Markers Of Replication Research Articles

HIV Antibody Level as a Marker of HIV Persistence and Low-Level Viral Replication.

Human immunodeficiency virus (HIV) antibodies are generated and maintained by ongoing systemic expression of HIV antigen. We investigated whether HIV antibody responses as measured by high-throughput quantitative and qualitative assays could be used to indirectly measure persistent HIV replication in individuals receiving antiretroviral therapy (ART). HIV antibody responses were measured over time in the presence or absence of suppressive ART and were compared to the HIV reservoir size and expression of antiviral restriction factors. Among untreated individuals, including both elite controllers (ie, persons with a viral load of ≤40 copies/mL) and noncontrollers, antibody parameters were stable over time and correlated with the individual viral load. Viral suppression with ART led to a progressive decline in antibody responses after treatment induction that persisted for 5-7 years. Higher levels of HIV antibodies during suppressive therapy were associated with later initiation of ART after infection, with higher DNA and cell-associated RNA levels, and with lower expression of multiple anti-HIV host restriction factors. These findings suggest that declining antibody levels during ART reflect lower levels of antigen production and/or viral replication in the persistent HIV reservoir. Results of relatively inexpensive and quantitative HIV antibody assays may be useful indirect markers that enable efficient monitoring of the viral reservoir and suppression during functional-cure interventions.

Sustained Inhibition of HBV Replication InVivo after Systemic Injection of AAVs Encoding Artificial Antiviral Primary MicroRNAs.

Chronic infection with hepatitis B virus (HBV) remains a problem of global significance and improving available treatment is important to prevent life-threatening complications arising in persistently infected individuals. HBV is susceptible to silencing by exogenous artificial intermediates of the RNA interference (RNAi) pathway. However, toxicity of Pol III cassettes and short duration of silencing by effectors of the RNAi pathway may limit anti-HBV therapeutic utility. To advance RNAi-based HBV gene silencing, mono- and trimeric artificial primary microRNAs (pri-miRs) derived from pri-miR-31 were placed under control of the liver-specific modified murine transthyretin promoter. The sequences, which target the X sequence of HBV, were incorporated into recombinant hepatotropic self-complementary adeno-associated viruses (scAAVs). Systemic intravenous injection of the vectors into HBV transgenic mice at a dose of 1 × 1011 per animal effected significant suppression of markers of HBV replication for at least 32 weeks. The pri-miRs were processed according to the intended design, and intrahepatic antiviral guide sequences were detectable for 40 weeks after the injection. There was no evidence of toxicity, and innate immunostimulation was not detectable following the injections. This efficacy is an improvement on previously reported RNAi-based inhibition of HBV replication and is important to clinical translation of the technology.

Serum vitamin D level not correlated with hepatitis B virus replication in patients with chronic hepatitis B virus infection

SummaryBackgroundsVitamin D is an important immune modulator in chronic liver diseases. The study aims to investigate the relationship between vitamin D and hepatitis B virus replication.MethodsThis was a prospective study. Hepatitis B virus (HBV) carriers were enrolled from May to Oct 2014 in Taipei Tzu Chi Hospital. All have positive hepatitis B surface antigen (HBsAg) for more than 6 months. Serum alanine aminotransferase (ALT), quantitative HBsAg (qHBsAg), HBV DNA and 25(OH)D3 were measured in each participant.Results204 patients (male 103; mean age 47.86 ± 10.22 years) with chronic HBV infection (94.12% HBeAg‐negative) were enrolled. Of them, 13 (6.4%) had severe deficiency (25(OH)D3 < 10 ng/mL), 176 (86.3%) insufficiency (25(OH)D3 ≥ 10 and < 30 ng/mL) and 15 (7.4%) adequate (25(OH)D3 ≧ 30 ng/mL) vitamin D levels. In the regard of age and sex, there was no difference in vitamin D levels. Using linear regression analysis, there was no correlation between serum vitamin D and HBV DNA/qHBsAg levels. While serum vitamin D levels were categorized by quartiles, there was no association between vitamin D levels and HBV DNA/qHBsAg levels using univariate and multivariate analyses.ConclusionsIn this study, 92.6% of patients with chronic HBV infection have inadequate vitamin D levels. Serum vitamin D levels do not correlate with the markers of HBV replication including serum HBV DNA and qHBsAg levels.Copyright © 2017, The Gastroenterological Society of Taiwan, The Digestive Endoscopy Society of Taiwan and Taiwan Association for the Study of the Liver.

Analysis of BZLF1 mRNA detection in saliva as a marker for active replication of Epstein-Barr virus

Monitoring replicative Epstein-Barr virus (EBV) infection still remains a challenge in modern laboratory routine. The immediate-early protein BZLF1 mediates the switch between latent and replicate forms of EBV infection. The aim of this study was to analyze the feasibility of BZLF1 mRNA detection in saliva as a marker for active replication of the virus. Various specimens (saliva, plasma, PBMC) from 17 patients with EBV-induced infectious mononucleosis (IM) and 4 control patients were examined for expression of viral BZLF1 mRNA by means of real-time PCR. BZLF1 expression was correlated to the amount of viral DNA in either compartment. Digestion of plasma and saliva samples with DNase I allowed distinguishing between encapsidated and naked viral DNA. BZLF1 transcripts were found in all different types of specimens in varying frequencies. BZLF1 expression in saliva, PBMC, and plasma correlated with viral load in each compartment. Interestingly, those patients with detectable BZLF1 expression in saliva had a more severe course of infection with longer duration of hospitalization. In conclusion, this study demonstrates the feasibility of BZLF1 mRNA detection in saliva specimens during replicative EBV infection. Its significance for the diagnosis of reactivated EBV infection, particularly under immunosuppression, has to be elucidated in further studies.

Serum lincRNA-p21 as a potential biomarker of liver fibrosis in chronic hepatitis B patients.

Serum long non-coding RNAs (lncRNAs) are emerging as promising biomarkers for various human diseases. The aim of this study was to investigate the feasibility of using serum long intergenic non-coding RNA-p21 (lincRNA-p21) as a biomarker for chronic hepatitis B patients. Serum lincRNA-p21 levels were quantified using real-time PCR in 417 CHB patients and 363 healthy controls. The promoter methylation level of lincRNA-p21 was detected using bisulphite-sequencing analysis in primary hepatic stellate cells (HSCs). Sera from hepatitis B-infected patients contained lower levels of lincRNA-p21 than sera from healthy controls. Serum lincRNA-p21 levels negatively correlated with stages of liver fibrosis in infected patients. Receiver operating characteristic (ROC) curve analyses suggested that serum lincRNA-p21 had a significant diagnostic value for liver fibrosis in these patients. It yielded an area under the curve of ROC of 0.854 with 100% sensitivity and 70% specificity in discriminating liver fibrosis from healthy controls. There was additionally a negative correlation between serum lincRNA-p21 level and the markers of liver fibrosis including α-SMA and Col1A1. However, there was no correlation of serum lincRNA-p21 level with the markers of viral replication, liver inflammatory activity, and liver function. Notably, during primary HSCs culture, loss of lincRNA-p21 expression was associated with promoter methylation. Serum lincRNA-p21 could serve as a potential biomarker of liver fibrosis in CHB patients. Down-regulation of lincRNA-p21 in liver fibrosis may be associated with promoter methylation.

THE RESULTS OF LONG-TERM STUDY OF HERPESVIRUS INFECTION AT THE DEPARTMENT OF INFECTIOUS DISEASES IN CHILDREN OF RUSSIAN NATIONAL RESEARCH MEDICAL UNIVERSITY

As a result of many years of research, the role of Herpesviridae IV, V and VI types in the structure of various pathologies in children (with infectious mononucleosis (MI), CNS damage, long-term subfebrile syndrome, vasculitis syndrome, hepatitis, etc.) has been proven. MI is a polyethological disease associated with the Epstein-Barr virus (EBV), cytomegalovirus (CMV) and human herpesvirus type 6 (HHV6), occurring in mono(58%) and mixed infections in various combinations of herpesviruses (42%). Clinical manifestations of MI are typical and do not depend on etiology. With CMV and HHV6 IM, the response to heterophilic antibodies is always negative. Markers of herpesvirus infections were detected in 87% of children observed with neurological pathology. Children with diagnoses such as convulsive syndrome, epilepsy, neuropathy need a comprehensive examination (ELISA, PCR, Indirect immunofluorescence reaction) to identify markers of active replication of herpes viruses, with an emphasis on the detection of HHV6. In 44% of cases, the role of active herpesvirus infection in the etiology of prolonged subfebrile fever in children was proven, with mixed forms (78%) prevailing over monoinfection (22%). To identify the active forms of herpesvirus infection, it is important to detect the markers of viral replication. For EBV, this is the DNA of the virus in the blood, a smear from the tonsils and saliva, antigens of the complete assembly of EBV in blood lymphocytes, antibodies to the capsid antigen (VCA) of IgM and IgG classes, and IgG to the early (EA) antigen. With CMV infection, complete virus assembly antigens (pp65 and pp72) in blood lymphocytes and/or DNA of the virus in the blood, virus DNA in saliva and urine, antibodies to the IgM class virus and high IgG class titer in the blood. With HHV6 infection, the DNA of the virus in the blood, a smear from tonsils and saliva, complete antigens of HHV6 in blood lymphocytes, specific IgM and/or IgG antibodies are 2—4 times higher than the diagnostic values.

Immunological Status to Hepatitis B Virus of Pregnant Women in Dakar, Senegal

Objective: Evaluate the immunological status to hepatitis B virus of Senegalese pregnant women by screening HBs antigen. Material and methods: The selection criteria of women were presence at the laboratory for biological exams of pregnancy follow-up. All volunteers for the study were screened for HBs antigen (HBs Ag). Investigation of chronic hepatitis B markers (HBe Ag, anti-HBe, viral quantification) was performed in HBs Ag positive participants. The concentration of anti-HBs antibodies was assessed in HBs Ag negative women. Results: One hundred and fifteen (115) pregnant women were included in the study from July to October 2014. The mean age was 29 ± 6 years, ranging from 16 to 47. The seroprevalence of HBs Ag was 12% and the majority of women (90%) were not vaccinated. Any of the 14 HBs Ag-positive subjects did not express serum HBe Ag, (marker of active viral replication) and all were positive for anti-HBe antibodies. Their viral load (HBV DNA) was undetectable and serum transaminases were normal. The anti-HBs antibodies titrated in HBs-Ag negative women serum revealed that only 46 had protective levels against HBV whilst 55 of them were unprotected.

Intrahepatic innate immune response pathways are downregulated in untreated chronic hepatitis B

Hepatitis B virus (HBV) persistence and the pathobiology of chronic HBV (CHB) infections result from the interplay between viral replication and host immune responses. We aimed to comprehensively analyse the expression of intrahepatic host genes as well as serum and liver HBV markers in a large cohort of untreated CHB patients. One-hundred and five CHB patients untreated at the time of liver biopsy (34 HBeAg[+] and 71 HBeAg[-]) were analysed for the intrahepatic expression profile of 67 genes belonging to multiple innate immunity pathways. Results were correlated to serological (quantification of HBsAg [qHBsAg] and HBV DNA) and intrahepatic viral markers (total HBV DNA, pre-genomic RNA and covalently closed circular HBV DNA). Intrahepatic gene expression profiling revealed a strong downregulation of antiviral effectors, interferon stimulated genes, Toll-like and pathogen recognition receptor pathways in CHB patients as compared to non-infected controls, which was not directly correlated to HBV replication. A subset of genes [CXCL10, GBP1, IFITM1, IFNB1, IL10, IL6, ISG15, TLR3, SOCS1, SOCS3] was more repressed in HBeAg(-) respect to HBeAg(+) patients (median of serum HBV DNA 7.9×103vs. 7.9×107IU/ml, respectively). Notably, HBeAg(-) patients with lower qHBsAg (<5×103IU/ml) showed a relief of repression of genes belonging to multiple pathways. Our results show a strong impairment of innate immune responses in the liver of CHB patients. The association of low levels of qHBsAg with gene repression, if confirmed, might prove useful for the identification of patients who would most benefit from immune-modulators and/or HBsAg targeting agents as strategies to restore immune responsiveness. Chronic hepatitis B virus (HBV) infections represent a major public health problem worldwide. Over 200 million people are chronically infected and at risk of developing chronic hepatitis, liver cirrhosis and cancer. Our work aimed to understand the molecular consequences of chronic hepatitis B in the infected liver. It was conducted in a large cohort of untreated chronically infected HBV patients and analysed the expression of immunity and liver disease-related genes in the liver, with respect to markers of viral replication and persistence. Our results indicate that chronic HBV infection has a suppressive effect on immune responses, which was more pronounced with high levels of hepatitis B virus surface antigen (HBsAg). These data provide novel insight into the mechanisms of HBV persistence in the liver and suggest that approaches aimed at reducing HBsAg levels, may restore immune responsiveness against the virus.

Influence of raltegravir intensification on viral load and 2-LTR dynamics in HIV patients on suppressive antiretroviral therapy

Antiretroviral therapy can suppress HIV-1 plasma viral load to below the detection limit but cannot eradicate the virus. Whether residual ongoing viral replication persists during suppressive therapy remains unclear. A few clinical studies showed that treatment intensification with an additional drug led to a lower viral load or an increase in 2-LTR (long terminal repeat), a marker for ongoing viral replication. However, some other studies found no change in the viral load and 2-LTR. In this paper, we developed multi-stage models to evaluate the influence of treatment intensification with the integrase inhibitor raltegravir on viral load and 2-LTR dynamics in HIV patients under suppressive therapy. We analyzed one model and obtained the local and global stability of the steady states. The model and its variation predict that raltegravir intensification induces a very minor decrease in the viral load and a minor increase in 2-LTR. We also compared modeling prediction with the 2-LTR data in a raltegravir intensification study. To achieve the 2-LTR increase observed in some patients, the level of viral replication needs to be substantially high, which is inconsistent with the sustained viral suppression in patients during treatment intensification. These modeling results, together with the theoretical estimate of the upper bound of the 2-LTR increase, suggest that treatment intensification with raltegravir has a minor effect on the plasma viremia and 2-LTR in patients under suppressive therapy. Other treatment strategies have to be developed for the cure or functional control of the infection.

Detection of viral RNA in tissues following plasma clearance from an Ebola virus infected patient

Purpose: An unprecedented Ebola outbreak occurred in 2014-2015 in West Africa. A better understanding of the EBOV life cycle is fundamental to develop new countermeasures, as well as to fully comprehend the pathways of inter-human transmission. We have explored the possibility of viral persistence in different body fluids’ samples obtained from a Health Care Worker (HCW) infected in Sierra Leone and treated at INMI L. Spallanzani, Italy. To evaluate whether the virus was in a replicative status or simply derived from blood spill over, we compared the trends of EBOV-specific negative sense genomic RNA (neg-RNA), positive sense RNA (pos-RNA) and total viral RNA in different clinical samples. Methods & Materials: Clinical samples were inactivated in BSL4 facility and RNA was extracted with QIAamp Viral-RNA Mini-Kit (Qiagen). Quantification of total viral RNA was performed with the reference test Altona-FilovirusScreen Kit1.0 (Altona Diagnostics). To measure EBOV-specific negative sense genomic RNA (neg-RNA) and positive sense RNA (pos-RNA) [including both replication intermediate (cRNA) and messenger RNA (mRNA)], L-gene specific reverse or forward primers were used in the reverse transcription step Results: The clinical samples analyzed (sputum, nasopharyngeal swab, ocular swab, urine and plasma) show different trends in neg-RNA, pos-RNA and total viral RNA levels. In Nasopharyngeal swab, Ocular Swab and Urine, total viral RNA is the only one detectable, until Day 12, 5 and 15 after hospitalization, respectively. On the contrary, in plasma, total viral RNA, neg-RNA and pos-RNA levels simultaneously decreased, starting from day 3 and becoming undetectable at Day 6. In Sputum, pos-RNA levels decreased since Day 8, persisting at detectable levels up to Day 10, coherently with total viral RNA and neg-RNA levels, which start decreasing at Day 10 and become undetectable at Day 11. Conclusion: The presence of viral RNA and replication markers (pos-RNA and neg-RNA) in sputum might be of relevance in future analysis regarding the persistence of the virus in the upper respiratory tract, even after viral clearance from plasma. These results should be taken under further investigation in order to better understand the role of the respiratory tract for possible involvement in viral shedding, viral replication site or as a viral reservoir.

HDV RNA replication is associated with HBV repression and interferon-stimulated genes induction in super-infected hepatocytes

Hepatitis D virus (HDV) super-infection of Hepatitis B virus (HBV)-infected patients is the most aggressive form of viral hepatitis. HDV infection is not susceptible to direct anti-HBV drugs, and only suboptimal antiviral responses are obtained with interferon (IFN)-alpha-based therapy. To get insights on HDV replication and interplay with HBV in physiologically relevant hepatocytes, differentiated HepaRG (dHepaRG) cells, previously infected or not with HBV, were infected with HDV, and viral markers were extensively analyzed. Innate and IFN responses to HDV were monitored by measuring pro-inflammatory and interferon-stimulated gene (ISG) expression. Both mono- and super-infected dHepaRG cells supported a strong HDV intracellular replication, which was accompanied by a strong secretion of infectious HDV virions only in the super-infection setting and despite the low number of co-infected cells. Upon HDV super-infection, HBV replication markers including HBeAg, total HBV-DNA and pregenomic RNA were significantly decreased, confirming the interference of HDV on HBV. Yet, no decrease of circular covalently closed HBV DNA (cccDNA) and HBsAg levels was evidenced. At the peak of HDV-RNA accumulation and onset of interference on HBV replication, a strong type-I IFN response was observed, with interferon stimulated genes, RSAD2 (Viperin) and IFI78 (MxA) being highly induced. We established a cellular model to characterize in more detail the direct interference of HBV and HDV, and the indirect interplay between the two viruses via innate immune responses. This model will be instrumental to assess molecular and immunological mechanisms of this viral interference.

Mesenchymal stromal cells from pooled mononuclear cells of multiple bone marrow donors as rescue therapy in pediatric severe steroid-refractory graft-versus-host disease: a multicenter survey.

To circumvent donor-to-donor heterogeneity which may lead to inconsistent results after treatment of acute graft-versus-host disease with mesenchymal stromal cells generated from single donors we developed a novel approach by generating these cells from pooled bone marrow mononuclear cells of 8 healthy "3(rd)-party" donors. Generated cells were frozen in 209 vials and designated as mesenchymal stromal cell bank. These vials served as a source for generation of clinical grade mesenchymal stromal cell end-products, which exhibited typical mesenchymal stromal cell phenotype, trilineage differentiation potential and at later passages expressed replicative senescence-related markers (p21 and p16). Genetic analysis demonstrated their genomic stability (normal karyotype and a diploid pattern). Importantly, clinical end-products exerted a significantly higher allosuppressive potential than the mean allosuppressive potential of mesenchymal stromal cells generated from the same donors individually. Administration of 81 mesenchymal stromal cell end-products to 26 patients with severe steroid-resistant acute graft-versus-host disease in 7 stem cell transplant centers who were refractory to many lines of treatment, induced a 77% overall response at the primary end point (day 28). Remarkably, although the cohort of patients was highly challenging (96% grade III/IV and only 4% grade II graft-versus-host disease), after treatment with mesenchymal stromal cell end-products the overall survival rate at two years follow up was 71±11% for the entire patient cohort, compared to 51.4±9.0% in graft-versus-host disease clinical studies, in which mesenchymal stromal cells were derived from single donors. Mesenchymal stromal cell end-products may, therefore, provide a novel therapeutic tool for the effective treatment of severe acute graft-versus-host disease.

Entry Sites of Venezuelan and Western Equine Encephalitis Viruses in the Mouse Central Nervous System following Peripheral Infection.

Venezuelan and western equine encephalitis viruses (VEEV and WEEV; Alphavirus; Togaviridae) are mosquito-borne pathogens causing central nervous system (CNS) disease in humans and equids. Adult CD-1 mice also develop CNS disease after infection with VEEV and WEEV. Adult CD-1 mice infected by the intranasal (i.n.) route, showed that VEEV and WEEV enter the brain through olfactory sensory neurons (OSNs). In this study, we injected the mouse footpad with recombinant WEEV (McMillan) or VEEV (subtype IC strain 3908) expressing firefly luciferase (fLUC) to simulate mosquito infection and examined alphavirus entry in the CNS. Luciferase expression served as a marker of infection detected as bioluminescence (BLM) by in vivo and ex vivo imaging. BLM imaging detected WEEV and VEEV at 12 h postinoculation (hpi) at the injection site (footpad) and as early as 72 hpi in the brain. BLM from WEEV.McM-fLUC and VEEV.3908-fLUC injections was initially detected in the brain's circumventricular organs (CVOs). No BLM activity was detected in the olfactory neuroepithelium or OSNs. Mice were also injected in the footpad with WEEV.McM expressing DsRed (Discosoma sp.) and imaged by confocal fluorescence microscopy. DsRed imaging supported our BLM findings by detecting WEEV in the CVOs prior to spreading along the neuronal axis to other brain regions. Taken together, these findings support our hypothesis that peripherally injected alphaviruses enter the CNS by hematogenous seeding of the CVOs followed by centripetal spread along the neuronal axis. VEEV and WEEV are mosquito-borne viruses causing sporadic epidemics in the Americas. Both viruses are associated with CNS disease in horses, humans, and mouse infection models. In this study, we injected VEEV or WEEV, engineered to express bioluminescent or fluorescent reporters (fLUC and DsRed, respectively), into the footpads of outbred CD-1 mice to simulate transmission by a mosquito. Reporter expression serves as detectable bioluminescent and fluorescent markers of VEEV and WEEV replication and infection. Bioluminescence imaging, histological examination, and confocal fluorescence microscopy were used to identify early entry sites of these alphaviruses in the CNS. We observed that specific areas of the brain (circumventricular organs [CVOs]) consistently showed the earliest signs of infection with VEEV and WEEV. Histological examination supported VEEV and WEEV entering the brain of mice at specific sites where the blood-brain barrier is naturally absent.

Long Terminal Repeat Circular DNA as Markers of Active Viral Replication of Human T Lymphotropic Virus-1 in Vivo.

Clonal expansion of human T-lymphotropic virus type-1 (HTLV-1) infected cells in vivo is well documented. Unlike human immunodeficiency virus type 1 (HIV-1), HTLV-1 plasma RNA is sparse. The contribution of the “mitotic” spread of HTLV-1 compared with infectious spread of the virus to HTLV-1 viral burden in established infection is uncertain. Since extrachromosomal long terminal repeat (LTR) DNA circles are indicators of viral replication in HIV-1 carriers with undetectable plasma HIV RNA, we hypothesised that HTLV-1 LTR circles could indicate reverse transcriptase (RT) usage and infectious activity. 1LTR and 2LTR DNA circles were measured in HTLV-1 cell lines and peripheral blood mononuclear cells (PBMC) of asymptomatic carriers (ACs) and patients with HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP) or adult T cell leukaemia/lymphoma (ATLL). 1LTR DNA circles were detected in 14/20 patients at a mean of 1.38/100 PBMC but did not differentiate disease status nor correlate with HTLV-1 DNA copies. 2LTR DNA circles were detected in 30/31 patients and at higher concentrations in patients with HTLV-1-associated diseases, independent of HTLV-1 DNA load. In an incident case the 2LTR DNA circle concentration increased 2.1 fold at the onset of HAM/TSP compared to baseline. Detectable and fluctuating levels of HTLV-1 DNA circles in patients indicate viral RT usage and virus replication. Our results indicate HTLV-1 viral replication capacity is maintained in chronic infection and may be associated with disease onset.

WSV399, a viral tegument protein, interacts with the shrimp protein PmVRP15 to facilitate viral trafficking and assembly

Viral responsive protein 15 (PmVRP15) has been identified as a highly up-regulated gene in the hemocyte of white spot syndrome virus (WSSV)-infected shrimp Penaeus monodon. However, the function of PmVRP15 in host–viral interaction was still unclear. To elucidate PmVRP15 function, the interacting partner of PmVRP15 from WSSV was screened by yeast two-hybrid assay and then confirmed by co-immunoprecipitation (Co-IP). Only WSV399 protein was identified as a PmVRP15 binding protein; however, the function of WSV399 has not been characterized. Localization of WSV399 on the WSSV virion was revealed by immunoblotting analysis (in vitro) and immunoelectron microscopy (in vivo). The results showed that WSV399 is a structural protein of the WSSV virion and is particularly located on the tegument. Gene silencing of wsv399 in WSSV-infected shrimp reduced the percentage of cumulative mortality by 74%, although the expression level of a viral replication marker gene, vp28, was not changed suggesting that WSV399 might not involved in viral replication but viral assembly. Because it has already been known that tegument proteins function in capsid transport during viral trafficking and assembly, interaction between PmVRP15 on hemocyte nuclear membrane and the WSV399 viral tegument protein suggests that PmVRP15 might be required for trafficking and assembly of WSSV during infection.

Quantification of serum HBV RNA allows differentiation of disease stages of hepatitis B virus (HBV) infections

Introduction: Inactive carriers of HBV infections are distinguished from individuals with active HBV infections by serum levels of HBV DNA. HBV RNA is another surrogate marker of HBV replication which can be quantified from serum. The aim of this study was to explore whether HBV RNA levels permits a better differentiation between the various stages of HBV infection. Methods: Serum samples from 12 patients with acute and 96 with chronic HBV infection (34 inactive HBsAg carrier with HBV DNA < 4 log10 copies/ml and ALT within normal ranges, 34 HBeAg negative and 28 HBeAg positive patients with chronic active hepatitis B (mean age 45.4 ± 15.6 (17 – 77) years, m/w = 73/35, mean ALT 8.7 ± 19.2 (0.22 – 100)µkat/ml, mean HBsAg 4.3 ± 0.9 (0.7 – 6.0) log10 ng/ml) were retrospectively analyzed. HBV DNA was measured by real-time PCR (limit of detection 2.6 log10 copies/ml). HBV-RNA levels were quantified based on a sensitive One-Step-RACE-PCR-Assay which was developed and validated (limit of detection 2.9 log10 copies/ml). Results: HBV RNA could be detected in 9 of 12 patients with acute HBV infection with a mean of 3.4 ± 1.6 (2.0 – 5.9) log10 copies/ml. HBV RNA was detectable in 29 out of 34 HBeAg negative patients and in all 28 patients with HBeAg positive chronic active HBV infection and showed a high correlation with HBV DNA levels (r = 0.93). Mean HBV DNA levels were significantly lower in HBeAg negative as compared to HBeAg positive patients (6.8 ± 1.3 (4.2 – 8.9) vs. 8.2 ± 1.4 (4.5 – 10.5) log10 copies/ml, p < 0.001), and so were mean HBV RNA levels (4.4 ± 1.5 (2.0 – 7.0) vs. 6.8 ± 1.2 (4.3 – 9.1) log10 copies/ml, p < 0.001). In contrast, HBV RNA was detectable in only in 2 out of 34 inactive HBsAg carriers (2.0 ± 0.2 (2.0 – 2.7) log10 copies/ml), while in 28 of those patients HBV DNA was still detectable with a mean of 3.1 ± 0.7 (2.0 – 4.6) log10 copies/ml. Conclusion: Serum levels of HBV RNA show a high correlation with HBV DNA levels in patients with active chronic HBV infection. However, in patients with inactive HBV infections, HBV RNA is less frequently detected than HBV DNA. It needs to be studied whether HBV RNA serum levels can identify patient inactive carrier status that have disease progression. Corresponding author: Krauel, Alexander E-Mail:Alexander.Krauel@medizin.uni-leipzig.de

Bone and cartilage changes in rabbit mandibular condyles after 1 injection of botulinum toxin.

Temporary paralysis of the masseter muscle caused by botulinum toxin is a common treatment for temporomandibular disorders, bruxism, and muscle hypertrophy. Loss of masseter force is associated with decreased mandibular mineral density. Our objectives were (1) to establish whether bone loss at the mandibular condyle is regionally specific and (2) to ascertain whether the treatment affects the condylar cartilage. Young adult female rabbits received a unilateral masseter injection of botulinum neurotoxin serotype A (BoNT/A, n = 31), saline solution (n = 19), or no injection (n = 3) and were also injected with bromodeoxyuridine (BrdU), a replication marker. The rabbits were killed at 4 or 12 weeks after treatment. The condyles were processed for paraffin histology. Cortical thickness, cartilage thickness, and trabecular bone areal density were measured, and replicating cells were counted after BrdU reaction. The BoNT/A rabbits exhibited a high frequency of defects in the condylar bone surface, occurring equally on the injected and uninjected sides. Bone loss was seen only on the side of the BoNT/A injection. Cortical as well as trabecular bone was severely affected. The midcondylar region lost the most bone. Recovery at 12 weeks was insignificant. Condylar cartilage thickness showed no treatment effect but did increase with time. The numbers of proliferating cells were similar in the treatment groups, but the BoNT/A animals showed more side asymmetry associated with the condylar defects. Bone loss may be a risk factor for the use of botulinum toxin in jaw muscles.

Glycoproteins and Gal-GalNAc cause Cryptosporidium to switch from an invasive sporozoite to a replicative trophozoite

The apicomplexan parasite Cryptosporidium causes cryptosporidiosis, a diarrheal disease that can become chronic and life threatening in immunocompromised and malnourished people. There is no effective drug treatment for those most at risk of severe cryptosporidiosis. The disease pathology is due to a repeated cycle of host cell invasion and parasite replication that amplifies parasite numbers and destroys the intestinal epithelium. This study aimed to better understand the Cryptosporidium replication cycle by identifying molecules that trigger the switch from invasive sporozoite to replicative trophozoite. Our approach was to treat sporozoites of Cryptosporidium parvum and Cryptosporidium hominis, the species causing most human cryptosporidiosis, with various media under axenic conditions and examine the parasites for rounding and nuclear division as markers of trophozoite development and replication, respectively. FBS had a concentration-dependent effect on trophozoite development in both species. Trophozoite development in C. parvum, but not C. hominis, was enhanced when RPMI supplemented with 10% FBS (RPMI-FBS) was conditioned by HCT-8 cells for 3h. The effect of non-conditioned and HCT-8 conditioned RPMI-FBS on trophozoite development was abrogated by proteinase K and sodium metaperiodate pretreatment, indicating a glycoprotein trigger. Cryptosporidium parvum and C. hominis trophozoite development also was triggered by Gal-GalNAc in a concentration-dependent manner. Cryptosporidium parvum replication was greatest following treatments with Gal-GalNAc, followed by conditioned RPMI-FBS and non-conditioned RPMI-FBS (P<0.05). Cryptosporidium hominis replication was significantly less than that in C. parvum for all treatments (P<0.05), and was greatest at the highest tested concentration of Gal-GalNAc (1mM).

Detection of Hepatitis B Virus Covalently Closed Circular DNA in the Plasma of Iranian HBeAg-Negative Patients With Chronic Hepatitis B.

Background:Covalently closed circular DNA (cccDNA) of hepatitis B virus (HBV) is a marker of HBV replication in the liver of patients infected with HBV.Objectives:This study aimed to investigate the association between the presence of cccDNA in the plasma samples of Iranian treatment-naive patients with chronic hepatitis B infection and HBV viral load and HBsAg levels.Patients and Methods:From April 2012 to May 2015, 106 treatment-naive patients with chronic hepatitis B infection were enrolled in this cross-sectional study. The HBsAg titer was measured by the Roche HBsAg II assay on the Cobas e411 system, and HBV DNA quantitation was performed using the COBAS TaqMan 48 kit. Real-time polymerase chain reaction was performed for the detection of HBV cccDNA.Results:The mean (SD) age of the patients was 41.1 ± 12.4 years (range, 20 - 62 years). From a total of 106 study participants, 67 (63.2%) were males. The HBV cccDNA was detected in plasma specimens in 19 (17.9%) out of the total 106 patients, and a significant relationship was found between the presence of cccDNA in plasma sample of males (23.9%) and females (7.7%) (P = 0.039). Also, a significant correlation was found between the presence of cccDNA in plasma sample of the patients and HBV viral load level (P < 0.0001) and HBsAg titer (P = 0.0043).Conclusions:This study showed that cccDNA can be detected in the plasma specimen of 17.9% of Iranian treatment-naive patients with chronic hepatitis B infection. Therefore, designing prospective studies focusing on the detection of cccDNA in these patients would provide more information.

Interferon-γ and Tumor Necrosis Factor-α Produced by T Cells Reduce the HBV Persistence Form, cccDNA, Without Cytolysis

Viral clearance involves immune cell cytolysis of infected cells. However, studies of hepatitis B virus (HBV) infection in chimpanzees have indicated that cytokines released by T cells also can promote viral clearance via noncytolytic processes. We investigated the noncytolytic mechanisms by which T cells eliminate HBV from infected hepatocytes. We performed a cytokine enzyme-linked immunosorbent assay of serum samples from patients with acute and chronic hepatitis B. Liver biopsy specimens were analyzed by in situ hybridization. HepG2-H1.3 cells, HBV-infected HepaRG cells, and primary human hepatocytes were incubated with interferon-γ (IFNγ) or tumor necrosis factor-α (TNF-α), or co-cultured with T cells. We measured markers of HBV replication, including the covalently closed circular DNA (cccDNA). Levels of IFNγ and TNF-α were increased in serum samples from patients with acute vs chronic hepatitis B and controls. In human hepatocytes with stably replicating HBV, as well as in HBV-infected primary human hepatocytes or HepaRG cells, IFNγ and TNF-α each induced deamination of cccDNA and interfered with its stability; their effects were additive. HBV-specific T cells, through secretion of IFNγ and TNF-α, inhibited HBV replication and reduced cccDNA in infected cells without the direct contact required for cytolysis. Blocking IFNγ and TNF-α after T-cell stimulation prevented the loss of cccDNA. Deprivation of cccDNA required activation of nuclear APOBEC3 deaminases by the cytokines. In liver biopsy specimens from patients with acute hepatitis B, but not chronic hepatitis B or controls, hepatocytes expressed APOBEC3A and APOBEC3B. IFNγ and TNF-α, produced by T cells, reduce levels of HBV cccDNA in hepatocytes by inducing deamination and subsequent cccDNA decay.