Analysis of Hepatitis B Virus Haplotype Diversity Detects Striking Sequence Conservation Across Genotypes and Chronic Disease Phase.

BACKGROUND AND AIMS: We conducted haplotype analysis of complete hepatitis B virus (HBV) genomes following deep sequencing from 368 patients across multiple phases of chronic hepatitis B (CHB) infection from four major genotypes (A-D), analyzing 4,110 haplotypes to identify viral variants associated with treatment outcome and disease progression. APPROACH AND RESULTS: Between 18.2% and 41.8% of nucleotides and between 5.9% and 34.3% of amino acids were 100% conserved in all genotypes and phases examined, depending on the region analyzed. Hepatitis B e antigen (HBeAg) loss by week 192 was associated with different haplotype populations at baseline. Haplotype populations differed across the HBV genome and CHB history, this being most pronounced in the precore/core gene. Mean number of haplotypes (frequency) per patient was higher in immune-active, HBeAg-positive chronic hepatitis phase 2 (11.8) and HBeAg-negative chronic hepatitis phase 4 (16.2) compared to subjects in the "immune-tolerant," HBeAg-positive chronic infection phase 1 (4.3, P< 0.0001). Haplotype frequency was lowest in genotype B (6.2, P< 0.0001) compared to the other genotypes (A = 11.8, C = 11.8, D = 13.6). Haplotype genetic diversity increased over the course of CHB history, being lowest in phase 1, increasing in phase 2, and highest in phase 4 in all genotypes except genotype C. HBeAg loss by week 192 of tenofovir therapy was associated with different haplotype populations at baseline. CONCLUSIONS: Despite a degree of HBV haplotype diversity and heterogeneity across the phases of CHB natural history, highly conserved sequences in key genes and regulatory regions were identified in multiple HBV genotypes that should be further investigated as targets for antiviral therapies and predictors of treatment response.

HBV variants are common in the 'immune-tolerant' phase of chronic hepatitis B.

Nucleos(t)ide analogues (NUC) treatment prevents progression of liver fibrosis in subjects with chronic hepatitis B (CHB). However, risk of hepatocellular carcinoma (HCC) persists despite viral suppression. Specific HBV variants have been associated with adverse outcomes, including HCC; however, the frequency of these variants during the seemingly benign immunotolerant (IT) phase is unknown. Next-generation sequencing and detailed virological characterization on a cohort of treatment-naïve IT subjects were performed to determine the frequency of clinically relevant viral variants. Samples from 97 subjects (genotype B/C 55%/45%, median HBV-DNA 8.5 log10 IU/mL, median HBsAg 4.8 log10  IU/mL, median HBeAg 3.6 log10  PEIU/mL) were analysed. Despite subjects being in the IT phase, clinically relevant HBV variants were common at baseline, particularly in the basal core promoter (BCP, overlaps the hepatitis B X (HBx) gene), precore and PreS regions. BCP/HBx variants were independently associated with lower baseline HBeAg, HBsAg and HBV-DNA titres. Precore variants were independently associated with higher baseline ALT. Increased viral diversity was associated with increased age and lower HBV-DNA, HBsAg and HBeAg levels. Low-level (<5%) drug resistance-associated amino acid substitutions in the HBV reverse transcriptase were detected in 9 (9%) subjects at pre-treatment but were not associated with reduced antiviral activity. Future studies should evaluate whether the detection of HBV variant during IT CHB is predictive of progression to immune clearance and poor prognosis, and whether early initiation of antiviral therapy during IT CHB to prevent the selection of HBV variants is clinically beneficial.

Deep sequencing shows that HBV basal core promoter and precore variants reduce the likelihood of HBsAg loss following tenofovir disoproxil fumarate therapy in HBeAg-positive chronic hepatitis B.

OBJECTIVE: Hepatitis B e antigen (HBeAg) seroconversion and hepatitis B surface antigen (HBsAg) loss are important clinical outcomes for patients with chronic hepatitis B (CHB) treated with antiviral therapy. To date, there have been few studies that have evaluated viral sequence markers predicting serological response to nucleos(t)ide analogue (NA) treatment. DESIGN: We used next-generation sequencing (NGS) and quantitative HBV serology (HBeAg and HBsAg) to identify viral sequence markers associated with serological response to long-term tenofovir disoproxil fumarate therapy among HBeAg-positive patients. In the GS-US-174-0103 study, approximately half the patients seroconverted to anti-HBe by week 192 and 11% of patients exhibited HBsAg loss, the closest outcome to functional cure. The frequency of HBV variants that have previously been associated with HBV clinical outcomes was evaluated. HBV viral diversity in baseline sequences generated by NGS was calculated using Shannon entropy. RESULTS: NGS analysis of HBV sequences from 157 patients infected with genotypes A to D showed the frequency of variants in the basal core promoter (BCP) and precore (PC) regions varied by genotype and that these mutations were associated with the absence of HBsAg loss. This was the case even when mutations were present at frequencies below the threshold of detection by population sequencing. Increased viral diversity across the HBV genome as determined by NGS was also associated with reduced likelihood of HBsAg loss. CONCLUSION: Patients with detectable BCP and/or PC variants and higher viral diversity have a lower probability of HBsAg loss during long-term NA therapy. Strategies to achieve functional cure of HBV infection through combination therapy should consider using NGS to stratify patients according to BCP/PC sequence. Consideration should also be given to earlier initiation of therapy prior to the emergence of BCP/PC variants. TRIAL REGISTRATION NUMBER: NCT00116805; Post result.

Immunosuppression increases JC polyomavirus large T antigen DNA load in the brains of patients without progressive multifocal leukoencephalopathy.

The relationship between latent JC polyomavirus (JCV) infection and progressive multifocal leukoencephalopathy (PML) remains unclear. In this study, JCV DNA was quantified by real-time polymerase chain reaction (qPCR) in brain and kidney tissue from patients without PML. Immunosuppressed patients had significantly higher JCV DNA levels in brain, compared with immunocompetent patients (P = .001). An inverse relationship was observed between CD4(+) T-cell counts and qPCR-determined brain JCV load among patients with HIV infection (r(2) = -0.9; P = .01; n = 7). Higher kidney JCV DNA load was strongly associated with higher brain JCV DNA load (Spearman ρ = 0.65; P = .004; n = 18). These findings highlight the importance of latent JVC brain infection to the pathogenesis of PML.

Advances in the molecular diagnosis of hepatitis B infection: providing insight into the next generation of disease.

Hepatitis B virus (HBV) infection continues to represent a significant health threat, affecting over 400 million people worldwide. Historically, the diagnosis and treatment of chronic hepatitis B (CHB) relied in detection of the hepatitis B surface antigen (HBsAg) and more recently realtime polymerase chain reaction (PCR) analysis. The advent of novel technologies and equipment for the identification and staging of the different stages of HBV infection has resulted in dramatic changes to patient monitoring and management. Through the use of rapid, quantitative HBsAg immunoassays, it is now possible to predict the likelihood of patient response to treatment as well as the clinical course of disease. Ultradeep sequencing technologies (also known as next-generation sequencing) overcome many of the traditional limitations associated with population-based sequencing approaches, and have provided significant insight into the viral response to therapeutic intervention and the molecular pathogenesis of CHB. The authors discuss recent developments in the molecular diagnosis of HBV infection, as well as potential advantages and caveats resultant of this rapid progression of technology.

Hepatitis B virus splicing is enhanced prior to development of hepatocellular carcinoma.

BACKGROUND & AIMS: The hepatitis B virus (HBV) genome encodes specific sequence elements which promote splicing of viral DNA. It has been previously suggested that spliced HBV (spHBV) variants promote viral replication and protein production, leading to hepatocellular carcinoma (HCC). In this study, we have analysed changes in spHBV over time; providing the first longitudinal analysis of spHBV in relation to the development of HCC. METHODS: Serial serum samples were collected from 165 patients with chronic HBV monoinfection, including 58 patients who later developed HCC. Real-time PCR was used to amplify and quantify wt and sp DNA loads. RESULTS: spHBV was detected in over 80% of patients with chronic HBV infection. Median serum spHBV levels were significantly higher in HCC patients than HCC-free control patients (p<0.001). Univariate analysis revealed a strong correlation between time to HCC diagnosis and spHBV DNA levels (τ=0.203; p=0.016). Asian HBV genotype (p=0.025) and increased viral load (p<0.001) were also significantly associated with increased spHBV DNA levels. Multiple regression analysis revealed time to diagnosis of HCC, Asian HBV genotypes, and viral load to be associated with increased spHBV DNA (model p<0.001; R(2)=0.189). CONCLUSIONS: HBV splicing is a common event during chronic infection and increases prior to diagnosis of HCC. Measurement of HBV splicing may prove a valuable adjunct to be used in the identification of chronically infected patients who are at increased risk of developing HCC.

Estimating Australian Hospitalization Ratios and Costs for Wildtype SARS-CoV-2 in 2020.

Utilizing a retrospective cohort study of SARS-CoV-2 wildtype (Wuhan) strain, we aimed to 1) utilize the unique Australian experience of temporarily eliminating SARS-CoV-2 to document and estimate the hospitalization demand; and 2) estimate the inpatient hospital costs associated with treatment. Case data was based on Victoria Australia from March 29 to December 31, 2020. Outcomes measures included hospitalization demand and case fatality ratio and inpatient hospitalization costs. Population adjusted results indicated that 10.2% (CI 9.9%-10.5%) required ward only admission, 1.0% (CI 0.9%-1.1%) required ICU admission plus 1.0% (CI 0.9%-1.1%) required ICU with mechanical ventilation. The overall case fatality ratio was 2.9% (CI 2.7%-3.1%). Mean ward only patient costs ranged from $22,714 to $57,100 per admission whilst ICU patient costs ranged from $37,228 to $140,455. With delayed, manageable outbreaks and public health measures leading to temporary elimination of community transmission, the Victorian COVID-19 data provides insight into initial pandemic severity and hospital costs.

Control of vaccine preventable diseases in Australian infants: reviewing a decade of experience with DTPa-HBV-IPV/Hib vaccine.

The combined vaccine against diphtheria, tetanus, pertussis, hepatitis B, poliomyelitis, and Haemophilus influenzae b (DTPa-HBV-IPV/Hib, Infanrix Hexa, GSK) has been used for childhood immunization in Australia according to a two-, four-, six-month schedule since 2009. We reviewed data available in the Australian National Notifiable Diseases Surveillance System, annual vaccination coverage reports, the Database of Adverse Event Notifications, and peer-reviewed literature to assess vaccine coverage rates, incidence of all six vaccine preventable diseases, and the safety profile of DTPa-HBV-IPV/Hib vaccine in Australian infants over a period of ten years of exclusive use. Between 2009 and 2018 vaccine coverage for infants aged 12 months increased from 91.7% to 94.0% and from 84.9% to 92.6% for all and for Indigenous infants, respectively. Over the same time period, there were no reports of poliomyelitis, diphtheria or tetanus in infants <12 months of age. The incidence of hepatitis B among Australian infants <12 months of age remains 10 to 20-fold lower than the national average. Control of Haemophilus influenzae b (Hib) and pertussis disease has continued to be challenging. Timely administration of the primary series, as well as increasing coverage rates, particularly among Indigenous children, has contributed to improvements in Hib and pertussis disease control. The incorporation of additional strategies such as adjustment of the first vaccination encounter to six weeks of age, parental cocooning, and most recently maternal vaccination has further reduced the burden of pertussis, particularly during the first six months of life. The frequency of the ten most common adverse events related to the DTPa-HBV-IPV/Hib vaccine demonstrates an acceptable safety profile. Data collected over ten years of consistent, exclusive use of the DTPa-HBV-IPV/Hib vaccine in Australia highlights combination vaccination as a cornerstone in maintaining infant health.

Perceptions of vaccine preventable diseases in Australian healthcare: focus on pertussis.

Adult vaccination in Australia is suboptimal. For instance, as few as one in nine people have received a pertussis vaccine in adolescence or adulthood, despite increasing disease burden and evidence of a positive correlation between older age and hospitalization rates. The objectives of this study were to describe general practitioners' (GPs) and adult consumers' knowledge and attitudes toward adult vaccination, with an emphasis on pertussis. Australian GPs and consumers were recruited in two nationally representative online surveys repeated annually between 2014 and 2018. Vaccination discussions occurred in a minority of adult/GP encounters. Pertussis was among the five most frequently identified vaccine preventable diseases but was unlikely to be proactively discussed with adults not in contact with young children. Among consumers, only one in three recalled ever receiving a pertussis vaccination. GPs are a strong predictor of adults receiving a pertussis vaccine. Possible factors contributing to low uptake are misconceptions around pertussis disease, vaccination requirements and lack of GP recommendation for adult vaccination. GPs have a key role to play in increasing adult vaccination coverage with their recommendation.

Increased vascular endothelial growth factor mRNA in endomyocardial biopsies from allografts demonstrating severe acute rejection: a longitudinal study.

Investigation into the contribution of the immune system and inflammatory cascade to acute rejection (AR) and cardiac allograft vasculopathy (CAV) has implicated vascular endothelial growth factor (VEGF). The endomyocardial biopsy (EB) has proved invaluable in the diagnosis of AR, and in providing information concerning the biological processes occurring following transplantation. The association between VEGF and AR and the development of CAV was examined in endomyocardial biopsies (EBs) from a cohort of 76 heart transplant recipients. VEGF mRNA levels were quantified through real time RT-PCR in 712 EBs, obtained at routine intervals during post-operative monitoring. VEGF and leukocyte and endothelial markers were assessed in a subset of biopsies through immunohistochemistry. The results of generalised linear modelling, adjusting for covariates, revealed VEGF mRNA expression was 19% greater during severe AR as compared to no rejection (p=0.007). Immunohistochemical results supported these findings. Mean VEGF mRNA levels were not significant predictors for the development of CAV (p=0.554). However the risk of cardiac related death increased 9-fold for a 1 unit increase in mean VEGF expression (p=0.006). Similarly, a single unit increase in mean AR severity equated to a 10-fold increase in the risk of cardiac related death (p<0.005). Our data suggest that increased VEGF expression is strongly associated with severe AR and cardiac related death.

Effects of long-term tenofovir-based combination antiretroviral therapy in HIV-hepatitis B virus coinfection on persistent hepatitis B virus viremia and the role of hepatitis B virus quasispecies diversity.

OBJECTIVE: Hepatitis B virus (HBV) can persist in some HIV-HBV coinfected individuals on tenofovir disoproxil fumarate (TDF)-containing combination antiretroviral therapy (cART) but HBV resistance to TDF has not been reported and the source of persistent HBV DNA on TDF is poorly understood. The aims of this study were to assess long-term HBV suppression in HIV-HBV coinfected individuals receiving TDF and investigate quasispecies variation using ultradeep pyrosequencing (UDPS). METHODS: Ninety-two HIV-HBV coinfected participants on, or about to commence, TDF-containing cART were enrolled [Australia (n = 40), Thailand (n = 52)] and followed for 2 years with study visits every 6 months. HBV reverse transcriptase sequencing was performed on samples with HBV DNA more than 400 IU/ml by population-based methods and UDPS. Quasispecies diversity was assessed using Shannon entropy. RESULTS: Over 24 months, viremia was detected at least once in 17% (n = 16) of the cohort. Novel mutations were not identified in on TDF samples tested by population-based sequencing (n = 19). Using UDPS, the median Shannon entropy value in samples prior to TDF in patients aviremic on TDF was not statistically different from those who were viremic on TDF (n = 50; 8.4 and 9.1, respectively, P = 0.9). Longitudinal Shannon entropy analysis of on TDF samples from five participants showed three individuals with significant changes in viral diversity over time. CONCLUSION: Persistent viremia on TDF-containing cART is common but TDF-resistance was not detected. In some individuals, changes in viral diversity over time were observed on TDF which could potentially be active viral replication. Further follow-up will be needed to determine the clinical significance of detectable HBV DNA on TDF-containing cART.

Long distance bicycle riding causes prostate-specific antigen to increase in men aged 50 years and over.

OBJECTIVES: To investigate whether bicycle riding alters total prostate-specific antigen (tPSA) serum concentrations in healthy older men. METHODS: 129 male participants, ranging in age from 50 to 71 years (mean 55 years), rode in a recreational group bicycle ride of between 55 and 160 kilometers. Blood samples for tPSA analysis were drawn within 60 minutes before starting, and within 5 minutes after completing the ride. The pre-cycling and post-cycling tPSA values were log transformed for normality and compared using paired t-tests. Linear regression was used to assess the relationship between changes in tPSA with age and distance cycled. RESULTS: Bicycle riding caused tPSA to increase by an average of 9.5% (95% CI = 6.1-12.9; p<0.001) or 0.23 ng/ml. The number of participants with an elevated tPSA (using the standard PSA normal range cut-off of 4.0 ng/ml) increased from two pre-cycle to six post-cycle (or from five to eight when using age-based normal ranges). Univariate linear regression analysis revealed that the change in tPSA was positively correlated with age and the distance cycled. CONCLUSIONS: Cycling causes an average 9.5% increase in tPSA, in healthy male cyclists ≥50 years old, when measured within 5 minutes post cycling. We considered the increase clinically significant as the number of participants with an elevated PSA, according to established cut-offs, increased post-ride. Based on the research published to date, the authors suggest a 24-48 hour period of abstinence from cycling and ejaculation before a PSA test, to avoid spurious results.

Frequency and large T (LT) sequence of JC polyomavirus DNA in oligodendrocytes, astrocytes and granular cells in non-PML brain.

Progressive multifocal leukoencephalopathy (PML) and JCV granular cell neuronopathy occur secondary to JCV polyomavirus (JCV) infection of oligodendrocytes and cerebellar granular cell neurons (CGNs) during immunosuppression. Pure populations of astrocytes, oligodendrocytes, CGNs and microglia from frontal cortex and cerebellum of 17 non-PML patients (9 immunocompetent; 8 immunosuppressed) were isolated by laser capture microdissection (LCM). JCV large T (LT) antigen DNA was detected by triple nested polymerase chain reaction (PCR). Sequence analysis was performed to assess LT gene variation. JCV DNA was detected in oligodendrocytes, astrocytes and CGNs of non-PML brains. The most common site for viral latency was cortical oligodendrocytes (65% of samples analyzed). Immunosuppressed patients were significantly more likely to harbor JCV DNA in CGN populations than immunocompetent patients (P = 0.01). Sequence analysis of the LT region revealed eight novel single nucleotide polymorphisms (SNPs) in four immunosuppressed patients. Of the eight novel SNPs detected, six were silent and two resulted in amino acid changes. JCV DNA is present within cells of the non-PML brain, known to be infected during PML and granular cell neuronopathy. This supports the argument for a brain only reservoir of JCV and supports the hypothesis that reactivation of latent brain JCV may be central to disease pathogenesis.

Immunosuppression increases latent infection of brain by JC polyomavirus.

AIMS: Progressive multifocal leukoencephalopathy (PML) is caused by reactivation of JC polyomavirus (JCV). Increased JCV reactivation in kidney, as indicated by JCV viruria is reported during immunosuppression; however, the relevance of systemic to neural reactivation remains unknown. METHODS: Brain and kidney tissue from 138 non-PML patients (78 immunocompetent; 60 immunosuppressed) was assessed for JCV large T (LT) and viral protein (VP)1 DNA with nested PCR. Immunohistochemistry was performed to detect presence of JCV protein. Autopsy findings were reviewed and all brains underwent neuropathological examination. RESULTS: JCV LT DNA was detected in 31% of kidney and 30% of brain from non-PML patients. Of non-PML patients with brain JCV LT DNA, 66% did not have kidney JCV LT DNA, indicating brain JCV LT DNA was independent of kidney JCV LT DNA (p = 0.69). JCV VP1 DNA was detected in 12% of non-PML kidney and 8% of non-PML brain. JCV LT DNA was more likely to be found in the kidney (p < 0.001) and brain (p = 0.009) of immunosuppressed than immunocompetent patients. HIV/AIDS patients with brain JCV LT DNA had lower CD4 counts than those without brain JCV LT DNA (p = 0.05). CONCLUSIONS: Immunosuppression drives increased brain JCV latency independent of systemic latency.

Characterisation of single nucleotide polymorphisms in the genome of JC polyomavirus using MALDI TOF mass spectrometry.

JC polyomavirus (JCV) infects 80% of the population. In immunosuppressed patients viral replication may be enhanced, resulting in progressive multifocal leukoencephalopathy. There are at least eight distinct strains of JCV of which distribution varies between ethnic groups however there remain issues with identifying viral strains due to the insufficient yield and sensitivity of Sanger sequencing (SS) protocols. In this study matrix assisted laser desorption/ionisation time of flight mass spectrometry (MALDI TOF MS) is presented as an alternative to SS. DNA from 19 urine samples was assessed. Successful amplification and strain identification was possible in all samples with >10 copies of starting material indicating a high sensitivity assay, in contrast to SS (>20 copies). The genotypes defined for each sample via MALDI TOF MS were identical where SS resulted in a readable trace. MALDI TOF MS identified eight novel SNPs in the VP1 gene. This study represents the first application of MALDI TOF MS to viral comparative sequence analysis.

Late onset antibody-mediated rejection and endothelial localization of vascular endothelial growth factor are associated with development of cardiac allograft vasculopathy.

BACKGROUND: Improvements in cardiac transplant practice and immunosuppressive treatment have done much to curb the incidence of acute cellular rejection (ACR); however, antibody-mediated rejection (AMR) and cardiac allograft vasculopathy (CAV) remain prevalent. Recent studies have shown that allograft rejection is governed by both allogeneic and nonallogeneic factors such as inflammation. Initial studies have suggested that vascular endothelial growth factor (VEGF), a leukocyte mitogen produced by activated endothelial cells and leukocytes, may play a specific role in not only leukocyte trafficking, but also in the augmentation of ACR and development of CAV. METHODS: We investigated the localization of VEGF protein using immunohistochemistry in a cohort of 76 heart transplant patients during periods of ACR and AMR and assessed the development of CAV. RESULTS: We showed a significant correlation between lymphocytic localization of VEGF protein and severe ACR (P<0.001). Antibody-mediated rejection positive biopsies taken at 12 months posttransplantation showed significantly greater endothelial localization of VEGF than time-matched AMR negative biopsies (P=0.006). Diffuse endothelial expression of VEGF was also associated with a 2.5-fold increase in the risk of developing CAV (P=0.001). CONCLUSIONS: These results show that localization of VEGF protein to the vascular endothelium during AMR is significantly increased in patients who develop CAV. This study also highlights the potential pathogenic role of the endothelial cell in late onset AMR and the development of CAV.