scholarly journals Clinical Resistance to Enfuvirtide Does Not Affect Susceptibility ofHuman Immunodeficiency Virus Type 1 to Other Classes of Entry Inhibitors

2007 ◽  
Vol 81 (7) ◽  
pp. 3240-3250 ◽  
Author(s):  
Neelanjana Ray ◽  
Jessamina E. Harrison ◽  
Leslie A. Blackburn ◽  
Jeffrey N. Martin ◽  
Steven G. Deeks ◽  
...  
Keyword(s):  
Virologic Failure ◽  
Viral Envelope ◽  
Fusion Inhibitor ◽  
Drug Resistant ◽  
Resistance Mutations ◽  
Entry Inhibitors ◽  
Clinical Resistance ◽  
Immunodeficiency Virus ◽  
Hiv 1

ABSTRACT The clinical use of the human immunodeficiency virus (HIV) fusion inhibitor enfuvirtide (ENF) can select for drug-resistant HIV-1 strains bearing mutations in the HR1 region of the viral envelope (Env) protein. We analyzed the properties of multiple Env proteins isolated from five patients who experienced an initial decline in viral load after ENF therapy followed by subsequent rebound due to emergence of ENF-resistant HIV-1. Prior to ENF therapy, each patient harbored genetically and phenotypically diverse Env proteins that used CCR5 and/or CXCR4 to elicit membrane fusion. Coreceptor usage patterns of the Envs isolated from two patients underwent homogenization following ENF therapy, whereas in the other three patients, recombination appeared to allow the introduction of a single HR1 sequence with ENF resistance mutations into phenotypically distinct Env proteins. Analysis of individual Env clones also revealed that prior to ENF therapy, there was sometimes marked heterogeneity in the susceptibility of individual Env proteins to coreceptor inhibitors. After virologic failure, all Envs acquired resistance to ENF but exhibited no consistent change in their sensitivity to the fusion inhibitor T-1249 or to coreceptor inhibitors. In summary, using patient-derived Env proteins, we found that ENF failure was associated with emergence of high-level resistance to ENF due largely to mutations in HR1 but that susceptibility to other entry inhibitors was unaffected, that in these late-stage patients there was greater clonal variability to coreceptor than to fusion inhibitors, and that recombination events in vivo could sometimes restore Env genotypic and phenotypic heterogeneity by introducing drug-resistant gp41 sequences into heterologous gp120 backgrounds.

scholarly journals Therapeutic Efficacy and Resistance Selection of a Lipopeptide Fusion Inhibitor in Simian Immunodeficiency Virus-Infected Rhesus Macaques

2020 ◽  
Vol 94 (15) ◽  
Author(s):  
Danwei Yu ◽  
Jing Xue ◽  
Huamian Wei ◽  
Zhe Cong ◽  
Ting Chen ◽  
...  
Keyword(s):  
Membrane Fusion ◽  
Therapeutic Efficacy ◽  
Rhesus Macaques ◽  
Heptad Repeat ◽  
Fusion Inhibitor ◽  
Resistance Mutations ◽  
Fusion Inhibitors ◽  
Immunodeficiency Virus ◽  
Hiv 1

ABSTRACT We recently reported a group of lipopeptide-based membrane fusion inhibitors with potent antiviral activities against human immunodeficiency virus type 1 (HIV-1), HIV-2, and simian immunodeficiency virus (SIV). In this study, the in vivo therapeutic efficacy of such a lipopeptide, LP-52, was evaluated in rhesus macaques chronically infected with pathogenic SIVmac239. In a pilot study with one monkey, monotherapy with low-dose LP-52 rapidly reduced the plasma viral loads to below the limit of detection and maintained viral suppression during three rounds of structurally interrupted treatment. The therapeutic efficacy of LP-52 was further verified in four infected monkeys; however, three out of the monkeys had viral rebounds under the LP-52 therapy. We next focused on characterizing SIV mutants responsible for the in vivo resistance. Sequence analyses revealed that a V562A or V562M mutation in the N-terminal heptad repeat (NHR) and a E657G mutation in the C-terminal heptad repeat (CHR) of SIV gp41 conferred high resistance to LP-52 and cross-resistance to the peptide drug T20 and two newly designed lipopeptides (LP-80 and LP-83). Moreover, we showed that the resistance mutations greatly reduced the stability of diverse fusion inhibitors with the NHR site, and V562A or V562M in combination with E657G could significantly impair the functionality of viral envelopes (Envs) to mediate SIVmac239 infection and decrease the thermostability of viral six-helical bundle (6-HB) core structure. In conclusion, the present data have not only facilitated the development of novel anti-HIV drugs that target the membrane fusion step, but also help our understanding of the mechanism of viral evolution to develop drug resistance. IMPORTANCE The anti-HIV peptide drug T20 (enfuvirtide) is the only membrane fusion inhibitor available for treatment of viral infection; however, it exhibits relatively weak antiviral activity, short half-life, and a low genetic barrier to inducing drug resistance. Design of lipopeptide-based fusion inhibitors with extremely potent and broad antiviral activities against divergent HIV-1, HIV-2, and SIV isolates have provided drug candidates for clinical development. Here, we have verified a high therapeutic efficacy for the lipopeptide LP-52 in SIVmac239-infected rhesus monkeys. The resistance mutations selected in vivo have also been characterized, providing insights into the mechanism of action of newly designed fusion inhibitors with a membrane-anchoring property. For the first time, the data show that HIV-1 and SIV can share a similar genetic pathway to develop resistance, and that a lipopeptide fusion inhibitor could have a same resistance profile as its template peptide.

scholarly journals Selective Excision of AZTMP by Drug-Resistant Human Immunodeficiency Virus Reverse Transcriptase

2001 ◽  
Vol 75 (10) ◽  
pp. 4832-4842 ◽  
Author(s):  
Paul L. Boyer ◽  
Stefan G. Sarafianos ◽  
Edward Arnold ◽  
Stephen H. Hughes
Keyword(s):  
Human Immunodeficiency Virus ◽  
Reverse Transcriptase ◽  
Nucleoside Analogs ◽  
Azido Group ◽  
Resistance Mutations ◽  
Immunodeficiency Virus ◽  
Azt Resistance ◽  
Hiv 1

ABSTRACT Two distinct mechanisms can be envisioned for resistance of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) to nucleoside analogs: one in which the mutations interfere with the ability of HIV-1 RT to incorporate the analog, and the other in which the mutations enhance the excision of the analog after it has been incorporated. It has been clear for some time that there are mutations that selectively interfere with the incorporation of nucleoside analogs; however, it has only recently been proposed that zidovudine (AZT) resistance can involve the excision of the nucleoside analog after it has been incorporated into viral DNA. Although this proposal resolves some important issues, it leaves some questions unanswered. In particular, how do the AZT resistance mutations enhance excision, and what mechanism(s) causes the excision reaction to be relatively specific for AZT? We have used both structural and biochemical data to develop a model. In this model, several of the mutations associated with AZT resistance act primarily to enhance the binding of ATP, which is the most likely pyrophosphate donor in the in vivo excision reaction. The AZT resistance mutations serve to increase the affinity of RT for ATP so that, at physiological ATP concentrations, excision is reasonably efficient. So far as we can determine, the specificity of the excision reaction for an AZT-terminated primer is not due to the mutations that confer resistance, but depends instead on the structure of the region around the HIV-1 RT polymerase active site and on its interactions with the azido group of AZT. Steric constraints involving the azido group cause the end of an AZT 5′-monophosphate-terminated primer to preferentially reside at the nucleotide binding site, which favors excision.

scholarly journals Relative Replicative Fitness of Human Immunodeficiency Virus Type 1 Mutants Resistant to Enfuvirtide (T-20)

2004 ◽  
Vol 78 (9) ◽  
pp. 4628-4637 ◽  
Author(s):  
Jing Lu ◽  
Prakash Sista ◽  
Françoise Giguel ◽  
Michael Greenberg ◽  
Daniel R. Kuritzkes
Keyword(s):  
Human Immunodeficiency Virus ◽  
Relative Order ◽  
Wild Type ◽  
Resistance Mutations ◽  
Recombinant Viruses ◽  
Immunodeficiency Virus ◽  
Hiv 1

ABSTRACT Resistance to enfuvirtide (ENF; T-20), a fusion inhibitor of human immunodeficiency virus type 1 (HIV-1), is conferred by mutations in the first heptad repeat of the gp41 ectodomain. The replicative fitness of recombinant viruses carrying ENF resistance mutations was studied in growth competition assays. ENF resistance mutations, selected in vitro or in vivo, were introduced into the env gene of HIV-1NL4-3 by site-directed mutagenesis and expressed in HIV-1 recombinants carrying sequence tags in nef. The doubling time of ENF-resistant viruses was highly correlated with decreasing ENF susceptibility (R 2 = 0.859; P < 0.001). Initial fitness experiments focused on mutants identified by in vitro selection in the presence of ENF (L. T. Rimsky, D. C. Shugars, and T. J. Matthews, J. Virol. 72:986-993, 1998). In the absence of drug, these mutants displayed reduced fitness compared to wild-type virus with a relative order of fitness of wild type > I37T > V38 M > D36S/V38 M; this order was reversed in the presence of ENF. Likewise, recombinant viruses carrying ENF resistance mutations selected in vivo displayed reduced fitness in the absence of ENF with a relative order of wild type > N42T > V38A > N42T/N43K ≈ N42T/N43S > V38A/N42D ≈ V38A/N42T. Fitness and ENF susceptibility were inversely correlated (r = −0.988; P < 0.001). Similar results were obtained with recombinants expressing molecularly cloned full-length env genes obtained from patient-derived HIV-1 isolates before and after ENF treatment. Further studies are needed to determine whether the reduced fitness of ENF-resistant viruses alters their pathogenicity in vivo.

scholarly journals SERINC5 Potently Restricts Retrovirus Infection In Vivo

mBio ◽  
2020 ◽  
Vol 11 (4) ◽  
Author(s):  
Uddhav Timilsina ◽  
Supawadee Umthong ◽  
Brian Lynch ◽  
Aimee Stablewski ◽  
Spyridon Stavrou
Keyword(s):  
Murine Leukemia Virus ◽  
Leukemia Virus ◽  
Viral Envelope ◽  
The Other ◽  
Immunodeficiency Virus ◽  
Retrovirus Infection ◽  
Hiv 1 ◽  
Murine Leukemia

ABSTRACT The serine incorporator (SERINC) proteins are multipass transmembrane proteins that affect sphingolipid and phosphatidylserine synthesis. Human SERINC5 and SERINC3 were recently shown to possess antiretroviral activity for a number of retroviruses, including human immunodeficiency virus (HIV), murine leukemia virus (MLV), and equine infectious anemia virus (EIAV). In the case of MLV, the glycosylated Gag (glyco-Gag) protein was shown to counteract SERINC5-mediated restriction in in vitro experiments and the viral envelope was found to determine virion sensitivity or resistance to SERINC5. However, nothing is known about the in vivo function of SERINC5. Antiretroviral function of a host factor in vitro is not always associated with antiretroviral function in vivo. Using SERINC5−/− mice that we had generated, we showed that mouse SERINC5 (mSERINC5) restriction of MLV infection in vivo is influenced not only by glyco-Gag but also by the retroviral envelope. Finally, we also examined the in vivo function of the other SERINC gene with known antiretroviral functions, SERINC3. By using SERINC3−/− mice, we found that the murine homologue, mSERINC3, had no antiretroviral role either in vivo or in vitro. To our knowledge, this report provides the first data showing that SERINC5 restricts retrovirus infection in vivo and that restriction of retrovirus infectivity in vivo is dependent on the presence of both glyco-Gag and the viral envelope. IMPORTANCE This study examined for the first time the in vivo function of the serine incorporator (SERINC) proteins during retrovirus infection. SERINC3 and SERINC5 (SERINC3/5) restrict a number of retroviruses, including human immunodeficiency virus 1 (HIV-1) and murine leukemia virus (MLV), by blocking their entry into cells. Nevertheless, HIV-1 and MLV encode factors, Nef and glycosylated Gag, respectively, that counteract SERINC3/5 in vitro. We recently developed SERINC3 and SERINC5 knockout mice to examine the in vivo function of these genes. We found that SERINC5 restriction is dependent on the absence of glycosylated Gag and the expression of a specific viral envelope glycoprotein. On the other hand, SERINC3 had no antiviral function. Our findings have implications for the development of therapeutics that target SERINC5 during retrovirus infection.

scholarly journals Combination of Drugs and Drug-Resistant Reverse Transcriptase Results in a Multiplicative Increase of Human Immunodeficiency Virus Type 1 Mutant Frequencies

2002 ◽  
Vol 76 (18) ◽  
pp. 9253-9259 ◽  
Author(s):  
Louis M. Mansky ◽  
Dennis K. Pearl ◽  
Lisa C. Gajary
Keyword(s):  
Human Immunodeficiency Virus ◽  
Drug Resistance ◽  
Reverse Transcriptase ◽  
Drug Resistant ◽  
Resistance Mutations ◽  
Drug Resistance Mutations ◽  
Immunodeficiency Virus ◽  
Hiv 1 ◽  
Mutant Frequency

ABSTRACT Replication of drug-resistant human immunodeficiency virus type 1 (HIV-1) in the presence of drug can lead to the failure of antiretroviral drug treatment. Drug failure is associated with the accumulation of drug resistance mutations. Previous studies have shown that 3′-azido-3′-deoxythymidine (AZT), (−)2′,3′-dideoxy-3′-thiacytidine (3TC), and AZT-resistant HIV-1 reverse transcriptase (RT) can increase the virus in vivo mutation rate. In this study, the combined effects of drug-resistant RT and antiretroviral drugs on the HIV-1 mutant frequency were determined. In most cases, a multiplicative effect was observed with AZT-resistant or AZT/3TC dually resistant RT and several drugs (i.e., AZT, 3TC, hydroxyurea, and thymidine) and led to increases in the odds of recovering virus mutants to over 20 times that of the HIV-1 mutant frequency in the absence of drug or drug-resistance mutations. This observation indicates that HIV-1 can mutate at a significantly higher rate when drug-resistant virus replicates in the presence of drug. These increased mutant frequencies could have important implications for HIV-1 population dynamics and drug therapy regimens.

scholarly journals Reverse transcriptase backbone can alter the polymerization and RNase activities of non-nucleoside reverse transcriptase mutants K101E+G190S

2013 ◽  
Vol 94 (10) ◽  
pp. 2297-2308
Author(s):  
Jiong Wang ◽  
Dongge Li ◽  
Robert A. Bambara ◽  
Carrie Dykes
Keyword(s):  
Reverse Transcriptase ◽  
Resistant Mutant ◽  
Rnase H ◽  
Drug Resistant ◽  
Resistance Mutations ◽  
Virus Growth ◽  
Immunodeficiency Virus ◽  
Drug Resistant Mutant ◽  
Replication Fitness ◽  
Hiv 1

Previous work by our group showed that human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) containing non-nucleoside RT inhibitor (NNRTI) drug resistance mutations has defects in RNase H activity as well as reduced amounts of RT protein in virions. These deficits correlate with replication fitness in the absence of NNRTIs. Viruses with the mutant combination K101E+G190S replicated better in the presence of NNRTIs than in the absence of drug. Stimulation of virus growth by NNRTIs occurred during the early steps of the virus life cycle and was modulated by the RT backbone sequence in which the resistance mutations arose. We wanted to determine what effects RT backbone sequence would have on RT content and polymerization and RNase H activities in the absence of NNRTIs. We compared a NL4-3 RT with K101E+G190S to a patient-isolate RT sequence D10 with K101E+G190S. We show here that, unlike the NL4-3 backbone, the D10 backbone sequence decreased the RNA-dependent DNA polymerization activity of purified recombinant RT compared to WT. In contrast, RTs with the D10 backbone had increased RNase H activity compared to WT and K101E+G190S in the NL4-3 backbone. D10 virions also had increased amounts of RT compared to K101E+G190S in the NL4-3 backbone. We conclude that the backbone sequence of RT can alter the activities of the NNRTI drug-resistant mutant K101E+G190S, and that identification of the amino acids responsible will aid in understanding the mechanism by which NNRTI drug-resistant mutants alter fitness and NNRTIs stimulate HIV-1 virus replication.

scholarly journals Characterisation of HIV-1 transmission clusters and drug-resistant mutations in Denmark, 2004 to 2016

2018 ◽  
Vol 23 (44) ◽  
Author(s):  
Andreas Petersen ◽  
Susan A Cowan ◽  
Jens Nielsen ◽  
Thea K Fischer ◽  
Jannik Fonager
Keyword(s):  
Drug Resistance ◽  
Drug Resistant ◽  
Resistance Mutations ◽  
Transmission Networks ◽  
Late Presenters ◽  
Drug Resistance Mutations ◽  
Hiv Epidemic ◽  
Immunodeficiency Virus ◽  
Sex With Men ◽  
Hiv 1

This study describes the prevalence of human immunodeficiency virus (HIV) drug resistance mutations among 1,815 patients in Denmark from 2004 to 2016 and characterises transmission clusters. POL sequences were analysed for subtype, drug resistance mutations and phylogenetic relationship. The prevalence of surveillance drug resistance mutations (SDRM) was 6.7%, while the prevalence of drug resistance mutations (DRM) with a clinical impact was 12.3%. We identified 197 transmission clusters with 706 patients. Patients 40 years or older were less likely to be members of a transmission cluster and patients in transmission clusters were less likely to be infected abroad. The proportion of late presenters (LP) was lower in active compared with inactive clusters. Large active clusters consisted of more men who have sex with men (MSM), had members more frequently infected in Denmark and contained a significantly lower proportion of LP and significantly fewer patients with DRM than small active clusters. Subtyping demonstrated that the Danish HIV epidemic is gradually becoming more composed of non-B subtypes/circulating recombinant forms. This study shows that active HIV-1 transmission has become increasingly MSM-dominated and that the recent increase in SDRM and DRM prevalence is not associated with more sustained transmission within identified transmission networks or clusters.

scholarly journals Characterization of a Human Immunodeficiency Virus Type 1 V3 Deletion Mutation That Confers Resistance to CCR5 Inhibitors and the Ability To Use Aplaviroc-Bound Receptor

2009 ◽  
Vol 83 (8) ◽  
pp. 3798-3809 ◽  
Author(s):  
Katrina M. Nolan ◽  
Gregory Q. Del Prete ◽  
Andrea P. O. Jordan ◽  
Beth Haggarty ◽  
Josephine Romano ◽  
...  
Keyword(s):  
Human Immunodeficiency Virus ◽  
Monoclonal Antibodies ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Entry Inhibitors ◽  
Coreceptor Binding ◽  
Immunodeficiency Virus ◽  
Hiv 1

ABSTRACT The human immunodeficiency virus type 1 (HIV-1) V3 loop is essential for coreceptor binding and principally determines tropism for the CCR5 and CXCR4 coreceptors. Using the dual-tropic virus HIV-1R3A, we previously made an extensive panel of V3 deletions and identified subdomains within V3 that could differentially mediate R5 and X4 tropism. A deletion of residues 9 to 12 on the N-terminal side of the V3 stem ablated X4 tropism while leaving R5 tropism intact. This mutation also resulted in complete resistance to several small-molecule CCR5 inhibitors. Here, we extend these studies to further characterize a variant of this mutant, Δ9-12a, adapted for growth in CCR5+ SupT1 cells. Studies using coreceptor chimeras, monoclonal antibodies directed against the CCR5 amino terminus (NT) and extracellular loops, and CCR5 point mutants revealed that, relative to parental R3A, R5-tropic Δ9-12a was more dependent on the CCR5 NT, a region that contacts the gp120 bridging sheet and V3 base. Neutralization sensitivity assays showed that, compared to parental R3A, Δ9-12a was more sensitive to monoclonal antibodies b12, 4E10, and 2G12. Finally, cross-antagonism assays showed that Δ9-12a could use aplaviroc-bound CCR5 for entry. These studies indicate that increased dependence on the CCR5 NT represents a mechanism by which HIV envelopes acquire resistance to CCR5 antagonists and may have more general implications for mechanisms of drug resistance that arise in vivo. In addition, envelopes such as Δ9-12a may be useful for developing new entry inhibitors that target the interaction of gp120 and the CCR5 NT.

scholarly journals Detection and Quantification of Minor Human Immunodeficiency Virus Type 1 Variants Harboring K103N and Y181C Resistance Mutations in Subtype A and D Isolates by Allele-Specific Real-Time PCR

2009 ◽  
Vol 53 (7) ◽  
pp. 2965-2973 ◽  
Author(s):  
Andrea Hauser ◽  
Kizito Mugenyi ◽  
Rose Kabasinguzi ◽  
Kerstin Bluethgen ◽  
Claudia Kuecherer ◽  
...  
Keyword(s):  
Human Immunodeficiency Virus ◽  
Single Dose ◽  
Population Based ◽  
Drug Resistant ◽  
Resistance Mutations ◽  
Immunodeficiency Virus ◽  
Allele Specific ◽  
Subtype A ◽  
Hiv 1

ABSTRACTNevirapine (single dose), commonly used to prevent the mother-to-child transmission of human immunodeficiency virus (HIV) in developing countries, frequently induces viral resistance. Even mutations which occur only in a minor population of the HIV quasispecies (<20%) are associated with subsequent treatment failure but cannot be detected by population-based sequencing. We developed sensitive allele-specific real-time PCR (ASPCR) assays for two key resistance mutations of nevirapine. The assays were specifically designed to analyze HIV-1 subtype A and D isolates accounting for the majority of HIV infections in Uganda. Assays were evaluated using DNA standards and clinical samples of Ugandan women having preventively taken single-dose nevirapine. Lower detection limits of drug-resistant HIV type 1 (HIV-1) variants carrying reverse transcriptase mutations were 0.019% (K103N [AAC]), 0.013% (K103N [AAT]), and 0.29% (Y181C [TGT]), respectively. Accuracy and precision were high, with coefficients of variation (the standard ratio divided by the mean) of 0.02 to 0.15 for intra-assay variability and those of 0.07 to 0.15 (K103N) and 0.28 to 0.52 (Y181C) for inter-assay variability. ASPCR assays enabled the additional identification of 12 (20%) minor drug-resistant HIV variants in the 20 clinical Ugandan samples (3 mutation analyses per patient; 60 analyses in total) which were not detectable by population-based sequencing. The individual patient cutoff derived from the clinical baseline sample was more appropriate than the standard-based cutoff from cloned DNA. The latter is a suitable alternative since the presence/absence of drug-resistant HIV-1 strains was concordantly identified in 92% (55/60) of the analyses. These assays are useful to monitor the emergence and persistence of drug-resistant HIV-1 variants in subjects infected with HIV-1 subtypes A and D.

scholarly journals Antiviral Breadth and Combination Potential of Peptide Triazole HIV-1 Entry Inhibitors

2011 ◽  
Vol 56 (2) ◽  
pp. 1073-1080 ◽  
Author(s):  
Karyn McFadden ◽  
Patricia Fletcher ◽  
Fiorella Rossi ◽  
Kantharaju ◽  
Muddagowda Umashankara ◽  
...  
Keyword(s):  
Transcriptase Inhibitor ◽  
Viral Envelope ◽  
Cell Infection ◽  
Entry Inhibitors ◽  
Antiviral Compounds ◽  
Subtype B ◽  
Immunodeficiency Virus ◽  
Glycoprotein Gp120 ◽  
Reverse Transcriptase Inhibitor ◽  
Hiv 1

ABSTRACTThe first stage of human immunodeficiency virus type 1 (HIV-1) infection involves the fusion of viral and host cellular membranes mediated by viral envelope glycoprotein gp120. Inhibitors that specifically target gp120 are gaining increased attention as therapeutics or preventatives to prevent the spread of HIV-1. One promising new group of inhibitors is the peptide triazoles, which bind to gp120 and simultaneously block its interaction with both CD4 and the coreceptor. In this study, we assessed the most potent peptide triazole, HNG-156, for inhibitory breadth, cytotoxicity, and efficacy, both alone and in combination with other antiviral compounds, against HIV-1. HNG-156 inhibited a panel of 16 subtype B and C isolates of HIV-1 in a single-round infection assay. Inhibition of cell infection by replication-competent clinical isolates of HIV-1 was also observed with HNG-156. We found that HNG-156 had a greater than predicted effect when combined with several other entry inhibitors or the reverse transcriptase inhibitor tenofovir. Overall, we find that HNG-156 is noncytotoxic, has a broad inhibition profile, and provides a positive combination with several inhibitors of the HIV-1 life cycle. These results support the pursuit of efficacy and toxicity analyses in more advanced cell and animal models to develop peptide triazole family inhibitors of HIV-1 into antagonists of HIV-1 infection.

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