High Levels Of Viral Replication Research Articles

Influenza A Virus Virulence Depends on Two Amino Acids in the N-Terminal Domain of Its NS1 Protein To Facilitate Inhibition of the RNA-Dependent Protein Kinase PKR.

The RNA-dependent protein kinase (PKR) has broad antiviral activity inducing translational shutdown of viral and cellular genes and is therefore targeted by various viral proteins to facilitate pathogen propagation. The pleiotropic NS1 protein of influenza A virus acts as silencer of PKR activation and ensures high-level viral replication and virulence. However, the exact manner of this inhibition remains controversial. To elucidate the structural requirements within the NS1 protein for PKR inhibition, we generated a set of mutant viruses, identifying highly conserved arginine residues 35 and 46 within the NS1 N terminus as being most critical not only for binding to and blocking activation of PKR but also for efficient virus propagation. Biochemical and Förster resonance energy transfer (FRET)-based interaction studies showed that mutation of R35 or R46 allowed formation of NS1 dimers but eliminated any detectable binding to PKR as well as to double-stranded RNA (dsRNA). Using in vitro and in vivo approaches to phenotypic restoration, we demonstrated the essential role of the NS1 N terminus for blocking PKR. The strong attenuation conferred by NS1 mutation R35A or R46A was substantially alleviated by stable knockdown of PKR in human cells. Intriguingly, both NS1 mutant viruses did not trigger any signs of disease in PKR+/+ mice, but replicated to high titers in lungs of PKR-/- mice and caused lethal infections. These data not only establish the NS1 N terminus as highly critical for neutralization of PKR's antiviral activity but also identify this blockade as an indispensable contribution of NS1 to the viral life cycle.IMPORTANCE Influenza A virus inhibits activation of the RNA-dependent protein kinase (PKR) by means of its nonstructural NS1 protein, but the underlying mode of inhibition is debated. Using mutational analysis, we identified arginine residues 35 and 46 within the N-terminal NS1 domain as highly critical for binding to and functional silencing of PKR. In addition, our data show that this is a main activity of amino acids 35 and 46, as the strong attenuation of corresponding mutant viruses in human cells was rescued to a large extent by lowering of PKR expression levels. Significantly, this corresponded with restoration of viral virulence for NS1 R35A and R46A mutant viruses in PKR-/- mice. Therefore, our data establish a model in which the NS1 N-terminal domain engages in a binding interaction to inhibit activation of PKR and ensure efficient viral propagation and virulence.

H7N9 Influenza A Virus Exhibits Importin-α7–Mediated Replication in the Mammalian Respiratory Tract

The acute respiratory distress syndrome (ARDS) is the leading cause of death in influenza A virus (IAV)-infected patients. Hereby, the cellular importin-α7 gene plays a major role. It promotes viral replication in the lung, thereby increasing the risk for the development of pneumonia complicated by ARDS. Herein, we analyzed whether the recently emerged H7N9 avian IAV has already adapted to human importin-α7 use, which is associated with high-level virus replication in the mammalian lung. Using a cell-based viral polymerase activity assay, we could detect a decreased H7N9 IAV polymerase activity when importin-α7 was silenced by siRNA. Moreover, virus replication was diminished in the murine cells lacking the importin-α7 gene. Consistently, importin-α7 knockout mice presented reduced pulmonary virus titers and lung lesions as well as enhanced survival rates compared to wild-type mice. In summary, our results show that H7N9 IAV have acquired distinct features of adaptation to human host factors that enable enhanced virulence in mammals. In particular, adaptation to human importin-α7 mediates elevated virus replication in the mammalian lung, which might have contributed to ARDS observed in H7N9-infected patients.

CXCR6-Mediated Simian Immunodeficiency Virus SIVagmSab Entry into Sabaeus African Green Monkey Lymphocytes Implicates Widespread Use of Non-CCR5 Pathways in Natural Host Infections.

Natural hosts of SIV do not progress to AIDS, in stark contrast to pathogenic human immunodeficiency virus type 1 (HIV-1)-human and SIVmac-macaque infections. Identifying how natural hosts avoid immunodeficiency can elucidate key mechanisms of pathogenesis. It is known that despite high viral loads, natural hosts have a low frequency of CD4+ cells expressing the SIV coreceptor CCR5. In this study, we demonstrate the efficient use of the coreceptor CXCR6 by SIVagmSab to infect sabaeus African green monkey lymphocytes. In conjunction with studies of SIVsmm, which infects sooty mangabeys, and SIVagmVer, which infects vervet monkeys, our data suggest a unifying model whereby in natural hosts, in which the CCR5 expression level is low, the use of CXCR6 or other coreceptors to mediate infection may target SIV toward distinct cell populations that are able to support high-level viral replication without causing a loss of CD4+ T cell homeostasis and lymphoid tissue damage that lead to AIDS in HIV-1 and SIVmac infections.

Circulating CXCR5+CXCR3+PD-1lo Tfh-like cells in HIV-1 controllers with neutralizing antibody breadth.

HIV-1-specific broadly neutralizing antibodies (bnAbs) typically develop in individuals with continuous high-level viral replication and increased immune activation, conditions that cannot be reproduced during prophylactic immunization. Understanding mechanisms supporting bnAb development in the absence of high-level viremia may be important for designing bnAb-inducing immunogens. Here, we show that the breadth of neutralizing antibody responses in HIV-1 controllers was associated with a relative enrichment of circulating CXCR5+CXCR3+PD-1lo CD4+ T cells. These CXCR3+PD-1lo Tfh-like cells were preferentially induced in vitro by functionally superior dendritic cells from controller neutralizers, and able to secrete IL-21 and support B cells. In addition, these CXCR3+PD-1lo Tfh-like cells contained higher proportions of stem cell-like memory T cells, and upon antigenic stimulation differentiated into PD-1hi Tfh-like cells in a Notch-dependent manner. Together, these data suggest that CXCR5+CXCR3+PD-1lo cells represent a dendritic cell-primed precursor cell population for PD-1hi Tfh-like cells that may contribute to the generation of bnAbs in the absence of high-level viremia.

Can research at the end of life be a useful tool to advance HIV cure?

OPINION Can research at the end of life be a useful tool to advance HIV cure? Sara Gianella a , Jeff Taylor b , Timothy R. Brown c , Andy Kaytes b , Cristian L. Achim a , David J. Moore a , Susan J. Little a , Ronald J. Ellis a and Davey M. Smith a,d AIDS 2017, 31:1–4 Keywords: end of life, eradication, HIV cure, last gift, legacy, peri-mortem research model, research Modern antiretroviral therapy (ART) has saved millions of years of life [1], but it cannot eradicate latently infected cells [2,3]. The replication-competent provirus that remains during ART represents the major barrier to curing HIV [4]. Most of this latent reservoir resides in solid tissues and not in circulating blood, and we have yet to fully define the sanctuary sites of HIV persistence [5]. Timothy Ray Brown (known by many as the ‘Berlin Patient’) may be the closest to an HIV cure after being treated with an allogeneic hematopoietic stem cell transplant from a donor who was homozygous for the CCR5D32 deletion [6]. Unfortunately, this remarkable success has not been reproduced, and robust viral replication resumes almost universally following treat- ment interruption [7–9]. Even if strategies currently in development succeed in purging HIV from circulating CD4 þ T cells, residual virus can remain in the central nervous system (CNS), gut-associated lymphoid tissue (GALT), genital tract, adipose tissue, and others [9–12]. Thus, cure efforts must tackle the eradication of HIV reservoirs in anatomic compartments and sanctuaries throughout the body. The earliest stages of HIV infection are characterized by high levels of viral replication and little immune response [13,14]. This window of uncontrolled replication allows the virus to seed reservoirs throughout the body, as early as within 2 weeks of HIV infection [15], and to persist indefinitely throughout the lifespan of HIV-infected individuals [10]. These proviral reservoirs continue to expand and diversify until viral replication is successfully suppressed with ART. Despite extensive investigations, we still do not fully understand the dynamics of the total body HIV reservoir and how sub-reservoirs in various compart- ments relate to one another. For example, it remains unclear what factors govern the size and the activity of replication-competent HIV DNA in the CNS compared to the genital tract and blood. Most studies that have explored nonblood reservoirs among persons living with HIV infection have been limited to small samples of cerebrospinal fluid (CSF), genital secretion, gut, and shallow lymph nodes [10,16]. Studies using macaque models with simian immunodeficiency viruses (SIVs) have been enlightening for reservoirs in different tissues [17–23], but while SIV shares a high degree of structural and sequence identity to HIV, studies of SIV will not suffice to test eradication strategies for humans. For example, the Merck adenovirus type 5 (Ad5) trivalent HIV-1 vaccine trial (STEP trial) did not show efficacy in preventing HIV infection or slowing disease progression [24], despite promising results in various macaques’ trials [25–27]. As the field tackles the ambitious goal of eradicating HIV from the human body, we will need to test the effectiveness of cure interventions in living persons. a University of California, San Diego, La Jolla, b Community Advisory Board (CAB) AntiViral Research Center (AVRC), c Las Vegas, Nevada, and d Veterans Affairs San Diego Healthcare System, San Diego, California, USA. Correspondence to Sara Gianella, University of California San Diego, 9500 Gilman Drive, MC 0679, La Jolla, CA 92093-0679, USA. Tel: +1 858 642 1620; fax: +1 858 552 7445; e-mail: gianella@ucsd.edu Received: 18 August 2016; revised: 23 September 2016; accepted: 27 September 2016. DOI:10.1097/QAD.0000000000001300 ISSN 0269-9370 Copyright Q 2016 Wolters Kluwer Health, Inc. All rights reserved. Copyright © 2016 Wolters Kluwer Health, Inc. All rights reserved.

Cytokine expression in three chicken host systems infected with H9N2 influenza viruses with different pathogenicities

ABSTRACTSD/818 and SD/196 are H9N2 influenza virus strains isolated from chickens from the same farm at different times that exhibited similar genetic evolution. However, strain SD/818 exhibited higher pathogenicity in chickens than strain SD/196 and other H9N2 influenza virus epidemic strains from China. The expression of cytokines is an important host defence mechanism following viral infection and their intensity is a major determinant of viral pathogenicity. To elucidate the mechanism underlying the increased pathogenicity of strain SD/818 from the host’s perspective, viral replication and cytokine expression were dynamically studied using real-time quantitative reverse transcription PCR in chickens infected with strain SD/818 compared with chickens infected with strain SD/196 in this study. The results showed that the replication of strain SD/818 and the expressions of IL-1β, IL-6, TNF-α, IFN-α and IFN-β induced by strain SD/818 were higher than those induced by strain SD/196 in the chicken host system. Expression of these cytokines in chickens coincided with or followed virus replication. These results suggested that high-level viral replication and pro-inflammatory cytokine expression (but not decreased type I IFN expression) were associated with the higher pathogenicity of strain SD/818 in chickens.

The Nature of Exposure Drives Transmission of Nipah Viruses from Malaysia and Bangladesh in Ferrets.

Person-to-person transmission is a key feature of human Nipah virus outbreaks in Bangladesh. In contrast, in an outbreak of Nipah virus in Malaysia, people acquired infections from pigs. It is not known whether this important epidemiological difference is driven primarily by differences between NiV Bangladesh (NiV-BD) and Malaysia (NiV-MY) at a virus level, or by environmental or host factors. In a time course study, ferrets were oronasally exposed to equivalent doses of NiV-BD or NiV-MY. More rapid onset of productive infection and higher levels of virus replication in respiratory tract tissues were seen for NiV-BD compared to NiV-MY, corroborating our previous report of increased oral shedding of NiV-BD in ferrets and suggesting a contributory mechanism for increased NiV-BD transmission between people compared to NiV-MY. However, we recognize that transmission occurs within a social and environmental framework that may have an important and differentiating role in NiV transmission rates. With this in mind, ferret-to-ferret transmission of NiV-BD and NiV-MY was assessed under differing viral exposure conditions. Transmission was not identified for either virus when naïve ferrets were cohoused with experimentally-infected animals. In contrast, all naïve ferrets developed acute infection following assisted and direct exposure to oronasal fluid from animals that were shedding either NiV-BD or NiV-MY. Our findings for ferrets indicate that, although NiV-BD may be shed at higher levels than NiV-MY, transmission risk may be equivalently low under exposure conditions provided by cohabitation alone. In contrast, active transfer of infected bodily fluids consistently results in transmission, regardless of the virus strain. These observations suggest that the risk of NiV transmission is underpinned by social and environmental factors, and will have practical implications for managing transmission risk during outbreaks of human disease.

Human Th17 Cells Lack HIV-Inhibitory RNases and Are Highly Permissive to Productive HIV Infection.

Human immunodeficiency virus (HIV) infects and depletes CD4(+) T cells, but subsets of CD4(+) T cells vary in their susceptibility and permissiveness to infection. For example, HIV preferentially depletes interleukin-17 (IL-17)-producing T helper 17 (Th17) cells and T follicular helper (Tfh) cells. The preferential loss of Th17 cells during the acute phase of infection impairs the integrity of the gut mucosal barrier, which drives chronic immune activation-a key determinant of disease progression. The preferential loss of Th17 cells has been attributed to high CD4, CCR5, and CXCR4 expression. Here, we show that Th17 cells also exhibit heightened permissiveness to productive HIV infection. Primary human CD4(+) T cells were sorted, activated under Th17- or Th0-polarizing conditions and infected, and then analyzed by flow cytometry. Th17-polarizing cytokines increased HIV infection, and HIV infection was disproportionately higher among Th17 cells than among IL-17(-) or gamma interferon-positive (IFN-γ(+)) cells, even upon infection with a replication-defective HIV vector with a pseudotype envelope. Further, Th17-polarized cells produced more viral capsid protein. Our data also reveal that Th17-polarized cells have diminished expression of RNase A superfamily proteins, and we report for the first time that RNase 6 inhibits HIV. Thus, our findings link Th17 polarization to increased HIV replication. Our study compares the intracellular replicative capacities of several different HIV isolates among different T cell subsets, providing a link between the differentiation of Th17 cells and HIV replication. Th17 cells are of key importance in mucosal integrity and in the immune response to certain pathogens. Based on our findings and the work of others, we propose a model in which HIV replication is favored by the intracellular environment of two CD4(+) T cell subsets that share several requirements for their differentiation: Th17 and Tfh cells. Characterizing cells that support high levels of viral replication (rather than becoming latently infected or undergoing cell death) informs the search for new therapeutics aimed at manipulating intracellular signaling pathways and/or transcriptional factors that affect HIV replication.

Non-Hepatic Adverse Effects of Antiretroviral Therapy for HIV Treatment and Care

High-level viral replication occurs when HIV enters the body. Due to frequent mutations, the virus becomes resistant in the body and persists in memory T cells making it incurable. Lifelong therapy is then required to suppress replication of the virus in the cells. The drugs available for HIV care such as Non-nucleoside reverse transcriptase inhibitors, Protease Inhibitors, Nucleoside reverse transcriptase inhibitors, Entry and Fusion Inhibitors and Integrase inhibitors have been documented to cause adverse effects in patients. Non-nucleoside reverse transcriptase inhibitors and protease inhibitors are metabolized by the cytochrome P450 system, resulting to numerous drug-drug interactions. Adverse effects of antiretroviral therapy should be monitored frequently. Monitoring should include complete blood counts and comprehensive metabolic profiles every three to six months. Lipid profiles and urinalysis for proteinuria should be done after every year. Long-term morbidity due to antiretroviral therapy includes renal, liver, glucose, and lipid abnormalities, and bone disease as well as cardiovascular disease. With some exceptions for lipid management, these morbidities can be managed. This review aims at describing different non-hepatic adverse effects of antiretrovirals as well as factors associated with them.

The response of aged mice to primary infection and re-infection with pneumonia virus of mice depends on their genetic background

The pneumonia virus of mice (PVM) model is used to study respiratory syncytial virus (RSV) pathogenesis. The outcome of PVM infection varies in different inbred mouse strains, BALB/c being highly susceptible and C57BL/6 more resistant. As the disease symptoms induced by RSV infection can become more severe as people age, we examined the primary and secondary immune responses to infection with PVM in aged BALB/c and C57BL/6 mice. Based on clinical parameters, aged C57BL/6 mice displayed less severe disease than young adult mice when infected with 3000pfu of PVM-15, while BALB/c mice were equally susceptible at both ages showing significant weight loss and high levels of virus replication. Furthermore, after primary infection the CD4+ T cell numbers in the lungs were higher in young adult mice, while the CD8+ T cell numbers were comparable in both age groups and strains. When either C57BL/6 or BALB/c mice were infected with PVM as young adults and then re-infected as aged mice, they were protected from clinical disease, while virus replication was reduced. In contrast to mice with a primary PVM-infection, re-infected mice did not have infiltration of neutrophils or inflammatory mediators in the lung. BALB/c mice had higher virus neutralizing antibody levels in the serum and lung than C57BL/6 mice upon re-infection. Re-infection with PVM led to significant influx of effector CD4+ T cells into the lungs when compared to aged mice with a primary infection, while this cell population was decreased in the lung draining lymph nodes in both mouse strains. After re-infection the effector CD8+ T cell population was also decreased in the lung draining lymph nodes in both mouse strain when compared to aged mice after primary infection. However, the central memory CD4+ and CD8+ T cells were significantly enhanced in numbers in the lungs and draining lymph nodes of both mouse strains after re-infection, and these numbers were higher for C57BL/6 mice.

Systemic Delivery of an Oncolytic Adenovirus Expressing Decorin for the Treatment of Breast Cancer Bone Metastases.

The development of novel therapies for breast cancer bone metastasis is a major unmet medical need. Toward that end, we have constructed an oncolytic adenovirus, Ad.dcn, and a nonreplicating adenovirus, Ad(E1-).dcn, both containing the human decorin gene. Our in vitro studies showed that Ad.dcn produced high levels of viral replication and the decorin protein in the breast tumor cells. Ad(E1-).dcn-mediated decorin expression in MDA-MB-231 cells downregulated the expression of Met, β-catenin, and vascular endothelial growth factor A, all of which are recognized decorin targets and play pivotal roles in the progression of breast tumor growth and metastasis. Adenoviral-mediated decorin expression inhibited cell migration and induced mitochondrial autophagy in MDA-MB-231 cells. Mice bearing MDA-MB-231-luc skeletal metastases were systemically administered with the viral vectors, and skeletal tumor growth was monitored over time. The results of bioluminescence imaging and X-ray radiography indicated that Ad.dcn and Ad(E1-).dcn significantly inhibited the progression of bone metastases. At the terminal time point, histomorphometric analysis, micro-computed tomography, and bone destruction biomarkers showed that Ad.dcn and Ad(E1-).dcn reduced tumor burden and inhibited bone destruction. A nonreplicating adenovirus Ad(E1-).luc expressing the luciferase 2 gene had no significant effect on inhibiting bone metastases, and in several assays, Ad.dcn and Ad(E1-).dcn were better than Ad.luc, a replicating virus expressing the luciferase 2 gene. Our data suggest that adenoviral replication coupled with decorin expression could produce effective antitumor responses in a MDA-MB-231 bone metastasis model of breast cancer. Thus, Ad.dcn could potentially be developed as a candidate gene therapy vector for treating breast cancer bone metastases.

Follicular regulatory T cells impair follicular T helper cells in HIV and SIV infection.

Human and simian immunodeficiency viruses (HIV and SIV) exploit follicular lymphoid regions by establishing high levels of viral replication and dysregulating humoral immunity. Follicular regulatory T cells (TFR) are a recently characterized subset of lymphocytes that influence the germinal centre response through interactions with follicular helper T cells (TFH). Here, utilizing both human and rhesus macaque models, we show the impact of HIV and SIV infection on TFR number and function. We find that TFR proportionately and numerically expand during infection through mechanisms involving viral entry and replication, TGF-β signalling, low apoptosis rates and the presence of regulatory dendritic cells. Further, TFR exhibit elevated regulatory phenotypes and impair TFH functions during HIV infection. Thus, TFR contribute to inefficient germinal centre responses and inhibit HIV and SIV clearance.

Filovirus pathogenesis and immune evasion: insights from Ebola virus and Marburg virus.

Ebola viruses and Marburg viruses, members of the filovirus family, are zoonotic pathogens that cause severe disease in people, as highlighted by the latest Ebola virus epidemic in West Africa. Filovirus disease is characterized by uncontrolled virus replication and the activation of host responses that contribute to pathogenesis. Underlying these phenomena is the potent suppression of host innate antiviral responses, particularly the type I interferon response, by viral proteins, which allows high levels of viral replication. In this Review, we describe the mechanisms used by filoviruses to block host innate immunity and discuss the links between immune evasion and filovirus pathogenesis.

The Combination of the R263K and T66I Resistance Substitutions in HIV-1 Integrase Is Incompatible with High-Level Viral Replication and the Development of High-Level Drug Resistance.

The R263K substitution in integrase has been selected in tissue culture with dolutegravir (DTG) and has been reported for several treatment-experienced individuals receiving DTG as part of salvage therapy. The R263K substitution seems to be incompatible with the presence of common resistance mutations associated with raltegravir (RAL), a different integrase strand transfer inhibitor (INSTI). T66I is a substitution that is common in individuals who have developed resistance against a different INSTI termed elvitegravir (EVG), but it is not known whether these two mutations might be compatible in the context of resistance against DTG or what impact the combination of these substitutions might have on resistance against INSTIs. E138K is a common secondary substitution observed with various primary resistance substitutions in RAL- and EVG-treated individuals. Viral infectivity, replicative capacity, and resistance against INSTIs were measured in cell-based assays. Strand transfer and 3' processing activities were measured biochemically. The combination of the R263K and T66I substitutions decreased HIV-1 infectivity, replicative capacity, and strand transfer activity. The addition of the E138K substitution partially compensated for these deficits and resulted in high levels of resistance against EVG but not against DTG or RAL. These findings suggest that the presence of the T66I substitution will not compromise the activity of DTG and may also help to prevent the additional generation of the R263K mutation. Our observations support the use of DTG in second-line therapy for individuals who experience treatment failure with EVG due to the T66I substitution. The integrase strand transfer inhibitors (INSTIs) elvitegravir and dolutegravir are newly developed inhibitors against human immunodeficiency virus type 1 (HIV-1). HIV drug-resistant mutations in integrase that can arise in individuals treated with elvitegravir commonly include the T66I substitution, whereas R263K is a signature resistance substitution against dolutegravir. In order to determine how different combinations of integrase resistance mutations can influence the outcome of therapy, we report here the effects of the T66I, E138K, and R263K substitutions, alone and in combination, on viral replicative capacity and resistance to integrase inhibitors. Our results show that the addition of R263K to the T66I substitution diminishes viral replicative capacity and strand transfer activity while not compromising susceptibility to dolutegravir. This supports the use of dolutegravir in second-line therapy for patients failing elvitegravir therapy who harbor the T66I resistance substitution.

Distinct Immune Responses in Resistant and Susceptible Strains of Mice during Neurovirulent Alphavirus Encephalomyelitis.

Susceptibility to alphavirus encephalomyelitis is dependent on a variety of factors, including the genetic background of the host. Neuroadapted Sindbis virus (NSV) causes uniformly fatal disease in adult C57BL/6 (B6) mice, but adult BALB/c (Bc) mice recover from infection. In B6 mice, fatal encephalomyelitis is immune mediated rather than a direct result of virus infection. To identify the immunological determinants of host susceptibility to fatal NSV-induced encephalomyelitis, we compared virus titers and immune responses in adult B6 and Bc mice infected intranasally with NSV. B6 mice had higher levels of virus replication, higher levels of type I interferon (IFN), and slower virus clearance than did Bc mice. B6 mice had more neuronal apoptosis, more severe neurologic disease, and higher mortality than Bc mice. B6 mice had more infiltration of inflammatory cells and higher levels of IL1b, IL-6, TNFa, Csf2, and CCL2 mRNAs and interleukin-6 (IL-6), tumor necrosis factor alpha (TNF-α), IFN-γ, and C-C motif ligand 2 (CCL2) protein in brains than Bc mice. However, Bc mice had more brain antibody at day 7 and a higher percentage of CD4(+) T cells. CD4(+) T cells in the brains of Bc mice included fewer Th17 cells and more regulatory T cells (Tregs) producing IL-10 than B6 mice, accompanied by higher levels of Il2 and Cxcl10 mRNAs. In the absence of IL-10, resistant Bc mice became susceptible to fatal encephalomyelitis after NSV infection. These studies demonstrate the importance of the immune response and its regulation in determining host survival during alphavirus encephalomyelitis. Mosquito-borne alphavirus infections are an important cause of encephalomyelitis in humans. The severity of disease is dependent both on the strain of the virus and on the age and genetic background of the host. A neurovirulent strain of Sindbis virus causes immune-mediated fatal encephalomyelitis in adult C57BL/6 mice but not in BALB/c mice. To determine the host-dependent immunological mechanisms underlying the differences in susceptibility between these two strains of mice, we compared their immune responses to infection. Resistance to fatal disease in BALB/c mice was associated with better antibody responses, more-rapid virus clearance, fewer Th17 cells, and more-potent regulatory T cell responses than occurred in susceptible C57BL/6 mice. In the absence of interleukin-10, a component of the regulatory immune response, resistant mice became susceptible to lethal disease. This study demonstrates the importance of the immune response and its regulation for host survival during alphavirus encephalomyelitis.

Genetic Characterization of Porcine Circovirus Type 2 (PCV2) in Pigs of Bhutan.

Porcine circovirus (PCV) is a small non-enveloped virus with a single-stranded circular DNA with two antigenically and genetically different species, PCV1 and PCV2. Among these two, PCV2 is responsible for multifactorial disease syndromes, the most important disease known as PCV2-systemic disease (PCV2-SD), previously known as post-weaning multisystemic wasting syndrome (PMWS). The epidemiological situation is dynamically changing and new strains including recombinant PCV2 have emerged in Asia. In Bhutan, pigs are important livestock and play a very important role in providing meat and income for rural farmers. Although high rate of pigs seropositive against PCV2 was described in Bhutan, there was no virological evidence for PCV2 infections. This study was conducted to confirm the presence of PCV2 through detection of PCV2 DNA and molecular characterization of PCV2 strains in tissue and blood samples collected from Bhutanese pigs. Porcine circovirus type 2 genome was detected in 16 of 34 tissue samples pigs from the government farm. In 9 pigs, very high level of viral replication indicated that PCV2-SD was detected. Phylogenetic analysis performed with a set of GenBank sequences revealed that the Bhutanese PCV2 strains belonged to the PCV2b genotype and grouped with cluster 1C.

Pandemic Swine-Origin H1N1 Influenza Virus Replicates to Higher Levels and Induces More Fever and Acute Inflammatory Cytokines in Cynomolgus versus Rhesus Monkeys and Can Replicate in Common Marmosets.

The close immunological and physiological resemblance with humans makes non-human primates a valuable model for studying influenza virus pathogenesis and immunity and vaccine efficacy against infection. Although both cynomolgus and rhesus macaques are frequently used in influenza virus research, a direct comparison of susceptibility to infection and disease has not yet been performed. In the current study a head-to-head comparison was made between these species, by using a recently described swine-origin pandemic H1N1 strain, A/Mexico/InDRE4487/2009. In comparison to rhesus macaques, cynomolgus macaques developed significantly higher levels of virus replication in the upper airways and in the lungs, involving both peak level and duration of virus production, as well as higher increases in body temperature. In contrast, clinical symptoms, including respiratory distress, were more easily observed in rhesus macaques. Expression of sialyl-α-2,6-Gal saccharides, the main receptor for human influenza A viruses, was 50 to 73 times more abundant in trachea and bronchus of cynomolgus macaques relative to rhesus macaques. The study also shows that common marmosets, a New World non-human primate species, are susceptible to infection with pandemic H1N1. The study results favor the cynomolgus macaque as model for pandemic H1N1 influenza virus research because of the more uniform and high levels of virus replication, as well as temperature increases, which may be due to a more abundant expression of the main human influenza virus receptor in the trachea and bronchi.

Can HIV Be Cured and Should We Try?

An estimated 35.0 million persons live with HIV; 2.1 million new infections occurred and 1.5 million persons died of HIV in 2013 (World Health Organization, http://www.who.int/hiv/data/epi_core_dec2014.png?ua=1). Despite effective combination antiretroviral therapy (cART), less than one-quarter of patients can access these life-prolonging medications; and despite its effectiveness, cART does not normalize life expectancy, as premature aging, metabolic complications and chronic inflammation complicate HIV therapy. HIV is incurable due to the presence of a latent viral reservoir. During the life cycle of the virus, HIV integrates into the host DNA. A subset of integrated HIV provirus remains transcriptionally silent, producing neither viral proteins nor viral progeny, until reactivation by various physiologic stimuli. This latency of HIV allows some infected cells to escape immune detection and elimination, and these latently infected cells constitute the viral reservoir. The latent viral reservoir allows viral rebound within weeks of interruption of cART, 1,2 where the magnitude of viral replication approaches that present pre-therapy. Although it was once thought that viral rebound occurred universally following therapy interruption, several recent reports challenge that paradigm. The “Berlin patient” successfully cleared HIV after two allogeneic transplants from a donor with homozygous CCR5Δ32 mutation,3 and he has not rebounded HIV after nearly seven years. This case likely represents a cure from HIV; yet other cases have been described where HIV rebound has been attenuated, or delayed. The “Mississippi” baby was a perinatally HIV-infected infant who initiated cART within hours of birth, and when interrupted eighteen months later, viremia remained undetectable for nearly two years.4 The Harvard BMT cases underwent allogeneic BMT, developed undetectable HIV DNA, yet rebounded viremia within only eight months after cART discontinuation.5 Together these cases demonstrate that control of viremia in the absence of cART is possible, if not durable.4 The VISCONTI cohort of 14 HIV-infected adults who initiated antiretroviral therapy during acute infection, and in whom high level rebound viremia had not occurred several years after cessation of therapy,6 similarly demonstrated that viral rebound following cART interruption can be attenuated. These cases have reinvigorated research toward finding a cure for HIV, which certainly will require an exceptional investment of time, talent and resources. Thus it is prudent to question whether such resources would be better devoted to more proximate needs with proven results (such as supplying cART to those without access to it, or supplying pre/post exposure prophylaxis to reduce the rate of new infections).

Maintenance of multiple Nef functions limits and dictates pathways of immune escape (VIR1P.1147)

Abstract CD8+ T lymphocytes (CD8TL) can effectively control SIV and HIV replication. CD8TL targeting of particular regions in the Nef globular core is associated with control of both viruses suggesting common mechanisms of control. Deep sequencing of SIV from nine Mamu-B*017:01+ macaques revealed distinct selection patterns in two Nef epitopes. Functional assays revealed that acute escape in one epitope impacted a conserved structural face of the Nef core and reduced MHC-I downregulation, rendering infected cells more susceptible to CD8TL against other epitopes. In contrast, acute mutations in a nearby epitope impacted Nef’s ability to downregulate Tetherin and CD28, both of which are modulated via Nef interactions with host clathrin adaptor proteins, with mutations impacting MHC-I anchor residues being most destructive. In fact, anchor residues in both epitopes are almost entirely conserved throughout primate lentiviral history, spanning more than 40 millennia suggesting selection for maintenance of function. These data suggest mechanisms by which Nef targeting CD8TL can contribute to viral control; by targeting conserved epitopes involved in fuctions that are critical for high-level viral replication in vivo. Our approach to understanding T cell mediated control of SIV revealed novel insights into Nef structure:function relationships and suggest that particular regions of the Nef protein might be attractive components of vaccines designed to induce potent CD8TL responses.

Lymphocytic Choriomeningitis Virus Differentially Affects the Virus-Induced Type I Interferon Response and Mitochondrial Apoptosis Mediated by RIG-I/MAVS.

Arenaviruses are important emerging human pathogens maintained by noncytolytic persistent infection in their rodent reservoir hosts. Despite high levels of viral replication, persistently infected carrier hosts show only mildly elevated levels of type I interferon (IFN-I). Accordingly, the arenavirus nucleoprotein (NP) has been identified as a potent IFN-I antagonist capable of blocking activation of interferon regulatory factor 3 (IRF3) via the retinoic acid inducible gene (RIG)-I/mitochondrial antiviral signaling (MAVS) pathway. Another important mechanism of host innate antiviral defense is represented by virus-induced mitochondrial apoptosis via RIG-I/MAVS and IRF3. In the present study, we investigated the ability of the prototypic Old World arenavirus lymphocytic choriomeningitis virus (LCMV) to interfere with RIG-I/MAVS-dependent apoptosis. We found that LCMV does not induce apoptosis at any time during infection. While LCMV efficiently blocked induction of IFN-I via RIG-I/MAVS in response to superinfection with cytopathic RNA viruses, virus-induced mitochondrial apoptosis remained fully active in LCMV-infected cells. Notably, in LCMV-infected cells, RIG-I was dispensable for virus-induced apoptosis via MAVS. Our study reveals that LCMV infection efficiently suppresses induction of IFN-I but does not interfere with the cell's ability to undergo virus-induced mitochondrial apoptosis as a strategy of innate antiviral defense. The RIG-I independence of mitochondrial apoptosis in LCMV-infected cells provides the first evidence that arenaviruses can reshape apoptotic signaling according to their needs. Arenaviruses are important emerging human pathogens that are maintained in their rodent hosts by persistent infection. Persistent virus is able to subvert the cellular interferon response, a powerful branch of the innate antiviral defense. Here, we investigated the ability of the prototypic arenavirus lymphocytic choriomeningitis virus (LCMV) to interfere with the induction of programmed cell death, or apoptosis, in response to superinfection with cytopathic RNA viruses. Upon viral challenge, persistent LCMV efficiently blocked induction of interferons, whereas virus-induced apoptosis remained fully active in LCMV-infected cells. Our studies reveal that the persistent virus is able to reshape innate apoptotic signaling in order to prevent interferon production while maintaining programmed cell death as a strategy for innate defense. The differential effect of persistent virus on the interferon response versus its effect on apoptosis appears as a subtle strategy to guarantee sufficiently high viral loads for efficient transmission while maintaining apoptosis as a mechanism of defense.