Characterization of hepatitis B virus surface antigen variability and impact on HBs antigen clearance under nucleos(t)ide analogue therapy.

For hepatitis B virus (HBV)-related chronic infection under treatment by nucleos(t)ide analogues (NUCs), HBsAg clearance is the ultimate therapeutic goal but very infrequent. We investigated how HBV envelope protein variability could lead to differential HBsAg clearance on NUCs. For 12 HBV genotype D patients receiving NUCs, six resolvers (HBsAg clearance) were compared to six matched nonresolvers (HBsAg persistence). PreS/S amino acid (aa) sequences were analysed with bioinformatics to predict HBV envelope antigenicity and aa covariance. To enrich our analyses on very rare resolvers, these were compared with other HBV genotype D strains in three characterized clinical cohorts including common chronically infected patients. The sT125M+sP127T combination was observed in four nonresolvers of six, corroborated by aa covariance analysis, associated with a lower predicted antigenicity than sT125T+sP127P. Concordant features within this HBV key functional domain, at positions 125 and 127, were reported from two of the three comparative cohorts. In our hands, a lower ELISA reactivity of HBV-vaccinated mice sera was observed against the sT125M mutant. In the S gene, 56 aa changes in minor variants were detected in non-resolvers, mainly in the major hydrophilic region, vs 28 aa changes in resolvers. Molecular features in patients showing HBsAg persistence on NUCs argue in favour of a different aa pattern in the HBV S gene compared to those showing HBsAg clearance. In nonresolvers, a decrease in HBs 'a' determinant antigenicity and more frequent mutations in the S gene suggest a role for the HBV envelope characteristics in HBsAg persistence.

Real-life rilpivirine resistance and potential emergence of an E138A-positive HIV strain in north-eastern France.

OBJECTIVES: To assess the prevalence of resistance to rilpivirine and mutations at position 138 in reverse transcriptase and to identify associated epidemiological and biological characteristics. METHODS: This retrospective study included 238 patients with available HIV-1 nucleotide sequences analysed at the Laboratory of Virology at the University Hospital of Nancy between January 2011 and June 2013. Resistance to non-nucleoside reverse transcriptase inhibitors (NNRTIs) was evaluated according to the ANRS algorithm (version 23) and correlated with clinico-epidemiological and therapeutic data. The virus strains were analysed by evaluating the distance and distribution of the phylogenetic tree (MEGAv5). RESULTS: Among previously treated patients (111/238, 46.6%), 68/111 (61.3%) had received NNRTIs; all were rilpivirine-naive. The prevalence of rilpivirine resistance in the whole cohort was 12.6% (30/238), and was 10.2% (13/127) and 15.3% (17/111) in naive and pre-treated patients, respectively. The E138A mutation was the most frequent mutation associated with resistance to rilpivirine (P < 0.0001). The prevalence of the E138A mutation tended to increase over time, from 3.6% (2/55) during the first half of 2011 to 9.3% (4/43) during the first half of 2013 (P = 0.0614). Seven viral strains from seven naive male patients positive for the E138A mutation appeared in the same cluster. CONCLUSIONS: In our cohort of patients, we observed significantly increased resistance to rilpivirine, mostly because of the E138A mutation, probably due to an E138A strain circulating in newly diagnosed men who have sex with men. Taken together, our results emphasize the need to investigate the prevalence of rilpivirine resistance-associated mutations in the coming years both in France and abroad.

Somatic coliphages are conservative indicators of SARS-CoV-2 inactivation during heat and alkaline pH treatments.

High concentrations of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) genome have been described in wastewater and sewage sludge. It raises the question of the security of land sludge disposal practices during a pandemic. This study aimed to compare SARS-CoV-2's resistance to the main inactivating factors in sludge treatments, pH and heat, to that of native wastewater somatic coliphages. The latest can be easily used as an indicator of treatment efficiency in the field. The effects of heat treatment and pH on the survival of SARS-CoV-2 and somatic coliphages were investigated in simple media. The T90 value (time required for a 90% reduction in the virus or a 1 × log10 decline) at 50 °C was about 4 min for infectious SARS-CoV-2, and around 133 min for infectious somatic coliphages, with no decrease in SARS-CoV-2 genome. For infectious SARS-CoV-2, a slight decrease (<1 log10 unit) was observed at pH 9 or 10 for 10 min; the decrease was over 5 log10 units at pH 11. However, both SARS-CoV-2 genome and infectious somatic coliphages decreased by less than 1 log10 unit at pH 12. All thermal or pH-based treatments that can remove or significantly reduce infectious somatic coliphages (>4 log10) can be considered efficient treatments for infectious SARS-CoV-2. We concluded that somatic coliphages can be considered highly conservative and easy to use indicators of the inactivation of SARS-CoV-2 during treatments based on heat and alkaline pH.

[Tropism of hepatitis C virus for leukocytes-- importance of the analysis of viral E1 and E2 envelope glycoprotein genes by sequencing]. / Tropisme leucocytaire du virus de l'hépatite C - intérêt de l'analyse des séquences des gènes des glycoprotéines d'enveloppe virales E1 et E2.

The ability of hepatitis C virus (HCV) to infect leukocytes could favour HCV pathogenesis. Although viral infection of these immunocompetent cells is poorly (or not) productive, the impact on their immunomodulatory functions could be important. Viral envelope glycoproteins E1 and E2, because of their crucial role in the recognition of viral receptors on permissive cells, could contribute to viral leukocytic tropism and, as a consequence, to the pathophysiology of HCV chronic infection.

Lack of effect of tumor necrosis factor-alpha -308 G/A polymorphism on severity of liver fibrosis in Tunisian hepatitis C virus (HCV)-infected patients.

OBJECTIVES: Tumor necrosis factor alpha (TNF-alpha) plays a key role in the immune response. An elevated plasma level of TNF-alpha was repeatedly observed in patients with active liver injury or cirrhosis regardless of the aetiology. The G/A transition at position -308 in the promoter region have been shown to influence TNF-alpha expression. In this study, we aimed to evaluate the impact of TNF-alpha -308 G/A functional polymorphism on fibrosis severity in Tunisian Hepatitis C Virus (HCV)-infected patients. METHODS: TNF-alpha -308 G/A polymorphism was evaluated by polymerase chain reaction (PCR) amplification followed by Restriction Fragment Length Polymorphism (RFLP) method in 53 chronic hepatitis C patients. Single-nucleotide polymorphism (SNP) frequencies were compared with regard to liver fibrosis severity as assessed by the METAVIR scoring system (F1-F2; n=22 versus F3-F4; n=31). RESULTS: The genotype distribution of the TNF-alpha -308 G/A polymorphism among the HCV-infected patients was as follows : GG : 67.9%, GA : 32.1%, AA : 0%. With regard to fibrosis score, no significant differences in TNF-alpha genotype distribution were observed between F1-F2 and F3-F4 patients (p=0.15). CONCLUSION: No significant association between TNF-alpha -308 polymorphism and and the severity of liver fibrosis was found in our Tunisian cohort.

Conservation of hepatitis C virus nonstructural protein 3 amino acid sequence in viral isolates during liver transplantation.

As a consequence of selective pressure exerted by the immune response during hepatitis C virus (HCV) infection, a high rate of nucleotide mutations in the viral genome is observed which leads to the emergence of viral escape mutants. The aim of this study was to evaluate the evolution of the amino acid (aa) sequence of the HCV nonstructural protein 3 (NS3) in viral isolates after liver transplantation. Six patients with HCV-induced liver disease undergoing liver transplantation (LT) were followed up for sequence analysis. Hepatitis C recurrence was observed in all patients after LT. The rate of synonymous (dS) nucleotide substitutions was much higher than that of nonsynonymous (dN) ones in the NS3 encoding region. The high values of the dS/dN ratios suggest no sustained adaptive evolution selection pressure and, therefore, absence of specific NS3 viral populations. Clinical genotype assignments were supported by phylogenetic analysis. Serial samples from each patient showed lower mean nucleotide genetic distance when compared with samples of the same HCV genotype and subtype. The NS3 samples studied had an N-terminal aa sequence with several differences as compared with reference ones, mainly in genotype 1b-infected patients. After LT, as compared with the sequences before, a few reverted aa substitutions and several established aa substitutions were observed at the N-terminal of NS3. Sites described to be involved in important functions of NS3, notably those of the catalytic triad and zinc binding, remained unaltered in terms of aa sequence. Rare or frequent aa substitutions occurred indiscriminately in different positions. Several cytotoxic T lymphocyte epitopes described for HCV were present in our 1b samples. Nevertheless, the deduced secondary structure of the NS3 protease showed a few alterations in samples from genotype 3a patients, but none were seen in 1b cases. Our data, obtained from patients under important selective pressure during LT, show that the NS3 protease remains well conserved, mainly in HCV 3a patients. It reinforces its potential use as an antigenic candidate for further studies aiming at the development of a protective immune response.

NS5A(ISDR-V3) region genetic variability of Tunisian HCV-1b strains: Correlation with the response to the combined interferon/ribavirin therapy.

In the non-structural protein 5A (NS5A) of hepatitis C virus (HCV), mutations within the interferon sensitivity-determining region (ISDR), the PKR-binding domain (PKR-BD), the variable region 3 (V3), and the interferon/ribavirin resistance-determining region (IRRDR) have been correlated with the IFN-based therapy response. In Tunisia, where a high prevalence of HCV-1b has been found, no data regarding the implication of NS5A in treatment response were available. The current study examined the relationship between the pre-treatment mutation number within ISDR, PKR-BD, V3, IRRDR, as well as in the entire ISDR-V3 region of NS5A (aa 2209-2379) and the response to the 48-week course of combined IFN plus ribavirin therapy in 15 HCV-1b-infected Tunisian patients. Referring to HCV-J sequence, a significant high genetic variability was observed within PKR-BD in the sustained virological responder patients compared to non-responders (P = 0.040). More importantly, when considering the entire region from ISDR to V3, referred to as NS5A(ISDR-V3), a clear difference in the mutation number was observed between sustained virological responders (19.6 +/- 3.16) and non-responders (15.0 +/- 1.41) (P = 0.002). Additionally, a more detailed analysis of NS5A(ISDR-V3) region revealed an elevated degree of mutation rate within the region located between amino acids 2282 and 2308 (P = 0.0006). Interestingly, an analysis of specific amino acid variations defined proline and serine at position 2300 as signature patterns for sensitive and resistant strains, respectively. The genetic variability within the NS5A region of HCV-1b strains was associated with the response to the combined IFN plus ribavirin therapy in our Tunisian cohort.

Transmission of hepatitis E virus by water: An issue still pending in industrialized countries.

Hepatitis E virus (HEV) is an enteric virus divided into eight genotypes. Genotype 1 (G1) and G2 are specific to humans; G3, G4 and G7 are zoonotic genotypes infecting humans and animals. Transmission to humans through water has been demonstrated for G1 and G2, mainly in developing countries, but is only suspected for the zoonotic genotypes. Thus, the water-related HEV hazard may be due to human and animal faeces. The high HEV genetic variability allows considering the presence in wastewater of not only different genotypes, but also quasispecies adding even greater diversity. Moreover, recent studies have demonstrated that HEV particles may be either quasi-enveloped or non-enveloped, potentially implying differential viral behaviours in the environment. The presence of HEV has been demonstrated at the different stages of the water cycle all over the world, especially for HEV G3 in Europe and the USA. Concerning HEV survival in water, the virus does not have higher resistance to inactivating factors (heat, UV, chlorine, physical removal), compared to viral indicators (MS2 phage) or other highly resistant enteric viruses (Hepatitis A virus). But the studies did not take into account genetic (genogroups, quasispecies) or structural (quasi- or non-enveloped forms) HEV variability. Viral variability could indeed modify HEV persistence in water by influencing its interaction with the environment, its infectivity and its pathogenicity, and subsequently its transmission by water. The cell culture methods used to study HEV survival still have drawbacks (challenging virus cultivation, time consuming, lack of sensitivity). As explained in the present review, the issue of HEV transmission to humans through water is similar to that of other enteric viruses because of their similar or lower survival. HEV transmission to animals through water and how the virus variability affects its survival and transmission remain to be investigated.

Hepatitis C virus structural proteins do not prevent human dendritic cell maturation.

AIM: Infection with hepatitis C virus (HCV) results in chronic hepatitis in more than 70% of cases. Alterations in the maturation of dendritic cells (DC) might play a role in the immune system's inability to eliminate the virus, although viral factors that could be involved have not been identified. This study in vitro investigated whether HCV structural proteins affect maturation of monocyte-derived DC. METHODS: HCV proteins (core, E1, E2) were expressed by transduction with recombinant adenoviruses of immature DC. The ability of these transduced DC to respond to a maturation stimulus was evaluated by measuring cell surface markers, allogenic lymphocyte stimulation and interleukin (IL)-12 production. RESULTS: Expression of HCV structural proteins did not modify DC maturation in the presence of lipopolysaccharide, as determined by their phenotype and stimulatory functioning. IL-12 secretion was not affected by HCV protein expression in mature DC. CONCLUSION: Our results suggest that HCV structural proteins do not affect maturation of monocyte-derived DC by lipopolysaccharide. These findings are important for further studies to clarify the pathogenesis of chronic HCV infection and towards the rational design of cellular vaccine approaches for immunotherapy against hepatitis C.

[Neutralizing antibodies in hepatitis C virus infection]. / Anticorps neutralisants dirigés contre le virus de l'hépatite C.

Hepatitis C virus (HCV) results in persistent infection in more than 70% of infected individuals despite the development of humoral and cellular immune responses. Following infection, although antibodies targeting epitopes of both structural and non structural proteins are elicited, the virus evades antibody-mediated neutralization. Studies of host neutralizing responses against HCV have been limited by the lack of a convenient tissue culture system for HCV infection. In the past five years in vitro models have been developed to characterize interaction of HCV glycoproteins with host cell entry factors and detect antibodies interfering with HCV entry and infection. These models have been used to characterize targets of neutralizing responses and better understand their impact on the pathogenesis of infection.

Molecular features of Hepatitis E Virus circulation in environmental and human samples.

BACKGROUND AND OBJECTIVES: Hepatitis E virus (HEV) is emerging but its circulation between humans and the environment remains misunderstood. HEV ORF2 gene encodes the capsid playing a key role in viral interactions with surfaces, ORF3 products are involved in the viral cycle. Our aim was to study the molecular characteristics of ORF2 and ORF3 which could favor HEV fitness in patients and the environment. STUDY DESIGN: Samples from 69 patients with hepatitis (blood/stools), 20 urban wastewaters, 20 effluents of a pig slaughterhouse, 22 farm pigs (stools), 20 wild boars (liver/stools) were collected in North-Eastern France. HEV strains were analyzed by direct sequencing within the ORF2 M region, of ORF2/ORF3, for phylogeny and physicochemical prediction and for ORF2 by ultra-deep sequencing. RESULTS: The results showed frequent HEV-positive samples: 9.1% of the patient bloods, 23.1% of their stools; 25.0% of wastewaters, 75.0% for the slaughterhouse, 10.0% of the boar livers, 5.3% of their stools. The strains were classified as HEV genotype 3. In ORF2, HEV highlighted one homogeneous major viral variant within quasispecies and a decrease in predicted antigenicity for two minor mutations (D442G, V402A). A cysteine signature at position 81 in ORF3 was observed in the boars. CONCLUSIONS: HEV RNA genotype 3 was detected in patients and in animals, in a slaughterhouse effluent and in wastewater. Moreover, the low variability of amino acids in the ORF2 M region and molecular features in ORF2 and ORF3 suggested that HEV strains could be advantageous for key properties.

Atypical symptoms in patients with herpesvirus DNA detected by PCR in cerebrospinal fluid.

BACKGROUND: Polymerase chain reaction (PCR) detection of herpesvirus DNA in cerebrospinal fluid (CSF) is an important tool in the diagnosis of central nervous system (CNS) syndromes. The corresponding viral infections present with diverse clinical signs, which are often classical although no sign can be considered as specific. This retrospective study aims to describe atypical symptoms in patients with herpesvirus DNA detected in CSF by PCR. A total of 3452 cerebrospinal fluid samples from patients with suspected herpesvirus infection of the CNS were investigated between 1998 and 2003 in our clinical virology laboratory. "In-house" PCRs for each herpesvirus [herpes simplex virus (HSV), varicella zoster virus (VZV), cytomegalovirus (CMV), Epstein Barr virus (EBV), or human herpes virus 6 (HHV6)] were used until 2001 and a commercially available "Herpes Consensus PCR" was used thereafter. One of the five herpesviruses investigated in this study was found in 71 (2.1%) of CSF samples (37 HSV, 14 VZV, 1 CMV, 9 EBV and 10 HHV6). These samples were obtained from 62 patients whose clinical findings were generally consistent with the PCR data. However, some little known features of herpesvirus-related symptoms, such as partial seizure associated with HSV infection, and unusual VZV or HHV6-related myelitis were also observed.

Impact of protease inhibitors on intrahepatic hepatitis C virus viral load.

During chronic hepatitis C, hepatitis C virus (HCV) load in plasma was shown to be higher in HIV-co-infected than in immunocompetent patients [1]. The reason for this increased HCV replication is not known. It may be as a result of HIV-induced immune deficiency [2], although some authors did not find any correlation with the CD4 cell count [3]. A direct interaction between HCV and HIV was also hypothesized [4]. Protease inhibitors (PI) used in highly active antiretroviral therapy (HAART) have no HCV reduction effect during the first months of treatment [5-8]. However, a decrease in HCV plasma load was recently described in patients treated with HAART for a year [9,10]. We therefore investigated the potential impact of HAART on intrahepatic HCV load.

Sequence analysis of env C2/V3, gag p17/p24, and pol protease regions of 25 HIV type 1 isolates from Ho Chi Minh City, Vietnam.

Env C2/V3, gag p17/p24, pol protease, and RT regions of HIV-1 isolates recently obtained from 25 HIV-1 seropositive individuals from Ho Chi Minh City (Vietnam) were studied, and genes subtypes were determined by DNA sequence analyses. Twenty-three isolates out of 25 were identified as belonging to subtype E, now recognized as circulating recombinant form 1 (CRF01_AE). The motif at the top of the V3 loop (generally GPGQ) was then preceded by an isoleucine or a methionine (M) residue; the M residue might be a local signature of Vietnamese E isolates compared to Thai E viruses. Two isolates (8%) were shown to be intersubtype recombinants: one E/B and one CRF02_AG(IBNG)/D. The polymorphism of pol protease was considered only for CRF01_AE isolates and is clearly different from that recorded for B viruses with substitutions at positions 13, 35, 36, 41, 69, and 89.

PCR detection of human enteric viruses in bathing areas, waste waters and human stools in Southwestern France.

Detection of human pathogenic viruses by molecular techniques might be suitable for identifying viral pollution in environmental waters and for improving diagnosis in patients. Environmental samples were taken from bathing areas and sewage treatment plants in southwestern France. Small volume samples (50 microL) were tested. Five groups of enteric pathogenic viruses were studied: enteroviruses, Norwalk-like viruses (NLVs), hepatitis A virus, rotaviruses and adenoviruses. Moreover, human samples were tested for NLV. After extraction of viral nucleic acids (Boom's procedure), a nested polymerase chain reaction was conducted before hybridization. Five bathing waters out of 26 were positive for one viral group, without systematic association with bacterial contamination. Eight sewage plant samples out of 13 were positive for at least one viral group. Seven patients out of 45 were NLV-positive. Molecular techniques allow efficient screening of viral contamination in environmental waters and the study of NLV molecular epidemiology.

Qualitative and quantitative molecular detection of enteroviruses in water from bathing areas and from a sewage treatment plant.

Pathogenic enteric viruses can be introduced into the environment as a result of human activities. Enteroviruses are regularly detected in environmental waters or shellfish and can provoke potentially serious diseases. Some authors believe that enteroviruses could represent an interesting indicator of viral contamination in the environment. Since molecular approaches seem to be promising for the detection of these viruses, we developed a simple qualitative RT-PCR procedure for enteroviruses, together with a quantitative RT-PCR assay using RNA internal standard. After one-tube-RT-PCR, this standard and wild enterovirus RNA were detected by differential hybridization with specific probes and a fluorimetric reaction. The quantification of enteroviruses, conducted in a sewage treatment plant, showed a decreasing number of genomic copies from the entrance to the exit (from 3.8 x 10(5) to 5.4 x 10(4) RNA copies/mL) but indicated the presence of enterovirus RNA in the neighboring river (2.2 x 10(3) RNA copies/mL). In bathing areas, enterovirus RNA was detected in 16 out of 226 samples, with copies numbers ranging from 3.7 x 10(2) RNA copies/mL to 7 x 10(4) RNA copies/mL.

A hospital outbreak of gastroenteritis possibly related to the contamination of tap water by a small round structured virus.

Small round structured viruses (SRSVs) are a major cause of gastroenteritis in institutions and sensitive new molecular techniques allow rapid diagnosis and the establishment of control measures. In January 1999, a 10 day-long outbreak of gastroenteritis in a re-education ward, was reported by a hospital hygiene department. A potential common source of contamination was tap water. The stools of six patients with gastroenteritis and seven tap water samples from the hospital ward, were tested for SRSV by reverse transcription and polymerase chain reaction (RT-PCR): three stools and four water samples, all bacteriologically negative, were SRSV-positive. Nucleotide sequencing of a fragment of the SRSV polymerase gene showed that the sequences of the positive samples (two patients and four water samples) were identical (genogroup II). We cannot exclude interhuman transmission of SRSV together with viral soiling of some taps in the ward, but this hospital infection was more likely due to the transient contamination of the ward supply of drinking water with a SRSV strain.

Hepatitis B exacerbation with a precore mutant virus following withdrawal of lamivudine in a human immunodeficiency virus-infected patient.

Chronic active hepatitis B exacerbations have been reported following development of resistance to or withdrawal of lamivudine in HIV-infected patients. A 38-year-old woman with HIV and chronic HBV infections was hospitalized because of acute hepatitis. The occurrence of cytolysis with replication of HBV 2 months after withdrawing lamivudine suggests that our patient experienced a severe reactivation of HBV infection due to the modification of her treatment. Sequencing of the HBV precore region showed the strain to be a mutant. We conclude that lamivudine should not be stopped in HIV- and HBV-infected patients, but could be continued at the dose of 100mg/day as used in isolated HBV infection.

[Commercial methods for the diagnosis of CMV infection after organ or bone marrow transplantation]. / Utilisation des tests commercialisés pour le diagnostic d'une infection a CMV chez les greffés de moelle et d'organe.

Cytomegalovirus infection is a major problem for transplant recipients. It has been recently shown that the rate of increase in viral load correlate with CMV disease in transplant recipients. The risk is very high in persons undergoing bone marrow transplantation and in some cases preemptive therapy is used as soon as viral replication is proven. In this context the assays based on the detection of CMV in leukocytes are found to be the most sensitive. After renal transplantation a number of studies have demonstrated that the amount of CMV DNA is significantly associated with disease development. But there is no consensus with regard to the cut-off level predictive of CMV disease because of the heterogeneity of the assays used. Recent studies carried out with commercial assays detecting CMV DNA in leukocytes or plasma have shown that the performances of both are equivalent and that by using a receiver operator curve analysis a cut-off viral load can be determined which maximizes the clinical utility of these assays.