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During 2011–2015, we conducted a Crimean-Congo hemorrhagic fever virus (CCHFV) survey in captured ticks that were feeding mainly on wild and domestic ungulates in Spain, where presence of this virus had been reported previously. We detected CCHFV RNA in Hyalomma lusitanicum and H. marginatum ticks for 3 of the 5 years. The rate of infected ticks was 2.78% (44/1,579), which was similar to those for other countries in Europe with endemic foci for CCHFV (Kosovo, Bulgaria, and Albania). These data confirm the established spread of CCHFV into western Europe. Phylogenetic study of the small RNA segment showed Africa-3 clade as the only genotype identified, although we observed cocirculation of genetic variants during 2011 and 2015. We could not rule out genetic reassortments because of lack of sequence data for the medium and large RNA segments of the virus genome.

Although Lloviu virus (LLOV) was discovered in the carcasses of insectivorous Schreiber&rsquo, s Bent-winged bats in the caves of Northern Spain in 2002, its infectivity and pathogenicity remain unclear. We examined the seroprevalence of LLOV in potentially exposed Schreiber&rsquo, s Bent-winged bats (n = 60), common serotine bats (n = 10) as controls, and humans (n = 22) using an immunoblot assay. We found antibodies against LLOV GP2 in all of Schreiber&rsquo, s Bent-winged bats serum pools, but not in any of the common serotine bats and human pools tested. To confirm this seroreactivity, 52 serums were individually tested using Domain Programmable Arrays (DPA), a phage display based-system serology technique for profiling filovirus epitopes. A serological signature against different LLOV proteins was obtained in 19/52 samples tested (36.5%). The immunodominant response was in the majority specific to LLOV-unique epitopes, confirming that the serological response detected was to LLOV. To our knowledge, this is the first serological evidence of LLOV exposure in live captured Schreiber&rsquo, s Bent-winged bats, dissociating LLOV circulation as the cause of the previously reported die-offs.

Certain host genetic variants, especially in the human leucocyte antigen (HLA) region, are associated with different progression of HIV-1-induced diseases and AIDS. Long term non progressors (LTNP) represent only the 2% of infected patients but are especially relevant because of their efficient HIV control. In this work we present a global analysis of genetic data in the large national multicenter cohort of Spanish LTNP, which is compared with sero-negative individuals and HIV-positive patients. We have analyzed whether several single-nucleotide polymorphisms (SNPs) including in key genes and certain HLA-A and B alleles could be associated with a specific HIV phenotype. A total of 846 individuals, 398 HIV-1positive patients (213 typical progressors, 55 AIDS patients, and 130 LTNPs) and 448 HIV-negative controls, were genotyped for 15 polymorphisms and HLA-A and B alleles. Significant differences in the allele frequencies among the studied populations identified 16 LTNP-associated genetic factors, 5 of which were defined for the first time as related to LTNP phenotype: the protective effect of HLA-B39, and the detrimental impact of HLA-B18, -A24, -B08 and –A29. The remaining eleven polymorphisms confirmed previous publications, including the protective alleles HLA-B57, rs2395029 (HCP5), HLA bw4 homozygosity, HLA-B52, HLA-B27, CCR2 V64I, rs9264942 (HLA-C) and HLA-A03; and the risk allele HLA bw6 homozygosity. Notably, individual Spanish HIV-negative individuals had an average of 0.12 protective HLA alleles and SNPs, compared with an average of 1.43 protective alleles per LTNP patient, strongly suggesting positive selection of LTNP. Finally, stratification of LTNP according to viral load showed a proportional relationship between the frequency of protective alleles with control of viral load. Interestingly, no differences in the frequency of protection/risk polymorphisms were found between elite controllers and LTNPs maintaining viral loads, Instituto de Salud Carlos III from the Spanish Ministry of Health, through Red Temática de Investigación Cooperativa en SIDA (RIS) [G03/173(2), PI050528 and RD06/0006/0033 to MDV, RD06/0006/0035 and RD12/0017/0037 to the HIV BioBank, and C03/173 and RD12/0017/0018 to CoRIS], cofinanced by ISCIII-Subdirección General de Evaluación and Fondo Europeo de Desarrollo Regional (FEDER), and also financed by Plan Nacional I+D+I from the Spanish Ministry of Science and Technology [SAF2007-60934, SAF2010-18917 and SAF2013-48754-C2-1-R to MDV], by Instituto de Salud Carlos III [Intrasalud PI12/0056 to JA] and by Fundación para la Investigación y Prevención del SIDA en España (FIPSE) [to HIV Biobank]. Institutional grants from the Fundación Ramón Areces and Banco de Santander to the CBMSO

The use of ribavirin to treat Crimean-Congo hemorrhagic fever virus (CCHFV) infection has been controversial, based on uncertainties about its antiviral efficacy in clinical case studies. We studied the effect of ribavirin treatment on viral populations in a recent case by deep-sequencing analysis of plasma samples obtained from a CCHFV-infected patient before, during, and after a 5-day regimen of ribavirin treatment. The CCHFV load dropped during ribavirin treatment, and subclonal diversity (transitions) and indels increased in viral genomes during treatment. Although the results are based on a single case, these data demonstrate the mutagenic effect of ribavirin on CCHFV in vivo.

Crimean-Congo hemorrhagic fever (CCHF) is a widely distributed, viral, tickborne disease. In Europe, cases have been reported only in the southeastern part of the continent. We report two autochthonous cases in Spain. The index patient acquired the disease through a tick bite in the province of Ávila - 300 km away from the province of Cáceres, where viral RNA from ticks was amplified in 2010. The second patient was a nurse who became infected while caring for the index patient. Both were infected with the African 3 lineage of this virus. (Funded by Red de Investigación Cooperativa en Enfermedades Tropicales [RICET] and Efficient Response to Highly Dangerous and Emerging Pathogens at EU [European Union] Level [EMERGE].). Supported by Red de Investigación Cooperativa en Enferme-dades Tropicales (RICET grant, RD12/0018/0023) and EMERGE (Efficient Response to Highly Dangerous and Emerging Patho-gens at EU [European Union] Level), a joint action funded under the third EU Health Program (677066),.Disclosure forms provided by the authors are available with the full text of this article at NEJM.org.We thank Julio Farias of the Pathology Department at Gregorio Marañón University General Hospital and Maria José Buitrago and the rest of the members of the Grupo de Respuesta Rápida of the Centro Nacional de Microbiología for their help. Sí

Since the first documented autochthonous transmission of chikungunya virus in the Caribbean island of Saint Martin in 2013, the infection has been reported within the Caribbean region as well as North, Central and South America. The risk of autochthonous transmission of chikungunya virus becoming established in Spain may be elevated due to the large numbers of travellers returning to Spain from countries affected by the 2013 epidemic in the Caribbean and South America, as well as the existence of the Aedes albopictus vector in certain parts of Spain. We retrospectively analysed the laboratory diagnostic database of the National Centre for Microbiology, Institute of Health Carlos III (CNM-ISCIII) from 2008 to 2014. During the study period, 264 confirmed cases, of 1,371 suspected cases, were diagnosed at the CNM-ISCIII. In 2014 alone, there were 234 confirmed cases. The highest number of confirmed cases were reported from the Dominican Republic (n = 136), Venezuela (n = 30) and Haiti (n = 11). Six cases were viraemic in areas of Spain where the vector is present. This report highlights the need for integrated active case and vector surveillance in Spain and other parts of Europe where chikungunya virus may be introduced by returning travellers. We would like to thank: Virginia Jimenez Planet (National Library of Health, ISCIII), Hospital of Navarra, Hospital Gregorio Marañon, Hospital German Trials iPujol and BR Salud Laboratory and other hospitals belonging to the national health system. In addition we thank the two anonymous reviewers. Financial support was provided by the Fondo de Investigaciones Sanitarias (FIS) PI10/00069 and FIS PI08/0834 and partially covered by DengueTools project under the Seventh Framework Programme of the European Community, Grant Agreement Number: 282589. LF was under contract by European Network of Imported Viral Disease-Collaborative Laboratory Response Network (ENIVD-CLRN) project funded by the European Centre for Disease Prevention and Control (ECDC) under the Framework Service Contract Ref. No. ECDC/2013/012. Sí

West Nile virus (WNV) is the most widespread arbovirus in the world. Several recent outbreaks and epizootics have been reported in Europe and the Mediterranean basin with increased virulence. In contrast to the well-characterized American and Australian strains, little is known about the virulence determinants of the WNV European-Mediterranean strains. To investigate the viral factors involved in the virulence of these strains, we generated chimeras between the highly neuropathogenic Israel 1998 (IS-98-ST1, IS98) strain and the non-pathogenic Malaysian Kunjin virus (KJMP-502). In vivo analyses in a mouse model of WNV pathogenesis shows that chimeric virus where KJMP-502 E glycoprotein was replaced by that of IS98 is neuropathogenic, demonstrating that this protein is a major virulence determinant. Presence of the N-glycosylation site had limited impact on virus virulence and the 5'UTR does not seem to influence pathogenesis. Finally, mice inoculated with KJMP-502 virus were protected against lethal IS98 infection.

Genetic recombination and high rate of mutation increase HIV-1 diversity, allowing viruses to escape more easily from the host immune response or antiretroviral drugs. The recombinant nature of full-length HIV-1 genomic sequences derived from viruses infecting five epidemiologically unlinked individuals carrying HIV-1 non-B variants was investigated. Overlapping PCR amplifications followed by direct sequencing of viral products derived from plasma and phylogenetic analyses were carried out. Four viral sequences clustered with CRF06_cpx and one with CRF02_AG. However, subtyping of separate genes within the same genome revealed that four were recombinant forms involving different subtypes and/or CRFs with distinct breakpoints. Two specimens included CRF02_AG and CRF06_cpx sequences with several fragments from other HIV-1 clades along their genomes. Three rapid subtyping tools (Stanford, NCBI, and REGA) showed discrepant results when interpreting these viral sequences. This is the first description of CRF02_AG/CRF06_ cpx recombinants in Spain. The results highlight the tremendous heterogeneity of HIV-1 recombinant strains currently in circulation.

A distinct effectiveness of highly active antiretroviral therapy (HAART) in HIV-infected patients across ethnicities may reflect differences in drug adherence, host genetic factors, and/or predominant HIV subtypes. We investigated the immunologic outcome in 79 drug-naive HIV individuals living in Madrid, 39 of whom were African immigrants, who achieved and maintained undetectable viral load for up to 24 months following initiation of HAART. Overall, 90% of whites were infected with clade B viruses while 80% of Africans carried non-B subtypes. Gender, age, mean baseline viral load, and CD4 counts were comparable in both groups. The mean time to reach undetectable viremia did not differ significantly between groups. CD4 gains at 24 months following HAART initiation were also similar, although Africans showed higher CD4 gains than whites at month 12. In a multivariate analysis neither HIV subtype nor ethnicity was associated with different CD4 gains at any given time point, suggesting that reconstitution of CD4+ T cells under HAART is not influenced by ethnicity.

The long terminal repeat (LTR) region of HIV-1 promotes and modulates proviral transcription. LTR genetic variability might influence viral replication and disease progression. Proviral LTR sequences from 32 HIV-1-infected individuals showing different rates of disease progression were examined. Non-progressors (NP, n = 11) were individuals with high and stable CD4 counts and persistently low or undetectable plasma HIV-RNA. Slow progressors (SP, n = 6) were subjects with minimal CD4 decays over time and low plasma HIV-RNA. Typical progressors (TP, n = 15) were individuals with chronic infection showing CD4 counts repeatedly below 500 cells/mul. The mutation frequency within distinct LTR functional regions involved in HIV-1 transcription were compared in these three groups of patients. No significant differences were observed in the mutation frequency in most LTR regulatory sites when comparing the three groups. However, changes in USF regulatory binding sites were more frequent in TP than in SP/NP, while changes in Sp1 binding sites were less common in the former. This is the first study examining the genetic variability of the HIV-1 LTR region in long-term non-progressors showing further divergent outcomes.

Transcriptional activation of HIV-1 gene expression is controlled by the interaction of sequence-specific transcription factors with the long terminal repeat (LTR). Information about differences between LTR regions in distinct HIV-1 subtypes is scarce. LTR sequences, including regulatory, enhancer, promoter, and TAR regions, were genetically characterized and compared in 59 HIV-1-infected individuals known to be infected with non-B subtypes. Phylogenetic analyses ascribed the LTR regions to the following subtypes: 10A, 2B, 6C, 1D, 2E, 2F, 16G, 3J, 2H, and 2U. Up to 34% of the samples were LTR/PR recombinants. The LTR region revealed a high degree of genetic variability among distinct HIV-1 subtypes and showed several subtype-specific markers, which hypothetically could influence the interactions with cellular transcription factors, leading to different transcriptional levels among distinct HIV-1 clades. To our knowledge, this is the first characterization of LTR sequences belonging to subtypes J and H.

La activacion transcripcional de los genes del virus de la inmunodeficiencia humana tipo 1 (VIH-1) esta modulada por la interaccion de factores celulares y proteinas virales con secuencias especificas de los extremos largos repetidos (LTR) del genoma proviral. Existen diferencias en las secuencias LTR de los distintos subtipos del VIH-1 que pueden afectar la capacidad de union de los distintos factores de transcripcion y a su funcionalidad en el inicio de la transcripcion viral. La repercusion de esta variabilidad sobre la biologia de los diferentes subtipos del VIH-1 y sobre la progresion de la inmunodeficiencia en los individuos infectados es controvertida. Sin embargo, en determinadas situaciones, existe una clara asociacion entre la capacidad replicativa in vitro y cambios en las secuencias de union de factores de transcripcion en las secuencias reguladoras del virus.

INTRODUCTION: Human immunodeficiency virus type 1 (HIV-1) dual infection (DI) in long-term nonprogressor-elite controller patients (LTNP-EC) has been described only in sporadic cases and then, consequences in disease progression are not clearly established. To fill-up this limited knowledge, we analyzed, for the first time, the prevalence, host genetic polymorphisms, and clinical consequences of HIV-1 DI in a group of LTNP-EC. METHODS: For DI detection, nucleotide sequences in env gene from viruses from 20 LTNP-EC were analyzed by maximum likelihood. Epidemiological and clinical parameters and host factors of patients were also studied. RESULTS: DI was detected in 4 (20%) of the 20 LTNP-EC, of which 3 maintained the elite controller status. CD4 T-cell counts were not different between single and DI patients although higher CD8 T-cell counts were observed in DI patients, and, consequently, the CD4/CD8 ratios were lower in LTNP-EC DI patients. CONCLUSIONS: Prevalence of HIV-1 DIs in LTNP-EC is similar to other groups of HIV-1 patients; in addition, DI was not associated with loss of disease control in the patients. These DI LTNP-EC patients showed, in comparison with single infected patients, higher numbers of CD8 T cells and lower CD4/CD8 ratios. Copyright © 2013 by Lippincott Williams & Wilkins.

Susceptibility to HIV infection and disease progression are complex traits modulated by environmental and genetic factors, affecting innate and adaptive immune responses, among other cellular processes. A single nucleotide polymorphism (SNP) 35. kb upstream of the HLA-C gene locus (-35C/T) was previously shown to correlate with increased HLA-C expression and improved control of HIV-1. Here, we genotyped the -35C/T SNP in 639 subjects (180 uninfected patients, 304 HIV progressors and 155 LTNP) and confirmed the association of the -35C/T variant with the LTNP phenotype. The genotype frequencies in the general population subjects did not differ significantly from those seen in HIV progressors (p-value = 0.472). However, a significant higher frequency of the protective CC genotype was identified when LTNP were compared either with HIV progressors alone (p-value < 0.0001) or progressors and uninfected subjects together (p-value < 0.0001). When considering aviremic LTNP alone (elite controllers; viral load below 50. copies/ml), the -35 CC genotype was not overrepresented compared to HIV progressors. Conversely, a significant association was found with the viremic LTNP groups (viral loads below 10,000. copies/ml). These results suggest that other factors alone or in combination with the -35 CC genotype may play an important role in differentiating the elite controller status from LTNP. Combination of different genetic variants may have additive or epistatic effects determining the HIV course of infection. © 2012 Elsevier GmbH., Ministerio de Ciencia e Innovación (BFU2009-06958, SAF2010-18917, RD06/0006/0033); FIPSE 360783-09 and FIPSE 360737-09 projects; the Gala contra la SIDA 2011; Bill and Melinda Gates Foundation (CEvac); Fondo de Investigación Sanitaria (FIS)

Transcriptional activation of HIV-1 gene expression is partially controlled by the interaction between viral and cellular transcription factors acting at HIV-1 long terminal repeat (LTR) sequences. HIV-1 subtyping at LTR region and nucleotide LTR variability from clinical samples in 48 subjects carrying different HIV-1 subtypes (9A, 5C, 3D, 3F, 21G, 2H, 3J and 2 undefined) at the protease (PR) gene, were performed. LTR sequences from each HIV-1 clade were cloned in luciferase-expression vectors to determine basal and Tat-induced transcriptional activities in the presence and absence of PMA stimulation. A high number (37.8%) of recombinants at LTR/PR regions were identified. All HIV-1 promoters presented low basal transcriptional activity that was nevertheless induced by Tat and PMA. LTR activity was similar across the majority of HIV-1 variants in response to Tat and cell activation. Only subtype C and CRF01_AE LTRs presented higher basal and induced-PMA transcription activities than HXB2 clade B promoter. No basal or Tat/PMA induced activity was found in those promoters presenting G to A hypermutation compared to the wild type promoter activities. G to A hypermutation at some important transcription binding-factor sites within LTR compromised the activity of the viral promoter, decreasing the in vitro viral transcription of the luciferase gene.

Information about gp41 variability across distinct HIV-1 subtypes is scarce, and yet such knowledge would be desirable for designing new drugs targeting this viral protein. Conserved gp41 residues in viruses derived from 79 individuals infected with distinct HIV-1 subtypes (29 A, 25 B, 8 C, 3 D, 4 F, 4 G, 2 H, 1 J, 1 U, and 2 CRF06_cpx) and naive for entry inhibitors were examined. Conservation of gp41 was also examined in 908, 56, and 3 HIV-1 group M, O, and N sequences, respectively, available at the Los Alamos HIV Sequence Database. Among the 345 residues in the full gp41 protein, 36% showed up to 90% conservation in all 987 group M sequences, as did 40% of 56 group O sequences and 49% of 3 group N sequences. The HR1 region (residues 29-82) showed a higher proportion of highly conserved residues than did the HR2 region (residues 116-161) in all groups (65 vs. 34% in group M, 57 vs. 46% in group O, and 80 vs. 52% in group N). Some secondary resistance mutations to enfuvirtide were found as natural polymorphisms (A30V and Q56K/R in group M, Q56R and S138A in group O, and S138A in group N). In fact, A30V was a signature change in clade G and CRF06_cpx, whereas Q56K/R was a signature change for clades A and J, as well as for CRF04_cpx, CRF09_cpx, CRF11_cpx, and CRF13_cpx. The relative conservation of amino acids in gp41 across HIV-1 variants indirectly highlights the critical role of this protein for HIV infectivity and makes it feasible to design new entry inhibitors with activity against diverse HIV-1 variants.

Transcriptional activation of gene expression in HIV-1 is controlled by the interaction of sequence-specific transcription factors with the long terminal repeat (LTR) of the provirus. The identification and characterization of cellular proteins involved in the process has provided a basic understanding about both general eukaryotic and HIV-1 proviral transcription regulation. The HIV-1 epidemic is expanding worldwide with an increasing number of distinct viral subtypes as well as intersubtype recombinant viruses. LTR-specific sequence variability among different HIV-1 variants could affect LTR binding to cellular and/or viral factors, influencing the extent of transcription. In vitro assays have demonstrated subtype-specific functional differences between the LTR regions of distinct HIV-1 subtypes. This observation could have consequences on the biology of the different HIV-1 clades and influence HIV-1 disease progression. Finally, the knowledge of the molecular mechanisms of transcription regulation events could help in the search for new compounds targeting the critical steps of viral transcription.

Transcriptional activation of HIV-1 gene expression is controlled in part by the interaction of viral and cellular transcription factors with the HIV-1 long terminal repeat (LTR) sequences. LTR variability among different HIV-1 subtypes could affect LTR binding of either cellular or viral elements, influencing the transcription level. This effect, in turn, may have consequences on the biology of the different HIV-1 clades and on disease progression. In some circumstances, a relationship between replication capacity in vitro and changes in binding sequences for transcription factors located at the LTR has been proven.

Lloviu virus (LLOV), a phylogenetically divergent filovirus, is the proposed etiologic agent of die-offs of Schreibers׳s long-fingered bats (Miniopterus schreibersii) in western Europe. Studies of LLOV remain limited because the infectious agent has not yet been isolated. Here, we generated a recombinant vesicular stomatitis virus expressing the LLOV spike glycoprotein (GP) and used it to show that LLOV GP resembles other filovirus GP proteins in structure and function. LLOV GP must be cleaved by endosomal cysteine proteases during entry, but is much more protease-sensitive than EBOV GP. The EBOV/MARV receptor, Niemann–Pick C1 (NPC1), is also required for LLOV entry, and its second luminal domain is recognized with high affinity by a cleaved form of LLOV GP, suggesting that receptor binding would not impose a barrier to LLOV infection of humans and non-human primates. The use of NPC1 as an intracellular entry receptor may be a universal property of filoviruses.