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Background Virus neutralization by antibodies is an important prognostic factor in many viral diseases. To easily and rapidly measure titers of neutralizing antibodies in serum or plasma, we developed pseudovirion particles composed of the spike glycoprotein of SARS-CoV-2 incorporated onto murine leukemia virus capsids and a modified minimal murine leukemia virus genome encoding firefly luciferase. This assay design is intended for use in laboratories with biocontainment level 2 and therefore circumvents the need for the biocontainment level 3 that would be required for replication-competent SARS-CoV-2 virus. To validate the pseudovirion assay, we set up comparisons with other available antibody tests including those from Abbott, Euroimmun and Siemens, using archived, known samples. Results 11 out of 12 SARS-CoV-2-infected patient serum samples showed neutralizing activity against SARS-CoV-2-spike pseudotyped MLV viruses, with neutralizing titers-50 (NT50) that ranged from 1:25 to 1:1,417. Five historical samples from patients hospitalized for severe influenza infection in 2016 tested negative in the neutralization assay (NT50 50 2 = 0.9344). Conclusions SARS-CoV-2 spike/MLV pseudovirions provide a practical means of assessing neutralizing activity of antibodies in serum or plasma from infected patients under laboratory conditions consistent with biocontainment level 2. This assay offers promise also in evaluating immunogenicity of spike glycoprotein-based candidate vaccines in the near future.

Coinfection with hepatitis C virus (HCV) influences HIV reservoir size. However, it is unknown whether this coinfection also induces a higher provirus transcription. Viral transcription is promoted by synergy between cellular factors such as NF-&kappa, B and the viral regulator Tat. The impact of HCV coinfection on HIV provirus transcription was analyzed in resting (r)CD4 T+ cells (CD3+CD4+CD25-CD69-HLADR-) and rCD4 T cells-depleted PBMCs (rCD4 T- PBMCs) from a multicenter cross-sectional study of 115 cART-treated HIV patients: 42 HIV+/HCV+ coinfected individuals (HIV+/HCV+), 34 HIV+ patients with HCV spontaneous clearance (HIV+/HCV&minus, ) and 39 HIV patients (HIV+). Viral transcription was assessed in total RNA through the quantification of unspliced, single spliced, and multiple spliced viral mRNAs by qPCR. Linear correlations between viral reservoir size and viral splicing were determined. A 3-fold increase of multiple spliced transcripts in rCD4 T+ cells of HIV+/HCV+ patients was found compared to HIV+ individuals (p &lt, 0.05). As Tat is synthesized by multiple splicing, the levels of Tat were also quantified in these patients. Significant differences in single and multiple spliced transcripts were also observed in rCD4 T- PBMCs. Levels of multiple spliced mRNAs were increased in rCD4 T+ cells isolated from HIV+/HCV+ subjects, which could indicate a higher Tat activity in these cells despite their resting state.

The causative mutation responsible for limb girdle muscular dystrophy 1F (LGMD1F) is one heterozygous single nucleotide deletion in the stop codon of the nuclear import factor Transportin 3 gene (TNPO3). This mutation causes a carboxy-terminal extension of 15 amino acids, producing a protein of unknown function (TNPO3_mut) that is co-expressed with wild-type TNPO3 (TNPO3_wt). TNPO3 has been involved in the nuclear transport of serine/arginine-rich proteins such as splicing factors and also in HIV-1 infection through interaction with the viral integrase and capsid. We analyzed the effect of TNPO3_mut on HIV-1 infection using PBMCs from patients with LGMD1F infected ex vivo. HIV-1 infection was drastically impaired in these cells and viral integration was reduced 16-fold. No significant effects on viral reverse transcription and episomal 2-LTR circles were observed suggesting that the integration of HIV-1 genome was restricted. This is the second genetic defect described after CCR5Δ32 that shows strong resistance against HIV-1 infection., Author summary TNPO3 has been described as a key factor in the infection by the human immunodeficiency virus (HIV-1), the causative agent of AIDS. In 2013, a relationship between a genetic defect in TNPO3 gene and a rare muscle disease named Limb Girdle Muscular Dystrophy 1F (LGMD1F), with an autosomal dominant transmission, was discovered. LGMD1F patients show a heterozygous single nucleotide deletion in the TNPO3 gene that generates a TNPO3_mut protein. Our results demonstrate that cells from patients with this mutation in TNPO3 are resistant to HIV-1 infection in vitro. We are faced with an in vivo situation in which the genetic defect that causes this rare disease confers resistance to HIV infection. Therefore, TNPO3 mutation represents a natural model to understand the pathogenesis of both diseases. Cells from LGMD1F patients can be used to understand the mechanisms of action of TNPO3 in HIV infection and to design new therapeutic strategies for the treatment of both diseases. The use of HIV-1 as a methodological tool will permit a better understanding of the physiopathological mechanisms derived from the mutation in TNPO3 that causes the muscle disease.

This work was supported by the Spanish Ministry of Economy and Competitiveness (SAF2013-44677-R, SAF2016-78480-R); the Instituto de Salud Carlos III (ISCIII-FIS PI16CIII/00034); and the Spanish AIDS Research Network RD16CIII/0002/0001-ISCIII-FEDER. JAM developed this work in the frame of a ‘Juan de la Cierva 2012’ post-doctoral program, Ministerio de Competitividad, Spain. JMM received a personal intensification research grant #INT15/00168 during 2016 from Instituto de Salud Carlos III, Madrid, Spain.

Monográfico con los resúmenes del Ninth International Workshop on HIV Persistence during Therapy 10-13 December 2019, Miami, Florida, USA Sí

Dendritic cells (DCs) are professional antigen-presenting cells whose functions are dependent on their degree of differentiation. In their immature state, DCs capture pathogens and migrate to the lymph nodes. During this process, DCs become resident mature cells specialized in antigen presentation. DCs are characterized by a highly limiting environment for human immunodeficiency virus type 1 (HIV-1) replication due to the expression of restriction factors such as SAMHD1 and APOBEC3G. However, uninfected DCs capture and transfer viral particles to CD4 lymphocytes through a trans-enhancement mechanism in which chemokines are involved. We analyzed changes in gene expression with whole-genome microarrays when immature DCs (IDCs) or mature DCs (MDCs) were productively infected using Vpx-loaded HIV-1 particles. Whereas productive HIV infection of IDCs induced expression of interferon-stimulated genes (ISGs), such induction was not produced in MDCs, in which a sharp decrease in ISG- and CXCR3-binding chemokines was observed, lessening trans-infection of CD4 lymphocytes. Similar patterns of gene expression were found when DCs were infected with HIV-2 that naturally expresses Vpx. Differences were also observed under conditions of restrictive HIV-1 infection, in the absence of Vpx. ISG expression was not modified in IDCs, whereas an increase of ISG- and CXCR3-binding chemokines was observed in MDCs. Overall these results suggest that sensing and restriction of HIV-1 infection are different in IDCs and MDCs. We propose that restrictive infection results in increased virulence through different mechanisms. In IDCs avoidance of sensing and induction of ISGs, whereas in MDCs increased production of CXCR3-binding chemokines, would result in lymphocyte attraction and enhanced infection at the immune synapse.IMPORTANCE In this work we describe for the first time the activation of a different genetic program during HIV-1 infection depending on the state of maturation of DCs. This represents a breakthrough in the understanding of the restriction to HIV-1 infection of DCs. The results show that infection of DCs by HIV-1 reprograms their gene expression pattern. In immature cells, productive HIV-1 infection activates interferon-related genes involved in the control of viral replication, thus inducing an antiviral state in surrounding cells. Paradoxically, restriction of HIV-1 by SAMHD1 would result in lack of sensing and IFN activation, thus favoring initial HIV-1 escape from the innate immune response. In mature DCs, restrictive infection results in HIV-1 sensing and induction of ISGs, in particular CXCR3-binding chemokines, which could favor the transmission of HIV to lymphocytes. Our data support the hypothesis that genetic DC reprograming by HIV-1 infection favors viral escape and dissemination, thus increasing HIV-1 virulence. We thank Olga Palao (AIDS Immunopathogenesis Unit) and A. Zaballos (Genomics Unit, Instituto de Salud Carlos III) for their secretarial and technical assistance, respectively. We also greatly appreciate our patients for their willingness to participate.Funding for this research was provided by: Ministerio de Economía y Competitividad (SAF2013-44677-R, PI16CIII/00034, ISCIII-FIS Nº) Instituto de Salud Carlos III (Spanish AIDS Research Network RD16CIII/0002/0001) Sí

Killing of CD4 lymphocytes and systemic immune suppression are the hallmarks of HIV infection. These milestones are produced by different mechanisms that draw a complex picture of AIDS immunopathogenesis. The role of the GALT system as a preferential target for HIV, chronic activation of the immune system and viral escape mechanisms are recent challenges that have changed our current view on the mechanisms leading to immune destruction and development of AIDS. In this article, the mechanisms of immune suppression, the evolution of immune response throughout the infection and the mechanisms of viral escape are analysed.

Massive activation of infected CD4+ T cells during acute HIV-1 infection leads to reservoir seeding and T-cell destruction. During T-cell activation, the antiviral effect of the innate factor SAMHD1 is neutralized through phosphorylation at T592, allowing HIV-1 infection. Dasatinib, a tyrosine kinase inhibitor currently used for treating chronic myeloid leukemia, has been described to control HIV-1 replication through its negative effect on T-cell proliferation and viral entry. We demonstrate that Dasatinib can actually interfere with SAMHD1 phosphorylation in human peripheral blood lymphocytes, preserving its antiviral activity against HIV-1. Dasatinib prevented SAMHD1 phosphorylation in vitro and ex vivo, impairing HIV-1 reverse transcription and proviral integration. This was the major mechanism of action because the presence of Vpx, which degrades SAMHD1, in HIV-1 virions impeded the inhibitory effect of Dasatinib on HIV-1 replication. In fact, infection with VSV-pseudotyped HIV-1 virions and fusion of BlaM-Vpr-containing HIV-1 viruses with activated PBMCs in the presence of Dasatinib suggested that Dasatinib was not acting at fusion level. Finally, PBMCs from patients on chronic treatment with Dasatinib showed a lower level of SAMHD1 phosphorylation in response to activating stimuli and low susceptibility to HIV-1 infection ex vivo. Consequently, Dasatinib is a compound currently used in clinic that preserves the antiviral function of SAMHD1. Using Dasatinib as adjuvant of antiretroviral therapy during early primary HIV-1 infection would contribute to reduce viral replication and spread, prevent reservoir seeding, and preserve CD4 counts and CTL responses. These events would create a more favorable virologic and immunologic environment for future interventional studies aiming at HIV-1 eradication. This work was supported by the Spanish Ministry of Economy and Competitiveness (SAF2010-18388, SAF2013-44677-R, FIS PI12/00506, and FIS PI12/00969); the SPANISH AIDS Research Network RD12/0017/0015 that is included in Acción Estratégica en Salud, Plan Nacional de Investigación Científica, Desarrollo e Innovación Tecnológica 2008-2011, Instituto de Salud Carlos III, European Region Development Fund (ERDF); Bristol-Myers Squibb (BMS AI471-041); AIM-HIV Network of Excellence of the EU, grant number HEALTH-F3-2012-305938; joined program of the Agence Nationale de Recherches sur le SIDA et les Hépatites Virales (ANRS AO2014-2); and the European FP7-HEALTH project HIT HIDDEN HIV. The work of Elena Mateos is supported by the Instituto de Salud Carlos III (Spain) (MPY 1371/12). The work of Sara Rodríguez-Mora is supported by a fellowship of Sara Borrell from Spanish Ministry of Economy and Competitiveness. The work of María Rosa López-Huertas is supported by a fellowship of the European Union Programme Health 2009 (CHAARM). The work of Dr Benjamin Descours is supported by the 31 European FP7-HEALTH project HIT HIDDEN HIV. Dr. Montserrat Plana is a researcher at the Institut d’Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS) and is supported by the Spanish Health Institute Carlos III (ISCIII) and the Health Department of the Catalan Government (Generalitat de Catalunya). Dr. Juan Ambrosioni developed this work in the framework of a ‘Juan de la Cierva 2012’ post- doctoral program, Ministerio de Competitividad, Madrid, Spain. Sí

PKCθ is essential for the activation of CD4(+) T cells. Upon TCR/CD28 stimulation, PKCθ is phosphorylated and migrates to the immunological synapse, inducing the activation of cellular transcription factors such as NF-κB and kinases as ERK that are critical for HIV-1 replication. We previously demonstrated that PKCθ is also necessary for HIV-1 replication but the precise mechanism is unknown. Efficient HIV-1 transcription and elongation are absolutely dependent on the synergy between NF-κB and the viral regulator Tat. Tat exerts its function by binding a RNA stem-loop structure proximal to the viral mRNA cap site termed TAR. Besides, due to its effect on cellular metabolic pathways, Tat causes profound changes in infected CD4(+) T cells such as the activation of NF-κB and ERK. We hypothesized that the aberrant upregulation of Tat-mediated activation of NF-κB and ERK occurred through PKCθ signaling. In fact, Jurkat TetOff cells with stable and doxycycline-repressible expression of Tat (Jurkat-Tat) expressed high levels of mRNA for PKCθ. In these cells, PKCθ located at the plasma membrane was phosphorylated at T(538) residue in undivided cells, in the absence of stimulation. Treatment with doxycycline inhibited PKCθ phosphorylation in Jurkat-Tat, suggesting that Tat expression was directly related to the activation of PKCθ. Both NF-κB and Ras/Raf/MEK/ERK signaling pathway were significantly activated in Jurkat-Tat cells, and this correlated with high transactivation of HIV-1 LTR promoter. RNA interference for PKCθ inhibited NF-κB and ERK activity, as well as LTR-mediated transactivation even in the presence of Tat. In addition to Tat-mediated activation of PKCθ in the cytosol, we demonstrated by sequential ChIP that Tat and PKCθ coexisted in the same complex bound at the HIV-1 LTR promoter, specifically at the region containing TAR loop. In conclusion, PKCθ-Tat interaction seemed to be essential for HIV-1 replication in CD4(+) T cells and could be used as a therapeutic target. This work was supported by the Spanish Ministry of Economy and Competitiveness (SAF2010-18388, SAF2013-44677-R, FIS PI12/00506); FIPSE (360924/10); the SPANISH AIDS Research Network RD12/0017/0015 that is included in the Spanish I + D + I Plan and is cofinanced by ISCIII-Subdirección General de Evaluacion and European Funding for Regional Development (FEDER); EUROPRISE Network of Excellence of the EU, grant number LSHP CT-2006-037611, and Agence nationale de recherches sur le sida et les hépatites virales (ANRS 2014-2). The work of EM is supported by a contract of the Instituto de Salud Carlos III (Spain) (MPY 1371/12). The work of SR-M is supported by a fellowship of Sara Borrell from Spanish Ministry of Economy and Competitiveness. The work of ML-H is supported by a fellowship of the European Union Programme Health 2009 (CHAARM). Sí

The interest in developing new diagnostic methods based on arrays of multiple probes to detect and type simultaneously a wide range of different infectious agents is increasing. This becomes a necessity in the case of infectious agents such as respiratory viruses that cause diseases with very similar signs and symptoms. Such tools will permit rapid and accurate diagnosis of different agents causing respiratory infection leading to the most adequate prevention and/or treatment measures. In this article a reverse-line blot hybridization (RLB) assay for the detection of a wide range of respiratory viruses is presented and evaluated for its usefulness in routine diagnosis. This assay employs an array of 18 oligonucleotide probes immobilized on a nylon membrane. Biotin-labeled PCR products obtained with two multiplex reverse transcription (RT)-polymerase chain reaction (PCR) assays described previously, which allow for the detection of fourteen different groups of respiratory viruses, were hybridized to the oligonucleotide array. Detection was performed using a chemiluminescent method. The standardization of the method showed that the RLB assay could be an alternative to the nested PCR assay for enhancing the sensitivity in the detection of the amplified products, avoiding the problem of cross-over contamination, increasing the specificity, and therefore simplifying the method. This is of main interest in laboratories with few facilities. The feasibility and accuracy of the RT-PCR-RLB assay for detecting respiratory viruses proves that such approach could be a first stage to develop a microarray assay for routine diagnosis of infectious diseases.

HIV-1 replication results in mitochondrial damage that is enhanced during antiretroviral therapy (ART). The onset of HIV-1 replication is regulated by viral protein Tat, a 101-residue protein codified by two exons that elongates viral transcripts. Although the first exon of Tat (aa 1–72) forms itself an active protein, the presence of the second exon (aa 73–101) results in a more competent transcriptional protein with additional functions. Results: Mitochondrial overall functions were analyzed in Jurkat cells stably expressing full-length Tat (Tat101) or one-exon Tat (Tat72). Representative results were confirmed in PBLs transiently expressing Tat101 and in HIV-infected Jurkat cells. The intracellular expression of Tat101 induced the deregulation of metabolism and cytoskeletal proteins which remodeled the function and distribution of mitochondria. Tat101 reduced the transcription of the mtDNA, resulting in low ATP production. The total amount of mitochondria increased likely to counteract their functional impairment. These effects were enhanced when Tat second exon was expressed. Conclusions: Intracellular Tat altered mtDNA transcription, mitochondrial content and distribution in CD4+ T cells. The importance of Tat second exon in non-transcriptional functions was confirmed. Tat101 may be responsible for mitochondrial dysfunctions found in HIV-1 infected patients. We greatly appreciate the secretarial assistance of Mrs Olga Palao. This work was supported by FIPSE (360924/10), Spanish Ministry of Economy and Competitiveness (SAF2010-18388), Spanish Ministry of Health (EC11- 285), AIDS Network ISCIII-RETIC (RD12/0017/0015), Instituto de Salud Carlos III, Spanish Ministry of Economy and Competitiveness (FIS PI12/00506). The work of Sara Rodríguez-Mora is supported by a fellowship of Sara Borrell from Spanish Ministry of Economy and Competitiveness (2013). The work of María Rosa López-Huertas is supported by a fellowship of the European Union Programme Health 2009 (CHAARM). Sí

Background Control of RNA polymerase II (RNAPII) release from pausing has been proposed as a checkpoint mechanism to ensure optimal RNAPII activity, especially in large, highly regulated genes. HIV-1 gene expression is highly regulated at the level of elongation, which includes transcriptional pausing that is mediated by both viral and cellular factors. Here, we present evidence for a specific role of the elongation-related factor TCERG1 in regulating the extent of HIV-1 elongation and viral replication in vivo. Results We show that TCERG1 depletion diminishes the basal and viral Tat-activated transcription from the HIV-1 LTR. In support of a role for an elongation mechanism in the transcriptional control of HIV-1, we found that TCERG1 modifies the levels of pre-mRNAs generated at distal regions of HIV-1. Most importantly, TCERG1 directly affects the elongation rate of RNAPII transcription in vivo. Furthermore, our data demonstrate that TCERG1 regulates HIV-1 transcription by increasing the rate of RNAPII elongation through the phosphorylation of serine 2 within the carboxyl-terminal domain (CTD) of RNAPII and suggest a mechanism for the involvement of TCERG1 in relieving pausing. Finally, we show that TCERG1 is required for HIV-1 replication. Conclusions Our study reveals that TCERG1 regulates HIV-1 transcriptional elongation by increasing the elongation rate of RNAPII and phosphorylation of Ser 2 within the CTD. Based on our data, we propose a general mechanism for TCERG1 acting on genes that are regulated at the level of elongation by increasing the rate of RNAPII transcription through the phosphorylation of Ser2. In the case of HIV-1, our evidence provides the basis for further investigation of TCERG1 as a potential therapeutic target for the inhibition of HIV-1 replication, This work was supported by grants from the Spanish Ministry of Science and Innovation (BFU2011-24577), the Foundation for Research and Prevention of AIDS in Spain (FIPSE-36768/08), and the Andalusian Government (Excellence Project CVI-4626/2009) to C.S.; by the Spanish Ministry of Science and Innovation (BFU2009-08796), and the Andalusian Government (Excellence Project CTS-6587) to C.H.M; and by FIPSE (360924/10), the Spanish Ministry of Economy and Competitiveness (SAF2010-18388; FIS PI0120506), the Spanish Ministry of Health (EC11-285, -278), Instituto de Salud Carlos III, AIDS Network ISCIII-RETIC (RD12/0017/0015), and the Health Programme 2009 on Combined Highly Active Anti-Retroviral Microbicides (CHAARM) to M.C. and J.A. Support from the European Region Development Fund, ERDF (FEDER) is also acknowledged. M.M. was supported by a fellowship from the Spanish Ministry of Education (FPU program). M.R.L.H was supported by a fellowship from the European Union (CHAARM). C.L.S. was funded by a fellowship from the Foundation for Medical Research (F.R.M., France) and by funds from NIH RO1 GM071037 (USA) to M.A.G-B.

BACKGROUND: Degradation of p65/RelA has been involved in both the inhibition of NF-kappaB-dependent activity and the onset of apoptosis. However, the mechanisms of NF-kappaB degradation are unclear and can vary depending on the cell type. Cleavage of p65/RelA can produce an amino-terminal fragment that was shown to act as a dominant-negative inhibitor of NF-kappaB, thereby promoting apoptosis. However, the opposite situation has also been described and the production of a carboxy-terminal fragment that contains two potent transactivation domains has also been related to the onset of apoptosis. In this context, a carboxy-terminal fragment of p65/RelA (DeltaNH2p65), detected in non-apoptotic human T lymphocytes upon activation, has been studied. T cells constitute one of the long-lived cellular reservoirs of the human immunodeficiency virus type 1 (HIV-1). Because NF-kappaB is the most important inducible element involved in initiation of HIV-1 transcription, an adequate control of NF-kappaB response is of paramount importance for both T cell survival and viral spread. Its major inhibitor IkappaBalpha constitutes a master terminator of NF-kappaB response that is complemented by degradation of p65/RelA. RESULTS AND CONCLUSIONS: In this study, the function of a caspase-3-mediated carboxy-terminal fragment of p65/RelA, which was detected in activated human peripheral blood lymphocytes (PBLs), was analyzed. Cells producing this truncated p65/RelA did not undergo apoptosis but showed a high viability, in spite of caspase-3 activation. DeltaNH2p65 lacked most of DNA-binding domain but retained the dimerization domain, NLS and transactivation domains. Consequently, it could translocate to the nucleus, associate with NF-kappaB1/p50 and IkappaBalpha, but could not bind -kappaB consensus sites. However, although DeltaNH2p65 lacked transcriptional activity by itself, it could increase NF-kappaB activity in a dose-dependent manner by hijacking IkappaBalpha. Thus, its expression resulted in a persistent transactivation activity of wild-type p65/RelA, as well as an improvement of HIV-1 replication in PBLs. Moreover, DeltaNH2p65 was increased in the nuclei of PMA-, PHA-, and TNFalpha-activated T cells, proving this phenomenon was related to cell activation. These data suggest the existence of a novel mechanism for maintaining NF-kappaB activity in human T cells through the binding of the carboxy-terminal fragment of p65/RelA to IkappaBalpha in order to protect wild-type p65/RelA from IkappaBalpha inhibition. This work was supported by the following projects: FIPSE 36633/07; ISCIII-RETIC RD06/0006; FIPSE 36584/06; FIS PI040614; Network of Excellence EUROPRISE; and VIRHOST Network from Comunidad de Madrid, Spain. M.R. López-Huertas is a pre-doctoral fellow funded by FIPSE 36453/03 and "Plan Nacional del SIDA" (MVI 1434/05-5). Sí

Human parainfluenza virus types 1 and 3 (HPIV1 and HPIV3, respectively), members of the virus family Paramyxoviridae, are common causes of lower respiratory tract infections in infants, young children, the immunocompromised, the chronically ill, and the elderly. In order to synthesize recombinant HPIV1 and HPIV3 nucleocapsid proteins, the coding sequences were cloned into the yeast Saccharomyces cerevisiae expression vector pFGG3 under control of GAL7 promoter. A high level of recombinant virus nucleocapsid proteins expression (20-24 mg l(-1) of yeast culture) was obtained. Electron microscopy demonstrated the assembly of typical herring-bone structures of purified recombinant nucleocapsid proteins, characteristic for other paramyxoviruses. These structures contained host RNA, which was resistant to RNase treatment. The nucleocapsid proteins were stable in yeast and were easily purified by caesium chloride gradient ultracentrifugation. Therefore, this system proved to be simple, efficient and cost-effective, suitable for high-level production of parainfluenza virus nucleocapsids as nucleocapsid-like particles. When used as coating antigens in an indirect ELISA, the recombinant N proteins reacted with sera of patients infected with HPIV1 or 3. Serological assays to detect HPIV-specific antibodies could be designed on this basis.

Background In HIV-infected T lymphocytes, NF-κB/Rel transcription factors are major elements involved in the activation of LTR-dependent transcription from latency. Most NF-κB heterodimer p65/p50 is sequestered as an inactive form in the cytoplasm of resting T lymphocytes via its interaction with IκB inhibitors. In these cells, both absolute HIV latency and low level ongoing HIV replication have been described. These situations could be related to differences in the balance between NF-κB and IκBα ratio. Actually, control of IκBα by cellular factors such as Murr-1 plays a critical role in maintaining HIV latency in unstimulated T lymphocytes. Formerly, our group demonstrated the presence of nuclear IκBα in T cells after PMA activation. Now we attempt to determine the dynamics of NF-κB/IκBα nucleocytosolic transport in absence of activation as a mechanism to explain both the maintenance of latency and the existence of low level ongoing HIV replication in resting CD4 + T lymphocytes. Results and conclusion We show that the inhibition of the nuclear export by leptomycin B in resting CD4 + T cells resulted in nuclear accumulation of both IκBα and p65/RelA, as well as formation of NF-κB/IκBα complexes. This proves the existence of a rapid shuttling of IκBα between nucleus and cytosol even in absence of cellular activation. The nuclear accumulation of IκBα in resting CD4 + T lymphocytes results in inhibition of HIV-LTR dependent transcription as well as restrains HIV replication in CD4 + T lymphocytes. On the other hand, basal NF-κB activity detected in resting CD4 + T lymphocytes was related to low level HIV replication in these cells.

The rapid development of new molecular biology methods has improved infectious disease diagnosis, which is increasingly important to clinical management and public health. A wide variety of new methods which are more specific, sensitive and robust, such as combination of PCR and microarray technology, has gradually replaced the conventional assays usually used in routine diagnosis. Both methods have the advantage of speed and sensitivity but tend to be expensive and technically demanding. Therefore, it is necessary to develop more simple assays that could be available for all diagnostic laboratories. To this aim, a simple microarray assay for detection and typing of adenoviruses causing acute respiratory disease in humans was developed. The absence of effective therapeutic or alternative prophylactic treatment for this infection makes essential its rapid diagnosis to implement fast control measures. Moreover, the family Adenoviridae includes numerous serotype groups and constitutes an ideal model system to develop diagnostic methods for other human pathogens. For their precise identification, an amplicon retrieval software that simplifies and accelerates the processing of all sequences necessary to perform this type of experiments has also been developed. This new technology was applied for the simultaneous detection and serotyping of acute respiratory disease-causing adenoviruses in laboratory and clinical samples with efficiency and accuracy.

A simple molecular technique was used for the rapid preliminary genetic characterization of human influenza A H3N2 viruses isolated in Spain from 1996 to 2000. Subtyping, based on RT-PCR, was followed by subtype-specific restriction enzyme fragment length polymorphism (RFLP) analyses of an amplified region of the HA1 domain of the H3 haemagglutinin (HA) gene to distinguish variants differentiated by common amino acid substitutions in HA1. The approach was tested using 135 Spanish H3N2 isolates and included nucleotide sequencing and phylogenetic analyses of a region of the HA1 domain of 41 representative isolates. The viruses were distinguished by haemagglutination inhibition (HI) assays into two antigenically discernible groups, the A/Wuhan/359/95-like and A/Sydney/5/97-like viruses. The results of PCR-RFLP analysis allowed a finer classification into five genetic variant subgroups, corresponding to those distinguished by phylogenetic analyses. This rapid, simple and variant-specific procedure could, therefore, be used to rapidly screen clinical specimens prior to more detailed antigenic and genetic analyses.

Methods For years, the molecular mechanisms leading to HIV-1 reactivation have been characterised in detail but the study of latency has remained elusive due to the technical limitations that arise when a negative phenomenon, like the absence of replication, is studied. Development of new techniques for studying HIV-1 latency, the identification of factors that restrict retroviral infections, the characterisation of chromatin structure in the setting of viral integration, and the discovery of new systems regulating gene expression

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