On the Preorganization of the Active Site of Choline Oxidase for Hydride Transfer and Tunneling Mechanism

Choline oxidase catalyzes the two-step oxidation of choline to glycine betaine, one of limited osmoprotectants, with the formation of betaine aldehyde as an enzyme bound intermediate. Glycine betaine accumulates in the cytoplasm of plants and bacteria as a defensive mechanism to withstand hyperosmolarity and elevated temperatures. This makes the genetic engineering of relevant plants which lack the property of salt accumulation of economic interest, and the biosynthetic pathway of the osmolyte a potential drug target in microbial infections. The reaction of alcohol oxidation occurs via a hydride ion tunneling transfer from the substrate donor to a flavin acceptor within a highly preorganized active site environment in which choline and FAD are in a rigidly close proximity. In this dissertation, factors contributing to the enzyme-substrate preorganization which is required for the hydride ion tunneling reaction mechanism in choline oxidase have been investigated. Crystallographic studies of wild-type choline oxidase revealed a covalent linkage between C8M atom of the FAD isoalloxazine ring and the N(3) atom of the side chain of a histidine at position 99, and a solvent excluded cavity in the substrate binding domain containing glutamic acid at position 312 as the only negatively charged amino acid residue in the active site of the enzyme. The role of the histidine residue and the contribution of the 8á-N(3)-histidyl covalent linkage of the flavin cofactor to the reaction of alcohol oxidation was investigated in a variant form of choline oxidase in which the histidine residue was replaced with an asparagine. The role of the glutamate residue and the importance of the spatial location of the negative charge at position 312 was investigated in variant forms of choline oxidase in which the negatively charged residue was replaced with glutamine and aspartate. Mechanistic data obtained for the variant enzymes and their comparison to previous data obtained for wild-type choline oxidase are consistent with the residues at positions 99 and 312 being important for relative positioning of the hydride ion donor and acceptor. The residues are important for the enzyme-substrate preorganization that is required for the hydride tunneling reaction in choline oxidase

Egress of non-enveloped enteric RNA viruses

A long-standing paradigm in virology was that non-enveloped viruses induce cell lysis to release progeny virions. However, emerging evidence indicates that some non-enveloped viruses exit cells without inducing cell lysis, while others engage both lytic and non-lytic egress mechanisms. Enteric viruses are transmitted via the faecal-oral route and are important causes of a wide range of human infections, both gastrointestinal and extra-intestinal. Virus cellular egress, when fully understood, may be a relevant target for antiviral therapies, which could minimize the public health impact of these infections. In this review, we outline lytic and non-lytic cell egress mechanisms of non-enveloped enteric RNA viruses belonging to five families: Picornaviridae, Reoviridae, Caliciviridae, Astroviridae and Hepeviridae. We discuss factors that contribute to egress mechanisms and the relevance of these mechanisms to virion stability, infectivity and transmission. Since most data were obtained in traditional two-dimensional cell cultures, we will further attempt to place them into the context of polarized cultures and in vivo pathogenesis. Throughout the review, we highlight numerous knowledge gaps to stimulate future research into the egress mechanisms of these highly prevalent but largely understudied viruses

Full genomic characterization of a porcine rotavirus strain detected in an asymptomatic piglet in Accra, Ghana

Abstract: Background: The introduction of rotavirus A vaccination across the developing world has not proved to be as efficacious as first hoped. One cause of vaccine failure may be infection by zoonotic rotaviruses that are very variable antigenically from the vaccine strain. However, there is a lack of genomic information about the circulating rotavirus A strains in farm animals in the developing world that may be a source of infection for humans. We therefore screened farms close to Accra, Ghana for animals sub-clinically infected with rotavirus A and then sequenced the virus found in one of these samples. Results: 6.1% of clinically normal cows and pigs tested were found to be Rotavirus A virus antigen positive in the faeces. A subset of these (33.3%) were also positive for virus RNA. The most consistently positive pig sample was taken forward for metagenomic sequencing. This gave full sequence for all open reading frames except segment 5 (NSP1), which is missing a single base at the 5′ end. The virus infecting this pig had genome constellation G5-P[7]-I5-R1-C1-M1-A8-N1-T7-E1-H1, a known porcine genotype constellation. Conclusions: Farm animals carry rotavirus A infection sub-clinically at low frequency. Although the rotavirus A genotype discovered here has a pig-like genome constellation, a number of the segments most closely resembled those isolated from humans in suspected cases of zoonotic transmission. Therefore, such viruses may be a source of variable gene segments for re-assortment with other viruses to cause vaccine breakdown. It is recommended that further human and pig strains are characterized in West Africa, to better understand this dynamic

Full genomic characterization of a porcine rotavirus strain detected in an asymptomatic piglet in Accra, Ghana

Abstract: Background: The introduction of rotavirus A vaccination across the developing world has not proved to be as efficacious as first hoped. One cause of vaccine failure may be infection by zoonotic rotaviruses that are very variable antigenically from the vaccine strain. However, there is a lack of genomic information about the circulating rotavirus A strains in farm animals in the developing world that may be a source of infection for humans. We therefore screened farms close to Accra, Ghana for animals sub-clinically infected with rotavirus A and then sequenced the virus found in one of these samples. Results: 6.1% of clinically normal cows and pigs tested were found to be Rotavirus A virus antigen positive in the faeces. A subset of these (33.3%) were also positive for virus RNA. The most consistently positive pig sample was taken forward for metagenomic sequencing. This gave full sequence for all open reading frames except segment 5 (NSP1), which is missing a single base at the 5′ end. The virus infecting this pig had genome constellation G5-P[7]-I5-R1-C1-M1-A8-N1-T7-E1-H1, a known porcine genotype constellation. Conclusions: Farm animals carry rotavirus A infection sub-clinically at low frequency. Although the rotavirus A genotype discovered here has a pig-like genome constellation, a number of the segments most closely resembled those isolated from humans in suspected cases of zoonotic transmission. Therefore, such viruses may be a source of variable gene segments for re-assortment with other viruses to cause vaccine breakdown. It is recommended that further human and pig strains are characterized in West Africa, to better understand this dynamic

Connexin Genes Variants Associated with Non-Syndromic Hearing Impairment: A Systematic Review of the Global Burden

Mutations in connexins are the most common causes of hearing impairment (HI) in many populations. Our aim was to review the global burden of pathogenic and likely pathogenic (PLP) variants in connexin genes associated with HI. We conducted a systematic review of the literature based on targeted inclusion/exclusion criteria of publications from 1997 to 2020. The databases used were PubMed, Scopus, Africa-Wide Information, and Web of Science. The protocol was registered on PROSPERO, the International Prospective Register of Systematic Reviews, with the registration number “CRD42020169697”. The data extracted were analyzed using Microsoft Excel and SPSS version 25 (IBM, Armonk, New York, United States). A total of 571 independent studies were retrieved and considered for data extraction with the majority of studies (47.8% (n = 289)) done in Asia. Targeted sequencing was found to be the most common technique used in investigating connexin gene mutations. We identified seven connexin genes that were associated with HI, and GJB2 (520/571 publications) was the most studied among the seven. Excluding PLP in GJB2, GJB6, and GJA1 the other connexin gene variants (thus GJB3, GJB4, GJC3, and GJC1 variants) had conflicting association with HI. Biallelic GJB2 PLP variants were the most common and widespread variants associated with non-syndromic hearing impairment (NSHI) in different global populations but absent in most African populations. The most common GJB2 alleles found to be predominant in specific populations include; p.Gly12ValfsTer2 in Europeans, North Africans, Brazilians, and Americans; p.V37I and p.L79Cfs in Asians; p.W24X in Indians; p.L56Rfs in Americans; and the founder mutation p.R143W in Africans from Ghana, or with putative Ghanaian ancestry. The present review suggests that only GJB2 and GJB3 are recognized and validated HI genes. The findings call for an extensive investigation of the other connexin genes in many populations to elucidate their contributions to HI, in order to improve gene-disease pair curations, globally

Filovirus disease outbreaks : a chronological overview

CITATION: Languon, S. & Quaye, O. 2019. Filovirus disease outbreaks : a chronological overview. Virology: Research and Treatment, 10:1-12, doi:10.1177/1178122X19849927.The original publication is available at https://journals.sagepub.comFiloviruses cause outbreaks which lead to high fatality in humans and non-human primates, thus tagging them as major threats to public health and species conservation. In this review, we give account of index cases responsible for filovirus disease outbreaks that have occurred over the past 52 years in a chronological fashion, by describing the circumstances that led to the outbreaks, and how each of the outbreaks broke out. Since the discovery of Marburg virus and Ebola virus in 1967 and 1976, respectively, more than 40 filovirus disease outbreaks have been reported; majority of which have occurred in Africa. The chronological presentation of this review is to provide a concise overview of filovirus disease outbreaks since the discovery of the viruses, and highlight the patterns in the occurrence of the outbreaks. This review will help researchers to better appreciate the need for surveillance, especially in areas where there have been no filovirus disease outbreaks. We conclude by summarizing some recommendations that have been proposed by health and policy decision makers over the years.Filoviruses cause outbreaks which lead to high fatality in humans and non-human primates, thus tagging them as major threats to public health and species conservation. In this review, we give account of index cases responsible for filovirus disease outbreaks that have occurred over the past 52 years in a chronological fashion, by describing the circumstances that led to the outbreaks, and how each of the outbreaks broke out. Since the discovery of Marburg virus and Ebola virus in 1967 and 1976, respectively, more than 40 filovirus disease outbreaks have been reported; majority of which have occurred in Africa. The chronological presentation of this review is to provide a concise overview of filovirus disease outbreaks since the discovery of the viruses, and highlight the patterns in the occurrence of the outbreaks. This review will help researchers to better appreciate the need for surveillance, especially in areas where there have been no filovirus disease outbreaks. We conclude by summarizing some recommendations that have been proposed by health and policy decision makers over the years.https://journals.sagepub.com/doi/10.1177/1178122X19849927Publisher's versio

Yellow Fever Virus Down-Regulates mRNA Expression of SOCS1 in the Initial Phase of Infection in Human Cell Lines

Flaviviruses are constantly evolving diverse immune evasion strategies, and the exploitation of the functions of suppressors of cytokine signalling (SOCS) and protein inhibitors of activated STATs (PIAS) to favour virus replication has been described for Dengue and Japanese encephalitis viruses but not for yellow fever virus (YFV), which is still of global importance despite the existence of an effective vaccine. Some mechanisms that YFV employs to evade host immune defence has been reported, but the expression patterns of SOCS and PIAS in infected cells is yet to be determined. Here, we show that SOCS1 is down-regulated early in YFV-infected HeLa and HEK 293T cells, while SOCS3 and SOCS5 are not significantly altered, and PIAS mRNA expression appears to follow a rise-dip pattern akin to circadian-controlled genes. We also demonstrate that YFV evades interferon-β application to produce comparable viral titres. This report provides initial insight into the in vitro expression dynamics of SOCS and PIAS upon YFV infection and a basis for further investigation into SOCS/PIAS expression and how these modulate the immune response in animal models

Altered Faecal Microbiota Composition and Structure of Ghanaian Children with Acute Gastroenteritis.

Acute gastroenteritis (AGE) is a disease of global public health importance. Recent studies show that children with AGE have an altered gut microbiota relative to non-AGE controls. Yet, how the gut microbiota differs in Ghanaian children with and without AGE remains unclear. Here, we explore the 16S rRNA gene-based faecal microbiota profiles of Ghanaian children five years of age and younger, comprising 57 AGE cases and 50 healthy controls. We found that AGE cases were associated with lower microbial diversity and altered microbial sequence profiles relative to the controls. The faecal microbiota of AGE cases was enriched for disease-associated bacterial genera, including Enterococcus, Streptococcus, and Staphylococcus. In contrast, the faecal microbiota of controls was enriched for potentially beneficial genera, including Faecalibacterium, Prevotella, Ruminococcus, and Bacteroides. Lastly, distinct microbial correlation network characteristics were observed between AGE cases and controls, thereby supporting broad differences in faecal microbiota structure. Altogether, we show that the faecal microbiota of Ghanaian children with AGE differ from controls and are enriched for bacterial genera increasingly associated with diseases