Characterization of Yersinia pestis Phage Lytic Activity in Human Whole Blood for the Selection of Efficient Therapeutic Phages.

The global increase in multidrug-resistant (MDR) pathogenic bacteria has led to growing interest in bacteriophage ("phage") therapy. Therapeutic phages are usually selected based on their ability to infect and lyse target bacteria, using in vitro assays. In these assays, phage infection is determined using target bacteria grown in standard commercial rich media, while evaluation of the actual therapeutic activity requires the presence of human blood. In the present work, we characterized the ability of two different Yersinia pestis lytic phages (ϕA1122 and PST) to infect and kill a luminescent Y. pestis EV76 strain suspended in Brain Heart Infusion (BHI)-rich medium or in human whole blood, simulating the host environment. We found that the ability of the phages to infect and lyse blood-suspended Y. pestis was not correlated with their ability to infect and lyse BHI-suspended bacteria. While the two different phages exhibited efficient infective capacity in a BHI-suspended culture, only the PST phage showed efficient lysis ability against blood-suspended bacteria. Therefore, we recommend that for personalized phage therapy, selection of phage(s) for efficient treatment of patients suffering from MDR bacterial infections should include prior testing of the candidate phage(s) for their lysis ability in the presence of human blood.

Genetic and molecular features for hepadnavirus and plague infections in the Himalayan marmot.

The Himalayan marmot (Marmota himalayana), a natural host and transmitter of plague, is also susceptible to the hepadnavirus infection. To reveal the genetic basis of the hepadnavirus susceptibility and the immune response to plague, we systematically characterized the features of immune genes in Himalayan marmot with those of human and mouse. We found that the entire major histocompatibility complex region and the hepatitis B virus pathway genes of the Himalayan marmot were conserved with those of humans. A Trim (tripartite motif) gene cluster involved in immune response and antiviral activity displays dynamic evolution, which is reflected by the duplication of Trim5 and the absence of Trim22 and Trim34. Three key regions of Ntcp, which is critical for hepatitis B virus entry, had high identity among seven species of Marmota. Moreover, we observed a severe alveolar hemorrhage, inflammatory infiltrate in the infected lungs and livers from Himalayan marmots after infection of EV76, a live attenuated Yersinia pestis strain. Lots of immune genes were remarkably up-regulated, which several hub genes Il2rγ, Tra29, and Nlrp7 are placed at the center of the gene network. These findings suggest that Himalayan marmot is a potential animal model for study on the hepadnavirus and plague infection.

[The "Black Death" : Crimean-Congo hemorrhagic fever]. / Der "schwarze Tod" : Krim-Kongo-Hämorrhagisches-Fieber.

The Crimean-Congo hemorrhagic fever (CCHF) is a tick-borne viral disease that has been known for centuries. In the last years more frequent cases reflect the effects of climate change, globalization and the increasing encroachment of humans into previously unexploited areas. Humans acquire the infection by tick bites or through the slaughtering and processing of infected animals. The course of the disease can be severe and the average mortality reaches up to 30 %. It is transmissible from human to human and there is no causal treatment. Thus, CCHF meets the criteria for a highly contagious life-threatening disease. In the following current data on the virus, its vector, the distribution and transmission will be presented, as well as information on the diagnosis, the disease, the underlying pathophysiology and consequences in dealing with patients and deceased.

Understanding the persistence of plague foci in Madagascar.

Plague, a zoonosis caused by Yersinia pestis, is still found in Africa, Asia, and the Americas. Madagascar reports almost one third of the cases worldwide. Y. pestis can be encountered in three very different types of foci: urban, rural, and sylvatic. Flea vector and wild rodent host population dynamics are tightly correlated with modulation of climatic conditions, an association that could be crucial for both the maintenance of foci and human plague epidemics. The black rat Rattus rattus, the main host of Y. pestis in Madagascar, is found to exhibit high resistance to plague in endemic areas, opposing the concept of high mortality rates among rats exposed to the infection. Also, endemic fleas could play an essential role in maintenance of the foci. This review discusses recent advances in the understanding of the role of these factors as well as human behavior in the persistence of plague in Madagascar.

Mutated and bacteriophage T4 nanoparticle arrayed F1-V immunogens from Yersinia pestis as next generation plague vaccines.

Pneumonic plague is a highly virulent infectious disease with 100% mortality rate, and its causative organism Yersinia pestis poses a serious threat for deliberate use as a bioterror agent. Currently, there is no FDA approved vaccine against plague. The polymeric bacterial capsular protein F1, a key component of the currently tested bivalent subunit vaccine consisting, in addition, of low calcium response V antigen, has high propensity to aggregate, thus affecting its purification and vaccine efficacy. We used two basic approaches, structure-based immunogen design and phage T4 nanoparticle delivery, to construct new plague vaccines that provided complete protection against pneumonic plague. The NH2-terminal ß-strand of F1 was transplanted to the COOH-terminus and the sequence flanking the ß-strand was duplicated to eliminate polymerization but to retain the T cell epitopes. The mutated F1 was fused to the V antigen, a key virulence factor that forms the tip of the type three secretion system (T3SS). The F1mut-V protein showed a dramatic switch in solubility, producing a completely soluble monomer. The F1mut-V was then arrayed on phage T4 nanoparticle via the small outer capsid protein, Soc. The F1mut-V monomer was robustly immunogenic and the T4-decorated F1mut-V without any adjuvant induced balanced TH1 and TH2 responses in mice. Inclusion of an oligomerization-deficient YscF, another component of the T3SS, showed a slight enhancement in the potency of F1-V vaccine, while deletion of the putative immunomodulatory sequence of the V antigen did not improve the vaccine efficacy. Both the soluble (purified F1mut-V mixed with alhydrogel) and T4 decorated F1mut-V (no adjuvant) provided 100% protection to mice and rats against pneumonic plague evoked by high doses of Y. pestis CO92. These novel platforms might lead to efficacious and easily manufacturable next generation plague vaccines.

Bacteriophage-resistant mutants in Yersinia pestis: identification of phage receptors and attenuation for mice.

BACKGROUND: Bacteriophages specific for Yersinia pestis are routinely used for plague diagnostics and could be an alternative to antibiotics in case of drug-resistant plague. A major concern of bacteriophage therapy is the emergence of phage-resistant mutants. The use of phage cocktails can overcome this problem but only if the phages exploit different receptors. Some phage-resistant mutants lose virulence and therefore should not complicate bacteriophage therapy. METHODOLOGY/PRINCIPAL FINDINGS: The purpose of this work was to identify Y. pestis phage receptors using site-directed mutagenesis and trans-complementation and to determine potential attenuation of phage-resistant mutants for mice. Six receptors for eight phages were found in different parts of the lipopolysaccharide (LPS) inner and outer core. The receptor for R phage was localized beyond the LPS core. Most spontaneous and defined phage-resistant mutants of Y. pestis were attenuated, showing increase in LD50 and time to death. The loss of different LPS core biosynthesis enzymes resulted in the reduction of Y. pestis virulence and there was a correlation between the degree of core truncation and the impact on virulence. The yrbH and waaA mutants completely lost their virulence. CONCLUSIONS/SIGNIFICANCE: We identified Y. pestis receptors for eight bacteriophages. Nine phages together use at least seven different Y. pestis receptors that makes some of them promising for formulation of plague therapeutic cocktails. Most phage-resistant Y. pestis mutants become attenuated and thus should not pose a serious problem for bacteriophage therapy of plague. LPS is a critical virulence factor of Y. pestis.

[Current state of problem of improving tools for plague vaccine prophylaxis].

Literature data on main immunobiological characteristics of 1st generation plague vaccines as well as ways of development of new tools for specific prophylaxis of plague: recombinant live, chemical, antiidiotypic, and DNA vaccines are presented in the review. Their expected advantages and disadvantages, perspectives of development and practical use in system of antiepidemic measures are assessed.

Diagnostic bioluminescent phage for detection of Yersinia pestis.

Yersinia pestis is the etiological agent of the plague. Because of the disease's inherent communicability, rapid clinical course, and high mortality, it is critical that an outbreak, whether it is natural or deliberate, be detected and diagnosed quickly. The objective of this research was to generate a recombinant luxAB ("light")-tagged reporter phage that can detect Y. pestis by rapidly and specifically conferring a bioluminescent signal response to these cells. The bacterial luxAB reporter genes were integrated into a noncoding region of the CDC plague-diagnostic phage phiA1122 by homologous recombination. The identity and fitness of the recombinant phage were assessed through PCR analysis and lysis assays and functionally verified by the ability to transduce a bioluminescent signal to recipient cells. The reporter phage conferred a bioluminescent phenotype to Y. pestis within 12 min of infection at 28 degrees C. The signal response time and signal strength were dependent on the number of cells present. A positive signal was obtained from 10(2) cells within 60 min. A signal response was not detectable with Escherichia coli, although a weak signal (100-fold lower than that with Y. pestis) was obtained with 1 (of 10) Yersinia enterocolitica strains and 2 (of 10) Yersinia pseudotuberculosis strains at the restrictive temperature. Importantly, serum did not prevent the ability of the reporter phage to infect Y. pestis, nor did it significantly quench the resulting bioluminescent signal. Collectively, the results indicate that the reporter phage displays promise for the rapid and specific diagnostic detection of cultivated Y. pestis isolates or infected clinical specimens.

Did medieval trade activity and a viral etiology control the spatial extent and seasonal distribution of Black Death mortality?

Recent research into the world's greatest recorded epidemic, the Medieval Black Death (MBD), has cast doubt on Bubonic Plague as the etiologic agent. Prior research has recently culminated in outstanding advances in our understanding of the spatio-temporal pattern of MBD mortality, and a characterization of the incubation, latent, infectious, and symptomatic periods of the MBD. However, until now, several mysteries remained unexplained, including perhaps the biggest quandary of all: why did the MBD exhibit inverse seasonal peaks in mortality from diseases recorded in modern times, such as seasonal Influenza or the Indian Plague Epidemics of the early 1900 s? Although some have argued that climate changes likely explain the observed differences between modern clinical Bubonic Plague seasonality and MBD mortality accounts, we believe that another factor explains these dissimilarities. Here, we provide a synthetic hypothesis which builds upon previous theories developed in the last ten years or so. Our all-encompassing theory explains the causation, dissemination, and lethality of the MBD. We theorize that the MBD was a human-to-human transmitted virus, originating in East-Central Asia and not Africa (as some recent work has proposed), and that its areal extent during the first great epidemic wave of 1347-1350 was controlled hierarchically by proximity to trade routes. We also propose that the seasonality of medieval trade controlled the warm-weather mortality peaks witnessed during 1347-1350; during the time of greatest market activity, traders, fairgoers, and religious pilgrims served as unintentional vectors of a lethal virus with an incubation period of approximately 32 days, including a largely asymptomatic yet infectious period of roughly three weeks. We include a description of the rigorous research agenda that we have proposed in order to subject our theory to scientific scrutiny and a description of our plans to generate the first publicly available georeferenced GIS dataset pertaining to MBD mortality, as far as we are aware. This proposed theory, if supported by our aggressive and statistically robust proposed research activities, finally contains all of the elements necessary to convincingly reanalyze both the greatest historical epidemic of the last millennium, and the risk to modern populations in light of such findings.