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Transmission of excreted vaccine-derived infectious virus from vaccinated to unvaccinated individuals is possible within close contacts. This randomized (1:1), double-blind study evaluated the potential for transmission of human rotavirus vaccine strain, HRV (Rotarix™) from vaccine recipients to unvaccinated close contacts (twins). 100 pairs of healthy twins aged 6–14 weeks at the time of Dose 1 of HRV vaccine/placebo were enrolled and one randomly selected twin from each pair received two vaccine doses and the other received placebo doses (at 2 and 4 months of age). Presence of vaccine strain in the stool samples of placebo recipients was an indicator of transmission. Serial stool samples were tested for rotavirus using ELISA at pre-determined time points; rotavirus positive stool samples were tested with RT-PCR and reverse hybridization assay to identify G1P[8] vaccine strain. If G1P[8] vaccine strain was detected, the complete genome was sequenced to assess the similarity between viral isolates. Immunogenicity and safety of HRV vaccine in transmission cases was assessed. 15 transmission cases were reported in 80 evaluable twins who received placebo and the transmission rate was 18.8% (95% CI: 10.9–29.0%). None of the transmission cases was associated with gastroenteritis symptoms. Anti-rotavirus IgA seroconversion was 62.5% (95% CI: 51.0–73.1%) (HRV) and 21.3% (95% CI: 12.9–31.8%) (placebo) 7-weeks post-Dose 2; seroconversion in transmission cases was 26.7% (95% CI: 7.8–55.1%). Genetic variations or amino acid substitutions in transmission cases were similar to that seen in corresponding vaccine recipients. Transmission of HRV vaccine strain to unvaccinated twins living in close contact occurred, however, they were not associated with increased of gastroenteritis. Whether transmission leads to indirect protection among unvaccinated individuals remains unknown at this stage.

The Yersinia Ysc apparatus is made of more than 20 proteins, 11 of which have homologues in many type III systems. Here, we characterize YscP from Yersinia enterocolitica. This 515-residue protein has a high proline content, a large tandem repetition and a slow migration in SDS-PAGE. Unlike the products of neighbouring genes, it has a counterpart only in Pseudomonas aeruginosa and it varies even between Yersinia Ysc machineries. An yscPDelta97-465 mutant was unable to secrete any Yop, even under conditions overcoming feedback inhibition of Yop synthesis. Interestingly, a cloned yscPDelta57-324 from Yersinia pestis introduced in the yscPDelta97-465 mutant can sustain a significant Yop secretion and thus partially complemented the mutation. This explains the leaky phenotype observed with the yscP mutant of Y. pestis. In accordance with this secretion deficiency, YscP is required for the delivery of Yop effectors into macrophages. Mechanical shearing, immunolabelling and electron microscopy experiments showed that YscP is exposed at the bacterial surface when bacteria are incubated at 37 degrees C in the presence of Ca2+ and thus do not secrete Yops. At 37 degrees C, when Ca2+ ions are chelated, YscP is released like a Yop protein. We conclude that YscP is a part of the Ysc injectisome which is localized at the bacterial surface and is destabilized by Ca2+ chelation.

SUMMARY The 70-kb virulence plasmid enables Yersinia spp. (Yersinia pestis, Y. pseudotuberculosis, and Y. enterocolitica) to survive and multiply in the lymphoid tissues of their host. It encodes the Yop virulon, an integrated system allowing extracellular bacteria to disarm the cells involved in the immune response, to disrupt their communications, or even to induce their apoptosis by the injection of bacterial effector proteins. This system consists of the Yop proteins and their dedicated type III secretion apparatus, called Ysc. The Ysc apparatus is composed of some 25 proteins including a secretin. Most of the Yops fall into two groups. Some of them are the intracellular effectors (YopE, YopH, YpkA/YopO, YopP/YopJ, YopM, and YopT), while the others (YopB, YopD, and LcrV) form the translocation apparatus that is deployed at the bacterial surface to deliver the effectors into the eukaryotic cells, across their plasma membrane. Yop secretion is triggered by contact with eukaryotic cells and controlled by proteins of the virulon including YopN, TyeA, and LcrG, which are thought to form a plug complex closing the bacterial secretion channel. The proper operation of the system also requires small individual chaperones, called the Syc proteins, in the bacterial cytosol. Transcription of the genes is controlled both by temperature and by the activity of the secretion apparatus. The virulence plasmid of Y. enterocolitica and Y. pseudotuberculosis also encodes the adhesin YadA. The virulence plasmid contains some evolutionary remnants including, in Y. enterocolitica, an operon encoding resistance to arsenic compounds.

Background Conventional techniques for diagnosing influenza based on viral cell culture or disease serology have limitations, and molecular assays, such as real-time polymerase chain reaction (rtPCR) are increasingly used. Objectives To evaluate the use of rtPCR as a diagnostic tool for the determination of influenza virus infection. Study design This prospective, double-blind, placebo-controlled, randomised efficacy study was conducted in persons aged 18–64 years. Cases of influenza-like-illness (ILI), defined as at least one systemic symptom [fever ≥37.8 °C and/or myalgia] and at least one respiratory symptom [cough and/or sore throat] were identified by active and passive surveillance. For each case of suspected ILI, nasal and throat swabs were collected and analysed by viral culture and rtPCR. Results 227 ILI cases were positive by rtPCR while 64% (145/227) were positive by both rtPCR and culture. For both assays, the maximum percentage of swabs that tested positive was on Day 0, thereafter positive samples by rtPCR remained constant until Day 5 but decreased progressively by culture. All rtPCR positive cases with a viral load of below 4.5 log 10 copies/sample were negative by culture. There were however culture negative cases with high viral loads. Vaccine efficacy for influenza was estimated as 54.7% by rtPCR (culture positive or negative) and 61.6% by culture irrespective of match to vaccine strain. Clinical severity was not significantly different between culture positive cases and culture negative but rtPCR positive cases. Conclusions rtPCR is a sensitive and specific diagnostic tool for influenza vaccine efficacy studies.

The genus Yersinia includes three species that are pathogenic for rodents and humans. Yersinia pestis, the agent of black death, is generally inoculated by a flea bite. I.: pseudotuberculosis is an agent of mesenteric adenitis and septicemia, and Y enterocolitica, the most prevalent species in humans, causes gastrointestinal syndromes, ranging from an acute enteritis to mesenteric lymphadenitis. Although these three Yersinia species infect their hosts by different routes and cause diseases of different severity, they present a common tropism for lymphoid tissues and a common capacity to resist the non-specific immune response 111. The Yersinia Yop (Yersinia outer protein) virulon (integrated virulence system) is the archetype of a new virulence mechanism, sometimes referred to as ‘type 111’ secretion. This mechanism allows extracellular bacteria which are in close contact with the surface of eukaryotic cells to inject virulence proteins into the cytosol of these cells in order to disarm them or disrupt their communication systems. This kind of mechanism is encountered in other animal pathogens, such as Salmonella spp., Shigella spp., enteropathogenic Esckerichia coli (EPEC) and Pseudoinonas aeruginosa, and also in the bacterial plant pathogens that elicit the ‘hypersensitive response’ (for review, see [2]). The Yop virulon of Yersinia is encoded by a 70-kb plasmid called pYV for ‘plasmid involved in Yersinia virulence’. It consists basically of a dozen secreted proteins, called Yop proteins, and their ‘contact’-or ‘type 111’-secretion apparatus, called Ysc. The Yop proteins form two distinct groups. The first group includes YopE, YopH, YopM, YopO, YopP and YopT, which are intracellular effectors, while the second one, including at least YopB, YopD and low calcium response virulence gene (LcrV), forms the translocation apparatus that is deployed at the bacterial surface to deliver the effectors into the target cell. The assembly of this translocation machinery is triggered by the contact between the bacterium and eukaryotic cell and controlled by proteins of the virulon, including YopN. Our present knowledge of the Yersinia system leads to the model presented in Figure 1 and described in this short paper. At 37”C, the host body temperature, the system is switched on. In these conditions, transcription of the yop genes is activated by VirF, a transcriptional activator of the AraC family [3,4]. The Ysc secretion machinery is synthesized and assembled in the bacterial membranes. It contains a putative ATPase, called YscN, which is probably the energizer of the system 151, and a set of integral membrane proteins. Among these, the YscC protein, an outer-membrane protein, forms a multimeric complex with a ring-shaped structure with an external diameter of about 200 A and an apparent central pore of about 50 A. This structure allows the passage of the Yop proteins across the external membrane of the bacterium [6]. Secretion of Yop proteins also requires some individual cytosolic chaperones, named Syc for ‘specific Yop chaperone’. These small acidic proteins bind to their cognate Yop proteins to prevent premature associations, to limit their toxicity and also to pilot them to the secretion machinery 171. Secretion of Yop proteins does not involve the cleavage of an N-terminal signal sequence, as is usually observed for secretion in bacteria. The secretion signal is nevertheless located in the N-terminal part of the Yop [8]. Recently, it has been suggested that the signal could be in the 5’ mRNA rather than in the N-terminus, but more information is needed to characterize this signal 191. The actual model for secretion of Yop proteins is

Pathogenic strains of Yersinia spp. inject a set of Yop effector proteins into eukaryotic cells by using a plasmid-encoded type III secretion system. In this study, we analyzed the inflammatory response of human umbilical vein endothelial cells (HUVECs) after infection with different Yersinia enterocolitica strains. We found that both expression of intercellular adhesion molecule 1 and release of the cytokines interleukin-6 (IL-6) and IL-8 by HUVECs are downregulated in a YopP-dependent way, demonstrating that YopP plays a major role in the inflammatory response of these cells. Infection of HUVECs with several low-virulence (biotype 2, 3, and 4) and high-virulence (biotype 1B) Y. enterocolitica strains showed that biotype 1B isolates are more efficient in inhibiting the inflammatory response than low-virulence Y. enterocolitica strains and that this effect depends on the time of contact. We extended the results of Ruckdeschel et al. and found that on the basis of the presence or absence of arginine-143 of YopP (K. Ruckdeschel, K. Richter, O. Mannel, and J. Heesemann, Infect. Immun. 69: 7652-7662, 2001) all the Y. enterocolitica strains used fell into two groups, which correlate with the low- and high-virulence phenotypes. In addition, we found that high-virulence strains inject more YopP into the cytosol of eukaryotic target cells than do low-virulence strains.

Synthesis of the Yop proteins by yersiniae is downregulated when secretion is prevented by closure or destruction of the contact (type III) secretion channel. In Yersinia pseudotuberculosis, a mutation in the IcrQ gene, encoding a secreted protein, reduces this feedback inhibition mechanism. Surprisingly, mutation in the yscM gene, the IcrQ homologue in Y. enterocolitica, does not lead to the same deregulated phenotype. In this paper, we addressed the question of this discrepancy. We found a new gene on the Y. enterocolitica pYV plasmid that encodes a protein with 57% identity to YscM (now called YscM1). Overexpression of this gene, called yscM2, like overexpression of IcrQ and yscM1, blocked Yop secretion. A double yscM1, yscM2 mutant had the same phenotype as that of the IcrQ mutant. The discrepancy can thus be explained by the existence of two functionally equivalent copies of yscM in Y. enterocolitica. Overexpression of yscM1 drastically reduced the expression of a yopH-cat reporter gene when tested in a pYV+ background. However, no effect could be observed in the absence of a pYV plasmid, indicating that YscM1 does not act directly as a transcriptional repressor or as an anti-VirF factor. We have also ruled out that YscM acts by obstructing the secretion channel.

Yersinia enterocolitica is an enterobacterium responsible for gastrointestinal syndromes. Its pathogenicity depends on the presence of the 70-kb pYV plasmid, which directs Yop secretion. The Yop secretion machinery, consisting of the YscA-U and LcrD proteins, presents some structural similarity with the flagellum assembly machinery characterized in other bacteria. Flagellum assembly requires sigma 28, an alternative sigma factor. The region upstream of the lcrD gene resembles promoters recognized by sigma 28, suggesting that the similarity between Yop secretion and flagellum assembly could extend to their regulation. The chromosome of Y. enterocolitica also contains pathogenicity determinants such as myfA, which encodes the Myf antigen subunit. The promoter region of myfA also resembles promoters recognized by sigma 28. In an attempt to clarify the role of sigma 28 in the expression of lcrD, myfA, and flagellar genes, we cloned, sequenced, and mutagenized the fliA gene encoding the sigma 28 homolog in Y. enterocolitica. As is the case in other bacteria, fliA was required for motility. However, it was involved neither in fibrilla synthesis nor in Yop secretion. The fliA mutant allowed us to monitor the role of motility in pathogenesis. At least in the mouse model, motility seemed not to be required for Y. enterocolitica pathogenesis.