Identification of New Microfoci and Genetic Characterization of Tick-Borne Encephalitis Virus Isolates from Eastern Germany and Western Poland.

(1) Background: Tick-borne encephalitis (TBE) is the most important tick-borne viral disease in Eurasia, although effective vaccines are available. Caused by the tick-borne encephalitis virus (TBEV, syn. Orthoflavivirus encephalitidis), in Europe, it is transmitted by ticks like Ixodes ricinus and Dermacentor reticulatus. TBEV circulates in natural foci, making it endemic to specific regions, such as southern Germany and northeastern Poland. Our study aimed to identify new TBEV natural foci and genetically characterize strains in ticks in previously nonendemic areas in Eastern Germany and Western Poland. (2) Methods: Ticks were collected from vegetation in areas reported by TBE patients. After identification, ticks were tested for TBEV in pools of a maximum of 10 specimens using real-time RT-PCR. From the positive TBEV samples, E genes were sequenced. (3) Results: Among 8400 ticks from 19 sites, I. ricinus (n = 4784; 56.9%) was predominant, followed by D. reticulatus (n = 3506; 41.7%), Haemaphysalis concinna (n = 108; 1.3%), and I. frontalis (n = 2; <0.1%). TBEV was detected in 19 pools originating in six sites. The phylogenetic analyses revealed that TBEV strains from Germany and Poland clustered with other German strains, as well as those from Finland and Estonia. (4) Conclusions: Although there are still only a few cases are reported from these areas, people spending much time outdoors should consider TBE vaccination.

Effects of antibiotics ceftriaxone and levofloxacin on the growth of Protophormia terraenovae (Diptera: Calliphoridae).

Protophormia terraenovae is a colonizer of decomposing bodies and is known to cause pre-mortem myiasis as the female flies lay eggs in uncleaned wounds. In this study the effects of different concentrations of antibiotics levofloxacin and ceftriaxone on maggot development, weight, length, and mortality were examined. The maggot length and weight were significantly increased by therapeutical doses of levofloxacin and ceftriaxone. The maggot development time was significantly decreased in every levofloxacin treatment compared to the control. The time to start pupation was significantly increased in the control compared to the antibiotic treatments. Levofloxacin significantly increased the survivability of the maggots. Every levofloxacin treatment significantly improved the rearing conditions for the maggots. Reaching the third instar was delayed by 24 h in the control compared to the Levo 3.57 treatment. The Pupation in the control was delayed by an average of 48 h compared to the Levo 3.57 treatment. The significantly reduced development time of the maggots in the antibiotic treatments might lead to an overestimation of the post-mortem interval and therefore an incorrect time of death determination. The improved rearing conditions may be an indication of the potential of a combined application of antibiotics and maggot therapy.

Effects of antibiotics ceftriaxone and levofloxacin on the growth of Lucilia sericata (Diptera: Calliphoridae).

Lucilia sericata is one of the most studied species in forensic entomology due to its widespread distribution, forensic importance as well as medical use. The growth and development stage of maggots is often used to determine the post-mortem interval in forensic cases. L. sericata can cause myiasis in humans who are not able to maintain personal hygiene due to age or medical condition and can therefore be used to determine the time period of neglect. The influence of the temperature on the maggot development has been examined in various studies. Different examinations on the effects of toxic substances on the maggot development and survival have been conducted in order to test the influence and resulting deviations. In this study, the effects of different therapeutical doses of the antibiotics ceftriaxone and levofloxacin were examined on L. sericata in order to determine and compare deviations in maggot development and survival. The used antibiotics did not significantly influence the maggot weight and length. A significant delay in the time of pupation has been determined in the treatments with high concentrations of levofloxacin. The mortality was significantly increased in all treatments with antibiotics compared to the control, whereby the survivability of all treatments remained over 80%. Because of the high doses needed to produce an effect, we concluded that an overestimation of the post-mortem interval is unlikely but higher antibiotic concentrations or possible interactions with other medications could increase the maggot development time.

Effects of antibiotics ceftriaxone and levofloxacin on the growth of Calliphora vomitoria L. (Diptera: Calliphoridae) and effects on the determination of the post-mortem interval.

The determination of the post-mortem interval (PMI) is one of the main tasks of forensic entomology, where growth and stages of development of arthropods are used for PMI determination. It is well acknowledged that maggot development is significantly influenced by temperature. Attention has also been paid to the microbial populations of the cadaver, because toxic substances contained in the substrate can influence the microorganisms and affect arthropods growth and development. However, little is known about the influence of antibiotics taken during lifetime of a person on insect development after that persons death. The aim of this study was to test the hypothesis that the antibiotics ceftriaxone and levofloxacin cause inhibition of growth and delay of pupation of the blow fly Calliphora vomitoria, which would then lead to an incorrect determination of the post-mortem interval in forensic cases. It was found that maggot development was delayed by levofloxacin mixed in minced pork, where a mixture of both antibiotics increased this effect. The maggot growth in the samples with ceftriaxone was not delayed. Pupation was delayed in treatments with a mixture of both antibiotics. The mortality was reduced by separate or combined application of ceftriaxone and levofloxacin, which we attribute to a bactericidal effect of the antibiotics on maggot pathogens. Depending on the concentration of the antibiotics, an underestimation of the post-mortem interval between 24 and 48 h could be suspected. We conclude that antibiotics need to be considered if instar stages are to be used to determine the PMI and that some antibiotics may improve the breeding conditions of maggots.

Sterility release testing of peripheral blood stem cells for transplantation: impact of culture bottles and incubation temperature.

BACKGROUND: Sterility testing of peripheral blood stem cells (PBSCs) is mandatory before release. As antibiotic treatment of the PBSC donor may result in false-negative results, PBSC matrix validation must be carried out. STUDY DESIGN AND METHODS: Three spiked PBSCs and a buffy coat (BC; control matrix) were analyzed using the blood culture device BacT/ALERT 3D with the low-temperature module. Samples were spiked with Staphylococcus aureus, Bacillus subtilis, Pseudomonas aeruginosa, Candida albicans, Aspergillus brasiliensis, Clostridium sporogenes, and Propionibacterium acnes. Standard iAST/iNST culture bottles and iFA/iFN Plus bottles, which include resorbing polymers, were incubated for 14 days. All aerobic bottles were incubated at 22.5°C and for a direct comparison also at 35°C while all anaerobic bottles were incubated at 35°C. RESULTS: The BacT/ALERT 3D system detected all microbes in iAST/iNST culture bottles according to their growth behavior in the BC matrix. Detection of microbes differed significantly in PBSC products using standard iAST/iNST culture bottles and iFA/iFN Plus bottles with resorbing polymers: In Graft 1 no growth was detected in spiked bottles with S. aureus (iAST), B. subtilis (iAST/iNST), C. sporogenes (iNST), and P. acnes (iNST) compared to iFA Plus and iFN Plus bottles wherein growth of spiked microbes was confirmed. Graft 2, with another antibiotic treatment, showed no growth in iAST/iNST bottles spiked with P. aeruginosa, B. subtilis, and C. sporogenes. However, using iFA/iFN Plus bottles all spiked microbes were detectable. The comparison of incubation temperature showed an expected slower growth at 22.5°C. CONCLUSION: The use of iFA/iFN Plus culture bottles incubated at different temperatures safely detected microbes in spiked PBSCs.

Detection of the relatively slow-growing Propionibacterium acnes in seven matrices of blood components and advanced therapeutical medicinal products.

BACKGROUND: Relatively slow-growing bacteria like Propionibacterium acnes represent a challenge for quality control investigations in sterility release testing of blood components and advanced therapeutic medicinal products (ATMPs). METHODS: A convenient validation with 7 matrices was performed using buffy coat, stem cells, islet cells, natural killer cells, red blood cells, platelets and plasma in the microbial detection system Bact/Alert®3D incubator. All matrix samples were spiked twofold with Propionibacterium acnes with approximately 50 colony forming units (CFUs) per bottle in iAST and iNST culture bottles for 14days using a multishot bioball. Additionally, the stem cell preparations were also incubated in iFAplus and iFNplus culture bottles, which include neutralizing polymers. RESULTS: The Bact/Alert®3D-System detected Propionibacterium acnes in anaerobic culture bottles in buffy coat [3.3 d (=positive signal day to detection as mean value)], red blood cells [3.2 d], platelets [3.3], plasma [3.7 d], natural killer cells [3.3 d] and islet cells [4.9 d], resp. No growth of Propionibacterium was found in autologous stem cells using iAST and iNST culture bottles. However, Propionibacterium was safely detected in the iFNplus culture bottle with polymers in the stem cell matrix. A successful validation of media was performed. CONCLUSIONS: Our study shows that Bact/Alert®3D-System safely detects the relatively slow-growing bacterium Propionibacterium acnes in different matrices in a practical way except stem cells. Using the iFNplus culture bottle for stem cell products positive signals were observed.

Molecular characterization of macrolide resistance of a Mycoplasma pneumoniae strain that developed during therapy of a patient with pneumonia.

The development of macrolide resistance that occurred during 3 days of therapy with azithromycin to treat Mycoplasma pneumoniae pneumonia in a paediatric patient is reported. After extended molecular characterization of strains, the parallel occurrence of clones showing the non-mutated wild-type 23S rRNA sequence as well as mutations A2063G and A2064G, which are both responsible for phenotypic resistance, was confirmed for the first time.

Overexpression of the ICL1 gene changes the product ratio of citric acid production by Yarrowia lipolytica.

The yeast Yarrowia lipolytica secretes high amounts of various organic acids, like citric (CA) and isocitric (ICA) acids, triggered by growth limitation caused by different factors and an excess of carbon source. Depending on the carbon source used, Y. lipolytica strains produce a mixture of CA and ICA in a characteristic ratio. To examine whether the CA/ICA product ratio can be influenced by gene-dose-dependent overexpression or by disruption of the isocitrate lyase (ICL)-encoding gene ICL1, recombinant Y. lipolytica strains were constructed, which harbour multiple ICL1 copies or a defective icl1 allele. The high-level expression of ICL in ICL1 multicopy integrative transformants resulted in a strong shift of the CA/ICA ratio into direction of CA. On glycerol, glucose and sucrose, the ICA proportion decreased from 10-12% to 3-6%, on sunflower oil or hexadecane even from 37-45% to 4-7% without influencing the total amount of acids (CA and ICA) produced. In contrast, the loss of ICL activity in icl1-defective strains resulted in a moderate 2-5% increase in the ICA proportion compared to ICL wild-type strains on glucose or glycerol.

A simple and rapid single-step multiplex RT-PCR to detect Norovirus, Astrovirus and Adenovirus in clinical stool samples.

A single-step multiplex reverse transcription-polymerase chain reaction (RT-PCR) assay that detects and identifies Norovirus, Astrovirus and Adenovirus in clinical stool samples is described. Four hundred sixty stool samples were tested from patients with non-rotavirus acute gastroenteritis, that were either stored at -80 degrees C and tested retrospectively, or tested immediately after viral nucleic acid extraction in a prospective manner, including outbreaks of gastroenteritis that occurred in Germany during the winter of 2003. The multiplex RT-PCR was validated against simplex RT-PCR with published primers for Norovirus (JV12/JV13 and p289/p290) and Astrovirus (Mon340/348), and against simplex PCR for Adenovirus. In both retrospective and prospective settings, the multiplex RT-PCR was equally sensitive and specific in detecting non-rotavirus infections compared with simplex RT-PCR/PCR. The specificity of the multiplex RT-PCR was assessed by sequencing of the amplicons that showed high nucleotide identities to Norovirus genogroup I/1, I/4, II/2, or II/4 clades, as well as to Astrovirus serotypes 1, 2, 4, or 8. The multiplex RT-PCR was also more sensitive than Astrovirus and Norovirus antigen enzyme immunoassays (IDEIA, Dako), as well as Astrovirus isolation in cell culture. This novel multiplex RT-PCR is an attractive technique for the rapid, specific, and cost-effective laboratory diagnosis of non-rotavirus acute gastroenteritis.

Foamy virus integration.

It had been suggested that during integration of spumaretroviruses (foamy viruses) the right (U5) end of the cDNA is processed, while the left (U3) remains uncleaved. We confirmed this hypothesis by sequencing two-long terminal repeat (LTR) circle junctions of unintegrated DNA. Based on an infectious foamy virus molecular clone, a set of constructs harboring mutations at the 5' end of the U3 region in the 3' LTR was analyzed for particle export, reverse transcription, and replication. Following transient transfection some mutants were severely impaired in generating infectious virus, while others replicated almost like the wild type. The replication competence of the mutants was unrelated to the cleavability of the newly created U3 end. This became obvious with two mutants both belonging to the high-titer type. One mutant containing a dinucleotide artificially transferred from the right to the left end was trimmed upon integration, while another one with an unrelated dinucleotide in that place was not. The latter construct in particular showed that the canonical TG motif at the beginning of the provirus is not essential for foamy virus integration.

Feline foamy virus genome and replication strategy.

Crucial aspects of the foamy virus (FV) replication strategy have so far only been investigated for the prototypic FV (PFV) isolate, which is supposed to be derived from nonhuman primates. To study whether the unusual features of this replication pathway also apply to more-distantly related FVs, we constructed feline FV (FFV) infectious molecular clones and vectors. It is shown by quantitative RNA and DNA PCR analysis that FFV virions contain more RNA than DNA. Full-length linear DNA was found in extracellular FFV by Southern blot analysis. Similar to PFV, azidothymidine inhibition experiments and the transfection of nucleic acids extracted from extracellular FFV indicated that DNA is the functional relevant FFV genome. Unlike PFV, no evidence was found indicating that FFV recycles its DNA into the nucleus.

Retrotransposition and cell-to-cell transfer of foamy viruses.

A remarkable feature of the prototype foamy virus (PFV) replication pathway has been reported to consist of the ability to retrotranspose intracellularly with high efficiency (M. Heinkelein, T. Pietschmann, G. Jármy, M. Dressler, H. Imrich, J. Thurow, D. Lindemann, M. Bock, A. Moebes, J. Roy, O. Herchenröder, and A. Rethwilm, EMBO J. 19:3436-3345, 2000). PFV intracellular retrotransposition (IRT) was reported to be enhanced by coexpression of fusion-defective envelope protein. To investigate the possibility of cell-to-cell transfer of PFV genomes, which could mimic IRT, we performed cocultivation experiments with cells transfected with an IRT-competent and marker gene-expressing PFV vector together with cells expressing a different marker and measured cells positive for both markers. The findings corroborated the initial report on IRT of Env-deficient PFV. Furthermore, they indicated that viral cores that have incorporated fusion-deficient Env can be transferred from cell to cell in a cell type-specific manor. One possible explanation consists of a minor alternative cleavage site in Env that can be used to expose the fusion peptide of the Env transmembrane protein, which appears to be required for virus uptake.

Replication-competent hybrids between murine leukemia virus and foamy virus.

Replication-competent chimeric retroviruses constructed of members of the two subfamilies of Retroviridae, orthoretroviruses and spumaretroviruses, specifically murine leukemia viruses (MuLV) bearing hybrid MuLV-foamy virus (FV) envelope (env) genes, were characterized. All viruses had the cytoplasmic tail of the MuLV transmembrane protein. In ESL-1, a truncated MuLV leader peptide (LP) was fused to the complete extracellular portion of FV Env, and ESL-2 to -4 contained the complete MuLV-LP followed by N-terminally truncated FV Env decreasing in size. ESL-1 to -4 had an extended host cell range compared to MuLV, induced a cytopathology reminiscent of FVs, and exhibited an ultrastructure that combined the features of the condensed core of MuLV with the prominent surface knobs of FVs. Replication of ESL-2 to -4 resulted in the acquisition of a stop codon at the N terminus of the chimeric Env proteins. This mutation rendered the MuLV-LP nonfunctional and indicated that the truncated FV-LP was sufficient to direct Env synthesis into the secretory pathway. Compared to the parental viruses, the chimeras replicated with only moderate cell-free titers.