TBK1 is part of a galectin 8 dependent membrane damage recognition complex and drives autophagy upon Adenovirus endosomal escape.

Intracellular pathogens cause membrane distortion and damage as they enter host cells. Cells perceive these membrane alterations as danger signals and respond by activating autophagy. This response has primarily been studied during bacterial invasion, and only rarely in viral infections. Here, we investigate the cellular response to membrane damage during adenoviral entry. Adenoviruses and their vector derivatives, that are an important vaccine platform against SARS-CoV-2, enter the host cell by endocytosis followed by lysis of the endosomal membrane. We previously showed that cells mount a locally confined autophagy response at the site of endosomal membrane lysis. Here we describe the mechanism of autophagy induction: endosomal membrane damage activates the kinase TBK1 that accumulates in its phosphorylated form at the penetration site. Activation and recruitment of TBK1 require detection of membrane damage by galectin 8 but occur independently of classical autophagy receptors or functional autophagy. Instead, TBK1 itself promotes subsequent autophagy that adenoviruses need to take control of. Deletion of TBK1 reduces LC3 lipidation during adenovirus infection and restores the infectivity of an adenovirus mutant that is restricted by autophagy. By comparing adenovirus-induced membrane damage to sterile lysosomal damage, we implicate TBK1 in the response to a broader range of types of membrane damage. Our study thus highlights an important role for TBK1 in the cellular response to adenoviral endosome penetration and places TBK1 early in the pathway leading to autophagy in response to membrane damage.

Bronchial epithelia from adults and children: SARS-CoV-2 spread via syncytia formation and type III interferon infectivity restriction.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections initiate in the bronchi of the upper respiratory tract and are able to disseminate to the lower respiratory tract, where infections can cause an acute respiratory distress syndrome with a high degree of mortality in elderly patients. We used reconstituted primary bronchial epithelia from adult and child donors to follow the SARS-CoV-2 infection dynamics. We show that, in epithelia from adult donors, infections initiate in multiciliated cells and spread within 24 to 48 h throughout the whole epithelia. Syncytia formed of ciliated and basal cells appeared at the apical side of the epithelia within 3 to 4 d and were released into the apical lumen, where they contributed to the transmittable virus dose. A small number of reconstituted epithelia were intrinsically more resistant to virus infection, limiting virus spread to different degrees. This phenotype was more frequent in epithelia derived from children versus adults and correlated with an accelerated release of type III interferon. Treatment of permissive adult epithelia with exogenous type III interferon restricted infection, while type III interferon gene knockout promoted infection. Furthermore, a transcript analysis revealed that the inflammatory response was specifically attenuated in children. Taken together, our findings suggest that apical syncytia formation is an underappreciated source of virus propagation for tissue or environmental dissemination, whereas a robust type III interferon response such as commonly seen in young donors restricted SARS-CoV-2 infection. Thus, the combination of interferon restriction and attenuated inflammatory response in children might explain the epidemiological observation of age-related susceptibility to COVID-19.

Multi-layered control of Galectin-8 mediated autophagy during adenovirus cell entry through a conserved PPxY motif in the viral capsid.

Cells employ active measures to restrict infection by pathogens, even prior to responses from the innate and humoral immune defenses. In this context selective autophagy is activated upon pathogen induced membrane rupture to sequester and deliver membrane fragments and their pathogen contents for lysosomal degradation. Adenoviruses, which breach the endosome upon entry, escape this fate by penetrating into the cytosol prior to autophagosome sequestration of the ruptured endosome. We show that virus induced membrane damage is recognized through Galectin-8 and sequesters the autophagy receptors NDP52 and p62. We further show that a conserved PPxY motif in the viral membrane lytic protein VI is critical for efficient viral evasion of autophagic sequestration after endosomal lysis. Comparing the wildtype with a PPxY-mutant virus we show that depletion of Galectin-8 or suppression of autophagy in ATG5-/- MEFs rescues infectivity of the PPxY-mutant virus while depletion of the autophagy receptors NDP52, p62 has only minor effects. Furthermore we show that wildtype viruses exploit the autophagic machinery for efficient nuclear genome delivery and control autophagosome formation via the cellular ubiquitin ligase Nedd4.2 resulting in reduced antigenic presentation. Our data thus demonstrate that a short PPxY-peptide motif in the adenoviral capsid permits multi-layered viral control of autophagic processes during entry.

Resistance mutations in subtype C HIV type 1 isolates from Indian patients of Mumbai receiving NRTIs plus NNRTIs and experiencing a treatment failure: resistance to AR.

We present here the first data available on resistance to nucleoside reverse transcriptase inhibitors (NRTIs) and nonnucleoside reverse transcriptase inhibitors (NNRTIs) in India. In these subtype C isolates, we have observed most of the mutations noted in reverse transcriptase (RT) for subtype B with some additional substitutions (at positions 98, 203, 208, and 221) that will warrant attention in the algorithms used.

Hepatitis B virus genotypes: a retrospective survey in Southwestern France, 1999-2004.

OBJECTIVE: To determine the prevalence of HBV genotypes in Southwestern France and the association between HBV genotypes and patients characteristics. METHODS: 194 HBsAg-positive patients (median age: 45 yrs, range: 7-77, male: 78%) followed in Bordeaux Hospital in 1999-2004 were included. HBV genotype, pre-core (PC) and core promoter (CP) mutations were determined by sequencing. RESULTS: Genotype distribution was A 51%, B 6.7%, C 5.7%, D 26.3%, E 7.7%, F 0.5%, G 2.1%. Among the 146 patients documented, 71.2% were Caucasians, 15.8% Africans, 13.0% Asians. Fifty-seven patients (36%) were HIV-infected. Eighty-two (42.3%) patients were HBeAg-positive. Genotype A was almost exclusively carried by Caucasians (96%), Africans were most frequent among genotype E (82%), and Asians were most prevalent among genotypes B and C (82% and 80%, respectively). Genotype A was associated with a higher prevalence of HBeAg than genotype D (53% versus 35.3%, P=0.03). PC variant was detected in 35% and CP variant in 43% of patients. PC variant was uncommon in genotype A patients (7.3%). CONCLUSION: Distribution of HBV genotypes differs according to ethnic origin, genotypes A and D being the most frequently found. Genotype A was more frequently associated with HBeAg-positivity and genotype D with HBeAg-negativity.

Outbreak of hepatitis C virus infection during sclerotherapy of varicose veins: long-term follow-up of 196 patients (4535 patient-years).

BACKGROUND/AIMS: The aim of this study was to describe the natural history of a HCV infection outbreak in 196 patients who had sclerotherapy by a same physician and to confirm patient-to-patient transmission using phylogenetic analysis in a large series of patients. METHODS: Demographic information included clinical and biological parameters. Fibrosis evaluation was performed using liver biopsy or transient elastography. Follow-up was maintained until death, or the end of the observation period. In order to determine if the virus had been transmitted between the HCV genotype 2 patients, sequence analysis was undertaken of a part of the NS5b region of the genomes in samples of patients. RESULTS: The mean duration of follow-up was 23.1+/-6.7 years (4535 patient-years). In patients with fibrosis evaluation, 55.7% had no or mild fibrosis and 44.3% had significant fibrosis. No patient died from HCV-related disease. Nucleotide sequence analysis of a part of the NS5b region revealed that patients were all infected with the same HCV subtype (genotype 2d). The most evident feature of the tree is the clustering of all patients involved in the outbreak without any unrelated isolates. CONCLUSION: This study emphasizes the risk for nosocomial spread of HCV during intravenous therapy.

Plasma and liver hepatitis C virus variability in patients coinfected with human immunodeficiency virus.

Liver and plasma hepatitis C virus (HCV) variability was compared by E2 cloning and sequencing in three patients coinfected with HCV and human immunodeficiency virus (HIV) before and after interferon treatment and in three patients solely infected with HCV. The plasma and liver samples contained unique sequences. In the patients coinfected with HIV, accumulated random mutations produced mostly nonsynonymous substitutions in contrast to the reduced HCV genetic variability seen after treatment.

An additive/subtractive genotypic score as a determinant of the virological response to didanosine in HIV-1 infected patients.

OBJECTIVE: To assess the genotypic determinants of the virological response (VR) to didanosine (ddI) in nucleoside reverse transcriptase inhibitors (NRTI)-experienced patients. METHODS: Human immunodeficiency virus type 1 (HIV-1) genotype was determined at baseline in 74 ddI-naive-patients with baseline viral load >500 copies/ml and receiving ddI as part of a new regimen. VR was defined as a plasma HIV-1 RNA <50 copies/ml after three months on ddI. NRTI resistance mutations associated with higher or lower frequencies of VR with a p-value<0.25 were retained in different sets of mutations, where the mutations associated with a worse VR were added, whereas the mutations associated with a better VR were subtracted. The most significant mutation scores were then studied in a multivariate analysis. RESULTS: Changes at three codons (M41L, L210W, T215Y/F/D/C/E) were associated with a worse VR and three mutations (K70R, M184V, K219Q) with a better VR. The strongest association with the VR was obtained with the score M41L+L210W+T215Y/F/D/C/E-K70R-K219Q. The score was independently associated with the VR in the multivariate analysis. CONCLUSION: Taking into account the mutations associated with a better VR may improve genotypic resistance algorithms. Our results are of interest for the management of antiretroviral therapy in NRTI-experienced patients.

Clonal analysis of HIV-1 variants in proviral DNA during treatment interruption in patients with multiple therapy failures.

BACKGROUND: Treatment interruption (TI) in antiretroviral-treated patients on virological failure (VF) often results in a shift from resistant to wild-type HIV-1 in plasma. A clonal analysis was set out to determine the importance of archiving of resistant HIV-1 variants during TI and its relationship with the occurrence of a VF after treatment resumption. METHODS: A fragment of the pol gene was cloned and sequenced from peripheral blood mononuclear cells DNA sampled at the end of TI. Clonal sequences were compared to bulk plasma sequences determined before TI, after TI and at VF. RESULTS: Four patients were enrolled; all had a complete reversion to wild-type HIV-1 at the end of TI. In two patients with subsequent VF, archiving of minority resistant variants was detected in proviral DNA. Archived resistant variants were found to be phylogenetically linked to sequences detected before TI and at VF, suggesting the re-expansion of resistant HIV-1 from archived quasi-species. CONCLUSION: In patients having a TI in the context of VF, archiving of resistant HIV-1 variants in proviral DNA can be involved into the mechanisms of further VF after treatment resumption.

Epidemiological and phylogenetic evidence for patient-to-patient hepatitis C virus transmission during sclerotherapy of varicose veins.

The aim of this study was to provide evidence for patient-to-patient nosocomial hepatitis C virus (HCV) transmission during sclerotherapy of varicose veins. Forty-three patients who had evidence of current infection by genotype 2 HCV have had sclerotherapy by the same physician. Based on this observation, a detailed epidemiological questionnaire on risk factors for HCV in genotype 2 infected patients was conducted. Seventeen sequences in the hypervariable region 1 (HVR1) of the HCV genome obtained from 17 HCV RNA positive patients with a past history of sclerotherapy, were compared with 17 sequences derived from genotype 2 patients with no past history of sclerotherapy, and with 25 sequences sampled from GenBank. Two hundred seven genotype 2 HCV infected patients were included. The main risk factors for HCV infection were transfusion (n = 76), drug use (n = 6), and sclerotherapy of varicose veins (n = 62 including 43 (20.8%) by the same physician), other or unknown (n = 76). These sclerotherapy sessions were carried out in the 1980s for many years. Five of these 43 patients had jaundice within a few weeks after a sclerotherapy session. Sequence analysis of HVR1 from 17 patients who had sclerotherapy by the same physician revealed that they were all infected with HCV genotype 2c. The phylogenetic tree indicated clustering of the patients with a past history of sclerotherapy. The method by which infection was likely to have been transmitted was probably the use of a single vial for multiple patients. This study provides strong evidence that sclerotherapy of varicose veins is a risk factor for HCV infection.

Molecular characterization of HIV type 1 isolates from untreated patients of Mumbai (Bombay), India, and detection of rare resistance mutations.

The molecular characterization of HIV-1 isolates in drug-naive cases in the early stages of HIV disease was studied in 128 cases from Mumbai (Bombay), India. Subtype C was largely predominant followed by A-C intersubtype recombinants, one subtype A and one CRF01 AE. Compared to subtype B, subtype C exhibited an important polymorphism; the percentages of substitutions could reach more than 90%. Two isolates showed M184V substitution the reverse transcriptase indicating resistance to 3TC.

HIV type 1 diversity in France, 1999-2001: molecular characterization of non-B HIV type 1 subtypes and potential impact on susceptibility to antiretroviral drugs.

Non-B HIV-1 samples collected in France between 1999 and 2001 were sequenced in the env, reverse transcriptase (RT), and protease genes (1) to characterize further the non-B strains circulating in the country, (2) to assess the importance of recombination, and (3) to describe the polymorphism of RT and protease genes and appreciate a possible impact on susceptibility to antiretroviral (ARV) drugs. The results show that, within a background of CRF02_AG predominance, there is a high genetic diversity of non-B isolates, including intersubtype recombinants. There is an extensive polymorphism of protease and RT genes compared with B consensus sequences; we have so far no data indicating that these non-B isolates may have reduced sensitivity to ARV drugs.