Antiviral Therapy

Porcine reproductive and respiratory syndrome (PRRS) is one of the most serious diseases affecting the swine industry worldwide; however, there are no efficient control strategies against PRRS virus (PRRSV) at present. Therefore, development of new antiviral treatment strategies is urgently needed. As reported, lithium chloride (LiCl) can efficiently impair the replication of a variety of viruses, including infectious bronchitis coronavirus (IBV) and transmissible gastroenteritis coronavirus (TGEV). In this report, we explored whether LiCl had the potential to inhibit PRRSV infection.

MARC-145 cells were treated with LiCl at various stages of PRRSV life cycle. Virus titration assay was performed to determine the virus infectivity. The expression of viral mRNA and protein was measured by real-time PCR and indirect immunofluorescence assay, respectively. The transcript levels of cytokines were evaluated by real-time PCR.

LiCl significantly suppressed the synthesis of viral RNA and protein; however, it did not block PRRSV binding and entry. Further studies confirmed that LiCl inhibited PRRSV replication at an early stage and TNF-α, an antiviral cytokine, was significantly increased after LiCl treatment. Thus, we suggested that LiCl inhibited PRRSV infection by up-regulating the level of antiviral cytokine TNF-α at an early infection stage.

Our findings imply that the LiCl has the potential to be used as anti-PRRSV therapy.

Porcine reproductive and respiratory syndrome virus (PRRSV) causes severe diseases affecting the swine industry worldwide. Currently, no vaccination regimen has proven sustained success in preventing PRRSV infection. Therefore, there is an urgent need for new antiviral strategies to control PRRSV. In this study, the inhibitory effects and molecular mechanism of antimicrobial peptide protegrin-1 (PG-1) isolated from porcine leukocytes against PRRSV were evaluated in vitro.

Marc-145 cells or porcine alveolar macrophages (PAMs) were infected with PRRSV in the presence or absence of PG-1 for 36 h. The inhibitory effects of PG-1 were assessed by measuring the transcript and protein level of PRRSV ORF7 in cells and virus titres in the supernatants. Virus attachment and entry assays were performed to explore the molecular mechanism of PG-1 action.

We demonstrated that PG-1 strongly inhibited PRRSV infection and replication by suppressing virus RNA and protein synthesis, virus progeny production and viral particles release. Furthermore, in the PRRSV life cycle, PG-1 mainly blocked viral attachment in Marc-145 cells. However, in PAMs, PG-1 could neither inhibit PRRSV replication nor elevate antiviral cytokine expression.

Our findings for the first time show that PG-1 is an antiviral peptide with effective inhibitory effects on PRRSV infection and replication in Marc-145 cells.

Cytokines are crucial factors in the non-cytolytic antiviral process to inhibit HBV gene expression and replication. Interleukin (IL)-21 has been suggested to play an important role in HBV infection, but it remains unknown whether IL-21 can inhibit HBV replication or how it inhibits HBV replication.

In this study, we investigated the influence of IL-21 on HBV replication based on human hepatoma Huh7.93 cells co-cultured with human peripheral blood mononuclear cells (PBMCs) and the possible correlation among IL-21, interferon-γ, tumour necrosis factor-α and IL-10.

We demonstrated that the decrease of IL-21 expression and the increase of IL-10 expression in PBMCs could promote HBV replication in vitro. We further revealed that IL-21 is not only able to effectively suppress HBV replication directly but also reduce HBV replication by inhibition of IL-10 secretion.

Our results provide important evidence for the non-cytolytic antiviral mechanism mediated by cytokines and their interactions in chronic hepatitis B.

There is increasing evidence that host immune responses influence antiviral efficacy in chronic hepatitis B (CHB). The aim of this study was to characterize the immunological features responsible for improved treatment responses with pegylated interferon (PEG-IFN)-α2a in entecavir (ETV)-suppressed patients with CHB.

Peripheral natural killer (NK) cells, Toll-like receptors (TLRs), T-cell subsets, regulatory T-cells (Tregs) and programmed death 1 (PD-1) were evaluated dynamically in 77 patients undergoing a clinical trial (OSST trial, NCT00940485) by flow cytometry. Response was defined as hepatitis B e antigen (HBeAg) seroconversion, hepatitis B surface antigen (HBsAg) loss and HBsAg seroconversion (either as singular events or in combination at week 48).

Compared with ETV responders or PEG-IFN-α non-responders, PEG-IFN-α responders exhibited a significant decline in HBsAg during treatment (P=0.033 and P<0.001, respectively) and a significant decline in Treg proportions from week 12 to week 24 (P=0.036 and P=0.004, respectively). Moreover, PEG-IFN-α responders showed a significantly higher increase in the NKG2C⁺ NK cell proportions from baseline to week 12 (P=0.0073) and of TLR2⁺ monocytes at week 12 than PEG-IFN-α non-responders (P=0.039).

Successful response to PEG-IFN-α correlates with an early significant restoration of impaired immune responses. Although antiviral treatment response can be achieved by both IFN and ETV, the underlying immunological features vary which may explain the generally observed difference in off-treatment durability of response between the two treatments, as well as effects on HBsAg.

Adefovir dipivoxil (ADV) nephrotoxicity is well known at a dose of 60 mg day^-1 or 120 mg day^-1. However, renal toxicity at a low-dose of 10 mg ADV for HBV-infected patients is not fully described. Our objective was to analyse the clinical features and outcomes of ADV-related Fanconi's syndrome in the Chinese population.

This was a retrospective study. A total of 35 patients with ADV-related Fanconi's syndrome were studied. Clinical manifestations and biochemical parameters were analysed. 19 patients were from Peking Union Medical College Hospital (PUMCH) included from August 2010 to December 2012. A total of 16 patients were eligible from case reports in the Chinese population retrieved in PUBMED, WANFANG and CNKI database. Bone mineral density and biochemical parameters including serum phosphate, calcium, creatinine, alkaline phosphatase (ALP) were measured before and after ADV cessation and during the follow-up.

All recruited patients had hypophosphataemia, increased urinary phosphate excretion and elevated alkaline phosphatase. Serum phosphate levels rapidly increased especially within the 4 weeks after ADV cessation. Serum creatinine remained high or at the upper limit of normal range even after ADV cessation for 1 year. ALP increased in the first three months of ADV cessation and decreased at the 24th week. Bone mineral density was significantly improved after 6 months cessation of ADV.

ADV can be nephrotoxic at prolonged low doses of 10 mg. For those who take ADV long term, regular monitoring of serum phosphate, creatinine levels and urine routine tests are required.

To conduct an economic evaluation of the three commonly used interventions that reduce sexual HIV transmission when an HIV-negative female aims to conceive with an HIV-positive male on combination anti-retroviral therapy (condomless sex restricted to timed ovulation [CS], sperm washing with intrauterine insemination [SW] and condomless sex restricted to timed ovulation with pre-exposure prophylaxis [CS-PrEP]). As SW and CS-PrEP are only privately available for pregnancy planning for this population in Canada, this study was conducted to inform policy decisions concerning potential public health insurance coverage, as well as to inform fertility counselling in settings with adequate combination antiretroviral therapy access globally.

We developed a cohort Markov model with a lifetime horizon and used the perspective of Ontario's Ministry of Health (MOH). Input parameters were drawn from literature, the MOH's Schedule of Benefits and a time trade-off questionnaire designed for this study. Outcome measures included quality-adjusted life-years and incremental cost-effectiveness. Costs and benefits were discounted at annual rates of 3%. Costs were reported in Canadian 2013 dollars and an exchange rate of 1 USD to 1.066 CND was applied where necessary. Sensitivity analysis assessed the uncertainty of model parameters.

The base case analysis found that CS-PrEP and SW were each more costly and less effective at conception than CS. The results were robust in the sensitivity analysis and suggest that CS is the dominant conception strategy in this population.

Neither CS-PrEP nor SW represent better value for money relative to CS as a conception option for HIV-discordant couples with positive male partners. Based on these findings, CS-PrEP and SW cannot be recommended for public-funding in developed countries.

Mutations in HBV core promoter (CP) are suggested to affect viral replication and disease progression. We investigated CP deletion/insertion mutations (Del/Ins) in hepatitis B e antigen (HBeAg)-positive chronic hepatitis B (CHB) patients before and during antiviral treatment.

Direct and clone sequencings were used for detection of CP Del/Ins in 12 patients. The dynamic changes of CP Del/Ins were tracked in these cases until week 48 of treatment. The effects of Del/Ins on CP activities and hepatitis B X protein (HBx) were analysed using luciferase assay and sequence comparison, respectively. Furthermore, 292 untreated HBeAg-positive CHB cases were also analysed.

Twelve cases with multi-peak PCR direct sequencing electropherograms at baseline were con-firmed to have CP Del/Ins by clone sequencing, with detection rates varying from 14.8% to 93.3% of clones analysed. Follow-up studies showed the detection rates of CP Del/Ins in patients decreased from 100% (12/12) at baseline to 16.7% (2/12) at week 48 of treatment (P<0.001), in parallel with a decline in HBV DNA, hepatitis B surface antigen (HBsAg), alanine aminotransferase (ALT) and aspartate transaminase (AST) levels along with an increase in HBeAg loss. Luciferase assay results showed distinct promoter activities among Del/Ins-harbouring CP sequences. Importantly, 71.8% (148/206) of Del/Ins sequences potentially resulted in HBx carboxy-terminal truncations. CP Del/Ins mutations were also found in 27.4% (80/292) of untreated cases.

Naturally occurring complex of CP Del/Ins mutants existed in untreated HBeAg-positive CHB patients. These mutations would affect HBV transcription activities and integrity of HBx, which might correlate with disease progression. Their prevalence decreases on antiviral therapy in parallel with the decline in HBV DNA, HBsAg and ALT and AST levels.

Immunocompromised patients are at greater risk of complicated influenza and may be more likely to develop antiviral resistance. This observational sub-study of the Influenza Resistance Information Study (NCT00884117) aimed to study antiviral resistance in immunocompromised patients with influenza and characterize its effect on clinical and virological outcomes.

Eligible immunocompromised patients were aged ≥1 year with a local rapid diagnostic or PCR test positive for influenza ≤96 h after diagnosis and with influenza symptoms. Nasal and throat swabs were taken for RT-PCR analysis on day 1 and then every 3 days until patients were virus-free. Resistance was assessed by mutation-specific RT-PCR, phenotypic susceptibility analysis and Sanger sequencing.

Of 42 patients enrolled, 29 (69%) were influenza-positive (RT-PCR) on day 1: 18 adults and 11 children aged 1–12 years. Six patients were severely immunocompromised. On days 3, 6 and 9, most patients tested (18/24, 9/15 and 6/9, respectively) had not cleared the virus. Two of five patients assessed after day 9 continued shedding virus until day 15. H1N1pdm09 viruses harbouring H275Y mutations were detected in post-baseline samples from four patients (aged 52–61 years), one of whom had prolonged viral shedding. No genotypic antiviral resistance was detected in the other 20 treated patients (prevalence of resistance, 17%). Correlation between level of immune compromise and resistance or outcomes could not be assessed. Ten patients (seven influenza-positive) were admitted to intensive care and three died.

In these patients with mild/moderate immunocompromise, emergence of oseltamivir-resistant viruses was not common. Severity of influenza symptoms ranged from mild to moderate, but correlation with level of compromise could not be determined.

In HIV–HBV-coinfected patients from sub-Saharan Africa, incidence of antiviral resistant HBV-mutations after initiating long-term antiretroviral therapy (ART) has only been evaluated in limited patient populations.

In this nested, prospective cohort study from two randomized controlled trials in Côte d'Ivoire, 168 ART-naive HIV–HBV-coinfected patients, starting lamivudine (LAM, n=82) or tenofovir/emtricitabine (TDF/FTC, n=86) containing ART were included. HBV DNA viral load (VL) was quantified using an in-house assay (detection limit: <12 copies/ml) while pol and preS/S regions of positive samples were sequenced.

At ART-initiation, 39 (23.2%) were hepatitis B e antigen-positive, 53 (31.5%) had alanine or aspartate aminotransferase levels >40 IU/ml and 98/100 (98.0%) harboured genotype E. Among the 127 (75.6%) patients with detectable baseline HBV VL (median 4.27 log₁₀ copies/ml, IQR 3.14–7.64), cumulative percentage achieving undetectable HBV DNA was 74.2% for patients undergoing LAM-containing ART and 94.2% for TDF/FTC-containing ART after a median 35.5 months (IQR 24.3-36.5). No baseline antiviral resistance mutations were observed. Among 28/127 (22.1%) patients with low-level persistent viraemia (last HBV VL: between 12 to <10⁵ copies/ml), no incident amino acid changes associated with antiviral resistance were observed. Among 11/127 (8.7%) patients with high-level persistent viraemia (last HBV VL: ≥10⁵ copies/ml), only two harboured incident LAM-resistance mutations at positions rtV173L+rtL180M+rtM204V with no patient exhibiting TDF/FTC-resistance. Two patients had transaminase fares >120 IU/ml (incidence rate =0.5/100 person-years).

Antiviral resistance, particularly to LAM, was remarkably rare in this cohort of HIV-HBV-coinfected patients. Further research is needed to determine which coinfected populations might benefit from LAM-containing ART with low risk of resistance.

We assessed the association of persistent low-level viraemia between 50–199 copies/ml (LLV) with the risk of virological failure (VF) among HIV-1-infected patients receiving combination antiretroviral therapy (ART).

ART-naive and ART-experienced patients followed up in the ANRS-CO3 Aquitaine Cohort were included if they started two nucleoside reverse transcriptase inhibitors (NRTIs) with either one non-nucleoside reverse transcriptase inhibitor (NNRTI) or one protease inhibitor boosted with ritonavir (PI/r) between 2000 and 2011 and achieved viral load (VL)<200 copies/ml 4–8 months after initiating ART. VF was defined as either two consecutive VL≥200 copies/ml or one VL≥200 followed by a modification of ART. LLV was defined as at least two consecutive VLs between 50–199 copies/ml for at least one month. We used Cox models to estimate the association of LLV with VF.

Among 2,374 patients with a median follow-up of 3 years, 205 (8.6%) experienced LLV. LLV was strongly associated with further VF (adjusted hazard ratio [aHR] 2.30, 95% CI 1.65, 3.20). LLV was associated with VF in ART-experienced patients (aHR 3.02, 95% CI 2.10, 4.33) but not in ART-naive patients. Neither type of ART regimen (PI/r- versus NNRTI-based regimen) nor cumulative duration of LLV was associated with VF.

Persistent LLV between 50–199 copies/ml was associated with VF among ART-experienced patients under ART. LLV between 50–199 copies/ml in ART-experienced patients should lead, after assessing patient's adherence and checking for drug interactions, to a closer monitoring and to consider ART optimization.

Different genotypic HIV resistance algorithms are based on different rules. They may therefore result in different drug-resistance interpretations for the same patient sample. In particular, for early periods of new retroviral inhibitors or classes, sequence interpretation is expected to vary. One would, however, assume that those differences between systems wane with growing experience and that different algorithms yield similar results for well-established drugs.

To assess the concordance of the Agence Nationale de Recherche sur le SIDA (ANRS), Rega and Stanford-HIVdb algorithms and their evolution over time, we analysed 284 routine samples with the current versions of each algorithm in 2004 and 2013. For 446 recent clinical sequences the differences for actual drugs were analysed. Scoring as ‘susceptible’ by one algorithm and ‘resistant’ by a second one defined a discordance.

The longitudinal analysis showed similar overall discordances for both time points as well as an evolution over time. The actual analysis demonstrated a higher overall discordance rate, mainly for certain drugs. Most deviations refected differences between the ANRS and the other two algorithms.

This study demonstrates discordances between three most commonly used interpretation tools even for long-available drugs. It thereby reveals a need for further adjustment and improvement of current interpretation tools and may point at a possibly crucial role of subtype-specific information.