High Levels Of Virus Research Articles

Long-Lasting T Cell-Independent IgG Responses Require MyD88-Mediated Pathways and Are Maintained by High Levels of Virus Persistence

ABSTRACTMany viruses induce acute T cell-independent (TI) B cell responses due to their repetitive epitopes and the induction of innate cytokines. Nevertheless, T cell help is thought necessary for the development of long-lasting antiviral antibody responses in the form of long-lived plasma cells and memory B cells. We found that T cell-deficient (T cell receptor β and δ chain [TCRβδ] knockout [KO]) mice persistently infected with polyomavirus (PyV) had long-lasting antiviral serum IgG, and we questioned whether they could generate TI B cell memory. TCRβδ KO mice did not form germinal centers after PyV infection, lacked long-lived IgG-secreting plasma cells in bone marrow, and did not have detectable memory B cell responses. Mice deficient in CD4+ T cells had a lower persisting virus load than TCRβδ KO mice, and these mice had short-lived antiviral IgG responses, suggesting that a high virus load is required to activate naive B cells continuously, and maintain the long-lasting serum IgG levels. Developing B cells in bone marrow encounter high levels of viral antigens, which can cross-link both their B cell receptor (BCR) and Toll-like receptors (TLRs), and this dual engagement may lead to a loss of their tolerance. Consistent with this hypothesis, antiviral serum IgG levels were greatly diminished in TCRβδ KO/MyD88−/− mice. We conclude that high persisting antigen levels and innate signaling can lead to the maintenance of long-lasting IgG responses even in the absence of T cell help.

Chronic Joint Disease Caused by Persistent Chikungunya Virus Infection Is Controlled by the Adaptive Immune Response

Chikungunya virus (CHIKV) is a reemerging mosquito-borne pathogen that causes incapacitating disease in humans characterized by intense joint pain that can persist for weeks, months, or even years. Although there is some evidence of persistent CHIKV infection in humans suffering from chronic rheumatologic disease symptoms, little is known about chronic disease pathogenesis, and no specific therapies exist for acute or chronic CHIKV disease. To investigate mechanisms of chronic CHIKV-induced disease, we utilized a mouse model and defined the duration of CHIKV infection in tissues and the associated histopathological changes. Although CHIKV RNA was readily detectable in a variety of tissues very early after infection, CHIKV RNA persisted specifically in joint-associated tissues for at least 16 weeks. Inoculation of Rag1(-/-) mice, which lack T and B cells, resulted in higher viral levels in a variety of tissues, suggesting that adaptive immunity controls the tissue specificity and persistence of CHIKV infection. The presence of CHIKV RNA in tissues of wild-type and Rag1(-/-) mice was associated with histopathological evidence of synovitis, arthritis, and tendonitis; thus, CHIKV-induced persistent arthritis is not mediated primarily by adaptive immune responses. Finally, we show that prophylactic administration of CHIKV-specific monoclonal antibodies prevented the establishment of CHIKV persistence, whereas therapeutic administration had tissue-specific efficacy. These findings suggest that chronic musculoskeletal tissue pathology is caused by persistent CHIKV infection and controlled by adaptive immune responses. Our results have significant implications for the development of strategies to mitigate the disease burden associated with CHIKV infection in humans.

Enhanced infectivity of H5N1 highly pathogenic avian influenza (HPAI) virus in pig ex vivo respiratory tract organ cultures following adaptation by in vitro passage

Pigs are thought to play a role in the adaptation of avian influenza (AI) viruses to mammalian hosts. To better understand this mechanism and to identify key mutations two highly pathogenic AI (HPAI) viruses (H5N1 and H7N7) were grown in pig cells, To mimic the pressure of an immune response, these viruses were grown in the presence of antiserum to the homologous virus or porcine IFN-γ. Mutations were identified in both viruses grown in vitro in the presence and absence of antisera or IFN-γ and included the PB2 mutations, E627K or 627E,D701N, described previously as requirements for the adaptation of AI viruses to mammalian species. Additional mutations were also identified in PB1, HA, NP and M genes for viruses passaged in the presence of immune pressure. The infectivity of these viruses was then assessed using ex vivo pig bronchi and lung organ cultures. For lung explants, higher levels of virus were detected in organ cultures infected with H5N1 HPAI viruses passaged in pig cell lines regardless of the presence or absence of homologous antisera or IFN-γ when compared with the wild-type parental viruses. No infection was observed for any of the H7N7 HPAI viruses. These results suggest that the mutations identified in H5N1 HPAI viruses may provide a replication or infection advantage in pigs in vivo and that pigs may continue to play an important role in the ecology of influenza A viruses including those of avian origin.

Infection of lymphoid tissues in the macaque upper respiratory tract contributes to the emergence of transmissible measles virus

Measles virus (MV), a member of the family Paramyxoviridae, remains a major cause of morbidity and mortality in the developing world. MV is spread by aerosols but the mechanism(s) responsible for the high transmissibility of MV are largely unknown. We previously infected macaques with enhanced green fluorescent protein-expressing recombinant MV and euthanized them at a range of time points. In this study a comprehensive pathological analysis has been performed of tissues from the respiratory tract around the peak of virus replication. Isolation of virus from nose and throat swab samples showed that high levels of both cell-associated and cell-free virus were present in the upper respiratory tract. Analysis of tissue sections from lung and primary bronchus revealed localized infection of epithelial cells, concomitant infiltration of MV-infected immune cells into the epithelium and localized shedding of cells or cell debris into the lumen. While high numbers of MV-infected cells were present in the tongue, these were largely encapsulated by intact keratinocyte cell layers that likely limit virus transmission. In contrast, the integrity of tonsillar and adenoidal epithelia was disrupted with high numbers of MV-infected epithelial cells and infiltrating immune cells present throughout epithelial cell layers. Disruption was associated with large numbers of MV-infected cells or cell debris 'spilling' from epithelia into the respiratory tract. The coughing and sneezing response induced by disruption of the ciliated epithelium, leading to the expulsion of MV-infected cells, cell debris and cell-free virus, contributes to the highly infectious nature of MV.

Role of Line Immunoassay in the Diagnosis of Early HIV Infection: A Diagnostic Case

Combined p24 antigen-HIV antibody fourth-generation assays that identify most of the early HIV infections have been used extensively worldwide for several years. This poses challenges for the traditional algorithm of line immunoassay (LIA) confirmation. LIA tests are useful methods with their high specificity and their ability to differentiate HIV-1 from HIV-2, but they are reactive days after the fourth generation enzyme immunoassays. With acute HIV infection, high levels of infectious virus are detectable in serum and genital secretions. The rate of transmission during acute HIV infection is higher than the established HIV infection, for this reason, new HIV testing strategies need to focus on sensitivity, especially for this highly contagious phase immediately after infection. Serum sample of a patient sent to Ege University Hospital Clinical Virology Laboratory was repeatedly reactive with low signal/cutoff ratios with two different commercial fourth generation enzyme immunoassays (Architect HIV Ag/Ab Combo Reagent Kit, Abbott, Germany and Vidas HIV Duo Quick, Biomerieux, France). The sample was non-reactive with the LIA (INNO-LIA HIV I/II Score, Innogenetics, Belgium) and HIV RNA (RealTime HIV-I Amplification Reagent Kit, Abbott, USA) result was positive (4.1 x 10(5) copies/ml). With the presentation of this case, the role of LIA in the diagnosis of early HIV infection and its place in test algorithms were questioned.

Epizootic hemorrhagic disease virus serotype 6 experimentation on adult cattle

Epizootic hemorrhagic disease virus (EHDV), an arthropod-borne orbivirus (family Reoviridae), is an emerging pathogen of wild and domestic ruminants closely related to bluetongue virus (BTV). EHDV serotype 6 (EHDV6) has recently caused outbreaks close to Europe in Turkey and Morocco and a recent experimental study performed on calves inoculated with these two EHDV6 strains showed that the young animals have remained clinically unaffected. The aim of this study was to investigate the pathogenicity of an EHDV6 strain from La Reunion Island in adult Holstein (18-month-old heifers). This EHDV6 strain has induced clinical signs in cattle in the field.Samples taken throughout the study were tested with commercially available ELISA and real-time RT-PCR kits. Very mild clinical manifestations were observed in cattle during the experiment although high levels of viral RNA and virus were found in their blood. EHDV was isolated from the blood of infected animals at 8dpi. Antibodies against EHDV were first detected by 7dpi and persisted up to the end of the study. Virus was detected in various tissue samples until 35dpi, but was not infectious.In view of the recent circulation of different arboviruses in Europe, this study demonstrates what the EHD induces a strong viraemia in adult Holstein cattle and shows that a spread of EHD on European livestock cattle is possible.

Oral direct-acting antiviral therapy to prevent reinfection of the liver graft after liver transplantation for hepatitis C virus-related cirrhosis

Hepatitis C virus (HCV) remains the most common indication for orthotopic liver transplantation (OLT) and universally reinfects the new liver graft; as a result, patients with HCV have poorer outcomes than patients undergoing OLT for non–HCV-related cirrhosis.1 Recently, new direct-acting antiviral (DAA) agents have been approved for genotype 1 HCV infections in combination with peginterferon/ribavirin (RBV).2, 3 However, these therapies may be associated with morbidity and mortality when they are given to patients with advanced liver disease.4 We report a sustained response with an oral DAA/RBV combination without interferon in a 61-year-old male with HCV genotype 1b (interleukin-28B TT) who underwent OLT at our center for HCV-related cirrhosis. He presented to our center for an OLT evaluation with a Model for End-Stage Liver Disease (MELD) score of 18 as well as ascites and encephalopathy, and interferon and RBV had previously failed in 1999. Once listed, he requested therapy with peginterferon/RBV and a DAA agent while he was awaiting OLT. Because of his ascites, encephalopathy, and MELD score, this was not offered. However, his HCV RNA viral level was found to be low at 887 IU/mL (Cobas TaqMan HCV assay), so telaprevir (TVR; 750 mg every 8 hours) and RBV (1200 mg daily) were initiated. At week 1 of therapy, his HCV RNA level was <43 IU/mL (target not detected). Because of gastrointestinal side effects at week 1 (nausea and diarrhea), he was converted to boceprevir (BOC; 800 mg every 8 hours) with RBV and underwent OLT 9 weeks after the initiation of therapy with a MELD score of 19. There were no additional episodes of decompensation, and he did not require transfusions, although an RBV dose reduction to 600 mg was required. Just before surgery, he took BOC (800 mg) as well as RBV (400 mg). Twenty-four weeks after OLT, he remained a sustained responder (Fig. 1). Recent reports have demonstrated that a sustained virological response (SVR) can be achieved in HCV patients without interferon, although no studies to date have included patients with decompensated cirrhosis.5 However, to our knowledge, this represents the first successful cure of HCV in which DAA therapy without interferon was used to suppress the virus before OLT in a patient with decompensated liver disease in whom interferon was contraindicated. It should be emphasized that this case was unique because of the very low viral level and the more favorable genotype 1b infection. In addition, our patient was able to undergo OLT with a MELD score of 19, which is a level below the national median MELD score for deceased transplants in 2011.6 These factors allowed the combination of TVR and RBV followed by BOC with RBV to achieve viral suppression without the emergence of viral resistance. Because BOC may be administered for up to 44 weeks, our patient could have continued to receive BOC with RBV if no donor had been available, although it is unknown whether a breakthrough would have occurred with a longer duration of therapy. The vast majority of patients with genotype 1 infections who present for listing for OLT will have a viral level significantly higher than our patient's level (typically 104−106 IU/mL), and this level in our opinion would contraindicate our approach in this report because of the risk of treatment futility and resistance with nonstructural protein 3 protease inhibitors and RBV. However, more effective, all-oral DAA combinations with high barriers to resistance are currently being studied that can achieve high SVR rates in patients with high viral levels, and we believe the approach of DAA combinations without interferon may serve as a model for preventing reinfection of the liver graft and improving the survival of HCV-infected individuals who require OLT. Paul Y. Kwo, M.D.1 A. Joseph Tector, M.D., Ph.D.2 Departments of 1Medicine and 2Transplant SurgeryIndiana University School of MedicineIndianapolis, IN

The L60V Variation in Hepatitis B Virus Core Protein Elicits New Epitope-Specific Cytotoxic T Lymphocytes and Enhances Viral Replication

Mutations in the core protein (HBc) of hepatitis B virus (HBV) are associated with aggressive hepatitis and advanced liver diseases in chronic hepatitis B (CHB). In this study, we identified the L60V variation in HBc that generates a new HLA-A2-restricted CD8(+) T cell epitope by screening an overlapping 9-mer peptide pool covering HBc and its variants. The nonameric epitope V60 was determined by structural and immunogenic analysis. The HBc L60V variation is correlated with hepatic necroinflammation and higher viral levels, and it may be associated with a poor prognosis in CHB patients. Immunization with the defined HBV epitope V60 peptide elicited specific cytotoxic T lymphocyte (CTL)-induced liver injury in HLA-A2(+) HBV transgenic mice. In addition, in vitro and in vivo experiments both demonstrated that the HBc L60V variation facilitates viral capsid assembly and increases HBV replication. These data suggest that the HBc L60V variation can impact both HBV replication and HBV-specific T cell responses. Therefore, our work provides further dissection of the impact of the HBc L60V variation, which orchestrates HBV replication, viral persistence, and immunopathogenesis during chronic viral infection.

Understanding foot-and-mouth disease virus transmission biology: identification of the indicators of infectiousness.

The control of foot-and-mouth disease virus (FMDV) outbreaks in non-endemic countries relies on the rapid detection and removal of infected animals. In this paper we use the observed relationship between the onset of clinical signs and direct contact transmission of FMDV to identify predictors for the onset of clinical signs and identify possible approaches to preclinical screening in the field. Threshold levels for various virological and immunological variables were determined using Receiver Operating Characteristic (ROC) curve analysis and then tested using generalized linear mixed models to determine their ability to predict the onset of clinical signs. In addition, concordance statistics between qualitative real time PCR test results and virus isolation results were evaluated. For the majority of animals (71%), the onset of clinical signs occurred 3–4 days post infection. The onset of clinical signs was associated with high levels of virus in the blood, oropharyngeal fluid and nasal fluid. Virus is first detectable in the oropharyngeal fluid, but detection of virus in the blood and nasal fluid may also be good candidates for preclinical indicators. Detection of virus in the air was also significantly associated with transmission. This study is the first to identify statistically significant indicators of infectiousness for FMDV at defined time periods during disease progression in a natural host species. Identifying factors associated with infectiousness will advance our understanding of transmission mechanisms and refine intra-herd and inter-herd disease transmission models.

Pattern of HIV-1 Drug Resistance among Adults on ART in Nigeria

Background: The development of anitiretroviral drug resistance may limit the benefit of antiretroviral therapy. Therefore the need to closely monitor these mutations, especially the use of ART is increasing. This study was therefore designed to determine the ARV drug resistance pattern among ART na?ve and expose individuals attending a PEPFAR supported by antiretroviral clinic in Nigeria. Methodology: The study participants included patients attending the PEPFAR supported by University College Hospital (UCH), Ibadan ART clinic who have been on HIV treatment for at least one year with consecutive viral load of over 2000 copies/ml as well some ART Na?ve individuals with high (>50,000 copies/ml) baseline viral level attending the hospital for pre-ART assessment. Blood sample was collected from each individual for CD4 enumeration, viral load level determination and DNA sequencing for genotypic typing. Antiretroviral drug resistance mutations (DRM) were determined by using the Viroseq software and drug mutations generated by using a combination of Viroseq and Stanford algorithm. DRM were classified as major or minor mutations based on the June 2013 Stanford DR database. Results: The most common major NRTI, NNRTI and PI mutation were D67N (33.3%), Y181C (16.7%) and M46L/I (55.6%) respectively. Lamivudine (3TC) and emtricitabine (FTC); nevirapine (NVP) and nelfinavir (NFV) were the most common NRTI, NNRTI, and PI drugs to which the virus in the infected individuals developed resistance. Isolates from 4 patients were resistant to triple drug class, including at least one NRTI, NNRTI and a PI. Only one (4.8%) of the isolates from drug Na?ve individuals had major DRM that conferred resistance to any drug. Conclusion: Demonstration of high rates of antiretroviral DRM among patients on 1st and 2nd line ART and the presence of DRM in drug Na?ve individuals in this study show the importance of surveillance for resistance to ARV in line with the magnitude of scaling up of treatment program in the country.

Correlation of Viral Loads with HCV Genotypes: Higher Levels of Virus Were Revealed among Blood Donors Infected with 6a Strains

BackgroundBoth HCV genotypes and viral loads are predictors of therapeutic outcomes among patients treated with α-interferon plus ribavirin; however, such correlation has only been studied for genotypes 1, 2, and 3 but not for genotype 6.Methodology/Findings299 voluntary blood donors were recruited who were HCV viremic. Their mean age was 31.8; the male/female ratio was 3.82 (225/59). The viral loads of HCV were measured using the COBAS AmpliPrep/COBAS TaqMan test (CAP/CTM) while HCV genotypes were determined by direct sequencing the partial NS5B region. HCV genotypes 1, 2, 3, and 6 were determined in 48.9%, 8.7%, 12.3%, and 30.1% of the donors, respectively, and the levels of mean viral loads in genotype 1 and 6 significantly higher than that of 2 and 3 (P<0.001). As a whole, the viral loads in male donors were higher than in female (P = 0.006). Moreover, the donors' gender and HCV genotypes are independently correlated with the measured viral loads.ConclusionHCV genotype 1 and 6 had significantly higher viral loads than genotype 2 and 3.

Persistence and chronic urinary shedding of the aphthovirus equine rhinitis A virus

Equine rhinitis A virus (ERAV) is a member of the Aphthovirus genus, and has many physical and structural similarities to the prototype Aphthovirus foot-and-mouth disease virus (FMDV). The pathogenesis of FMDV has been extensively studied, however, the similarities in the pathogenesis of ERAV and FMDV disease has not been well documented. This study describes and compares the pathogenesis of ERAV both in the natural host and a small animal model alternative (CBA mice). Distinct parallels in the pathogenesis of the acute infection of these two viruses are described where infection in the upper respiratory tract precedes shedding of high levels of virus from the nasopharynx and a transient viraemic phase before dissemination to distal sites. The finding that ERAV is maintained at high levels in the urine of infected horses for at least 37 days post infection, however, is a feature unique to ERAV amongst all of the picornaviruses.

A Review of Known and Hypothetical Transmission Routes for Noroviruses

Human noroviruses (NoVs) are considered a worldwide leading cause of acute non-bacterial gastroenteritis. Due to a combination of prolonged shedding of high virus levels in feces, virus particle shedding during asymptomatic infections, and a high environmental persistence, NoVs are easily transmitted pathogens. Norovirus (NoV) outbreaks have often been reported and tend to affect a lot of people. NoV is spread via feces and vomit, but this NoV spread can occur through several transmission routes. While person-to-person transmission is without a doubt the dominant transmission route, human infective NoV outbreaks are often initiated by contaminated food or water. Zoonotic transmission of NoV has been investigated, but has thus far not been demonstrated. The presented review aims to give an overview of these NoV transmission routes. Regarding NoV person-to-person transmission, the NoV GII.4 genotype is discussed in the current review as it has been very successful for several decades but reasons for its success have only recently been suggested. Both pre-harvest and post-harvest contamination of food products can lead to NoV food borne illness. Pre-harvest contamination of food products mainly occurs via contact with polluted irrigation water in case of fresh produce or with contaminated harvesting water in case of bivalve molluscan shellfish. On the other hand, an infected food handler is considered as a major cause of post-harvest contamination of food products. Both transmission routes are reviewed by a summary of described NoV food borne outbreaks between 2000 and 2010. A third NoV transmission route occurs via water and the spread of NoV via river water, ground water, and surface water is reviewed. Finally, although zoonotic transmission remains hypothetical, a summary on the bovine and porcine NoV presence observed in animals is given and the presence of human infective NoV in animals is discussed.

Persistence and chronic urinary shedding of the aphthovirus equine rhinitis A virus

Equine rhinitis A virus (ERAV) is a member of the Aphthovirus genus, and has many physical and structural similarities to the prototype Aphthovirus foot-and-mouth disease virus (FMDV). The pathogenesis of FMDV has been extensively studied, however, the similarities in the pathogenesis of ERAV and FMDV disease has not been well documented. This study describes and compares the pathogenesis of ERAV both in the natural host and a small animal model alternative (CBA mice). Distinct parallels in the pathogenesis of the acute infection of these two viruses are described where infection in the upper respiratory tract precedes shedding of high levels of virus from the nasopharynx and a transient viraemic phase before dissemination to distal sites. The finding that ERAV is maintained at high levels in the urine of infected horses for at least 37 days post infection, however, is a feature unique to ERAV amongst all of the picornaviruses.

Surveillance for Influenza Viruses in Poultry and Swine, West Africa, 2006–2008

To determine the extent of animal influenza virus circulation in Côte d'Ivoire, Benin, and Togo, we initiated systematic year-round active influenza surveillance in backyard birds (predominantly chickens, guinea fowl, and ducks) and pigs. A total of 26,746 swab specimens were screened by using reverse transcription PCR. Animal influenza prevalence was estimated at 0 (95% CIs for each of the 2 study years 0-0.04% to 0-1.48% [birds] and 0-0.28% to 0-5% [pigs]). In addition, 2,276 serum samples from the same populations were negative for influenza-specific antibodies. These data indicate that the environments and host populations previously identified as harboring high levels of influenza virus in Southeast Asia do not do so in these 3 countries. The combination of climate and animal density factors might be responsible for what appears to be the absence of influenza virus in the backyard sector of the 3 countries.

In Vitro HIV Infection Impairs the Capacity of Myeloid Dendritic Cells to Induce Regulatory T Cells

Myeloid dendritic cells (mDCs) are the antigen-presenting cells best capable of promoting peripheral induction of regulatory T cells (Tregs), and are among the first targets of HIV. It is thus important to understand whether HIV alters their capacity to promote Treg conversion. Monocyte-derived DCs (moDCs) from uninfected donors induced a Treg phenotype (CD25+FOXP3+) in autologous conventional T cells. These converted FOXP3+ cells suppressed the proliferation of responder T cells similarly to circulating Tregs. In contrast, the capacity of moDCs to induce CD25 or FOXP3 was severely impaired by their in vitro infection with CCR5-utilizing virus. MoDC exposure to inactivated HIV was sufficient to impair FOXP3 induction. This DC defect was not dependent on IL-10, TGF-β or other soluble factors, but was due to preferential killing of Tregs by HIV-exposed/infected moDCs, through a caspase-dependent pathway. Importantly, similar results were obtained with circulating primary myeloid DCs. Upon infection in vitro, these mDCs also killed Treg through mechanisms at least partially caspase-dependent, leading to a significantly lower proportion of induced Tregs. Taken together, our data suggest that Treg induction may be defective when DCs are exposed to high levels of virus, such as during the acute phase of infection or in AIDS patients.

Experimental Infection of Four Aquacultured Species with Viral Hemorrhagic Septicemia Virus Type IVb

Viral hemorrhagic septicemia virus (VHSV) is the etiologic agent of a disease of great concern to aquaculture. We performed experimental infections with intraperitoneal VHSV IVb injections at 106 pfu/fish in four species: tiger muskellunge, ♂Esox lucius×♀Esox masquinongy; Atlantic salmon, Salmo salar; channel catfish, Ictalurus punctatus; and walleye, Sander vitreus. The fish were thermal shocked concurrent with infection to cause immunosuppression and increase the probability of eliciting disease. Each species was observed for morbidity and mortality and periodic samples taken. Virus was detected using virus isolation and real‐time quantitative reverse‐transcription polymerase chain reaction (qRT‐PCR). Histopathology confirmed lesions in tiger muskellunge and Atlantic salmon, and immunohistochemistry showed virus associated with some lesions. Tiger muskellunge were highly susceptible to VHSV IVb, and high viral levels were detected in exposed fish. A single mortality was observed in exposed Atlantic salmon with a short viral replication period. Channel catfish and walleye were resistant to developing clinical signs of VHS. The results show that these species vary in the development of VHS under these conditions, and qRT‐PCR testing enabled virus detection in subclinically affected fish. Basic concordance statistics between the qRT‐PCR test results and the virus isolation results were evaluated.

Hepatitis B

Hepatitis B remains a significant health issue and a leading cause of cirrhosis and hepatocellular carcinoma worldwide. Transmission of the hepatitis B virus, despite the availability of the vaccine, still occurs, particularly in the perinatal setting. Studies have shown that mothers with active viremia especially those with very high viral levels (>10⁸ copies/mL or >10⁷ IU/mL) are at increased risk of hepatitis B virus transmission, even with appropriate immunoprophylaxis at birth. With the development of new and relatively safe oral antiviral therapies, assessment with the mother about the risks and benefits of antiviral therapy should be discussed in those with highest transmission risk.

Maraviroc and Other HIV-1 Entry Inhibitors Exhibit a Class-Specific Redistribution Effect That Results in Increased Extracellular Viral Load

HIV entry inhibitors, such as maraviroc (MVC), prevent cell-free viruses from entering the cells. In clinical trials, patients who were treated with MVC often displayed viral loads that were above the limit of conventional viral load detection compared to efavirenz-based regimens. We hypothesize that viruses blocked by entry inhibitors may be redistributed to plasma, where they artificially increase viral load measurements compared to those with the use of antiretroviral drugs (ARVs) that act intracellularly. We infected PM-1 cells with CCR5-tropic HIV-1 BaL or CXCR4-tropic HIV-1 NL4-3 in the presence of inhibitory concentrations of efavirenz, raltegravir, enfuvirtide, maraviroc, and AMD3100, the latter three being entry inhibitors. Supernatant viral load, reverse transcriptase enzyme activity, and intracellular nucleic acid levels were measured at times up to 24 h postinfection. Infectivity of redistributed dual-tropic HIV-1 was assessed using TZM-bl cells. Extracellular viral load analysis revealed that entry inhibitor-treated cells had higher levels of virus in the supernatant than the cells treated with other ARVs at 8 h postinfection. By 24 h, the supernatant viral load was still higher for entry inhibitors than other ARVs. We observed a correlation between viral load and the step of entry inhibition. Dual-tropic virus infectivity was undiminished utilizing the CCR5 coreceptor following redistribution by CXCR4 entry inhibition. This in vitro model indicates that entry inhibitors exhibit a redistribution effect unseen with intracellular ARV drugs. Based on these results, the effectiveness of some entry inhibitors may be underestimated if plasma viral load is used as a sole indicator of clinical success.

Interruption with TNFα/TNFR1 function impairs host immune response against cutaneous vaccinia virus infection (52.8)

Abstract One strategy adopted by vaccinia virus (VV) to evade the host immune system is to encode homologs of TNF receptors (TNFR) that block TNFα function. The response to VV skin infection under conditions of TNFα deficiency, however, has not been reported. We found that TNFR1-/- developed larger skin lesions with higher virus levels after VV scarification. Following their recovery, these TNFR1-/- mice were fully protected against lethal VV intranasal challenge, suggesting their effective memory immune response. A functional systemic immune response of TNFR1-/- mice was further demonstrated by enhanced production of VV-specific IFNγ and CD8+ T cells in spleens and draining lymph nodes as well as efficient T cell skin homing. Interestingly, bone marrow (BM) reconstitution studies using WT BM →TNFR1-/- mice, but not TNFR1-/- BM → WT mice, reproduced the original results seen in TNFR1-/- mice, indicating that TNFR1 deficiency in resident skin cells, rather than hematopoietic cells, accounts for the impaired cutaneous immune response. Similar phenotype was also found in in TNFα-/- mice following VV scarification while both WT BM→ TNFα-/- mice and TNFα-/-BM →WT mice developed significantly larger skin lesions with higher virus counts than the control chimeric mice.Therefore, while the target of TNFα action is restricted to skin stromal components, the source of TNFα produced in response to VV infection would appear to include both hematopoietic cells and radioresistant skin elements.