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Zika virus (ZIKV) is a re-emerging mosquito-borne flavivirus that can have devastating health consequences. The developmental and neurological effects of a ZIKV infection arise in part from the virus triggering cellular stress pathways and perturbing transcriptional programs. To date, the underlying mechanisms of transcriptional control directing viral restriction and virus-host interaction are understudied. Activating Transcription Factor 3 (ATF3) is a stress-induced transcriptional effector that modulates the expression of genes involved in a myriad of cellular processes, including inflammation and antiviral responses, to restore cellular homeostasis. While ATF3 is known to be upregulated during ZIKV infection, the mode by which ATF3 is activated, and the specific role of ATF3 during ZIKV infection is unknown. In this study, we show via inhibitor and RNA interference approaches that ZIKV infection initiates the integrated stress response pathway to activate ATF4 which in turn induces ATF3 expression. Additionally, by using CRISPR-Cas9 system to delete ATF3, we found that ATF3 acts to limit ZIKV gene expression in A549 cells. We also determined that ATF3 enhances the expression of antiviral genes such as STAT1 and other components in the innate immunity pathway to induce an ATF3-dependent anti-ZIKV response. Our study reveals crosstalk between the integrated stress response and innate immune response pathways and highlights an important role for ATF3 in establishing an antiviral effect during ZIKV infection., Importance: Zika virus (ZIKV) is a re-emerging mosquito-borne flavivirus that co-opts cellular mechanisms to support viral processes that can reprogram the host transcriptional profile. Such viral-directed transcriptional changes and the pro- or anti-viral outcomes remain understudied. We previously showed that ATF3, a stress-induced transcription factor, is significantly upregulated in ZIKV-infected mammalian cells, along with other cellular and immune response genes. We now define the intracellular pathway responsible for ATF3 activation and elucidate the impact of ATF3 expression on ZIKV infection. We show that during ZIKV infection, the integrated stress response pathway stimulates ATF3 which enhances the innate immune response to antagonize ZIKV infection. This study establishes a link between viral-induced stress response and transcriptional regulation of host defense pathways and thus expands our knowledge of virus-mediated transcriptional mechanisms and transcriptional control of interferon-stimulated genes during ZIKV infection., Competing Interests: The authors declare no conflict of interest.

Zika virus (ZIKV), a re-emerging flavivirus, is associated with devasting developmental and neurological disease outcomes particularly in infants infected in utero. Towards understanding the molecular underpinnings of the unique ZIKV disease pathologies, numerous transcriptome-wide studies have been undertaken. Notably, these studies have overlooked the assimilation of RNA-seq analysis from ZIKV-infected patients with cell culture model systems. In this study we find that ZIKV-infection of human lung adenocarcinoma A549 cells, mirrored both the transcriptional and alternative splicing profiles from previously published RNA-seq data of peripheral blood mononuclear cells collected from pediatric patients during early acute, late acute, and convalescent phases of ZIKV infection. Our analyses show that ZIKV infection in cultured cells correlates with transcriptional changes in patients, while the overlap in alternative splicing profiles was not as extensive. Overall, our data indicate that cell culture model systems support dissection of select molecular changes detected in patients and establishes the groundwork for future studies elucidating the biological implications of alternative splicing during ZIKV infection.

Zika virus (ZIKV), a re-emerging flavivirus, is associated with devasting developmental and neurological disease outcomes particularly in infants infected in utero . Towards understanding the molecular underpinnings of the unique ZIKV disease pathologies, numerous transcriptome-wide studies have been undertaken. Notably, these studies have overlooked the assimilation of RNA-seq analysis from ZIKV-infected patients with cell culture model systems. In this study we find that ZIKV-infection of human lung adenocarcinoma A549 cells, mirrored both the transcriptional and alternative splicing profiles from previously published RNA-seq data of peripheral blood mononuclear cells collected from pediatric patients during early acute, late acute, and convalescent phases of ZIKV infection. Our analyses show that ZIKV infection in cultured cells correlates with transcriptional changes in patients, while the overlap in alternative splicing profiles was not as extensive. Overall, our data indicate that cell culture model systems support dissection of select molecular changes detected in patients and establishes the groundwork for future studies elucidating the biological implications of alternative splicing during ZIKV infection.

We propose a novel approach for building a classification/identification framework based on the full complement of RNA post-transcriptional modifications (rPTMs) expressed by an organism at basal conditions. The approach relies on advanced mass spectrometry techniques to characterize the products of exonuclease digestion of total RNA extracts. Sample profiles comprising identities and relative abundances of all detected rPTM were used to train and test the capabilities of different machine learning (ML) algorithms. Each algorithm proved capable of identifying rigorous decision rules for differentiating closely related classes and correctly assigning unlabeled samples. The ML classifiers resolved different members of the Enterobacteriaceae family, alternative Escherichia coli serotypes, a series of Saccharomyces cerevisiae knockout mutants, and primary cells of the Homo sapiens central nervous system, which shared very similar genetic backgrounds. The excellent levels of accuracy and resolving power achieved by training on a limited number of classes were successfully replicated when the number of classes was significantly increased to escalate complexity. A dendrogram generated from ML-curated data exhibited a hierarchical organization that closely resembled those afforded by established taxonomic systems. Finer clustering patterns revealed the extensive effects induced by the deletion of a single pivotal gene. This information provided a putative roadmap for exploring the roles of rPTMs in their respective regulatory networks, which will be essential to decipher the epitranscriptomics code. The ubiquitous presence of RNA in virtually all living organisms promises to enable the broadest possible range of applications, with significant implications in the diagnosis of RNA-related diseases.

Detection of viruses is critical for controlling disease spread. Recent emerging viral threats, including Zika virus, Ebola virus, and SARS-CoV-2 responsible for coronavirus disease 2019 (COVID-19) highlight the cost and difficulty in responding rapidly. To address these challenges, we develop a platform for low-cost and rapid detection of viral RNA with DNA nanoswitches that mechanically reconfigure in response to specific viruses. Using Zika virus as a model system, we show nonenzymatic detection of viral RNA with selective and multiplexed detection between related viruses and viral strains. For clinical-level sensitivity in biological fluids, we paired the assay with sample preparation using either RNA extraction or isothermal preamplification. Our assay requires minimal laboratory infrastructure and is adaptable to other viruses, as demonstrated by quickly developing DNA nanoswitches to detect SARS-CoV-2 RNA in saliva. Further development and field implementation will improve our ability to detect emergent viral threats and ultimately limit their impact., (Copyright © 2020 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works. Distributed under a Creative Commons Attribution License 4.0 (CC BY).)

The alternative splicing of pre-mRNAs expands a single genetic blueprint to encode multiple, functionally diverse protein isoforms. Viruses have previously been shown to interact with, depend on, and alter host splicing machinery. The consequences, however, incited by viral infection on the global alternative slicing (AS) landscape are under-appreciated. Here, we investigated the transcriptional and alternative splicing profile of neuronal cells infected with a contemporary Puerto Rican Zika virus (ZIKV PR ) isolate, an isolate of the prototypical Ugandan ZIKV (ZIKV MR ), and dengue virus 2 (DENV2). Our analyses revealed that ZIKV PR induced significantly more differential changes in expressed genes compared to ZIKV MR or DENV2, despite all three viruses showing equivalent infectivity and viral RNA levels. Consistent with the transcriptional profile, ZIKV PR induced a higher number of alternative splicing events compared to ZIKV MR or DENV2, and gene ontology analyses highlighted alternative splicing changes in genes associated with mRNA splicing. In summary, we show that ZIKV affects cellular RNA homeostasis not only at the transcriptional levels but also through the alternative splicing of cellular transcripts. These findings could provide new molecular insights into the neuropathologies associated with this virus.

Epitranscriptomics, the study of posttranscriptional chemical moieties placed on RNA, has blossomed in recent years. This is due in part to the emergence of high-throughput detection methods as well as the burst of discoveries showing biological function of select chemical marks. RNA modifications have been shown to affect RNA structure, localization, and functions such as alternative splicing, stabilizing transcripts, nuclear export, cap-dependent and cap-independent translation, microRNA biogenesis and binding, RNA degradation, and immune regulation. As such, the deposition of chemical marks on RNA has the unique capability to spatially and temporally regulate gene expression. The goal of this article is to present the exciting convergence of the epitranscriptomic and virology fields, specifically the deposition and biological impact of N7-methylguanosine, ribose 2'-O-methylation, pseudouridine, inosine, N6-methyladenosine, and 5-methylcytosine epitranscriptomic marks on gene expression of RNA viruses. This article is categorized under: RNA in Disease and Development > RNA in Disease RNA Interactions with Proteins and Other Molecules > Protein-RNA Interactions: Functional Implications., (© 2019 Wiley Periodicals, Inc.)

Flaviviruses limit the cell stress response by preventing the formation of stress granules (SGs) and modulate viral gene expression by subverting different proteins involved in the stress granule pathway. In this study, we investigated the formation of stress granules during Zika virus (ZIKV) infection and the role stress granule proteins play during the viral life cycle. Using immunofluorescence and confocal microscopy, we determined that ZIKV disrupted the formation of arsenite-induced stress granules and changed the subcellular distribution, but not the abundance or integrity, of stress granule proteins. We also investigated the role of different stress granule proteins in ZIKV infection by using target-specific short interfering RNAs to deplete Ataxin2, G3BP1, HuR, TIA-1, TIAR, and YB1. Knockdown of TIA-1 and TIAR affected ZIKV protein and RNA levels but not viral titers. Conversely, depletion of Ataxin2 and YB1 decreased virion production despite having only a small effect on ZIKV protein expression. Notably, however, depletion of G3BP1 and HuR decreased and increased ZIKV gene expression and virion production, respectively. Using an MR766 Gaussia Luciferase reporter genome together with knockdown and overexpression assays, G3BP1 and HuR were found to modulate ZIKV replication. These data indicate that ZIKV disrupts the formation of stress granules by sequestering stress granule proteins required for replication, where G3BP1 functions to promote ZIKV infection while HuR exhibits an antiviral effect. The results of ZIKV relocalizing and subverting select stress granule proteins might have broader consequences on cellular RNA homeostasis and contribute to cellular gene dysregulation and ZIKV pathogenesis. IMPORTANCE Many viruses inhibit SGs. In this study, we observed that ZIKV restricts SG assembly, likely by relocalizing and subverting specific SG proteins to modulate ZIKV replication. This ZIKV-SG protein interaction is interesting, as many SG proteins are also known to function in neuronal granules, which are critical in neural development and function. Moreover, dysregulation of different SG proteins in neurons has been shown to play a role in the progression of neurodegenerative diseases. The likely consequences of ZIKV modulating SG assembly and subverting specific SG proteins are alterations to cellular mRNA transcription, splicing, stability, and translation. Such changes in cellular ribostasis could profoundly affect neural development and contribute to the devastating developmental and neurological anomalies observed following intrauterine ZIKV infection. Our study provides new insights into virus-host interactions and the identification of the SG proteins that may contribute to the unusual pathogenesis associated with this reemerging arbovirus., (Copyright © 2019 Bonenfant et al.)

More than 140 post-transcriptional modifications (PTMs) are known to decorate cellular RNAs, but their incidence, identity and significance in viral RNA are still largely unknown. We have developed an agnostic analytical approach to comprehensively survey PTMs on viral and cellular RNAs. Specifically, we used mass spectrometry to analyze PTMs on total RNA isolated from cells infected with Zika virus, Dengue virus, hepatitis C virus (HCV), poliovirus and human immunodeficiency virus type 1. All five RNA viruses significantly altered global PTM landscapes. Examination of PTM profiles of individual viral genomes isolated by affinity capture revealed a plethora of PTMs on viral RNAs, which far exceeds the handful of well-characterized modifications. Direct comparison of viral epitranscriptomes identified common and virus-specific PTMs. In particular, specific dimethylcytosine modifications were only present in total RNA from virus-infected cells, and in intracellular HCV RNA, and viral RNA from Zika and HCV virions. Moreover, dimethylcytosine abundance during viral infection was modulated by the cellular DEAD-box RNA helicase DDX6. By opening the Pandora's box on viral PTMs, this report presents numerous questions and hypotheses on PTM function and strongly supports PTMs as a new tier of regulation by which RNA viruses subvert the host and evade cellular surveillance systems.

Hepatitis C virus (HCV) subverts the cellular DEAD-box RNA helicase DDX6 to promote virus infection. Using polysome gradient analysis and the subgenomic HCV Renilla reporter replicon genome, we determined that DDX6 does not affect HCV translation. Rather expression of the subgenomic HCV Renilla luciferase reporter at late times, as well as labeling of newly synthesized viral RNA with 4-thiouridine showed that DDX6 modulates replication. Because DDX6 is an effector protein of the microRNA pathway, we also investigated its role in miR-122-directed HCV gene expression. Similar to sequestering miR-122, depletion of DDX6 modulated HCV RNA stability. Interestingly, miR-122-HCV RNA interaction assays with mutant HCV genomes sites and compensatory exogenous miR-122 showed that DDX6 affects the function of miR-122 at one particular binding site. We propose that DDX6 facilitates the miR-122 interaction with HCV 5' UTR, which is necessary for stabilizing the viral genome and the switch between translation and replication., (Copyright © 2017 Elsevier Inc. All rights reserved.)

During infection, positive-strand RNA viruses subvert cellular machinery involved in RNA metabolism to translate viral proteins and replicate viral genomes to avoid or disable the host defense mechanisms. Cytoplasmic RNA granules modulate the stabilities of cellular and viral RNAs. Understanding how hepatitis C virus and other flaviviruses interact with the host machinery required for protein synthesis, localization, and degradation of mRNAs is important for elucidating how these processes occur in both virus-infected and uninfected cells., (Copyright © 2016, American Society for Microbiology. All Rights Reserved.)

The transcriptional regulator deformed epidermal autoregulatory factor 1 (DEAF1) has been suggested to play a role in maintaining peripheral tolerance by controlling the transcription of peripheral tissue antigen genes in lymph node stromal cells (LNSCs). Here, we demonstrate that DEAF1 also regulates the translation of genes in LNSCs by controlling the transcription of the poorly characterized eukaryotic translation initiation factor 4 gamma 3 (Eif4g3) that encodes eIF4GII. Eif4g3 gene expression was reduced in the pancreatic lymph nodes of Deaf1-KO mice, non-obese diabetic mice, and type 1 diabetes patients, where functional Deaf1 is absent or diminished. Silencing of Deaf1 reduced Eif4g3 expression, but increased the expression of Caspase 3, a serine protease that degrades eIF4GII. Polysome profiling showed that reduced Eif4g3 expression in LNSCs resulted in the diminished translation of various genes, including Anpep, the gene for aminopeptidase N, an enzyme involved in fine-tuning antigen presentation on major histocompatibility complex (MHC) class II. Together these findings suggest that reduced DEAF1 function, and subsequent loss of Eif4g3 transcription may affect peripheral tissue antigen (PTA) expression in LNSCs and contribute to the pathology of T1D.

Components of cytoplasmic processing bodies (P-bodies) and stress granules can be subverted during viral infections to modulate viral gene expression. Because hepatitis C virus (HCV) RNA abundance is regulated by P-body components such as microRNA miR-122, Argonaute 2 and RNA helicase RCK/p54, we examined whether HCV infection modulates P-bodies and stress granules during viral infection. It was discovered that HCV infection decreased the number of P-bodies, but induced the formation of stress granules. Immunofluorescence studies revealed that a number of P-body and stress granule proteins co-localized with viral core protein at lipid droplets, the sites for viral RNA packaging. Depletion of selected P-body proteins decreased overall HCV RNA and virion abundance. Depletion of stress granule proteins also decreased overall HCV RNA abundance, but surprisingly enhanced the accumulation of infectious, extracellular virus. These data argue that HCV subverts P-body and stress granule components to aid in viral gene expression at particular sites in the cytoplasm., (Copyright © 2012 Elsevier Inc. All rights reserved.)

The paramyxovirus family includes important human pathogens such as measles, mumps, respiratory syncytial virus, and the recently emerged, highly pathogenic Hendra and Nipah viruses. The viral fusion (F) protein plays critical roles in infection, promoting both the virus-cell membrane fusion events needed for viral entry as well as cell-cell fusion events leading to syncytia formation. We describe the surprising finding that addition of the short epitope HA tag to the cytoplasmic tail (CT) of the Hendra virus F protein leads to a significant increase in the extent of cell-cell membrane fusion. This increase was not due to alterations in surface expression, cleavage state, or association with lipid microdomains. Addition of a Myc tag of similar length did not alter Hendra F protein fusion activity, indicating that the observed stimulation was not solely a result of lengthening the CT. Three tyrosine residues within the HA tag were critical for the increase in the extent of fusion, suggesting C-terminal tyrosines may modulate Hendra fusion activity. The effects of addition of the HA tag varied with other fusion proteins, as parainfluenza virus 5 F-HA showed a decreased level of surface expression and no stimulation of fusion. These results indicate that additions to the C-terminal end of the F protein CT can modulate protein function in a sequence specific manner, reinforcing the need for careful analysis of epitope-tagged glycoproteins. In addition, our results implicate C-terminal tyrosine residues in the modulation of the membrane fusion reaction promoted by these viral glycoproteins.

The protein DDX3X is a DEAD-box RNA helicase that is essential for the hepatitis C virus (HCV) life cycle. The HCV core protein has been shown to bind to DDX3X both in vitro and in vivo. However, the specific interactions between these two proteins and the functional importance of these interactions for the HCV viral life cycle remain unclear. We show that amino acids 16-36 near the N-terminus of the HCV core protein interact specifically with DDX3X both in vitro and in vivo. Replication of HCV replicon NNeo/C-5B RNA (genotype 1b) is significantly suppressed in HuH-7-derived cells expressing green fluorescent protein (GFP) fusions to HCV core protein residues 16-36, but not by GFP fusions to core protein residues 16-35 or 16-34. Notably, the inhibition of HCV replication due to expression of the GFP fusion to HCV core protein residues 16-36 can be reversed by overexpression of DDX3X. These results suggest that the protein interface on DDX3X that binds the HCV core protein is important for replicon maintenance. However, infection of HuH-7 cells by HCV viruses of genotype 2a (JFH1) was not affected by expression of the GFP fusion protein. These results suggest that the role of DDX3X in HCV infection involves aspects of the viral life cycle that vary in importance between HCV genotypes.

MicroRNAs are 20-21 nucleotides-long noncoding RNAs that function as posttranscriptional regulators of gene expression in a variety of organisms ranging from plants to mammalian cells. These regulators are encoded by approximately 800 genes in the mammalian genome and target half of the mRNAs in mammalian cells. While the biogenesis of microRNAs is fairly well understood, the mechanism by which target genes are regulated remains controversial. The recent discoveries that viruses encode microRNAs or subvert host cell microRNAs has enhanced our knowledge about biological functions of microRNAs during disease and has suggested that microRNAs could be used as targets in antiviral gene therapy. This review will provide a brief history of microRNA research, discuss the biogenesis and mechanisms of microRNAs, and summarize findings that have employed inhibitors of microRNA miR-122 to treat hepatitis C virus-induced liver disease., (Copyright © 2009 Elsevier Inc. All rights reserved.)

The Nipah virus fusion (F) protein is proteolytically processed to F1 + F2 subunits. We demonstrate here that cathepsin L is involved in this important maturation event. Cathepsin inhibitors ablated cleavage of Nipah F. Proteolytic processing of Nipah F and fusion activity was dramatically reduced in cathepsin L shRNA-expressing Vero cells. Additionally, Nipah virus F-mediated fusion was inhibited in cathepsin L-deficient cells, but coexpression of cathepsin L restored fusion activity. Both purified cathepsin L and B could cleave immunopurified Nipah F protein, but only cathepsin L produced products of the correct size. Our results suggest that endosomal cathepsins can cleave Nipah F, but that cathepsin L specifically converts Nipah F to a mature and fusogenic form.

Proteolytic processing of paramyxovirus fusion (F) proteins is essential for the generation of a mature and fusogenic form of the F protein. Although many paramyxovirus F proteins are proteolytically processed by the cellular protease furin at a multibasic cleavage motif, cleavage of the newly emerged Hendra virus F protein occurs by a previously unidentified cellular protease following a single lysine at residue 109. We demonstrate here that the cellular protease cathepsin L is involved in converting the Hendra virus precursor F protein (F(0)) to the active F(1) + F(2) disulfide-linked heterodimer. To initially identify the class of protease involved in Hendra virus F protein cleavage, Vero cells transfected with pCAGGS-Hendra F or pCAGGS-SV5 F (known to be proteolytically processed by furin) were metabolically labeled and chased in the absence or presence of serine, cysteine, aspartyl, and metalloprotease inhibitors. Nonspecific and specific protease inhibitors known to decrease cathepsin activity inhibited proteolytic processing of Hendra virus F but had no effect on simian virus 5 F processing. We next designed shRNA oligonucleotides to cathepsin L which dramatically reduced cathepsin L protein expression and enzyme activity. Cathepsin L shRNA-expressing Vero cells transfected with pCAGGS-Hendra F demonstrated a nondetectable amount of cleavage of the Hendra virus F protein and significantly decreased membrane fusion activity. Additionally, we found that purified human cathepsin L processed immunopurified Hendra virus F(0) into F(1) and F(2) fragments. These studies introduce a novel mechanism for primary proteolytic processing of viral glycoproteins and also suggest a previously unreported biological role for cathepsin L.

The Hendra virus fusion (F) protein contains five potential sites for N-linked glycosylation in the ectodomain. Examination of F protein mutants with single asparagine-to-alanine mutations indicated that two sites in the F(2) subunit (N67 and N99) and two sites in the F(1) subunit (N414 and N464) normally undergo N-linked glycosylation. While N-linked modification at N414 is critical for protein folding and transport, F proteins lacking carbohydrates at N67, N99, or N464 remained fusogenically active. As N464 lies within heptad repeat B, these results contrast with those seen for several paramyxovirus F proteins.

Proteolytic cleavage of the Hendra virus fusion (F) protein results in the formation of disulfide-linked F1 and F2 subunits, with cleavage occurring after residue K109 in the sequence GDVK/L. This unusual cleavage site and efficient propagation of Hendra virus in a furin-deficient cell line indicate that the Hendra F protein is not cleaved by furin, the protease responsible for proteolytic activation of many viral fusion proteins. To identify the subcellular site of Hendra F processing, Vero cells transfected with pCAGGS-Hendra F or pCAGGS-SV5 F were metabolically labeled and chased in the absence and presence of inhibitors of exocytosis. The addition of carbonyl-cyanide-3-chlorophenylhydrazone, monensin, brefeldin A, or NaF-AlCl3 or incubation of cells at 20 degrees C all inhibited processing of the Hendra F protein, suggesting that cleavage of Hendra F occurs either in secretory vesicles budding from the trans-Golgi network or at the cell surface. In contrast to proteolytic cleavage of the simian virus 5 (SV5) F protein by the Ca(2+)-dependent protease furin, proteolytic cleavage of the Hendra F protein was not significantly inhibited by decreases in Ca2+ levels following incubation with EGTA or A23187. However, in the presence of weak amines and H+ V-ATPase inhibitors, known to raise intracellular pH, cleavage of Hendra F protein was inhibited while processing of the SV5 F protein was not significantly affected. The subcellular location, sensitivity to pH changes, and decreased Ca2+ requirement suggest that the protease responsible for cleavage of Hendra F protein differs from proteases previously shown to be involved in the processing of other viral glycoproteins.

Despite the clear importance of the left-handed polyproline II (PPII) helical conformation in many physiologically important processes as well as its potential significance in protein unfolded states, little is known about the physical determinants of this conformation. We present here a scale of relative PPII helix-forming propensities measured for all residues, except tyrosine and tryptophan, in a proline-based host peptide system. Proline has the highest measured propensity in this system, a result of strong steric interactions that occur between adjacent prolyl rings. The other measured propensities are consistent with backbone solvation being an important component in PPII helix formation. Side chain to backbone hydrogen bonding may also play a role in stabilizing this conformation. The PPII helix-forming propensity scale will prove useful in future studies of the conformational properties of proline-rich sequences as well as provide insights into the prevalence of PPII helices in protein unfolded states., (Copyright 2003 Wiley-Liss, Inc.)

Rotavirus infection is associated with acute infantile gastroenteritis in infants and young children globally. In South Africa, rotavirus infection has been shown to be associated with approximately one-quarter of all diarrhoeal admissions to hospital. Rotavirus infection predominantly occurs in infants less than 12 months of age (75%) and has a peak of shedding during the cooler, drier months of the year. A secondary peak during the spring has been observed. Multiple infections with rotavirus and at least one other microbial agent are common. The circulating VP7 serotypes and VP4 genotypes have been determined in various regions of South Africa and show a geographic specific distribution. A decade previously, P[8]G1 or G4 strains predominated, and P[4]G2 strains occurred in an epidemic pattern in one region. More recently, rotavirus strains with P[6] genotype have become common and novel VP7/VP4 genotype combinations are occurring across the country. G9 strains have been reported from Cape Town to Vendaland. The circulating rotavirus types observed in this study add to the knowledge of the natural history of rotavirus infection and provide the groundwork to consider future vaccine strategies.

Astrovirus has been shown to be an important aetiological agent associated with gastroenteritis in children, although few studies have been conducted in Africa. In this study, stool specimens were obtained from 375 young children less than 5 years of age with acute gastroenteritis presenting at Ahmadu Bello University Hospital, and from a control group of 122 children without diarrhoeal illness. The specimens were examined for the presence of human astroviruses using a monoclonal antibody-based ELISA (Astrovirus IDEIATM, Dako, UK). Negative staining electron microscopy was performed on specimens to confirm the presence of astrovirus particles. Astrovirus was detected in 6.7 per cent (25/375) of the diarrhoeal stools compared to 5.7 per cent (7/122) of the control specimens. Astrovirus seemed to infect older children and more than half the children were between 1 and 4 years of age (15/25). Only four children were less than 6 months old. A winter peak of shedding was observed.

Sixty-seven rotavirus-positive fecal samples, collected between January and April 1999, from children with diarrhea in the Upper East Region of Ghana were examined for rotavirus VP7 and VP4 types. Sufficient viral RNA could be obtained from 46 (68.7%) of the samples and all the isolates had short electrophoretic pattern and typed as subgroup I rotaviruses by subgroup ELISA. Three rotavirus strains with G8 specificity were identified for the first time in Ghana. G and P typing by PCR identified two distinct strains, P[6]G2 (50%) and P[6]G8 (4.3%). Eighty-two percent of the isolates (n = 38) were of the "putative" neonatal P[6] genotype. Two of these G8 isolates carried the VP4 P[6] genotype whereas the third could not be assigned a P type. Mixed infections of G1, G2, G3 and G8 were detected amongst the stool samples. The presence of these unusual strains, especially the high incidence of G2 rotavirus strains in Ghana, reinforces the need to put in place a surveillance system for the detection of new and exotic rotavirus strains, that will provide information on the spread of these strains in West Africa as well as useful data for the formulation of the next generation of rotavirus vaccines.

Over the past decade, a G4P[6] strain has been found to be circulating in different neonatal wards in the Pretoria area. This endemic strain was associated with both asymptomatic and symptomatic infection, providing the opportunity to undertake a molecular study of some of the putative "virulence" genes. The genes encoding NSP4, VP8*, and VP7 of two asymptomatic and two Symptomatic strains were sequenced and compared with ST3. Within each of these genes, amino acid substitutions unique to South African strains were recorded. Four conserved amino acid differences between asymptomatic and symptomatic strains at aa 82 (serine to leucine), aa 114 (aspartic acid to glutamic acid), aa 138 (proline to threonine), and aa 169 (leucine to serine) were identified within the NSP4 gene. The hypervariable region of VP8* exhibited 10 specific amino acid differences (at aa 73, 78, 98, 111, 116, 142, 145, 167, 169, and 188) between asymptomatic and symptomatic strains, while three amino acid substitutions within VP7 were noted. These changes to VP7 occurred within the glycosylation site at aa 70 (leucine to serine), at antigenic region A (aa 96, asparagine to threonine), and at aa 318 (aspartic acid to glycine). It may be speculated that these changes are specific to G4P[6] strains. Furthermore, the observed substitutions may also be particular to South African strains. NSP4, VP8*, and VP7 have been associated with virulence and the amino acid substitutions within these genes correlate with both asymptomatic and symptomatic infection observed in neonates., (Copyright 2000 Wiley-Liss, Inc.)

Epidemiological studies on the VP7 serotype prevalence of human rotaviruses in South Africa and the United Kingdom identified several strains which could not be serotyped as G1-G4 by monoclonal antibodies. Further analysis of these strains with a G8-specific monoclonal antibody and with probes for human rotaviruses confirmed them as G8 rotaviruses. These G8 strains exhibited a high degree of sequence identity when compared with each other and with other rotavirus G8 strains. Five South African strains were further characterized as VP6 subgroup I, but with a long RNA electropherotype, which is similar to the G8 strains previously isolated in Finland. In the UK strains, one was VP6 subgroup II with a long RNA electropherotype (similar to the Italian G8 strain). The other two were subgroup I with a short RNA electropherotype. None of these strains exhibited the super-short RNA electropherotype described in the prototype G8 strains recovered from Indonesia (69M).