Nonstructural Research Articles

Rapid detection of H146-like goose calicivirus using a TaqMan-based real-time PCR assay

H146-like goose-origin calicivirus (H146-like GCV) is a novel Caliciviridae family member in the Sanovirus genus that was recently discovered and proposed to cause runting-stunting syndrome and urate deposition in geese. At present, however, there is a lack of epidemiological information pertaining to the dynamics and distribution of H146-like GCV. The development of novel molecular diagnostic approaches capable of rapidly and accurately detecting this virus would support the strengthening, the prevention, and control of H146-like GCV infection. In the present study, we therefore used a TaqMan probe and primers specific for the viral nonstructural (NS) gene to develop a highly sensitive and specific PCR assay capable of detecting this H146-like GCV. The assay reproducibly detected 5.07 × 102 copies of a recombinant DNA plasmid containing the NS gene, with a dynamic range of 8 orders of magnitude (102–109 copies). Importantly, no cross-reactivity was observed with common viruses that affected waterfowl, and when we used this assay to evaluate clinical samples, we found it to be more sensitive and faster than traditional PCR. In summary, herein, we developed a novel TaqMan-based real-time PCR approach that could reliably detect and diagnose H146-like GCV. This tool will allow for the real-time diagnosis of H146-like GCV infections, enabling researchers to better understand the epidemiology and clinical presentation of this disease.

Identification of Novel Yellow Fever Class II Epitopes in YF-17D Vaccinees.

Yellow fever virus (YFV) is a mosquito-borne member of the genus flavivirus, including other important human-pathogenic viruses, such as dengue, Japanese encephalitis, and Zika. Herein, we report identifying 129 YFV Class II epitopes in donors vaccinated with the live attenuated YFV vaccine (YFV-17D). A total of 1156 peptides predicted to bind 17 different common HLA-DRB1 allelic variants were tested using IFNγ ELISPOT assays in vitro re-stimulated peripheral blood mononuclear cells from twenty-six vaccinees. Overall, we detected responses against 215 YFV epitopes. We found that the capsid and envelope proteins, as well as the non-structural (NS) proteins NS3 and NS5, were the most targeted proteins by CD4+ T cells from YF-VAX vaccinated donors. In addition, we designed and validated by flow cytometry a CD4+ mega pool (MP) composed of structural and non-structural epitopes in an independent cohort of vaccinated donors. Overall, this study provides a comprehensive prediction and validation of YFV epitopes in a cohort of YF-17D vaccinated individuals. With the design of a CD4 epitope MP, we further provide a useful tool to detect ex vivo responses of YFV-specific CD4 T cells in small sample volumes.

Meta-Transcriptomic Discovery of a Divergent Circovirus and a Chaphamaparvovirus in Captive Reptiles with Proliferative Respiratory Syndrome

Viral pathogens are being increasingly described in association with mass morbidity and mortality events in reptiles. However, our knowledge of reptile viruses remains limited. Herein, we describe the meta-transcriptomic investigation of a mass morbidity and mortality event in a colony of central bearded dragons (Pogona vitticeps) in 2014. Severe, extensive proliferation of the respiratory epithelium was consistently found in affected dragons. Similar proliferative lung lesions were identified in bearded dragons from the same colony in 2020 in association with increased intermittent mortality. Total RNA sequencing identified two divergent DNA viruses: a reptile-infecting circovirus, denoted bearded dragon circovirus (BDCV), and the first exogeneous reptilian chaphamaparvovirus—bearded dragon chaphamaparvovirus (BDchPV). Phylogenetic analysis revealed that BDCV was most closely related to bat-associated circoviruses, exhibiting 70% amino acid sequence identity in the Replicase (Rep) protein. In contrast, in the nonstructural (NS) protein, the newly discovered BDchPV showed approximately 31%–35% identity to parvoviruses obtained from tilapia fish and crocodiles in China. Subsequent specific PCR assays revealed BDCV and BDchPV in both diseased and apparently normal captive reptiles, although only BDCV was found in those animals with proliferative pulmonary lesions and respiratory disease. This study expands our understanding of viral diversity in captive reptiles.

Ribosome Pausing at Inefficient Codons at the End of the Replicase Coding Region Is Important for Hepatitis C Virus Genome Replication.

Hepatitis C virus (HCV) infects liver cells and often causes chronic infection, also leading to liver cirrhosis and cancer. In the cytoplasm, the viral structural and non-structural (NS) proteins are directly translated from the plus strand HCV RNA genome. The viral proteins NS3 to NS5B proteins constitute the replication complex that is required for RNA genome replication via a minus strand antigenome. The most C-terminal protein in the genome is the NS5B replicase, which needs to initiate antigenome RNA synthesis at the very 3′-end of the plus strand. Using ribosome profiling of cells replicating full-length infectious HCV genomes, we uncovered that ribosomes accumulate at the HCV stop codon and about 30 nucleotides upstream of it. This pausing is due to the presence of conserved rare, inefficient Wobble codons upstream of the termination site. Synonymous substitution of these inefficient codons to efficient codons has negative consequences for viral RNA replication but not for viral protein synthesis. This pausing may allow the enzymatically active replicase core to find its genuine RNA template in cis, while the protein is still held in place by being stuck with its C-terminus in the exit tunnel of the paused ribosome.

NS5-V372A and NS5-H386Y variations are responsible for differences in interferon α/β induction and co-contribute to the replication advantage of Japanese encephalitis virus genotype I over genotype III in ducklings.

Japanese encephalitis virus (JEV) genotype I (GI) replicates more efficiently than genotype III (GIII) in birds, and this difference is considered to be one of the reasons for the JEV genotype shift. In this study, we utilized duck embryo fibroblasts and domestic ducklings as in vitro and in vivo models of a JEV amplifying avian host to identify the viral determinants of the differing replication efficiency between the GI and GIII strains in birds. GI strains induced significantly lower levels of interferon (IFN)-α and β production than GIII strains, an effect orrelated with the enhanced replication efficiency of GI strains over GIII strains. By using a series of chimeric viruses with exchange of viral structural and non-structural (NS) proteins, we identified NS5 as the viral determinant of the differences in IFN-α and β induction and replication efficiency between the GI and III strains. NS5 inhibited IFN-α and β production induced by poly(I:C) stimulation and harbored 11 amino acid variations, of which the NS5-V372A and NS5-H386Y variations were identified to co-contribute to the differences in IFN-α and β induction and replication efficiency between the strains. The NS5-V372A and NS5-H386Y variations resulted in alterations in the number of hydrogen bonds formed with neighboring residues, which were associated with the different ability of the GI and GIII strains to inhibit IFN-α and β production. Our findings indicated that the NS5-V372A and NS5-H386Y variations enabled GI strains to inhibit IFN-α and β production more efficiently than GIII strains for antagonism of the IFN-I mediated antiviral response, thereby leading to the replication and host adaption advantages of GI strains over GIII strains in birds. These findings provide new insight into the molecular basis of the JEV genotype shift.

Mutations in the cytoplasmic domain of dengue virus NS4A affect virus fitness and interactions with other non-structural proteins.

The dengue virus (DENV) replication complex is made up of its non-structural (NS) proteins and yet-to-be identified host proteins, but the molecular interactions between these proteins are not fully elucidated. In this work, we sought to uncover the interactions between DENV NS1 and its fellow NS proteins using a yeast two-hybrid (Y2H) approach, and found that domain II of NS1 binds to an N-terminal cytoplasmic fragment of NS4A. Mutations in amino acid residues 41 and 43 in this cytoplasmic region of NS4A disrupted the interaction between NS1 and the NS4A-2K-4B precursor protein. When the NS4A Y41F mutation was introduced into the context of the virus via a DENV2 infectious clone, this mutant virus exhibited impaired viral fitness and decreased infectious virus production. The NS4A Y41F mutant virus triggered a significantly muted transcriptional activation of interferon-stimulated genes compared to wild-type virus that is independent of NS4A's ability to antagonize type I interferon signalling. Taken together, we have identified a link between DENV NS1 and the cytoplasmic domain in NS4A that is important for its cellular and viral functions.

Mitochondrial Import of Dengue Virus NS3 Protease and Cleavage of GrpEL1, a Cochaperone of Mitochondrial Hsp70.

Dengue virus infections, which have been reported in nearly 140 countries, pose a significant threat to human health. The genome of dengue virus encodes three structural and seven nonstructural (NS) proteins along with two untranslated regions, one each on both ends. Among them, dengue protease (NS3) plays a pivotal role in polyprotein processing and virus multiplication. NS3 is also known to regulate several host proteins to induce and maintain pathogenesis. Certain viral proteins are known to interact with mitochondrial membrane proteins and interfere with their functions, but the association of a virus-coded protein with the mitochondrial matrix is not known. In this report, by using in silico analysis, we show that NS3pro alone is capable of mitochondrial import; however, this is dependent on its innate mitochondrial transport signal (MTS). Transient-transfection and protein import studies confirm the import of NS3pro to the mitochondrial matrix. Similarly, NS3pro-helicase (amino acids 1 to 464 of NS3) also targets the mitochondria. Intriguingly, reduced levels of matrix-localized GrpE protein homolog 1 (GrpEL1), a cochaperone of mitochondrial Hsp70 (mtHsp70), were noticed in NS3pro-expressing, NS3pro-helicase-expressing, and virus-infected cells. Upon the use of purified components, GrpEL1 undergoes cleavage, and the cleavage sites have been mapped to KR81A and QR92S. Importantly, GrpEL1 levels are seriously compromised in severe dengue virus-infected clinical samples. Our studies provide novel insights into the import of NS3 into host mitochondria and identify a hitherto unknown factor, GrpEL1, as a cleavage target, thereby providing new avenues for dengue virus research and the design of potential therapeutics.IMPORTANCE Approximately 40% of the world's population is at risk of dengue virus infection. There is currently no specific drug or potential vaccine for these infections. Lack of complete understanding of the pathogenesis of the virus is one of the hurdles that must be overcome in developing antivirals for this virus infection. In the present study, we observed that the dengue virus-coded protease imports to the mitochondrial matrix, and our report is the first ever of a virus-coded protein, either animal or human, importing to the mitochondrial matrix. Our analysis indicates that the observed mitochondrial import is due to an inherited mitochondrial transport signal. We also show that matrix-localized GrpE protein homolog 1 (GrpEL1), a cochaperone of mitochondrial Hsp70 (mtHsp70), is also the substrate of dengue virus protease, as observed in vitro and ex vivo in virus-infected cells and dengue virus-infected clinical samples. Hence, our studies reveal an essential aspect of the pathogenesis of dengue virus infections, which may aid in developing antidengue therapeutics.

Rapid detection of H146-like goose calicivirus using real-time RT-PCR with a Taqman minor groove binder probe

H146-like goose-origin calicivirus (H146-like GCV) is a novel Caliciviridae family member in the Sanovirus genus that was associated with gosling growth retardation syndrome growth retardation syndrome complicated by visceral urate deposition. However, there is no accurate and high throughput real-time quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) available for the rapid and highly sensitive identification of H146-like GCV. In this study, a pair of specific primers and a TaqMan minor groove binder (MGB) probe were designed based on a conserved region in the nonstructural (NS) gene sequence. The TaqMan-MGB probe-based one-step qRT-PCR assay was capable of detecting quite low number of targeting nucleic acid as low as 5.07 copies/μL and had excellent intra-assay and inter-assay repeatability with the coefficient of variation (CV) value from 0.558% to 1.293%. The assay was highly specific for H146-like GCV, without cross-reactions with other non-targeted goose-origin viruses, and 62 suspicious tissue samples infected with H146-like GCV from different regions of Fujian Province were used in this study to verify the feasibility and effectiveness of this assay in clinical diagnosis. The results indicated that our assay for the diagnosis and quantification of H146-like GCV was highly sensitive and specific, and should provide a reliable real-time tool for epidemiological and pathogenetic study of H146-like GCV infection, enabling researchers to better understand the epidemiology and clinical presentation of this disease.

Evaluation of Bluetongue Virus (BTV) Antibodies for the Immunohistochemical Detection of BTV and Other Orbiviruses.

The detection of bluetongue virus (BTV) antigens in formalin-fixed tissues has been challenging; therefore, only a limited number of studies on suitable immunohistochemical approaches have been reported. This study details the successful application of antibodies for the immunohistochemical detection of BTV in BSR variant baby hamster kidney cells (BHK-BSR) and infected sheep lungs that were formalin-fixed and paraffin-embedded (FFPE). BTV reactive antibodies raised against non-structural (NS) proteins 1, 2, and 3/3a and viral structural protein 7 (VP7) were first evaluated on FFPE BTV-infected cell pellets for their ability to detect BTV serotype 1 (BTV-1). Antibodies that were successful in immunolabelling BTV-1 infected cell pellets were further tested, using similar methods, to determine their broader immunoreactivity against a diverse range of BTV and other orbiviruses. Antibodies specific for NS1, NS2, and NS3/3a were able to detect all BTV isolates tested, and the VP7 antibody cross-reacted with all BTV isolates, except BTV-15. The NS1 antibodies were BTV serogroup-specific, while the NS2, NS3/3a, and VP7 antibodies demonstrated immunologic cross-reactivity to related orbiviruses. These antibodies also detected viral antigens in BTV-3 infected sheep lung. This study demonstrates the utility of FFPE-infected cell pellets for the development and validation of BTV immunohistochemistry.

Spatial and Temporal Evolutionary Patterns in Puumala Orthohantavirus (PUUV) S Segment.

The S segment of bank vole (Clethrionomys glareolus)-associated Puumala orthohantavirus (PUUV) contains two overlapping open reading frames coding for the nucleocapsid (N) and a non-structural (NSs) protein. To identify the influence of bank vole population dynamics on PUUV S segment sequence evolution and test for spillover infections in sympatric rodent species, during 2010–2014, 883 bank voles, 357 yellow-necked mice (Apodemus flavicollis), 62 wood mice (A. sylvaticus), 149 common voles (Microtus arvalis) and 8 field voles (M. agrestis) were collected in Baden-Wuerttemberg and North Rhine-Westphalia, Germany. In total, 27.9% and 22.3% of bank voles were positive for PUUV-reactive antibodies and PUUV-specific RNA, respectively. One of eight field voles was PUUV RNA-positive, indicating a spillover infection, but none of the other species showed evidence of PUUV infection. Phylogenetic and isolation-by-distance analyses demonstrated a spatial clustering of PUUV S segment sequences. In the hantavirus outbreak years 2010 and 2012, PUUV RNA prevalence was higher in our study regions compared to non-outbreak years 2011, 2013 and 2014. NSs amino acid and nucleotide sequence types showed temporal and/or local variation, whereas the N protein was highly conserved in the NSs overlapping region and, to a lower rate, in the N alone coding part.

Hepatitis C in 2020: A North American Society for Pediatric Gastroenterology, Hepatology, and Nutrition Position Paper.

In 1989, a collaboration between the Centers for Disease Control (CDC) and a California biotechnology company identified the hepatitis C virus (HCV, formerly known as non-A, non-B hepatitis virus) as the causative agent in the epidemic of silent posttransfusion hepatitis resulting in cirrhosis. We now know that, the HCV genome is a 9.6 kb positive, single-stranded RNA. A single open reading frame encodes a 3011 amino acid residue polyprotein that undergoes proteolysis to yield 10 individual gene products, consisting of 3 structural proteins (core and envelope glycoproteins E1 and E2) and 7 nonstructural (NS) proteins (p7, NS2, NS3, NS4A, NS4B, NS5A, and NS5B), which participate in posttranslational proteolytic processing and replication of HCV genetic material. Less than 25 years later, a new class of medications, known as direct-acting antivirals (DAAs) which target these proteins, were introduced to treat HCV infection. These highly effective antiviral agents are now approved for use in children as young as 3 years of age and have demonstrated sustained virologic responses exceeding 90% in most genotypes. Although tremendous scientific progress has been made, the incidence of acute HCV infections has increased by 4-fold since 2005, compounded in the last decade by a surge in opioid and intravenous drug use. Unfortunately, awareness of this deadly hepatotropic virus among members of the lay public remains limited. Patient education, advocacy, and counseling must, therefore, complement the availability of curative treatments against HCV infection if this virus is to be eradicated.

Extensive genetic diversity and host range of rodent-borne coronaviruses.

To better understand the genetic diversity, host associations and evolution of coronaviruses (CoVs) in China we analyzed a total of 696 rodents encompassing 16 different species sampled from Zhejiang and Yunnan provinces. Based on reverse transcriptase PCR-based CoV screening of fecal samples and subsequent sequence analysis of the RNA-dependent RNA polymerase gene, we identified CoVs in diverse rodent species, comprising Apodemus agrarius, Apodemus chevrieri, Apodemus latronum, Bandicota indica, Eothenomys cachinus, Eothenomys miletus, Rattus andamanensis, Rattus norvegicus, and Rattus tanezumi. CoVs were particularly commonplace in A. chevrieri, with a detection rate of 12.44 per cent (24/193). Genetic and phylogenetic analysis revealed the presence of three groups of CoVs carried by a range of rodents that were closely related to the Lucheng Rn rat CoV (LRNV), China Rattus CoV HKU24 (ChRCoV_HKU24), and Longquan Rl rat CoV (LRLV) identified previously. One newly identified A. chevrieri-associated virus closely related to LRNV lacked an NS2 gene. This virus had a similar genetic organization to AcCoV-JC34, recently discovered in the same rodent species in Yunnan, suggesting that it represents a new viral subtype. Notably, additional variants of LRNV were identified that contained putative non-structural (NS)2b genes located downstream of the NS2 gene that were likely derived from the host genome. Recombination events were also identified in the open reading frame (ORF) 1a gene of Lijiang-71. In sum, these data reveal the substantial genetic diversity and genomic complexity of rodent-borne CoVs, and extend our knowledge of these major wildlife virus reservoirs.

MHC class II invariant chain-adjuvanted viral vectored vaccines enhances T cell responses in humans.

Strategies to enhance the induction of high magnitude T cell responses through vaccination are urgently needed. Major histocompatibility complex (MHC) class II-associated invariant chain (Ii) plays a critical role in antigen presentation, forming MHC class II peptide complexes for the generation of CD4+ T cell responses. Preclinical studies evaluating the fusion of Ii to antigens encoded in vector delivery systems have shown that this strategy may enhance T cell immune responses to the encoded antigen. We now assess this strategy in humans, using chimpanzee adenovirus 3 and modified vaccinia Ankara vectors encoding human Ii fused to the nonstructural (NS) antigens of hepatitis C virus (HCV) in a heterologous prime/boost regimen. Vaccination was well tolerated and enhanced the peak magnitude, breadth, and proliferative capacity of anti-HCV T cell responses compared to non-Ii vaccines in humans. Very high frequencies of HCV-specific T cells were elicited in humans. Polyfunctional HCV-specific CD8+ and CD4+ responses were induced with up to 30% of CD3+CD8+ cells targeting single HCV epitopes; these were mostly effector memory cells with a high proportion expressing T cell activation and cytolytic markers. No volunteers developed anti-Ii T cell or antibody responses. Using a mouse model and in vitro experiments, we show that Ii fused to NS increases HCV immune responses through enhanced ubiquitination and proteasomal degradation. This strategy could be used to develop more potent HCV vaccines that may contribute to the HCV elimination targets and paves the way for developing class II Ii vaccines against cancer and other infections.

The Andes Orthohantavirus NSs Protein Antagonizes the Type I Interferon Response by Inhibiting MAVS Signaling.

The small messenger RNA (SmRNA) of the Andes orthohantavirus (ANDV), a rodent-borne member of the Hantaviridae family of viruses of the Bunyavirales order, encodes a multifunctional nucleocapsid (N) protein and for a nonstructural (NSs) protein of unknown function. We have previously shown the expression of the ANDV-NSs, but only in infected cell cultures. In this study, we extend our early findings by confirming the expression of the ANDV-NSs protein in the lungs of experimentally infected golden Syrian hamsters. Next, we show, using a virus-free system, that the ANDV-NSs protein antagonizes the type I interferon (IFN) induction pathway by suppressing signals downstream of the melanoma differentiation-associated protein 5 (MDA5) and the retinoic acid-inducible gene 1 (RIG-I) and upstream of TBK1. Consistent with this observation, the ANDV-NSs protein antagonized mitochondrial antiviral-signaling protein (MAVS)-induced IFN-β, NF-κB, IFN-regulatory factor 3 (IRF3), and IFN-sensitive response element (ISRE) promoter activity. Results demonstrate that ANDV-NSs binds to MAVS in cells without disrupting the MAVS-TBK-1 interaction. However, in the presence of the ANDV-NSs ubiquitination of MAVS is reduced. In summary, this study provides evidence showing that the ANDV-NSs protein acts as an antagonist of the cellular innate immune system by suppressing MAVS downstream signaling by a yet not fully understand mechanism. Our findings reveal new insights into the molecular regulation of the hosts' innate immune response by the Andes orthohantavirus.IMPORTANCEAndes orthohantavirus (ANDV) is endemic in Argentina and Chile and is the primary etiological agent of hantavirus cardiopulmonary syndrome (HCPS) in South America. ANDV is distinguished from other hantaviruses by its unique ability to spread from person to person. In a previous report, we identified a novel ANDV protein, ANDV-NSs. Until now, ANDV-NSs had no known function. In this new study, we established that ANDV-NSs acts as an antagonist of cellular innate immunity, the first line of defense against invading pathogens, hindering the cellular antiviral response during infection. This study provides novel insights into the mechanisms used by ANDV to establish its infection.

Construction of an Escherichia coli expression vector for the non-structural (NS)-1 protein of avian infuenza virus H5N1

In the search for universal vaccine candidates for the prevention of avian influenza, the non-structural (NS)-1 protein 1 of avian influenza virus (AIV) H5N1 has shown promising potential for its ability to effectively stimulate the host immunity. This study was aimed to produce a bacterial expression plasmid using pR SET B vector to harbour the NS1 gene of AIV H5N1 (A/Chicken/Malaysia/5858/2004 (H5N1)) for protein expression in Escherichia coli (E. coli) . The NS1 gene (687 bp) was initially amplified by polymerase chain reaction (PCR) and then cloned into a pGEM-T Easy TA vector. The NS1 gene was released from pGEM-T-NS1 using Eco RI and Xho I restriction enzymes (RE). The pR SET B vector was also linearized using the same RE. The digested NS1 gene and linearized pR SET B were ligated using T4 DNA ligase to form the expression plasmid, pR SET B-NS1. The NS1 gene sequence in pR SET B-NS1 was confirmed by DNA sequencing. To prepare recombinant bacterial cells for protein expression in the future, pR SET B-NS1 was transformed into E. coli strain BL21 (DE3) by heat-shock. Colonies bearing the recombinant plasmid were screened using PCR. The DNA sequencing analysis revealed that the NS1 gene sequence was 97% homologous to that of AIV H5N1 A/Chicken/Malaysia/5858/2004 (H5N1). These results indicated that the NS1 gene of influenza A/Chicken/Malaysia/5858/2004 (H5N1) was successfully amplified and cloned into a pR SET B vector. Bacterial colonies carrying pR SET B-NS1 can be used for the synthesis of NS1-based influenza vaccine in the future and thereby aid in the prevention of avian influenza. DOI: https://doi.org/10.17576/jskm-2020-1802-08

Understanding tricuspid valve remodelling in atrial fibrillation using three-dimensional echocardiography.

Atrial fibrillation (AF) has been associated with tricuspid annulus (TA) dilation in patients with severe functional tricuspid regurgitation (TR); however, the impact of AF is less clear in patients without severe TR. Our aim was to characterize TA remodelling in patients with AF in the absence of severe TR using 3D transoesophageal echocardiography (TOE). Ninety patients underwent clinically indicated transthoracic and TOE: non-structural (NS)-AF (n = 30); AF with left heart disease (LHD) (n = 30), and controls in sinus rhythm (n = 30). Three-dimensional TOE datasets were analysed to measure TA dimensions using novel dedicated tricuspid valve software. The NS-AF group showed biatrial dilatation and normal right ventricular (RV) size with decreased longitudinal function compared to controls, whereas the LHD-AF group showed biatrial dilatation, RV enlargement, decreased biventricular function, and higher systolic pulmonary artery pressure compared with the other groups. Indexed TA area, minimum diameter, maximum diameter, and total perimeter were significantly larger in the NS-AF group than in controls (measurements in end-diastole: 6.4 ± 1.1 vs. 5.0 ± 0.6 cm2/m2, 1.8 ± 0.3 vs. 1.6 ± 0.2 cm/m2, 2.1 ± 0.3 vs. 1.9 ± 0.2 cm/m2, and 6.6 ± 0.9 vs. 5.9 ± 0.7 cm/m2, respectively, all P < 0.05). There was no significant difference in any indexed TA parameter between AF groups. TA circularity index (ratio between minimum and maximal diameters) and TA fractional area change between end-diastole and end-systole were no different among the three groups. AF is associated with right atrial and tricuspid annular remodelling independent of the presence of LHD in patients with intrinsically normal tricuspid leaflets without severe TR.

T Cell Responses Induced by Attenuated Flavivirus Vaccination Are Specific and Show Limited Cross-Reactivity with Other Flavivirus Species.

Members of the flavivirus genus share a high level of sequence similarity and often circulate in the same geographical regions. However, whether T cells induced by one viral species cross-react with other related flaviviruses has not been globally addressed. In this study, we tested pools of epitopes derived from dengue (DENV), Zika (ZIKV), Japanese encephalitis (JEV), West Nile (WNV), and yellow fever (YFV) viruses by intracellular cytokine staining (ICS) using peripheral blood mononuclear cells (PBMCs) of individuals naturally exposed to DENV or immunized with DENV (TV005) or YF17D vaccine. CD8 T cell responses recognized epitopes from multiple flaviviruses; however, the magnitude of cross-reactive responses was consistently severalfold lower than those to the autologous epitope pools and was associated with lower expression of activation markers such as CD40L, CD69, and CD137. Next, we characterized the antigen sensitivity of short-term T cell lines (TCL) representing 29 different individual epitope/donor combinations. TCL derived from DENV monovalent vaccinees induced CD8 and CD4 T cells that cross-reacted within the DENV serocomplex but were consistently associated with >100-fold-lower antigen sensitivity for most other flaviviruses, with no cross-recognition of YFV-derived peptides. CD8 and CD4 TCL from YF17D vaccinees were associated with very limited cross-reactivity with any other flaviviruses and in five out of eight cases >1,000-fold-lower antigen sensitivity. Overall, our data suggest limited cross-reactivity for both CD4 and CD8 T cell responses between flaviviruses and have implications for understanding immunity elicited by natural infection and strategies to develop live attenuated vaccines against flaviviral species.IMPORTANCE The envelope (E) protein is the dominant target of neutralizing antibodies for dengue virus (DENV) and yellow fever virus (YFV). Accordingly, several DENV vaccine constructs use the E protein in a live attenuated vaccine format, utilizing a backbone derived from a heterologous flavivirus (such as YF) as a delivery vector. This backbone comprises the nonstructural (NS) and capsid (C) antigens, which are dominant targets of T cell responses. Here, we demonstrate that cross-reactivity at the level of T cell responses among different flaviviruses is very limited, despite high levels of sequence homology. Thus, the use of heterologous flavivirus species as a live attenuated vaccine vector is not likely to generate optimal T cell responses and might thus impair vaccine performance.

Reverse Genetics System for Shuni Virus, an Emerging Orthobunyavirus with Zoonotic Potential.

The genus Orthobunyavirus (family Peribunyaviridae, order Bunyavirales) comprises over 170 named mosquito- and midge-borne viruses, several of which cause severe disease in animals or humans. Their three-segmented genomes enable reassortment with related viruses, which may result in novel viruses with altered host or tissue tropism and virulence. One such reassortant, Schmallenberg virus (SBV), emerged in north-western Europe in 2011. Shuni virus (SHUV) is an orthobunyavirus related to SBV that is associated with neurological disease in horses in southern Africa and recently caused an outbreak manifesting with neurological disease and birth defects among ruminants in Israel. The zoonotic potential of SHUV was recently underscored by its association with neurological disease in humans. We here report a reverse genetics system for SHUV and provide first evidence that the non-structural (NSs) protein of SHUV functions as an antagonist of host innate immune responses. We furthermore report the rescue of a reassortant containing the L and S segments of SBV and the M segment of SHUV. This novel reverse genetics system can now be used to study SHUV virulence and tropism, and to elucidate the molecular mechanisms that drive reassortment events.

Structure-based screening and validation of bioactive compounds as Zika virus methyltransferase (MTase) inhibitors through first-principle density functional theory, classical molecular simulation and QM/MM affinity estimation

Recent Zika virus (ZIKV) outbreak and association with human diseases such as neurological disorders have raised global health concerns. However, in the absence of an approved anti-ZIKV drug has generated urgency for the drug development against ZIKV infection. Here, structure-based virtual screening of 8589 bioactive compounds, screened at the substrate-binding site of ZIKV nonstructural 5 (NS5)-based structure N-terminal methyltransferase (MTase) domain followed by ADMET (absorption, distribution, metabolism, excretion and toxicity) profiling concluded the four potential lead inhibitors, i.e. (4-acetylamino-benzenesulfonylamino)-acetic acid (F3342-0450), 3-(5-methylfuran-2-yl)-N-(4-sulfamoylphenyl)propanamide (F1736-0142), 8-(2-hydroxy-ethylamino)-1,3-dimethyl-7-(3-methyl-benzyl)-3,7-dihydro-purine-2,6-dione (F0886-0080) and N-[4-(aminosulfonyl)phenyl]-2,3-dihydro-1,4-benzodioxine-2-carboxamide (F0451-2187). Collectively, extra precision docking and Density Functional Theory(DFT) calculations studies identified the F3342-0450 molecule, having strong interactions on the active site of MTase, further supported by molecular dynamics simulation, binding affinity and hybrid QM/MM calculations, suggest a new drug molecule for the antiviral drug development against ZIKV infection. Communicated by Ramaswamy H. Sarma

A Chimeric Japanese Encephalitis Vaccine Protects against Lethal Yellow Fever Virus Infection without Inducing Neutralizing Antibodies.

Recent outbreaks of yellow fever virus (YFV) in West Africa and Brazil resulted in rapid depletion of global vaccine emergency stockpiles and raised concerns about being unprepared against future YFV epidemics. Here we report that a live attenuated virus similar to the Japanese encephalitis virus (JEV) vaccine JE-CVax/Imojev that consists of YFV-17D vaccine from which the structural (prM/E) genes have been replaced with those of the JEV SA14-14-2 vaccine strain confers full protection in mice against lethal YFV challenge. In contrast to the YFV-17D-mediated protection against YFV, this protection is not mediated by neutralizing antibodies but correlates with YFV-specific nonneutralizing antibodies and T cell responses against cell-associated YFV NS1 and other YFV nonstructural (NS) proteins. Our findings reveal the potential of YFV NS proteins to mediate protection and demonstrate that chimeric flavivirus vaccines, such as Imojev, could confer protection against two flaviviruses. This dual protection may have implications for the possible off-label use of JE-CVax in case of emergency and vaccine shortage during YFV outbreaks. In addition, populations in Asia that have been vaccinated with Imojev may already be protected against YFV should outbreaks ever occur on that continent, as several countries/regions in the Asia-Pacific are vulnerable to international spread of the YFV.IMPORTANCE Efficient and safe vaccines against yellow fever (e.g., YFV-17D) that provide long-lasting protection by rapidly inducing neutralizing antibody responses exist. However, the vaccine supply cannot cope with an increasing demand posed by urban outbreaks in recent years. Here we report that JE-CVax/Imojev, a YFV-17D-based chimeric Japanese encephalitis vaccine, also efficiently protects against YFV infection in mice. In case of shortage of the YFV vaccine during yellow fever outbreaks, (off-label) use of JE-CVax/Imojev may be considered. Moreover, wider use of JE-CVax/Imojev in Asia may lower the risk of the much-feared YFV spillover to the continent. More generally, chimeric vaccines that combine surface antigens and replication machineries of two distinct flaviviruses may be considered dual vaccines for the latter pathogen without induction of surface-specific antibodies. Following this rationale, novel flavivirus vaccines that do not hold a risk for antibody-dependent enhancement (ADE) of infection (inherent to current dengue vaccines and dengue vaccine candidates) could be designed.