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• The protective effect of HLA-B*57:01/58:01 on HIV-1 CRF01_AE infection was examined. • The protective impact is impaired against CRF01_AE infection. • TW10-specific CD8+ T cells select for T242N even in CRF01_AE with viral fitness cost. • Less non-TW10 epitope presentation may contribute to the impaired protective impact. Human Leukocyte Antigen HLA-B*57:01 and B*58:01 are considered anti-HIV-1 protective alleles. HLA-B*57:01/58:01-restricted HIV-1 Gag TW10 (TSTLQEQIGW, Gag residues 240-249) epitope-specific CD8+ T cell responses that frequently select for a Gag escape mutation, T242N, with viral fitness cost are crucial for HIV-1 control. Although this finding has been observed in cohorts where HIV-1 subtype B or C predominates, the protective impact of HLA-B*57:01/58:01 has not been reported in Southeast Asian countries where HIV-1 CRF01_AE is the major circulating strain. Here, the effect of HLA-B*57:01/58:01 on CRF01_AE infection was investigated. The correlation of HLA-B*57:01/58:01 with viral load and CD4 counts were analyzed in the CRF01_AE-infected Vietnamese cohort (N = 280). The impact of the T242N mutation on CRF01_AE replication capacity was assessed. HLA-B*57:01/58:01 -positive individuals mostly had HIV-1 with T242N (62/63) but showed neither a significant reduction in viral load nor increased CD4 counts relative to B*57:01/58:01 -negative participants. In vitro and in vivo analyses revealed a significant reduction in viral fitness of CRF01_AE with T242N. In silico analysis indicated reduced presentation of epitopes in the context of CRF01_AE compared to subtype B or C in 10/16 HLA-B*57:01/58:01-restricted HIV-1 epitopes. The protective impact of HLA-B*57:01/58:01 on CRF01_AE infection is impaired despite strong suppressive pressure by TW10-specific CD8+ T cells. [ABSTRACT FROM AUTHOR]

Abstract: Human adenovirus serotype 14 (HAdV-14; agent de Wit), a subspecies B2 member, was first identified in The Netherlands in 1955 in a military training camp and last reported in Eurasia in 1963. This virus has been conspicuous by its absence in global serosurveys and surveillance in subsequent decades. In early 2006, HAdV-14 was described at five military training centers in the United States and was subsequently associated with civilian cases of severe respiratory disease and fatalities in New York and California. In 2007, 140 cases of HAdV-14 were identified in outbreak clusters across the U.S. (Oregon, Washington, and Texas), with 37% requiring hospitalization, 17% admittance to intensive care units, and a 5% mortality rate. Genetic characterization of this emerging HAdV-14 revealed a novel genome variant (termed HAdV-14p1), and an identical virus has recently been detected in Europe, also associated with morbidity and mortality in both immunocompromised and immunocompetent individuals. As a recently emerged virus, HAdV-14 has an increased potential for high rates of transmission and high attack rates due to low levels of herd immunity, simply because the vast majority of individuals are extremely likely to have never been exposed. Part I of this two-part article reviewed the classification of HAdV-14p1; the clinical symptoms associated with this virus, including case reports; and risk factors associated with these infections. Part II of this article reviews the epidemiology, diagnosis, prevention, and treatment modalities of HAdV-14p1. [Copyright &y& Elsevier]

• We describe 11 major mutation events which defined five major clades (G 614 , S 84 , V 251 , I 378 and D 392) of globally-circulating viral populations. • We have successfully developed a multiplexed sequencing-based, rapid genotyping protocol for high-throughput classification of major clade types of SARS-CoV-2 in clinical samples. • Several nonsynonymous mutations in the spike protein may have functional consequences: the G clade–defining mutation D614 G located in subdomain 1; the V367 F, G476S and V483A are localised in the receptor binding domain (RBD) of the spike protein. The SARS-CoV-2 pathogen has established endemicity in humans. This necessitates the development of rapid genetic surveillance methodologies to serve as an adjunct with existing comprehensive, albeit though slower, genome sequencing-driven approaches. A total of 21,789 complete genomes were downloaded from GISAID on May 28, 2020 for analyses. We have defined the major clades and subclades of circulating SARS-CoV-2 genomes. A rapid sequencing-based genotyping protocol was developed and tested on SARS-CoV-2-positive RNA samples by next-generation sequencing. We describe 11 major mutations which defined five major clades (G 614 , S 84 , V 251 , I 378 and D 392) of globally circulating viral populations. The clades can specifically identify using an 11-nucleotide genetic barcode. An analysis of amino acid variation in SARS-CoV-2 proteins provided evidence of substitution events in the viral proteins involved in both host entry and genome replication. Globally circulating SARS-CoV-2 genomes could be classified into 5 major clades based on mutational profiles defined by an 11-nucleotide barcode. We have successfully developed a multiplexed sequencing-based, rapid genotyping protocol for high-throughput classification of major clade types of SARS-CoV-2 in clinical samples. This barcoding strategy will be required to monitor decreases in genetic diversity as treatment and vaccine approaches become widely available. [ABSTRACT FROM AUTHOR]

Chikungunya fever is a re-emerging zoonotic disease caused by chikungunya virus (CHIKV), a member of the Alphavirus genus in the Togaviridae family. Only a few studies have reported on the host factors required for intracellular CHIKV trafficking. Here, we conducted an imaging-based siRNA screen to identify human host factors for intracellular trafficking that are involved in CHIKV infection, examined their interactions with CHIKV proteins, and investigated the contributions of these proteins to CHIKV infection. The results of the siRNA screen revealed that host endosomal sorting complexes required for transport (ESCRT) proteins are recruited during CHIKV infection. Co-immunoprecipitation analyses revealed that both structural and nonstructural CHIKV proteins interact with hepatocyte growth factor-regulated tyrosine kinase substrate (HGS), a component of the ESCRT-0 complex. We also observed that HGS co-localizes with the E2 protein of CHIKV and with dsRNA, a marker of the replicated CHIKV genome. Results from gene knockdown analyses indicated that, along with other ESCRT factors, HGS facilitates both genome replication and post-translational steps during CHIKV infection. Moreover, we show that ESCRT factors are also required for infections with other alphaviruses. We conclude that during CHIKV infection, several ESCRT factors are recruited via HGS and are involved in viral genome replication and post-translational processing of viral proteins. [ABSTRACT FROM AUTHOR]

Dengue virus (DENV) is the causative agent of dengue fever and dengue hemorrhagic fever/dengue shock syndrome. At present, no antiviral drugs are available for treatment DENV infections. In this study, a screening system based on a DENV-infected cell-based assay identified a novel anti-DENV agent with a benzimidazole skeleton, named Compound-B, which demonstrated antiviral activity specific to four DENV serotypes (EC 50 : 1.32–4.12 μM). Analysis of a single amino acid substitution of Compound-B-resistant DENV2 revealed that mutation C87S in the non-structural protein 4A (NS4A) contributes to resistance to Compound-B. [ABSTRACT FROM AUTHOR]

Abstract: Background: Ireland is classified as an area of high measles incidence. A World Health Organisation-European Region strategic plan exists for measles elimination by 2015. Objectives: To retrospectively investigate measles outbreaks using all patient samples (sera and oral fluid) received for measles laboratory diagnosis and characterise the genetic diversity of circulating measles genotypes in Ireland. Study Design: 704 cases of acute measles infection as determined by the presence of measles specific IgM in sera and oral fluids were confirmed at the National Virus Reference Laboratory. Measles positive samples (n =116) were examined by genotyping, sequence analysis and phylogenetic characterisation. Results: Three measles outbreaks occurred over the study period: 2004, 2009/2010 and 2011. Measles IgM positivity ranged from 22–29% in outbreak years to 5–10% in the intervening years. Age profile analysis revealed that whereas individuals >10 years accounted for only 8% of cases in the 2004 outbreak, this increased to 33% and 29% in the 2009/2010 and 2011 outbreaks, respectively. The <1 year cohort accounted for 18–20% of cases in all outbreaks. Phylogenetic analysis demonstrated both indigenous transmission and also importation events. Clade D viruses were exclusively found circulating in Ireland, with autochthonous transmission of diverse genotype D4 strains associated with large outbreaks across Europe. More recently, genotype D8 was identified and these were associated with importation events. Conclusions: This study provides a comprehensive genetic analysis of circulating measles genotypes in Ireland and discriminated between indigenous and imported viral strains. Notably, an increase in laboratory-confirmed measles cases in the greater than 10 years of age group was seen over the study period. This information is valuable to inform vaccination strategies with a focus on those populations who remain susceptible to measles infection. [Copyright &y& Elsevier]

Abstract: Background: Congenital cytomegalovirus (cCMV) causes serious intrauterine infection and is the leading cause of sensorineural hearing loss. In the absence of routine screening, asymptomatic infections, which constitute approximately 90% of all cCMV cases, remain undiagnosed; however many clinical abnormalities manifest later in childhood. Objectives: The aims of the present study were to determine, for the first time, the incidence of cCMV infection in a large maternity hospital in Dublin city and the optimal sampling method for neonatal screening. Study design: A pilot screening study of asymptomatic infants born was conducted over a 12-month period. Mothers were consented and neonates screened for evidence of CMV infection (n =1044). Urine or saliva was tested for the presence of CMV DNA and reactive results were confirmed with follow-up testing and clinical evaluation. Results: cCMV incidence in the asymptomatic neonates screened was 0.19%. An overall cCMV incidence was extrapolated based on the total number of live-births and data from those infants routinely screened for cCMV during the study period, and estimated as 0.23%. Neonatal urine collection proved prohibitive to mass screening. However, testing of saliva for CMV DNA was rapid, sensitive and suitable for screening. Furthermore, in a low prevalence population, pooling of patient samples proved effective and cost-efficient. Conclusions: The present study concluded that there is a significant burden of undiagnosed cCMV infection in Ireland. The introduction of neonatal CMV DNA testing of saliva is viable, and could be considered as part of the national newborn screening programme, following a cost-benefit analysis. [Copyright &y& Elsevier]

Abstract: Rapid diagnosis is critical to minimize morbidity and mortality associated with infections of the central nervous system (CNS). In this study, we evaluated the performance of a multiplex polymerase chain reaction (PCR) and microarray-based method, Prove-it™ Herpes, in a routine clinical laboratory setting for the diagnostics of 7 herpesviruses in viral CNS infections. Cerebrospinal fluid samples (n = 495), which had arrived for diagnostics in the 5 participating laboratories, were analyzed for herpesvirus DNA both by the current PCR-based method of the laboratory and by the microarray assay. The sensitivity and specificity for the microarray assay were 93% and 99%, respectively. The microarray assay was considered as a rapid and robust diagnostic platform that was easily implemented into the laboratory workflow. The broad herpesvirus coverage and the small sample volume required by the assay could benefit the diagnostics and thus the treatment of life-threatening infections of the CNS, especially among immunocompromised patients. [Copyright &y& Elsevier]

Abstract: Background: Two recent mumps outbreaks have occurred in Ireland in 2004/2005 and 2008/2009. Objectives: To retrospectively investigate any potential shifts in the gender bias and age profile and to identify cohorts who are maintaining mumps virus in circulation. Study design: 2600 cases of acute mumps infection, as determined by the presence of mumps-specific IgM in sera and oral fluids, were confirmed at the National Virus Reference Laboratory. Results: Acute mumps infection occurred more frequently in males with a ratio of approximately 2:1 in the 1–9 and 10–19 years old age groups. A 3:2 ratio was observed in the 20–29 years old cohort and the 30+ age group did not show a gender bias. Serological evidence of prior immunological exposure to mumps virus, as determined by the presence of mumps-specific IgG, was high and similar in males and females of all age cohorts (93.1–100%). A significant increase in the number of acute mumps cases in the ≥30 years old age group was observed. This increase was most striking in the periods between the outbreaks (71.1% in 2007 and 56.2% in 2010). Conclusions: Acute mumps infection showed a male gender bias. The consistent and significant increase of mumps infection in the ≥30 years old age group which is also evident in the periods between outbreaks suggests that this may be the cohort maintaining the mumps virus in circulation. [Copyright &y& Elsevier]

Abstract: The nucleolus, site of ribosome biogenesis, is a dynamic subnuclear organelle involved in diverse cellular functions. The size, number and organisation of nucleoli are cell-specific and while it remains to be established, the nucleolar protein composition would be expected to reflect lineage-specific transcriptional regulation of rDNA genes and have cell-type functional components. Here, we describe the first characterisation of the human T-cell nucleolar proteome. Using the Jurkat T-cell line and a reproducible organellar proteomic approach, we identified 872 nucleolar proteins. In addition to ribosome biogenesis and RNA processing networks, network modeling and topological analysis of nucleolar proteome revealed distinct macromolecular complexes known to orchestrate chromatin structure and to contribute to the regulation of gene expression, replication, recombination and repair, and chromosome segregation. Furthermore, among our dataset, we identified proteins known to functionally participate in T-cell biology, including RUNX1, ILF3, ILF2, STAT3, LSH, TCF-1, SATB1, CTCF, HMGB3, BCLAF1, FX4L1, ZAP70, TIAM1, RAC2, THEMIS, LCP1, RPL22, TOPK, RETN, IFI-16, MCT-1, ISG15, and 14-3-3τ, which support cell-specific composition of the Jurkat nucleolus. Subsequently, the nucleolar localisation of RUNX1, ILF3, STAT3, ZAP70 and RAC2 was further validated by Western Blot analysis and immunofluorescence microscopy. Overall, our T-cell nucleolar proteome dataset not only further expands the existing repertoire of the human nucleolar proteome but support a cell type-specific composition of the nucleolus in T cell and highlights the potential roles of the nucleoli in lymphocyte biology. [Copyright &y& Elsevier]

Abstract: Background: Routine diagnosis of Human T Lymphotropic virus (HTLV) infection is primarily serologically based; however the proportion of unresolved and indeterminate Western blot results range from 0.02% to 50% in endemic areas. Objectives: To validate a sensitive in-house quantitative multiplex real-time assay (mqRT-PCR), capable of detecting and quantifying HTLV-1 and HTLV-2, and use it to differentiate unresolved serological profiles, and monitor infection in HTLV-1 infected patients. Study Design: The mqRT-PCR was designed as a single-tube assay. Quantitative results were reported as copy number of HTLV provirus per 106 cells and the numbers of cells were calculated based on the quantitative result for albumin, of which there are 2 copies/cell. Assay standards were amplified from HTLV-1 infected MT-2 cells and HTLV-2 transfected CEM cells. Blood samples were obtained from HTLV seropositive former blood donors. Results: The mqRT-PCR assay was efficient (98.8–101.2%), reproducible (coefficient of variance<5%) and sensitive to 1 copy for HTLV-1, HTLV-2 and Albumin. The assay resolved the infection profile in 16/17 patients, with undetermined subtype, all of which were reassigned as HTLV-1 infections. In addition, the average PVL detected in patients suffering from HTLV-1 associated HAM/TSP (n =23, 13,450 copies/106 cells) was significantly higher than those detected in asymptomatic carriers (n =21, 6665 copies/106 cells). Conclusions: We propose a new testing algorithm for the laboratory diagnosis of HTLV infection, which includes HTLV specific mqRT-PCR for resolving HTLV serological results. Furthermore, quantitation of PVL load by real-time PCR may be useful in assessing the link between infection and disease, and in monitoring patients undergoing therapy. [Copyright &y& Elsevier]

Abstract: Expression of genes for precursor M (prM) and envelope (E) proteins of West Nile virus (WNV) leads to the production of small, capsidless, and non-infectious virus-like particles (VLPs) possessing the E antigen which is responsible for viral entry and immune protection. It has been reported that processing of the secretion signal affects viral release. We examined the secretion efficiency of VLPs into the culture medium from RK13 or 293T cells transfected with expression vectors for prM and E proteins of WNV which were constructed to comprise different lengths of signal peptides upstream of the prM-E domain. The number of amino acid residues present in the segment markedly affected the production, processing, and secretion of VLPs. Secreted VLPs possessed both the processed M protein and the glycosylated E protein. In addition, immunization with VLPs induced neutralizing antibodies in C3H/HeN mice. These results indicate that the number of amino acid residues comprising the N-terminus of the signal segment controls the efficiency of assembly, maturation, and release of VLPs in the absence of viral protease, which in turn indicates the potential of VLPs as a candidate for an effective WNV subunit vaccine. [Copyright &y& Elsevier]

Abstract: The Human I-mfa domain-Containing protein, HIC, is a 246 amino acid protein that functions as a transcriptional regulator. Although the precise function of HIC remains to be clarified, the association of the HIC gene locus with myeloid neoplasms, its interactions with lymphotropic viruses such as EBV, HIV-1 and HTLV-1 and its expression in immune tissues suggest that HIC might have a modulatory role in immune cells. To further characterise the HIC functional relationship with the immune system, we sought to analyse the HIC gene expression profile in immune cells and to determine if immunomodulatory cytokines, such as interleukin (IL)-2, could regulate the expression of HIC mRNA. Relative quantitative real-time RT-PCR revealed that HIC mRNA is highly expressed in PBMCs and in various hematopoietic cell lines. The immunomodulatory cytokine IL-2 up-regulated HIC gene expression in PBMCs, CEM, MT-2 and U937 but markedly reduced HIC gene expression in Raji. Addition of cycloheximide indicated that the IL-2 effects were independent of de novo protein synthesis and that the HIC gene is a direct target of IL-2. Two cell lines (Jurkat and BJAB) displayed a distinct loss in HIC gene expression. However, when these cell lines were subjected to a combination of DNA methyltransferase and histone-deacetylase inhibitors, (5-aza-2-deoxycytidine and trichostatin A, respectively), HIC expression was de-repressed, indicating possible epigenetic control of HIC expression. Overall, our study describes that the immune expression of HIC is cell-specific, dynamic, and identifies the HIC gene as an IL-2 responsive gene. Furthermore, our de-repression studies support the hypothesis that HIC might represent a candidate tumor suppressor gene. Overall, this report provides new insights for a putative role of HIC in the modulation of immune and inflammatory responses and/or hematological malignancies. [Copyright &y& Elsevier]

HTLV-1 is the etiologic agent of the adult T cell leukemia-lymphoma (ATLL). The viral regulatory protein Tax plays a central role in leukemogenesis as a transcriptional transactivator of both viral and cellular gene expression, and this requires Tax activity in both the cytoplasm and the nucleus. In the present study, we have investigated the mechanisms involved in the nuclear localization of Tax. Employing a GFP fusion expression system and a range of Tax mutants, we could confirm that the N-terminal 60 amino acids, and specifically residues within the zinc finger motif in this region, are important for nuclear localization. Using an in vitro nuclear import assay, it could be demonstrated that the transportation of Tax to the nucleus required neither energy nor carrier proteins. Specific and direct binding between Tax and p62, a nucleoporin with which the importin beta family of proteins have been known to interact was also observed. The nuclear import activity of wild type Tax and its mutants and their binding affinity for p62 were also clearly correlated, suggesting that the entry of Tax into the nucleus involves a direct interaction with nucleoporins within the nuclear pore complex (NPC). The nuclear export of Tax was also shown to be carrier independent. It could be also demonstrated that Tax it self may have a carrier function and that the NF-κB subunit p65 could be imported into the nucleus by Tax. These studies suggest that Tax could alter the nucleocytoplasmic distribution of cellular proteins, and this could contribute to the deregulation of cellular processes observed in HTLV-1 infection. [ABSTRACT FROM AUTHOR]

Abstract: HIV-1 genome has an AU-rich sequence and requires rapid nuclear export by Rev activity to prevent multiple splicing. HIV-1 infection occurs in activated CD4+ T cells where the decay of mRNAs of cytokines and chemokines is regulated by the binding of AU-rich elements to the mRNA-destabilizing protein tristetraprolin. We here investigated the influence of tristetraprolin on the replication of HIV-1. Treatment of siRNA against tristetraprolin in a latently HIV-1 infected cell line increases HIV-1 production following stimulation. A chloramphenicol acetyltransferase and luciferase assay revealed that exogenous tristetraprolin reduced HIV-1 virion production and in contrast increased the multiply spliced products. Furthermore, quantitative RT–PCR analysis showed tristetraprolin increases the ratio of multiple-spliced RNAs to un-, single-spliced RNA. Moreover, an electrophoretic mobility shift assay showed that tristetraprolin binds to synthesized HIV-1 RNA with AU-rich sequence but not to RNA with less AU sequence. These results suggest that tristetraprolin is a regulator of HIV-1 replication and enhances splicing by direct binding to AU-rich sequence of HIV-1 RNAs. [Copyright &y& Elsevier]

Dengue virus (DENV) infection is one of the most important infectious diseases in tropical and subtropical regions around the world. Previously, we performed an initial phenotypic screening of 7000 compounds using DENV type 2 (DENV2)-infected BHK-21 cells to identify small molecules which could inhibit virus replication. In this study, we describe two novel compounds with anti-DENV2 activity, tentatively named Compound-X and Compound-Y. Both compounds possess a quinolone skeleton, and the EC 50 s of Compound-X and Compound-Y against DENV2 were 3.9 μM and 9.2 μM, respectively. Based on a DENV replicon assay, it was suggested that these compounds have anti-DENV2 activity by inhibition of a step in virus replication. Furthermore, using mutational analysis we obtained compounds-resistant to DENV2 infection and identified a mutation, V130A in the NS5 methyltransferase (MTase) domain. However, these compounds did not inhibit MTase activity. In addition, incorporation of an additional NS1 N246D mutation with the NS5 V130A mutation in DENV2 resulted in recovery of viral replication and a further reduction of the sensitivity to the quinolone compounds by an unknown mechanism. Therefore further investigations are required to clarify the antiviral mechanisms of these quinolone compounds. • Quinolone-containing Compound-X and Compound-Y have anti- DENV activity. • The DENV2 NS5 V130A mutant exhibited some resistance to Compound-X and Compound-Y. • The NS1 N246D/NS5 V130A DENV2 double mutant restored viral replication and was also resistant to Compound-X and Compound-Y. • Compound-X and Compound-Y were found not to inhibit methyltransferase (MTase) activity. [ABSTRACT FROM AUTHOR]