Virus Core Research Articles

Cyclophilin A Prevents HIV-1 Restriction in Lymphocytes by Blocking Human TRIM5α Binding to the Viral Core

Disruption of cyclophilin A (CypA)-capsid interactions affects HIV-1 replication in human lymphocytes. To understand this mechanism, we utilize human Jurkat cells, peripheral blood mononuclear cells (PBMCs), and CD4+ Tcells. Our results show that inhibition of HIV-1 infection caused by disrupting CypA-capsid interactions is dependent on human tripartite motif 5α (TRIM5αhu), showing that TRIM5αhu restricts HIV-1 in CD4+ Tcells. Accordingly, depletion of TRIM5αhu in CD4+ Tcells rescues HIV-1 that fail to interact with CypA, such as HIV-1-P90A. We found that TRIM5αhu binds to the HIV-1 core. Disruption of CypA-capsid interactions fail to affect HIV-1-A92E/G94D infection, correlating with the loss of TRIM5αhu binding to HIV-1-A92E/G94D cores. Disruption of CypA-capsid interactions in primary cells has a greater inhibitory effect on HIV-1 when compared to Jurkat cells. Consistent with TRIM5α restriction, disruption of CypA-capsid interactions in CD4+ Tcells inhibits reverse transcription. Overall, our results reveal that CypA binding to the core protects HIV-1 from TRIM5αhu restriction.

SOX2 Represses Hepatitis B Virus Replication by Binding to the Viral EnhII/Cp and Inhibiting the Promoter Activation.

Hepatitis B virus (HBV) replication is controlled by four promoters (preS1, preS2, Cp, and Xp) and two enhancers (EnhI and EnhII). EnhII stimulates Cp activity to regulate the transcriptions of precore, core, polymerase, and pregenomic RNAs, and therefore, EnhII/Cp is essential for the regulation of HBV replication. This study revealed a distinct mechanism underlying the suppression of EnhII/Cp activation and HBV replication. On the one hand, the sex determining region Y box2 (SOX2), a transcription factor, is induced by HBV. On the other hand, SOX2, in turn, represses the expression levels of HBV RNAs, HBV core-associated DNA, hepatitis B surface antigen (HBsAg), and hepatitis B e antigen (HBeAg), thereby playing an inhibitory role during HBV replication. Further studies indicated that SOX2 bound to the EnhII/Cp DNA and repressed the promoter activation. With the deletion of the high mobility group (HMG) domain, SOX2 loses the ability to repress EnhII/Cp activation, viral RNA transcription, HBV core-associated DNA replication, HBsAg and HBeAg production, as well as fails to enter the nucleus, demonstrating that the HMG domain is required for the SOX2-mediated repression of HBV replication. Moreover, SOX2 represses HBsAg and HBeAg secretion in BALB/c mice sera, and attenuates HBV 3.5 kb RNA transcription and hepatitis B virus core protein (HBc) production in the liver tissues, demonstrating that SOX2 suppresses HBV replication in mice. Furthermore, the results revealed that the HMG domain was required for SOX2-mediated repression of HBV replication in the mice. Taken together, the above facts indicate that SOX2 acts as a new host restriction factor to repress HBV replication by binding to the viral EnhII/Cp and inhibiting the promoter activation through the HMG domain.

Reovirus uses macropinocytosis-mediated entry and fast axonal transport to infect neurons.

Several barriers protect the central nervous system (CNS) from pathogen invasion. Yet viral infections of the CNS are common and often debilitating. Understanding how neurotropic viruses co-opt host machinery to overcome challenges to neuronal entry and transmission is important to combat these infections. Neurotropic reovirus disseminates through neural routes and invades the CNS to cause lethal encephalitis in newborn animals. To define mechanisms of reovirus neuronal entry and directional transport, we used primary neuron cultures, which reproduce in vivo infection patterns displayed by different reovirus serotypes. Treatment of neurons with small-molecule inhibitors of different endocytic uptake pathways allowed us to discover that the cellular machinery mediating macropinocytosis is required for reovirus neuronal entry. This mechanism of reovirus entry differs from clathrin-mediated endocytosis, which is used by reovirus to invade non-neuronal cells. Analysis of reovirus transport and release from isolated soma or axonal termini of neurons cultivated in microfluidic devices indicates that reovirus is capable of retrograde but only limited anterograde neuronal transmission. The dynamics of retrograde reovirus movement are consistent with fast axonal transport coordinated by dynein along microtubules. Further analysis of viral transport revealed that multiple virions are transported together in axons within non-acidified vesicles. Reovirus-containing vesicles acidify after reaching the soma, where disassembly of virions and release of the viral core into the cytoplasm initiates replication. These results define mechanisms of reovirus neuronal entry and transport and establish a foundation to identify common host factors used by neuroinvasive viruses. Furthermore, our findings emphasize consideration of cell type-specific entry mechanisms in the tailored design of neurotropic viruses as tracers, oncolytic agents, and delivery vectors.

HIV-1 uncoats in the nucleus near sites of integration

HIV-1 capsid core disassembly (uncoating) must occur before integration of viral genomic DNA into the host chromosomes, yet remarkably, the timing and cellular location of uncoating is unknown. Previous studies have proposed that intact viral cores are too large to fit through nuclear pores and uncoating occurs in the cytoplasm in coordination with reverse transcription or at the nuclear envelope during nuclear import. The capsid protein (CA) content of the infectious viral cores is not well defined because methods for directly labeling and quantifying the CA in viral cores have been unavailable. In addition, it has been difficult to identify the infectious virions because only one of ∼50 virions in infected cells leads to productive infection. Here, we developed methods to analyze HIV-1 uncoating by direct labeling of CA with GFP and to identify infectious virions by tracking viral cores in living infected cells through viral DNA integration and proviral DNA transcription. Astonishingly, our results show that intact (or nearly intact) viral cores enter the nucleus through a mechanism involving interactions with host protein cleavage and polyadenylation specificity factor 6 (CPSF6), complete reverse transcription in the nucleus before uncoating, and uncoat <1.5 h before integration near (<1.5 μm) their genomic integration sites. These results fundamentally change our current understanding of HIV-1 postentry replication events including mechanisms of nuclear import, uncoating, reverse transcription, integration, and evasion of innate immunity.

Inhibition of hepatitis C virus genotype 4 replication using siRNA targeted to the viral core region and the CD81 cellular receptor.

Hepatitis C virus (HCV) is one of the most important causative agents of hepatitis worldwide. The current study aimed to evaluate the silencing effect of the small interference RNA (siRNA) molecules designed against the core region of HCV genotype 4 (HCV-4) and the CD81 gene, which is the cellular receptor for HCV in the human hepatocytes. RT-PCR was used to measure the changes in both the viral HCV core and the cellular CD81 genes induced by the specific siRNA molecules. Additionally, the fluctuations in either the viral or the cellular proteins of the target regions were tested by flow cytometry and immunofluorescence. The results showed the effectiveness of the used siRNA molecules against the target genes in either RNA or protein levels. The effect of 100nM of siCD81 and 40nM of siCore was more evident at 24 and 48h post-transfection. The combination of the two siRNA molecules resulted in an extra inhibitory effect of the HCV core at both the RNA (85.6%) and protein (98.5%) levels. The current study suggested that targeting of the CD81 cellular receptor and/or the viral HCV core region by the small interference molecules might be a suitable choice in the suppression of HCV-4 replication. This might assist the development of new antiviral medications and provides a new alternative strategy for the targeting and treatment of HCV genotype 4.

Real-Time Condensation of Nanoconfined DNA by an Intrinsically Disordered Polycationic Protein

The Hepatitis C viral core protein (HCVcp) is an intrinsically disordered nucleocapsid protein that induces structural transformations in the viral RNA necessary for its efficient packaging within the viral particle. The mechanism of the nucleoprotein complex (NPC) formation is mainly driven by electrostatic interactions, where HCVcp locally screens repulsion charges by acting as a macromolecular counterion (+22). In the later stage of viral assembly, the number of HCVcp molecules increases above a critical threshold concentration after which all NPCs are transformed into nucleocapsid like particles (NLP). Herein, we have used single DNA molecule nanofluidics in combination with fluorescence microscopy to monitor the interaction between HCVcp and double stranded DNA. In our novel dynamic nanofluidic device, we can confine single DNA molecules in the nanochannels and visualize their condensation under constant inflow of proteins. We observed that NPC formation incur changes in the mechanical properties of stretched DNA molecules. These changes are primarily marked by appearance of a local compaction at either end of the stretched DNA, which serves as a nucleation site for global compaction. The length of the stretched DNA molecules gradually decreases and reaches a condensed state at above threshold concentrations of HCVcp. We were also able to follow unpacking of the condensed state in real time. Proteinase K was introduced from another inlet of the dynamic device and it gradually removed the HCVcp from DNA, which in turn lead to an increase in extension of condensed DNA. Our data allowed us to investigate the formation and dissociation of the DNA:HCVcp complex in real time. On a broader level, our results highlight the use of nanofluidic channels for studying complex and dynamic DNA-protein interactions.

Prolyl Isomerase Pin1 Regulates the Stability of Hepatitis B Virus Core Protein.

The dynamic interplay between virus and host proteins is critical for establishing efficient viral replication and virus-induced pathogenesis. Phosphorylation-dependent prolyl isomerization by Pin1 provides a unique mechanism of molecular switching to control both protein function and stability. We demonstrate here that Pin1 binds and stabilizes hepatitis B virus core protein (HBc) in a phosphorylation-dependent manner, and promotes the efficient viral propagation. Phos-tag gel electrophoresis with various site-directed mutants of HBc revealed that Thr160 and Ser162 residues within the C terminal arginine-rich domain are phosphorylated concomitantly. GST pull-down assay and co-immunoprecipitation analysis demonstrated that Pin1 associated with phosphorylated HBc at the Thr160-Pro and Ser162-Pro motifs. Chemical or genetic inhibition of Pin1 significantly accelerated the rapid degradation of HBc via a lysosome-dependent pathway. Furthermore, we found that the pyruvate dehydrogenase phosphatase catalytic subunit 2 (PDP2) could dephosphorylate HBc at the Pin1-binding sites, thereby suppressing Pin1-mediated HBc stabilization. Our findings reveal an important regulatory mechanism of HBc stability catalyzed by Pin1 and may facilitate the development of new antiviral therapeutics targeting Pin1 function.

Design of a novel rapid immunoassay for simultaneous detection of hepatitis C virus core antigen and antibodies.

HCV is a potential cause of viral hepatitis, which leads to blood-borne chronic hepatitis, liver cirrhosis and hepatocellular carcinoma. Anti-HCV antibody detection assays detect HCV infection after approximately 70 days. HCV core antigen can be detected much earlier than anti-HCV antibodies. However, it disappears soon after the appearance of anti-HCV antibodies. Thus, there is a need for a rapid assay for simultaneous detection of HCV core antigen and anti-HCV antibodies for early diagnosis of HCV infection. A rapid diagnostic assay (HCV Ag-Ab Combo) for simultaneous detection of HCV core antigen and anti-HCV antibodies for early diagnosis of HCV infection was developed. HCV Ag-Ab Combo was evaluated in order to determine its potential for detection of HCV infection earlier than anti-HCV antibody detection assays. We compared the sensitivity of the newly developed assay with anti-HCV antibody detection assays (ELISA [HCV Ab ELISA] and rapid test [HCV Ab Rapid]) and HCV core antigen/anti-HCV antibody detection ELISA (HCV Ag-Ab ELISA). This study included 11 samples that were found positive in HCV RNA detection and HCV Ag-Ab ELISA but negative in HCV antibody detection assays (HCV Ab ELISA and rapid), 10 samples that were found positive in HCV Ag-Ab ELISA and HCV Ab ELISA but negative in HCV Ab Rapid, 69 samples that were found positive in HCV Ag-Ab ELISA, HCV Ab ELISA, and HCV Ab Rapid, and 509 samples that were found negative in HCV Ag-Ab ELISA, HCV Ab ELISA, and HCV Ab Rapid. Three seroconversion panels, PHV 913, PHV 911 (M) and PHV904-00-1.0, and aHCV RNA genotype qualification panel (2400-0182) acquired from Seracare Life Sciences (USA) were also tested. HCV Ag-Ab Combo showed a combined sensitivity and specificity of 100% when tested with 90 samples that were positive for HCV by HCV Ag-Ab ELISA and 509 HCV-negative samples. Its positive predictive value (PPV) and negative predictive value (NPV) were found to be 100%. It detected HCV infection approximately 7 to 12 days earlier than the HCV Ab detection assays, and its performance was not affected when testing different genotypes of HCV. HCV Ag-Ab Combo did not detect HCV infection as early as HCV RNA or HCV Ag-Ab ELISA. HCV Ag-Ab Combo provided a significant improvement for the early detection of HCV infection during the preseroconversion phase when compared with anti-HCV antibody detection assays. It could be a useful screening assay, and an alternative to HCV RNA detection or HCV Ag-Ab ELISA when nucleic acid technologies cannot be implemented.

Influenza A virus uncoating.

Influenza A virus (IAV) is an enveloped virus of the Orthomyxoviridae with a negative-sense single-stranded RNA genome. During virus cell entry, viral and cellular cues are delivered in a stepwise manner within two distinct cellular compartments-the endosomes and the cytosol. Endosome maturation primes the viral core for uncoating by cytosolic host proteins and host-mediated virus disaggregation is essential for genome import and replication in the nucleus. Recent evidence shows that two well-known cellular proteins-histone deacetylase 6 (HDAC6) and karyopherin-β2 (kapβ2)-uncoat influenza virus. HDAC6 is 1 of 11 HDACs and an X-linked, cytosolic lysine deacetylase. Under normal cellular conditions HDAC6 is the tubulin deacetylase. Under proteasomal stress HDAC6 binds unanchored ubiquitin, dynein and myosin II to sequester misfolded protein aggregates for autophagy. Kapβ2 is a member of the importin β family that transports RNA-binding proteins into the nucleus by binding to disordered nuclear localization signals (NLSs) known as PY-NLS. Kapβ2 is emerging as a universal uncoating factor for IAV and human immunodeficiency virus type 1 (HIV-1). Kapβ2 can also reverse liquid-liquid phase separation (LLPS) of RNA-binding proteins by promoting their disaggregation. Thus, it is becoming evident that key players in the management of cellular condensates and membraneless organelles are potent virus uncoating factors. This emerging concept reveals implications in viral pathogenesis, as well as, the promise for cell-targeted therapeutic strategies to block universal virus uncoating pathways hijacked by enveloped RNA viruses.

Dietary Restriction Suppresses Steatosis-Associated Hepatic Tumorigenesis in Hepatitis C Virus Core Gene Transgenic Mice

Background and Aims: Dietary restriction (DR) is a preventive strategy for obesity, metabolic syndrome, cardiovascular disease, and diabetes. Although an interconnection between obesity, metabolic syndrome, fatty liver, and hepatocellular carcinoma has been documented, the mechanism and impact of DR on steatosis-derived hepatocarcinogenesis are not fully understood. This study aimed to evaluate whether DR can prevent hepatic tumorigenesis. Methods: Male hepatitis C virus core gene transgenic (HCVcpTg) mice that develop spontaneous age-dependent insulin resistance, hepatic steatosis, and ensuing liver tumor development without apparent hepatic fibrosis, were fed with either a control diet ad libitum (control group) or 70% of the same control diet (DR group) for 15 months, and liver phenotypes were investigated. Results: DR significantly reduced the number and volume of liver tumors. DR attenuated hepatic oxidative and endoplasmic reticulum stress and markedly suppressed nuclear factor-κB, signal transducer and activator of transcription 3 (STAT3) and STAT5, and phosphorylation of extracellular signal-regulated kinase, leading to downregulation of several pro-oncogenic mediators, such as cyclin D1. Serum insulin and insulin-like growth factor 1 levels, as well as hepatic expression of insulin receptor substrate 1/2, phosphatidylinositol-3 kinase, and serine/threonine-protein kinase AKT, were downregulated by DR. A transcriptome analysis revealed that STAT3 signaling and lipogenesis were the most suppressed hepatocarcinogenic pathways affected by DR. Additionally, DR stimulated autophagy and p62/sequestosome 1 degradation, enhanced phosphorylation of AMP-activated protein kinase α, increased fibroblast growth factor 21 expression, and attenuated expression of senescence-associated secretory phenotypes. Conclusion: DR suppressed steatosis-associated hepatic tumorigenesis in HCVcpTg mice, mainly due to attenuation of pathways involved in inflammation, cellular stress, cell proliferation, insulin signaling, and senescence. These findings support the notion that persistent 30% reduction of daily food intake is beneficial for preventing steatosis-associated hepatocarcinogenesis caused by HCV core protein.

Acute Hepatitis B or Chronic Hepatitis B with Acute Exacerbation: Differentiating Clinical, Biochemical, Immunonological, and Virological Parameters

Background and Aims: Acute hepatitis B virus (HBV) infection and chronic HBV infection presenting as acute illness have differing prognosis. Differentiation between acute viral hepatitis B (AVH-B) and chronic hepatitis B with an acute exacerbation (CHB-AE) is difficult if prior hepatitis B surface antigen (HBsAg) status is unknown. This prospective study was undertaken to screen various factors that could help with this differentiation. Methods: All consecutive patients presenting with AVH-B like illness were enrolled in this study and were evaluated as per predefined study protocol. Patients were divided into AVH-B and CHB-AE groups based on HBsAg status at the end of 6 months. Results: Significant differences in clinical and laboratory parameters were found between AVH-B and CHB-AE. No statistically significant difference in prodromal symptoms and jaundice was seen. Ascites (40%) and hepatic encephalopathy (10%) were seen only in patients with CHB-AE (P = 0.002 and 0.244, respectively). Anti-hepatitis B virus core antigen immunoglobulin M (IgM anti-HBc) levels ≥10.15 signal-cutoff-ratio (S/CO) had positive predictive value (PPV) and negative predictive value (NPV) of 90% for diagnosis of AVH-B. Hepatitis B virus deoxyriboNucleic acid (HBV DNA) levels ≥25032 IU/mLhas 62.5% PPV and 69% NPV for diagnosis of CHB-AE. Alpha-feto protein (AFP) at >22.5 ng/mLmL for diagnosing CHB-AE has PPV and NPV of 83% and 62%, respectively. All six mortalities were seen in CHB-AE group with median survival of 2 months. Conclusions: Differentiation of AVH-B and CHB-AE is important as management and prognosis differ. Low IgM anti-HBc levels ( 22.5 ng/mL) favor CHB-AE over AVH-B.

Structural studies on negative-strand RNA virus

Negative-strand RNA viruses do not possess a rigid viral shell, and their structures are flexible and fragile. We have applied various electron microscopies to analyze the morphologies of influenza and Ebola virus. Our studies have revealed the native interior and exterior ultrastructures of influenza virus as well as the assembly of Ebola virus core in atomic detail.

The enhancing impact of amino termini of hepatitis C virus core protein on activation of hepatic stellate cells.

To investigate the potential effects of carboxyl and amino termini of HCV core protein on the HSCs activation. The core protein is recognized as the most important fibrosis inducer of Hepatitis C virus (HCV). While the exogenous fibrotic effect of HCV core protein has been reported earlier, the endogenous effect and the role of two termini must still be investigated. Plasmids expressing full length, carboxyl-truncated (T1), or amino-truncated (T3) versions of the core were transfected into LX2 cells. MTT assay was performed to evaluate the cytotoxicity of the endogenous expression of different regions of core protein on these cells. Afterwards, the total RNA was reversely transcribed and introduced into quantitative polymerase chain reaction (qPCR) to measure the expression level of collagen type I (COL1A1), α-smooth muscle actin (-SMA), tissue metalloproteinase inhibitor1 (TIMP-1), and transforming growth factor-β1 (TGF-β1). In addition, TGF-β1 as a fibrotic factor, was also assessed in the supernatant of LX-2 cells using ELISA method. The full and T1 versions of the core exhibited a measurable proliferative effect on LX2 cells (P<0.05). Analysis of the gene expression was also showed that, in spite of amino-truncated version, these constructs represented a significant activation impact compared to the empty plasmid. Moreover, the result of TGFβ assay was in agreement with the results of mRNA expression analysis. The endogenous expression of the full and carboxyl-truncated versions of the core exhibited a significant activator effect on HSCs. Therefore, it can be concluded that, amino domain of HCV core protein performs a stellate cell activation role.

Detection of hepatitis C virus core antigen as an alternative method for diagnosis of hepatitis C virus infection in blood donors negative for hepatitis C virus antibody.

Screening of blood donors in many countries is based on the use of serologic assays to detect specific anti-HCV antibodies (HCV Ab), but it lacks detection sensitivity. So, HCV RNA detection using the current gold standard real-time PCR is a must to rule out HCV infection with the main disadvantage being of high cost. HCV core antigen (HCV-c-Ag) immunoassay is proposed as a more cost efficient alternative to HCV RNA detection with PCR. To evaluate the effectiveness of HCV-c-Ag detection as a cheap alternative to HCV RNA (PCR) in diagnosis of HCV infection in blood donors who are HCV Ab negative. One hundred eighty-six volunteer blood donors who tested negative for HCV Ab were examined for HCV-c-Ag. Seven cases out of these 186 cases were HCV-c-Ag positive (4%). HCV RNA detection (PCR technique) was done to 30 cases (seven cases who test positive for HCV-c-Ag and 23 cases who test negative). Six out of the seven cases who were HCV-c-Ag positive (86%) were HCV RNA positive. Twenty-two cases out of the 23 cases who were HCV-c-Ag negative (96%) were HCV RNA negative. HCV-c-Ag detection is an efficient method for diagnosis of HCV infection during screening of blood donors with high specificity (95.6%) and high negative predictive value (95.6%).

Disrupting the association of Autographa californica multiple nucleopolyhedrovirus Ac93 with cellular ESCRT-III/Vps4 hinders nuclear egress of nucleocapsids and intranuclear microvesicles formation

The endosomal sorting complex required for transport (ESCRT) pathway is required for efficient egress of Autographa californica multiple nucleopolyhedrovirus (AcMNPV). In this study, we found that Ac93, a baculovirus core protein, contains a conserved MIM1-like motif. Alanine substitutions for six leucine residues in MIM1-like motif revealed that L142, L145, L146, and L149 are required for association of Ac93 with the MIT domain of Vps4. Mutations of these residues also blocked self-association and the association of Ac93 with ESCRT-III proteins or other viral core proteins Ac76 and Ac103, and resulted in a substantial reduction of infectious virus production, less efficient nuclear egress of progeny nucleocapsids, and the defect of intranuclear microvesicles formation. Combined with the localization of the association of Ac93 with ESCRT-III/Vps4 and other viral proteins at the nuclear membrane, we propose that the coordinated action of these viral proteins and ESCRT-III/Vps4 may be involved in remodeling the nuclear membrane.

Structural Basis of Poxvirus Transcription: Vaccinia RNA Polymerase Complexes

Poxviruses encode a multisubunit DNA-dependent RNA polymerase (vRNAP) that carries out viral gene expression in the host cytoplasm. We report cryo-EM structures of core and complete vRNAP enzymes from Vaccinia virus at 2.8Å resolution. The vRNAP core enzyme resembles eukaryotic RNA polymerase II (Pol II) but also reveals many virus-specific features, including the transcription factor Rap94. The complete enzyme additionally contains the transcription factor VETF, the mRNA processing factors VTF/CE and NPH-I, the viral core protein E11, and host tRNAGln. This complex can carry out the entire early transcription cycle. The structures show that Rap94 partially resembles the Pol II initiation factor TFIIB, that the vRNAP subunit Rpo30 resembles the Pol II elongation factor TFIIS, and that NPH-I resembles chromatin remodeling enzymes. Together with the accompanying paper (Hillen etal., 2019), these results provide the basis for unraveling the mechanisms of poxvirus transcription and RNA processing.

Can hepatitis C virus core antigen replace quantitative RNA in the assessment of a sustained virologic response?

BackgroundThe widespread use of direct-acting antivirals (DAAs) in the treatment of hepatitis C virus (HCV) infection has reduced the need for monitoring HCV-RNA levels because viral kinetics do not predict a sustained virologic response (SVR). However, the performance of cheaper tests, such as the assay to quantify hepatitis C virus core antigen (HCV Ag), has not been determined. Our aim was to assess the accuracy of the HCV core Ag test in predicting the achievement of SVR and its predictive value as a monitoring test in the course of treatment.Patients and methodsThis prospective study was carried out on 90 patients, chronically infected with HCV, who received DAA therapy. Blood samples were collected and the levels of HCV core Ag and HCV-RNA were measured at baseline and at 12 weeks after the end of treatment. We compared the ability of these assays to predict which patients would achieve SVR12.ResultsThe baseline level of HCV-RNA was 1 688 529.6±994 697.3 IU/ml (range: 312 700–3 491 100 IU/ml) and HCV core Ag was 179.2±83.5 pg/ml (range: 33.5–315.6 pg/ml). HCV Ag became undetectable in 92.2% 12 weeks after the end of treatment, whereas HCV-RNA became undetectable in 87.8% at the end of treatment (P<0001). Seventy-nine out of 90 (87.8%) patients achieved SVR12; the test for HCV Ag identified 63.6% of these patients.ConclusionMeasurement of HCV core Ag can monitor the efficacy of DAA therapy for HCV infection. Thus, it can be applied in clinical practice.

What are the limits of the packaging capacity for genomic RNA in the cores of rotaviruses and of other members of the Reoviridae?

Species A rotaviruses (RVAs) can package additional homologous genomic sequences which have emerged by partial duplication events (genome rearrangements). Due to the availability of a plasmid only-based reverse genetics system for RVs, various heterologous sequences have been rescued as fusion genes into viable RVAs. Using cryo-electron microscopy, the native structures of the dsRNA genome of Cytoplasmic Polyhedrosis Virus, a member of the Cypovirus genus of the Reoviridae, and partially of Rotavirus and Orbivirus, have been determined. These structural and some functional data were used to calculate the volume occupied by the viral genome in relation to the volume available in viral cores. The native dsRNA genomes were estimated to occupy only one to two thirds of the space available in core particles of the genera Cypovirus, Rotavirus, Orbivirus, Orthoreovirus, and Coltivirus in the Reoviridae family. The findings raise the captivating question of the upper limit of the recombinant RNA packaging capacity of reovirus particles.

The emergence of subclades A1 and A3 avipoxviruses in India.

During the years 2010-2018, avipoxvirus (APV) outbreaks were observed in the domestic chickens and pigeons present in the eastern Indian state of Odisha. Based on typical pox lesions, followed by molecular techniques, the overall morbidity was found to be 18%-19.23% and 16.92%-23% in chickens and pigeons, respectively. The cutaneous forms of the disease were observed with varied rates of mortality, being 47.36%-52.77% in chickens and 39.13%-92% in pigeons. PCR amplification targeting the viral P4b core protein-coding gene and the DNA polymerase gene confirmed the presence of APV strains in 10 birds. Subsequent phylogenetic analysis of these two genes confirmed that the circulating strains were members of APV clade A. The subclade analysis revealed the introduction of A1 and A3 subclades in Indian chickens and pigeons, respectively. This study is the first molecular record of APVs circulating in eastern Indian birds (Odisha) and involves the first use of the polymerase gene to reveal the circulating clades of Indian APVs.

TMIC-04. CANCER STEM CELL GENETIC AND GENOMIC ALTERATIONS DIFFERENTIALLY DICTATE STROMAL CELL REGULATION OF LOW-GRADE GLIOMA GROWTH

Abstract BACKGROUND Children with the Neurofibromatosis type 1 (NF1) brain tumor predisposition syndrome are prone to the formation of low-grade gliomas (LGGs) of the optic pathway (optic pathway glioma; OPG). While all tumors harbor bi-allelic NF1 gene inactivation, there is significant clinical variability in affected children with respect to tumor growth, associated neurologic deficits, and response to therapy. In order to identify the potential factors that contribute to glioma biological variability, we leveraged a series of Nf1 mutant optic glioma mouse strains with different germline Nf1 gene mutations and secondary genomic alterations. METHODS Cancer stem cells (optic glioma stem cells; o-GSCs) were isolated from these Nf1 mutant optic glioma mouse strains, and their intrinsic growth properties and chemokine expression profiles determined. RESULTS Differences in the intrinsic o-GSC growth properties did not account for the observed differences in Nf1 optic glioma tumor growth. Instead, Nf1 optic glioma tumor growth correlated with the differential recruitment of T cells and microglia by chemokines produced by the o-GSCs. As such, the collective action of T cells and microglia dictated the level of Ccl5 expression, the key stromal factor that determines murine NF1-associated optic glioma growth. CONCLUSIONS Taken together, these observations demonstrate that genomic and genetic differences within the cancer cells create unique Nf1 optic glioma growth patterns through the differential elaboration of chemokines that attract T cells and microglia. GRANT SUPPORT: This work was funded by grants from the National Cancer Institute (1-R01-CA195692-01) and the National Institute of Neurological Disorders and Stroke (1-R35-NS07211-01) to D.H.G, as well as to the Hope Center Viral Vectors Core and the Genome Technology Access Center (UL1-TR000448). Y.P. was supported by a fellowship grant from the James S. McDonnell Foundation.