were exposed to firearm violence but did not sustain acute injury. Assessing and collecting data on exposure to firearm violence in ambulatory care settings can expand research and more fully frame the public health issue. OBJECTIVE: The aim of the study is to evaluate the demographic and clinical characteristics of patients who self-reported exposure to firearm violence during a behavioral health visit. METHODS: This study assessed early data from an initiative implemented in 2022 across a national network of ambulatory behavioral health centers to support trauma-informed care by integrating structured data fields on trauma exposure into an electronic health record behavioral health patient assessment form (SmartForm), as such variables are generally not included in standard outpatient medical records. We calculated descriptive statistics on clinic characteristics, patient demographics, and select clinical conditions among clinics that chose to implement the SmartForm and among patients who reported an exposure to firearm violence. Data on patient counts are limited to positive reports of exposure to firearm violence, and the representativeness of firearm exposure among all patients could not be calculated due to unknown variability in the implementation of the SmartForm. RESULTS: There were 323 of 629 (51%) clinics that implemented the SmartForm and reported at least 1 patient exposed to firearm violence. In the first 11 months of implementation, 3165 patients reported a recent or past exposure to firearm violence across the 323 clinics. Among patients reporting exposure, 52.7% (n=1669) were male, 38.8% (n=1229) were Black, 45.7% (n=1445) had posttraumatic stress disorder, 37.5% (n=1186) had a substance abuse disorder (other than nicotine), and 11.7% (n=371) had hypertension. CONCLUSIONS: Current research on firearm violence using standardized data is limited to acute care settings and death data. Early results from an initiative across a large network of behavioral health clinics demonstrate that a high number of clinics chose to implement the SmartForm, resulting in thousands of patients reporting exposure to firearm violence. This study demonstrates that collecting standardized data on firearm violence exposure in ambulatory care settings is feasible. This study further demonstrates that resultant data from ambulatory settings can be used for meaningful analysis in describing populations affected by firearm violence. The results of this study hold promise for further collection of structured data on exposure to firearm violence in ambulatory settings.

The (Re-)Emergence and Spread of Viral Zoonotic Disease: A Perfect Storm of Human Ingenuity and Stupidity.

Diseases that are transmitted from vertebrate animals to humans are referred to as zoonotic diseases. Although microbial agents such as bacteria and parasites are linked to zoonotic events, viruses account for a high percentage of zoonotic diseases that have emerged. Worryingly, the 21st century has seen a drastic increase in the emergence and re-emergence of viral zoonotic disease. Even though humans and animals have coexisted for millennia, anthropogenic factors have severely increased interactions between the two populations, thereby increasing the risk of disease spill-over. While drivers such as climate shifts, land exploitation and wildlife trade can directly affect the (re-)emergence of viral zoonotic disease, globalisation, geopolitics and social perceptions can directly facilitate the spread of these (re-)emerging diseases. This opinion paper discusses the "intelligent" nature of viruses and their exploitation of the anthropogenic factors driving the (re-)emergence and spread of viral zoonotic disease in a modernised and connected world.

Skeletal Muscle Mitochondrial Respiration and Exercise Intolerance in Patients With Heart Failure With Preserved Ejection Fraction.

Importance: The pathophysiology of exercise intolerance in patients with heart failure with preserved ejection fraction (HFpEF) remains incompletely understood. Multiple lines of evidence suggest that abnormal skeletal muscle metabolism is a key contributor, but the mechanisms underlying metabolic dysfunction remain unresolved. Objective: To evaluate the associations of skeletal muscle mitochondrial function using respirometric analysis of biopsied muscle fiber bundles from patients with HFpEF with exercise performance. Design, Setting, and Participants: In this cross-sectional study, muscle fiber bundles prepared from fresh vastus lateralis biopsies were analyzed by high-resolution respirometry to provide detailed analyses of mitochondrial oxidative phosphorylation, including maximal capacity and the individual contributions of complex I-linked and complex II-linked respiration. These bioenergetic data were compared between patients with stable chronic HFpEF older than 60 years and age-matched healthy control (HC) participants and analyzed for intergroup differences and associations with exercise performance. All participants were treated at a university referral center, were clinically stable, and were not undergoing regular exercise or diet programs. Data were collected from March 2016 to December 2017, and data were analyzed from November 2020 to May 2021. Main Outcomes and Measures: Skeletal muscle mitochondrial function, including maximal capacity and respiration linked to complex I and complex II. Exercise performance was assessed by peak exercise oxygen consumption, 6-minute walk distance, and the Short Physical Performance Battery. Results: Of 72 included patients, 50 (69%) were women, and the mean (SD) age was 69.6 (6.1) years. Skeletal muscle mitochondrial function measures were all markedly lower in skeletal muscle fibers obtained from patients with HFpEF compared with HCs, even when adjusting for age, sex, and body mass index. Maximal capacity was strongly and significantly correlated with peak exercise oxygen consumption (R = 0.69; P < .001), 6-minute walk distance (R = 0.70; P < .001), and Short Physical Performance Battery score (R = 0.46; P < .001). Conclusions and Relevance: In this study, patients with HFpEF had marked abnormalities in skeletal muscle mitochondrial function. Severely reduced maximal capacity and complex I-linked and complex II-linked respiration were associated with exercise intolerance and represent promising therapeutic targets.

Integrated complex care coordination for children with medical complexity: a mixed-methods evaluation of tertiary care-community collaboration.

BACKGROUND: Primary care medical homes may improve health outcomes for children with special healthcare needs (CSHCN), by improving care coordination. However, community-based primary care practices may be challenged to deliver comprehensive care coordination to complex subsets of CSHCN such as children with medical complexity (CMC). Linking a tertiary care center with the community may achieve cost effective and high quality care for CMC. The objective of this study was to evaluate the outcomes of community-based complex care clinics integrated with a tertiary care center. METHODS: A before- and after-intervention study design with mixed (quantitative/qualitative) methods was utilized. Clinics at two community hospitals distant from tertiary care were staffed by local community pediatricians with the tertiary care center nurse practitioner and linked with primary care providers. Eighty-one children with underlying chronic conditions, fragility, requirement for high intensity care and/or technology assistance, and involvement of multiple providers participated. Main outcome measures included health care utilization and expenditures, parent reports of parent- and child-quality of life [QOL (SF-36®, CPCHILD©, PedsQL™)], and family-centered care (MPOC-20®). Comparisons were made in equal (up to 1 year) pre- and post-periods supplemented by qualitative perspectives of families and pediatricians. RESULTS: Total health care system costs decreased from median (IQR) $244 (981) per patient per month (PPPM) pre-enrolment to $131 (355) PPPM post-enrolment (p=.007), driven primarily by fewer inpatient days in the tertiary care center (p=.006). Parents reported decreased out of pocket expenses (p<.0001). Parental QOL did not significantly change over the course of the study. Child QOL improved between baseline and 6 months in two PedsQL™ domains [Social (p=.01); Emotional (p=.003)], and between baseline and 1 year in two CPCHILD© domains [Health Standardization Section (p=.04); Comfort and Emotions (p=.03)], while total CPCHILD© score decreased between baseline and 1 year (p=.003). Parents and providers reported the ability to receive care close to home as a key benefit. CONCLUSIONS: Complex care can be provided in community-based settings with less direct tertiary care involvement through an integrated clinic. Improvements in health care utilization and family-centeredness of care can be achieved despite minimal changes in parental perceptions of child health.

Prevalence of hypothalamic-pituitary-adrenal axis suppression in children treated for asthma with inhaled corticosteroid.

OBJECTIVE: To determine the prevalence of hypothalamic-pituitary-adrenal (HPA) axis suppression in asthmatic children on inhaled corticosteroids (ICS). METHODS: Clinical and demographic variables were recorded on preconstructed, standardized forms. HPA axis suppression was measured by morning serum cortisol levels and confirmed by low-dose adrenocorticotropic hormone stimulation testing. RESULTS: In total, 214 children participated. Twenty children (9.3%, 95% CI 5.3% to 13.4%) had HPA axis suppression. Odds of HPA axis suppression increased with ICS dose (OR 1.005, 95% CI 1.003 to 1.009, P<0.001). All children with HPA axis suppression were on a medium or lower dose of ICS for their age (200 µg/day to 500 µg/day). HPA axis suppression was not predicted by drug type, dose duration, concomitant use of long-acting beta-agonist or nasal steroid, or clinical features. CONCLUSION: Laboratory evidence of HPA axis suppression exists in children taking ICS for asthma. Children should be regularly screened for the presence of HPA axis suppression when treated with high-dose ICS (>500 µg/day). Consideration should be given to screening children on medium-dose ICS.

OBJECTIF: Déterminer la prévalence de suppression de l'axe hypothalamo-hypophyso-surrénalien (HHA) chez les enfants asthmatiques traités au moyen de corticoïdes par aérosol (CPA). MÉTHODOLOGIE: Les chercheurs ont obtenu des variables cliniques et démographiques à l'aide de formulaires standardisés préétablis. Ils ont mesuré la suppression de l'axe HHA au moyen de taux de cortisol sérique le matin et l'ont confirmée grâce à un test de stimulation à l'hormone adrénocorticotrope (ACTH) à faible dose. RÉSULTATS: Sur les 214 enfants qui ont participé à l'étude, 20 (9,3 %, 95 % IC 5,3 % à 13,4 %) présentaient une suppression de l'axe HHA. Le risque d'une telle suppression augmentait proportionnellement à la dose de CPA (RRR 1,005, 95 % IC 1,003 à 1,009, P<0,001). Tous les enfants présentant une suppression de l'axe HHA prenaient une dose moyenne à faible de CPA compte tenu de leur âge (200 µg/jour à 500 µg/jour). La suppression de l'axe HHA ne pouvait être prédite selon les caractéristiques cliniques, le type de médicament, la durée de la dose ou l'utilisation concomitante de bêta-agonistes à longue durée d'action ou de stéroïdes par voie nasale. CONCLUSION: Il existe des preuves de laboratoire de suppression de l'axe HHA chez les enfants qui prenaient des CPA pour traiter leur asthme. Il faudrait procéder au dépistage régulier de la suppression de l'axe HHA chez des enfants traités au moyen de fortes doses de CPA (plus de 500 µg/jour). Il faut également envisager de dépister les enfants traités au moyen d'une dose moyenne de CPA.

Structural effects of HIV-1 subtype C integrase mutations on the activity of integrase strand transfer inhibitors in South African patients.

HIV-1 integrase enzyme is responsible for the integration of viral DNA into the host genomic DNA. Integrase strand transfer inhibitors (INSTIs) are highly potent antiretroviral agents that inhibit this process, and are internationally approved for the treatment of both naïve and treated HIV-1 patients. However, their long-term efficacy is threatened by development of drug resistance strains resulting in resistance mutations. This work aimed to examine the effect of INSTI resistance-associated mutations (RAMs) and polymorphisms on the structure of HIV-1 subtype C (HIV-1C) integrase. Genetic analysis was performed on seven HIV-1C infected individuals with virologic failure after at least 6 months of INSTI-based antiretroviral therapy, presenting at the King Edward VIII hospital in Durban, South Africa. These were compared with sequences from 41 INSTI-naïve isolates. Integrase structures of selected isolates were modeled on the SWISS model online server. Molecular docking and dynamics simulations were also conducted using AutoDock-Vina and AMBER 18 force fields, respectively. Only one INSTI-treated isolate (14.28%) harboured major mutations (G140A + Q148R) as well as the E157Q minor mutation. Interestingly, S119T and V151I were only found in patients failing raltegravir (an INSTI drug). Molecular modeling and docking showed that RAMs and polymorphisms associated with INSTI-based therapy affect protein stability and this is supported by their weakened hydrogen-bond interactions compared to the wild-type. To the best of our knowledge, this is the first study to identify a double mutant in the 140's loop region from South African HIV-1C isolates and study its effects on Raltegravir, Elvitegravir, and Dolutegravir binding.Communicated by Ramaswamy H. Sarma.

Understanding the co-evolutionary molecular mechanisms of resistance in the HIV-1 Gag and protease.

Due to high human immunodeficiency virus type 1 (HIV-1) subtype C infections coupled with increasing antiretroviral treatment failure, the elucidation of complex drug resistance mutational patterns arising through protein co-evolution is required. Despite the inclusion of potent protease inhibitors Lopinavir (LPV) and Darunavir (DRV) in second- and third-line therapies, many patients still fail treatment due to the accumulation of mutations in protease (PR) and recently, Gag. To understand the co-evolutionary molecular mechanisms of resistance in the HIV-1 PR and Gag, we performed 100 ns molecular dynamic simulations on multidrug resistant PR's when bound to LPV, DRV or a mutated A431V NC|p1 Gag cleavage site (CS). Here we showed that distinct changes in PR's active site, flap and elbow regions due to several PR resistance mutations (L10F, M46I, I54V, L76V, V82A) were found to alter LPV and DRV drug binding. However, binding was significantly exacerbated when the mutant PRs were bound to the NC|p1 Gag CS. Although A431V was shown to coordinate several residues in PR, the L76V PR mutation was found to have a significant role in substrate recognition. Consequently, a greater binding affinity was observed when the mutated substrate was bound to an L76V-inclusive PR mutant (Gbind: -62.46 ± 5.75 kcal/mol) than without (Gbind: -50.34 ± 6.28 kcal/mol). These data showed that the co-selection of resistance mutations in the enzyme and substrate can simultaneously constrict regions in PR's active site whilst flexing the flaps to allow flexible movement of the substrate and multiple, complex mechanisms of resistance to occur. Communicated by Ramaswamy H. Sarma.

Review of genome sequencing technologies in molecular characterization of influenza A viruses in swine.

The rapidly evolving antigenic diversity of influenza A virus (IAV) genomes in swine makes it imperative to detect emerging novel strains and track their circulation. We analyzed in our review the sequencing technologies used for subtyping and characterizing swine IAV genomes. Google Scholar, PubMed, and International Nucleotide Sequence Database Collaboration (INSDC) database searches identified 216 studies that have utilized Sanger, second-, and third-generation sequencing techniques to subtype and characterize swine IAV genomes up to 31 March 2021. Sanger dideoxy sequencing was by far the most widely used sequencing technique for generating either full-length (43.0%) or partial (31.0%) IAV genomes in swine globally; however, in the last decade, other sequencing platforms such as Illumina have emerged as serious competitors for the generation of whole-genome sequences of swine IAVs. Although partial HA and NA gene sequences were sufficient to determine swine IAV subtypes, whole-genome sequences were critical for determining reassortments and identifying unusual or less frequently occurring IAV subtypes. The combination of Sanger and second-generation sequencing technologies also greatly improved swine IAV characterization. In addition, the rapidly evolving third-generation sequencing platform, MinION, appears promising for on-site, real-time sequencing of complete swine IAV genomes. With a higher raw read accuracy, the use of the MinION could enhance the scalability of swine IAV testing in the field and strengthen the swine IAV disease outbreak response.

A systematic review of influenza A virus prevalence and transmission dynamics in backyard swine populations globally.

BACKGROUND: Backyard swine farming is critical to generating subsistence and food security in rural and peri-urban households in several developing countries. The objective of this systematic review was to analyze the molecular and serological prevalence of influenza A virus (IAV) in backyard swine populations globally. RESULTS: We identified 34 full-text research articles in NCBI-PubMed and Google Scholar databases that have reported IAV sero- and/or virological prevalence in backyard swine up to 11 July 2021. The highest number of studies were reported from Asia (n = 11) followed by North America (n = 10), South America (n = 6), Africa (n = 6), and Europe (n = 1). While the maximum number of studies (44.12%) reported human-to-swine transmission of IAV, swine-to-human (5.88%), poultry-to-swine (5.88%), and wild birds-to-swine (2.94%) transmissions were also reported. An overall higher IAV seroprevalence (18.28%) in backyard swine was detected compared to the virological prevalence (1.32%). The human-origin pandemic A(H1N1)pdm09 virus clade 1A.3.3.2 was the more frequently detected IAV subtype in virological studies (27.27%) than serological studies (18.92%). In addition, the avian-origin highly pathogenic H5N1 and H5N8 viruses were also detected, which further substantiated the evidence of avian-swine interactions in the backyards. CONCLUSION: Human-swine and avian-swine interactions in backyards may transmit IAV between species. Monitoring the circulation and evolution of IAV in backyard swine would help stakeholders make informed decisions to ensure sustainable backyard swine farming and public safety.

Molecular dynamic mechanism(s) of inhibition of bioactive antiviral phytochemical compounds targeting cytochrome P450 3A4 and P-glycoprotein.

P-glycoprotein (ABCB1) and cytochrome P450 3A4 (CYP3A4) metabolize almost all known human immunodeficiency virus' protease inhibitor drugs (PIs). Over induction of these proteins' activities has been linked to rapid metabolism of PIs which are then pumped out of the circulatory system, eventually leading to drug-resistance in HIV-positive patients. This study aims to determine, with the use of computational tools, the inhibitory potential of four phytochemical compounds (PCs) (epigallocatechin gallate (EGCG), kaempferol-7-glucoside (K7G), luteolin (LUT) and ellagic acid (EGA)) in inhibiting the activities of these drug-metabolizing proteins. The comparative analysis of the MM/GBSA results revealed that the binding affinity (ΔGbind) of EGCG and K7G for CYP3A4 and ABCB1 are higher than LUT and EGA and fall between the ΔGbind of the inhibitors of CYP3A4 and ABCB1 (Ritonavir (strong inhibitor) and Lopinavir (moderate inhibitor)). The structural analysis (RMSD, RMSF, RoG and protein-ligand interaction plots) also confirmed that EGCG and K7G showed similar inhibitory activities with the inhibitors. The study has shown that EGCG and K7G have inhibitory activities against the two proteins and assumes they could decrease intracellular efflux of PIs, consequently increasing the optimal concentration of PIs in the systemic circulation.Communicated by Ramaswamy H. Sarma.

Metagenomic Analysis of RNA Fraction Reveals the Diversity of Swine Oral Virome on South African Backyard Swine Farms in the uMgungundlovu District of KwaZulu-Natal Province.

Numerous RNA viruses have been reported in backyard swine populations in various countries. In the absence of active disease surveillance, a persistent knowledge gap exists on the diversity of RNA viruses in South African backyard swine populations. This is the first study investigating the diversity of oral RNA virome of the backyard swine in South Africa. We used three samples of backyard swine oral secretion (saliva) collected from three distantly located backyard swine farms (BSFs) in the uMgungundlovu District, KwaZulu-Natal, South Africa. Total viral RNA was extracted and used for the library preparation for deep sequencing using the Illumina HiSeq X instrument. The FASTQ files containing paired-end reads were analyzed using Genome Detective v 1.135. The assembled nucleotide sequences were analyzed using the PhyML phylogenetic tree. The genome sequence analysis identified a high diversity of swine enteric viruses in the saliva samples obtained from BSF2 and BSF3, while only a few viruses were identified in the saliva obtained from BSF1. The swine enteric viruses belonged to various animal virus families; however, two fungal viruses, four plant viruses, and five unclassified RNA viruses were also identified. Specifically, viruses of the family Astroviridae, according to the number of reads, were the most prevalent. Of note, the genome sequences of Rotavirus A (RVA) and Rotavirus C (RVC) at BSF2 and RVC and Hepatitis E virus (HEV) at BSF3 were also obtained. The occurrence of various swine enteric viruses in swine saliva suggests a high risk of diarrhoeic diseases in the backyard swine. Of note, zoonotic viruses in swine saliva, such as RVA, RVC, and HEV, indicate a risk of zoonotic spillover to the exposed human populations. We recommend the implementation of biosecurity to ensure sustainable backyard swine farming while safeguarding public health.