Identification and characterization of the herpes simplex virus type 2 gene encoding the essential capsid protein ICP32/VP19c.

We describe the characterization of the herpes simplex virus type 2 (HSV-2) gene encoding infected cell protein 32 (ICP32) and virion protein 19c (VP19c). We also demonstrate that the HSV-1 UL38/ORF.553 open reading frame (ORF), which has been shown to specify a viral protein essential for capsid formation (B. Pertuiset, M. Boccara, J. Cebrian, N. Berthelot, S. Chousterman, F. Puvian-Dutilleul, J. Sisman, and P. Sheldrick, J. Virol. 63: 2169-2179, 1989), must encode the cognate HSV type 1 (HSV-1) ICP32/VP19c protein. The region of the HSV-2 genome deduced to contain the gene specifying ICP32/VP19c was isolated and subcloned, and the nucleotide sequence of 2,158 base pairs of HSV-2 DNA mapping immediately upstream of the gene encoding the large subunit of the viral ribonucleotide reductase was determined. This region of the HSV-2 genome contains a large ORF capable of encoding two related 50,538- and 49,472-molecular-weight polypeptides. Direct evidence that this ORF encodes HSV-2 ICP32/VP19c was provided by immunoblotting experiments that utilized antisera directed against synthetic oligopeptides corresponding to internal portions of the predicted polypeptides encoded by the HSV-2 ORF or antisera directed against a TrpE/HSV-2 ORF fusion protein. The type-common immunoreactivity of the two antisera and comparison of the primary amino acid sequences of the predicted products of the HSV-2 ORF and the equivalent genomic region of HSV-1 provided evidence that the HSV-1 UL38 ORF encodes the HSV-1 ICP32/VP19c. Analysis of the expression of the HSV-1 and HSV-2 ICP32/VP19c cognate proteins indicated that there may be differences in their modes of synthesis. Comparison of the predicted structure of the HSV-2 ICP32/VP19c protein with the structures of related proteins encoded by other herpes viruses suggested that the internal capsid architecture of the herpes family of viruses varies substantially.

Genome sequence of Fusobacterium nucleatum subspecies polymorphum - a genetically tractable fusobacterium.

Fusobacterium nucleatum is a prominent member of the oral microbiota and is a common cause of human infection. F. nucleatum includes five subspecies: polymorphum, nucleatum, vincentii, fusiforme, and animalis. F. nucleatum subsp. polymorphum ATCC 10953 has been well characterized phenotypically and, in contrast to previously sequenced strains, is amenable to gene transfer. We sequenced and annotated the 2,429,698 bp genome of F. nucleatum subsp. polymorphum ATCC 10953. Plasmid pFN3 from the strain was also sequenced and analyzed. When compared to the other two available fusobacterial genomes (F. nucleatum subsp. nucleatum, and F. nucleatum subsp. vincentii) 627 open reading frames unique to F. nucleatum subsp. polymorphum ATCC 10953 were identified. A large percentage of these mapped within one of 28 regions or islands containing five or more genes. Seventeen percent of the clustered proteins that demonstrated similarity were most similar to proteins from the clostridia, with others being most similar to proteins from other gram-positive organisms such as Bacillus and Streptococcus. A ten kilobase region homologous to the Salmonella typhimurium propanediol utilization locus was identified, as was a prophage and integrated conjugal plasmid. The genome contains five composite ribozyme/transposons, similar to the CdISt IStrons described in Clostridium difficile. IStrons are not present in the other fusobacterial genomes. These findings indicate that F. nucleatum subsp. polymorphum is proficient at horizontal gene transfer and that exchange with the Firmicutes, particularly the Clostridia, is common.

Analysis of a human placental lactogen gene promoter

Human placental lactogen (hPL) and human growth hormone (hGH) comprise a multigene family that share $>$90% nucleic acid sequence homology including 500 bp of 5$\sp\prime$ flanking sequence. Despite these similarities, hGH is produced in the anterior pituitary while hPL is expressed in the placenta. For most genes studied to date, regulation of expression occurs by alterations at the level of transcriptional initiation. Nuclear proteins bind specific DNA sequences in the promoter to regulate gene expression. In this study, the hPL$\sb3$ promoter was analyzed for DNA sequences that contribute to its expression. The interaction between the hPL$\sb3$ promoter and nuclear proteins was examined using nuclear extracts from placental and non-placental cells.^ To identify regulatory elements in the promoter of the hPL$\sb3$ gene, 5$\sp\prime$ deletion mutants were constructed by cleaving 1200 bp of upstream sequence with various restriction enzymes. These DNA fragments were ligated 5$\sp\prime$ to a promoterless bacterial gene chloramphenicol acetyltransferase (CAT) and transfected into JEG-3 cells, a human placental choriocarcinoma cell line. The level of CAT activity reflects the ability of the promoter mutants to activate transcription. Deletion of the sequence between $-$142 bp and $-$129 bp, relative to the start of transcription, resulted in an 8-fold decrease in CAT activity. Nuclear proteins from JEG-3, HeLa, and HepG2 (human liver cells), formed specific binding complexes with this region of the hPL$\sb3$ promoter, as shown by gel mobility shift assay. The $-$142 bp to $-$129 bp region contains a sequence similar to that of a variant binding site for the transcription factor Sp1. Sp1-like proteins were identified by DNA binding assay, in the nuclear extracts of the three cell lines. A series of G nucleotides in the hPL$\sb3$ promoter regulatory region were identified by methylation interference assay to interact with the DNA-binding proteins and the pattern obtained is similar to that for other Sp1 binding sites that have been studied. This suggests that hPL$\sb3$ may be transcriptionally regulated by Sp1 or a Sp1-like transacting factor. ^

REPSA: A method for determining the sequence specificity of DNA binding ligands

We developed a novel combinatorial method termed restriction endonuclease protection selection and amplification (REPSA) to identify consensus binding sites of DNA-binding ligands. REPSA uses a unique enzymatic selection based on the inhibition of cleavage by a type IIS restriction endonuclease, an enzyme that cleaves DNA at a site distal from its recognition sequence. Sequences bound by a ligand are protected from cleavage while unprotected sequences are cleaved. This enzymatic selection occurs in solution under mild conditions and is dependant only on the DNA-binding ability of the ligand. Thus, REPSA is useful for a broad range of ligands including all classes of DNA-binding ligands, weakly binding ligands, mixed populations of ligands, and unknown ligands. Here I describe REPSA and the application of this method to select the consensus DNA-binding sequences of three representative DNA-binding ligands; a nucleic acid (triplex-forming single-stranded DNA), a protein (the TATA-binding protein), and a small molecule (Distamycin A). These studies generated new information regarding the specificity of these ligands in addition to establishing their DNA-binding sequences. ^

Molecular beacons with a homo-DNA stem: improving target selectivity

Molecular beacons (MBs) are stem-loop DNA probes used for identifying and reporting the presence and localization of nucleic acid targets in vitro and in vivo via target-dependent dequenching of fluorescence. A drawback of conventional MB design is present in the stem sequence that is necessary to keep the MBs in a closed conformation in the absence of a target, but that can participate in target binding in the open (target-on) conformation, giving rise to the possibility of false-positive results. In order to circumvent these problems, we designed MBs in which the stem was replaced by an orthogonal DNA analog that does not cross-pair with natural nucleic acids. Homo-DNA seemed to be specially suited, as it forms stable adenine-adenine base pairs of the reversed Hoogsteen type, potentially reducing the number of necessary building blocks for stem design to one. We found that MBs in which the stem part was replaced by homo-adenylate residues can easily be synthesized using conventional automated DNA synthesis. As conventional MBs, such hybrid MBs show cooperative hairpin to coil transitions in the absence of a DNA target, indicating stable homo-DNA base pair formation in the closed conformation. Furthermore, our results show that the homo-adenylate stem is excluded from DNA target binding, which leads to a significant increase in target binding selectivity

Influences of porcine circo-, arteriviruses and cathelicidins in plasmacytoid dendritic cell-derived interferon-alpha responses

Type I interferons (IFNs), mainly IFN-α/β play a crucial role in innate defense against viruses. In addition to their direct antiviral activity, type I IFNs have antitumoral and immunomodulatory effects. Although all cells are virtually able to induce IFN-α, the plasmacytoid dendritic cell (pDC) subset represents the ultimate producers of IFN-α as well as other proinflammatory cytokines. Due to the specific expression of TLR7 and TLR9 recognizing single-stranded (ss) RNA and unmethylated CpG motifs respectively, pDCs can secrete up to 1000 times more IFN-α than any cellular types. Additionally, it is well known that several cytokines including type I and II IFNs, Flt3-L, IL-4 and GM-CSF favor pDC-derived IFN-α responses to unmethylated CpG motifs. In a first step, we aimed to characterize and clarify the interactions of two porcine viruses with pDCs. The double-stranded DNA replicative forms of porcine circovirus type 2 (PCV2) were demonstrated to inhibit CpG-induced IFN- α by pDCs. Our study showed that none of the cytokines known to enhance pDC responsiveness can counter-regulate the PCV2-mediated inhibition of IFN-α induced by CpG, albeit IFN-γ significantly reduced the level of inhibition. Interestingly, the presence of IFN-γ enabled pDCs to induce IFN-α to low doses of PCV2. We also noted that after DNase treatment, PCV2 preparations were still able to stimulate pDCs. These data suggest that encapsulated viral ssDNA promotes the induction of IFN-α in pDCs treated with IFN-γ whereas free DNA, presumably as double-stranded forms, was responsible for inhibiting pDC responses. Regarding PRRSV, it has been reported that North American isolates did not induce and even inhibited IFN-α response in pDCs. However, PRRSV infection was also shown to lead to an induction of IFN-α in the serum and in the lungs suggesting that certain cells are responsive to the virus. Contrasting to previous reports we found that numerous PRRSV isolates directly induced IFN-α in pDCs. This response was still observed after UV-inactivation of viruses and required TLR7 signaling. The inhibition of CpG-induced IFN-α was weak and strain dependent, again contrasting with a previous report. We also observed that IFN-γ and IL-4 enhanced IFN-α response to two prototype strains, VR-2332 and LVP23. In summary, we demonstrated that both PCV2 and PRRSV promote IFN-α secretion in pDCs in vitro suggesting that IFN-α detected in PCV2- or PRRSV-infected animal might originate from pDCs. On the other hand, PRRSV replication is restricted to the macrophage (MΦ) lineage. These innate immune cells represent a heterogeneous population which can be induce to “classical” (M1) and “alternative” (M2) activated MΦ acquiring inflammatory or “wound-healing” functional properties, respectively. Nonetheless, little is known about the effect of polarization into M1 or M2 and the susceptibility of these cells to PRRSV. Thus, we examined the impact of cytokine on MΦ polarization into M1 or M2. Infections of these cells by several PRRSV isolates enabled the discrimination of PRRSV isolate in a genotype- and irulencedependent manner in M1 and IFN-β-activated MΦ. In contrast, the expression of PRRSV nucleocapsid in M2 or inactivated MΦ was indistinguishable among the PRRSV isolates tested. In the last part of my Thesis, we investigated the influence of three synthetic porcine cathelicidin peptides for their ability to deliver nucleic acid to pDCs. We reported that all cathelicidins tested can complex and quickly deliver nucleic acids resulting in IFN-α induction. Moreover, we show that the typical α- helical amphipathic conformation is required to mediate killing of bacteria but not for inducing IFN-α secretion by pDCs. Furthermore, we found that E.coli treated with one of these cathelicidins is able to induce significantly higher levels of IFN-α compared to a non-sense version of the peptide. These data suggest that cathelicidins could influence the immune response in a two-step process. First, these peptides target bacteria leading to cell lysis. In turn, cathelicidins form complexes and deliver extracellular microbial nucleic acids released into pDCs. These pDC-derived IFN-α responses could be of particular relevance in driving the adaptive immune responses against microbial infections.

A modular LHC built on the DNA three-way junction

Over the past years, in numerous studies the DNA double helix serves as a scaffold for the controlled arrangement of functional molecules, including a wide range of different chromophores. Other nucleic acid structures like the DNA three-way junction have been exploited for this purpose as well. Recently, the successful development of DNA-based light-harvesting antenna systems have been reported. Herein, we describe the use of the DNA three-way junction (3WJ) as a versatile scaffold for the modular construction of an artificial light harvesting complex (LHC). The LHC is based on a modular construction in which a phenanthrene antenna is located in one of the three stems and the acceptor is brought into proximity of the antenna through the annealing of the third strand. Phenanthrene excitation (320 nm) is followed by energy transfer to pyrene (resulting in exciplex emission), perylenediimide (quencher) or a cyanine dye (cyanine fluorescence).

The role of FUS in splicing regulation

Fused in sarcoma (FUS), also called translocated in liposarcoma (TLS), is a ubiquitously expressed DNA/RNA binding protein belonging to the TET family and predominantly localized in the nucleus. FUS is proposed to be involved in various RNA metabolic pathways including transcription regulation, nucleo-cytosolic RNA transport, microRNA processing or pre-mRNA splicing [1]. Mutations in the FUS gene were identified in patients with familial amyotrophic lateral sclerosis (ALS) type 6 and sporadic ALS [2, 3]. ALS, also termed Lou Gehrig's disease, is a fatal adult-onset neurodegenerative disease affecting upper and lower motor neurons in the brain and spinal cord. There is increasing evidence supporting the hypothesis that FUS might play an important role in pre-mRNA splicing regulation. Several splicing factors were identified to associate with FUS including hnRNPA2 and C1/C2 [4], Y-box binding protein 1 (YB-1) [5] and serine arginine (SR) proteins (SC35 and TASR) [6]. Additionally, FUS was identified as a constituent of human spliceosomal complexes [1]. Our recent results indicate that FUS has increased affinity for certain but not all snRNPs of the minor and major spliceosome. Furthermore, in vitro studies revealed that FUS directly interacts with a factor specific for one of those snRNPs. These findings might uncover the molecular mechanism by which FUS regulates splicing and could explain previously observed effects of FUS on the splicing of the adenovirus E1A minigene [7] and changes in splicing caused by ALS associated FUS mutations. [1] Lagier-Tourenne C et al. (2010) Human Molecular Genetics 19:46-64 [2] Kwiatkowski TJ Jr et al. (2009) Science 323:1205-8 [3] Vance C et al. (2009) Science 323:1208-11 [4] Zinser H et al. (1994) Genes Dev 8:2513-26 [5] Chansky, H.A., et al. (2001) Cancer Res. 61: 3586-90. [6] Yang L et al. (1998) J Biol Chem 273:27761-6 [7] Kino Y et al. (2010) Nucleic Acid Research 7:2781-2798

The FUS(s) about splicing

Fused in sarcoma (FUS), also called translocated in liposarcoma (TLS), is a ubiquitously expressed DNA/RNA binding protein belonging to the TET family and predominantly localized in the nucleus. FUS is proposed to be involved in various RNA metabolic pathways including transcription regulation, nucleo-cytosolic RNA transport, microRNA processing or pre-mRNA splicing [1]. Mutations in the FUS gene were identified in patients with familial amyotrophic lateral sclerosis (ALS) type 6 and sporadic ALS [2, 3]. ALS, also termed Lou Gehrig's disease, is a fatal adult-onset neurodegenerative disease affecting upper and lower motor neurons in the brain and spinal cord. There is increasing evidence supporting the hypothesis that FUS might play an important role in pre-mRNA splicing regulation. Several splicing factors were identified to associate with FUS including hnRNPA2 and C1/C2 [4], Y-box binding protein 1 (YB-1) [5] and serine arginine (SR) proteins (SC35 and TASR) [6]. Additionally, FUS was identified as a constituent of human spliceosomal complexes [1]. Our recent results indicate that FUS has increased affinity for certain but not all snRNPs of the minor and major spliceosome. Furthermore, in vitro studies revealed that FUS directly interacts with a factor specific for one of those snRNPs. These findings might uncover the molecular mechanism by which FUS regulates splicing and could explain previously observed effects of FUS on the splicing of the adenovirus E1A minigene [7] and changes in splicing caused by ALS associated FUS mutations. [1] Lagier-Tourenne C et al. (2010) Human Molecular Genetics 19:46-64 [2] Kwiatkowski TJ Jr et al. (2009) Science 323:1205-8 [3] Vance C et al. (2009) Science 323:1208-11 [4] Zinser H et al. (1994) Genes Dev 8:2513-26 [5] Chansky, H.A., et al. (2001) Cancer Res. 61: 3586-90. [6] Yang L et al. (1998) J Biol Chem 273:27761-6 [7] Kino Y et al. (2010) Nucleic Acid Research 7:2781-2798

Recent improvements in antigene technology

DNA triple helix based approaches to control and modulate cellular functions on the level of genomic DNA (antigene technology) suffered in the past from a stepmother like treatment in comparison to the flourishing field of oligonucleotide based control of translation (antisense technology). This was mostly due to lack of affinity of triplex forming oligonucleotides (TFOs) to their DNA target, to sequence restriciton constraints imposed by the triple helical recogniton motifs and by open questions to the accessibility of the target DNA. Recent developments in the area have brought about new bases that specifically recognize pyrimidine-purine inversion sites as well as sugar modifications, e.g. the 2'-aminoethoxy-oligonucleotides or oligonucleotides based on the locked nucleic acid (LNA) sugar unit, that greatly enhance triplex stability and alleviate in part the sequence restriction constraints. With this, sequence specific genomic DNA manipulation starts to become a useful tool in biotechnology

Warum Pentose- und nicht Hexose-Nucleinsäuren??. Teil V. (Purin-Purin)-Basenpaarung in der homo-DNS-Reihe: Guanin, Isoguanin, 2,6-Diaminopurin und Xanthin

Why Pentose- and Not Hexose-Nucleic Acids? Purine-Purine Pairing in homo-DNA: Guanine,Isoguanine, 2,6-Diaminopurine, and Xanthine This paper concludes the series of reports in this journal [1–4] on the chemistry of homo-DNA, the constitutionally simplifie dmodel system of hexopyranosyl-(6′ → 4′)-oligonucleotide systems stidued in our laboratory as potentially natural-nucleic-acid alternatives in the context of a chemical aetiology of nucleic-acid structure. The report describes the synthesis and pairing properties of homo-DNA oligonucleotides which contain as nucleobases exclusively purines, and gives, together with part III of the series [3], a survey of what we know today about purine-purine pairingin homo-DNA. In addition, the paper discusses those aspects of the chemistry of homo-DNA which, we think, influence the way how some of the structural features of DNA (and RNA) are to be interpreted on a qualitative level. Purine-purine pairing occurs in the homo-DNA domain in great variety. Most prominent is a novel tridentate Watson-Crick pair between guanine and isoguanine, as well as one between 2,6-diaminopurine and xanthinone, both giving rise to very stable duplexes containing the all-purine strands in antiparallel orientation. For the guanine-isoguanine pair, constitutional assignment is based on temperature-dependent UV and CD spectroscopy of various guanine- and isoguanine-containg duplexes in comparison with duplexes known to be paired in the reverse guanine is replaced by 7-carbauguanine. Isoguanine and 2,6-diaminopurine also have the capability of self-pariring in the reverse-Hoogsteen mode, as previously observed for adenine and guanine [3]. In this type of pairing, the interchangeably. Fig. 36 provides an overall survey of the relative strength of pairing in all possible purine-purine combinations. Watson-Crick pairing of isoguanine with guanine demands the former to participate in its 3H-tautomeric form; hitherto this specific tautomer had not been considered in the pairing chemistry of isoguanine. Whereas (cumulative) purine-purine pairing in DNA (reverse-Hoogsten or Hoogsteen) seems to occur in triplexes and tetrapalexes only, its occurrence in duplexes in a characteristic feature of homo-DNA chemistry. The occurrence of purine-purine Watson-Crick base pairs is probably a consequence of homo-DNA's quasi-linear ladder structure [1][4]. In a double helix, the distance between the two sugar C-atoms, on which a base pair is anchored, is expected to be constrained by the dimensions of the helix; in a linear duplex, however, there would be no restrictions with regard to base-pair length. Homo-DNA's ladder-like model also allows one to recognize one of the reasons why nucleic-acid duplexes prefer to pair in antiparallel, rather than parallel strand orientation: in homo-DNA duplexes, (averaged) backbone and base pair axes are strongly inclined toward one another [4]; the stronger this inclination, the higher the preference for antiparallel strand orientation is expected to be (Fig. 16). In retrospect, homo-DNA turns out to be one of the first artificial oligonucleotide systems (cf. Footnote 65) to demonstrate in a comprehensive way that informational base pairing involving purines and pyrimidines is not a capability unique to ribofuranosyl systems. Stability and helical shape of pairing complexes are not necessary conditions of one another; it is the potential for extensive conformational cooperativity of hte backbone structure with respect to the constellational demands of base pairing and base stacking that determines whether or nor a given type of base-carrying backbone structure is an informational pairing system. From the viewpoint of the chemical aetiology of nucleic-acid structure, which inspired our investigations on hexopyranosyl-(6′ → 4′)-oligonucleotide systems in the first place, the work on homo-DNA is only an extensive model study, because homo-DNA is not to be considered a potential natural-nucleic-acid altenratie. In retrospect, it seems fortunate that the model study was carried out, because without it we could hardly have comprehended the pairing behavior of the proper nucleic-acid alternatives which we have studied later and which will be discussed in Part VI of this series. The English footnotes to Fig. 1–49 provide an extension of this summary.

Structural elucidation and antisense properties of a sugar-modified DNA analogue

Antisense oligonucleotides deserve great attention as potential drug candidates for the treatment of genetic disorders. For example, muscle dystrophy can be treated successfully in mice by antisense-induced exon skipping in the pre-mRNA coding for the structural protein dystrophin in muscle cells. For this purpose a sugar- and backbone-modified DNA analogue was designed, in which a tricyclic ring system substitutes the deoxyribose. These chemical modifications stabilize the dimers formed with the targeted RNA relative to native nucleic acid duplexes and increase the biostability of the antisense oligonucleotide. While evading enzymatic degradation constitutes an essential property of antisense oligonucleotides for therapeutic application, it renders the oligonucleotide inaccessible to biochemical sequencing techniques and requires the development of alternative methods based on mass spectrometry. The set of sequences studied includes tcDNA oligonucleotides ranging from 10 to 15 nucleotides in length as well as their hybrid duplexes with DNA and RNA complements. All samples were analyzed on a LTQ Orbitrap XL instrument equipped with a nano-electrospray source. For tandem mass spectrometric experiments collision-induced dissociation was performed, using helium as collision gas. Mass spectrometric sequencing of tcDNA oligomers manifests the applicability of the technique to substrates beyond the scope of enzyme-based methods. Sequencing requires the formation of characteristic backbone fragments, which take the form of a-B- and w-ions in the product ion spectra of tcDNA. These types of product ions are typically associated with unmodified DNA, which suggests a DNA-like fragmentation mechanism in tcDNA. The loss of nucleobases constitutes the second prevalent dissociation pathway observed in tcDNA. Comparison of partially and fully modified oligonucleotides indicates a pronounced impact of the sugar-moiety on the base loss. As this event initiates cleavage of the backbone, the presented results provide new mechanistic insights into the fragmentation of DNA in the gas-phase. The influence of the sugar-moiety on the dissociation extends to tcDNA:DNA and tcDNA:RNA hybrid duplexes, where base loss was found to be much more prominent from sugar-modified oligonucleotides than from their natural complements. Further prominent dissociation channels are strand separation and backbone cleavage of the single strands, as well as the ejection of backbone fragments from the intact duplex. The latter pathway depends noticeably on the base sequence. Moreover, it gives evidence of the high stability of the hybrid dimers, and thus directly reflects the affinity of tcDNA for its target in the cell. As the cellular target of tcDNA is a pre-mRNA, the structure was designed to discriminate RNA from DNA complements, which could be demonstrated by mass spectrometric experiments.

Changes in chlamydia control activities in Europe between 2007 and 2012: a cross-national survey.

BACKGROUND In 2012, the levels of chlamydia control activities including primary prevention, effective case management with partner management and surveillance were assessed in 2012 across countries in the European Union and European Economic Area (EU/EEA), on initiative of the European Centre for Disease Control (ECDC) survey, and the findings were compared with those from a similar survey in 2007. METHODS Experts in the 30 EU/EEA countries were invited to respond to an online questionnaire; 28 countries responded, of which 25 participated in both the 2007 and 2012 surveys. Analyses focused on 13 indicators of chlamydia prevention and control activities; countries were assigned to one of five categories of chlamydia control. RESULTS In 2012, more countries than in 2007 reported availability of national chlamydia case management guidelines (80% vs. 68%), opportunistic chlamydia testing (68% vs. 44%) and consistent use of nucleic acid amplification tests (64% vs. 36%). The number of countries reporting having a national sexually transmitted infection control strategy or a surveillance system for chlamydia did not change notably. In 2012, most countries (18/25, 72%) had implemented primary prevention activities and case management guidelines addressing partner management, compared with 44% (11/25) of countries in 2007. CONCLUSION Overall, chlamydia control activities in EU/EEA countries strengthened between 2007 and 2012. Several countries still need to develop essential chlamydia control activities, whereas others may strengthen implementation and monitoring of existing activities.