Relation of genetic phylogeny and geographical distance of tick-borne encephalitis virus in central Europe.

Tick-borne encephalitis virus (TBEV) is the most important arboviral agent causing disease of the central nervous system in central Europe. In this study, 61 TBEV E gene sequences derived from 48 isolates from the Czech Republic, and four isolates and nine TBEV strains detected in ticks from Germany, covering more than half a century from 1954 to 2009, were sequenced and subjected to phylogenetic and Bayesian phylodynamic analysis to determine the phylogeography of TBEV in central Europe. The general Eurasian continental east-to-west pattern of the spread of TBEV was confirmed at the regional level but is interlaced with spreading that arises because of local geography and anthropogenic influence. This spread is reflected by the disease pattern in the Czech Republic that has been observed since 1991. The overall evolutionary rate was estimated to be approximately 8×10(-4) substitutions per nucleotide per year. The analysis of the TBEV E genes of 11 strains isolated at one natural focus in zdár Kaplice proved for the first time that TBEV is indeed subject to local evolution.

How we should respond to the Coronavirus SARS-CoV-2 outbreak: A German perspective.

BACKGROUND: In the early phase of the COVID-19 pandemic Germany missed to set up efficient containment measures. Consequently, the number of cases increased exponentially until a lockdown was implemented to suppress the spread of SARS-CoV-2. Fortunately, Germany has a high capability for coronavirus lab testing and more than 30,000 ICU beds. These capabilities and the lockdown turned out to be an advantage to combat the pandemic and to prevent a health-system overload. AIM: The aim was to predict the plateau day of SARS-CoV-2 infections or deaths. RESULTS: The effect on the viral spread of the German measures taken and the impact on the peak of new infection cases is shown. By normalizing daily case numbers, the plateau day of the current outbreak in Germany could be calculated to be reached at April 12, 2020 (day 103 of 2020). CONCLUSION: Normalized case number curves are helpful to predict the time point at which no further new infections will occur if the epidemic situation remains stable. Upon reaching the plateau day during a lockdown phase, a residual time-period of about 2-3 weeks can be utilized to prepare a safe unlocking period. As can be learned from Asian countries such as South Korea and Taiwan there must be strict rules to keep the risk of infection low. Those include social distancing, face mask wearing in combination with digital contact tracing and serosurveillance studies. Following those rules, a safe dance around the infection curve allows to keep the population at a reduced infection rate.

Herd immunity or suppression strategy to combat COVID-19.

Some months ago, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) broke out in Wuhan, China, and spread rapidly around the world. Some states, such as the Netherlands, Germany, Great Britain, Sweden and the USA initially focused on keeping the restrictions for economy and society as low as possible. The responsible authorities were of the opinion - and still are e.g. in Sweden - that it is sufficient enough to protect particularly vulnerable persons such as the elderly or people with pre-existing conditions. The idea behind this is that as soon as 60 to 70 percent of the population is infected with a pathogen, a so-called "herd immunity" has developed. However, the increasing numbers of deaths and modelling studies showed the expected overload of the hospitals. Therefore, most countries decided for a temporary lockdown with the exception of Sweden.Based on the number of the total population, three times more people died from COVID-19 in Sweden (2679 deaths per 10 million inhabitants) compared to Germany (6848 deaths per 80 million inhabitants). The comparison Sweden versus Taiwan is even worse because 1072 times more people died in Sweden based on the number of the population (6 deaths per 24 million inhabitants).In the face of the lack of an antiviral treatment and the lack of a protective vaccine one must state Taiwan has made the best out of the pandemic situation whereas Sweden failed completely.

A lab-on-a-chip for preconcentration of bacteria and nucleic acid extraction.

To improve detection sensitivity, molecular diagnostics require preconcentration of low concentrated samples followed by rapid nucleic acid extraction. This is usually achieved by multiple centrifugation, lysis and purification steps, for instance, using chemical reagents, spin columns or magnetic beads. These require extensive infrastructure as well as time consuming manual handling steps and are thus not suitable for point of care testing (POCT). To overcome these challenges, we developed a microfluidic chip combining free-flow electrophoretic (FFE) preconcentration (1 ml down to 5 µl) and thermoelectric lysis of bacteria as well as purification of nucleic acids by gel-electrophoresis. The integration of these techniques in a single chip is unique and enables fast, easy and space-saving sample pretreatment without the need for laboratory facilities, making it ideal for the integration into small POCT devices. A preconcentration efficiency of nearly 100% and a lysis/gel-electrophoresis efficiency of about 65% were achieved for the detection of E. coli. The genetic material was analyzed by RT-qPCR targeting the superfolder Green Fluorescent Protein (sfGFP) transcripts to quantify mRNA recovery and qPCR to determine DNA background.

Correction: A lab-on-a-chip for preconcentration of bacteria and nucleic acid extraction.

[This corrects the article DOI: 10.1039/C8RA02177E.].

Asymptomatic HIV infection is characterized by rapid turnover of HIV RNA in plasma and lymph nodes but not of latently infected lymph-node CD4+ T cells.

OBJECTIVES: To study the kinetics of plasma viraemia and HIV-infected lymph-node cells in stable asymptomatic HIV infection with high CD4+ T-cell counts. METHODS: Nine asymptomatic HIV-infected patients with stable CD4+ T-cell counts (510-1350 x 10(6)/l) were treated with a triple-drug combination. Plasma viraemia was determined at days 0, 3, 7, 10, 14, 21 and 28 of treatment [Roche polymerase chain reaction (PCR) and ultrasensitive PCR assay]. Sequential lymph-node biopsies were examined in four patients before and after 4 weeks of treatment. Productively infected cells were counted in lymph-node sections (in situ hybridization). The infection rates of FACS-sorted CD4+ lymph-node T cells and the expression of single-spliced, double-spliced and full-length HIV transcripts were determined. RESULTS: HIV plasma RNA half-lives ranged from 1.4 to 2.7 days. Viral turnover varied between 0.07 and 7.54 x 10(8) copies per day. The number of productively infected lymph-node cells as well as the amount of extracellular virus in germinal centres was markedly reduced during treatment, paralleled by a clearance of single-spliced, double-spliced and full-length HIV transcripts from CD4+ lymph-node T cells. Plasma viraemia remained detectable with an ultrasensitive PCR assay in three out of four patients. The percentage of lymph-node CD4+ T cells harbouring proviral DNA decreased only slightly. CONCLUSIONS: The kinetics of HIV replication are rapid in stable asymptomatic infection, and the magnitude of replication varies considerably. Productively infected lymph-node cells and extracellular virus in germinal centres undergo a rapid turnover, whereas latently infected CD4+ T cells have a lower rate of turnover. The latter may contribute substantially to viral persistence during therapy.

Germinal centre CD4+ T cells are an important site of HIV replication in vivo.

OBJECTIVE: CD4+ T cells are the main target for HIV. However, the highest HIV antigen concentration in infected subjects accumulates on the cell surface of follicular dendritic cells in the germinal centres of the lymphoid tissue. Germinal centres contain a T-helper cell subset which expresses CD57 molecules. Here we analysed virus replication and viral load in CD57+CD4+ germinal centre T cells and in the CD4+ T cells found mostly outside germinal centres (CD57-CD4+). METHODS: Peripheral blood mononuclear cells and lymph-node cells were prepared, stained for CD4 and CD57 and purified by FACS. Defined cell numbers of CD4+CD57+ cells and CD4+CD57- cells were sorted directly into polymerase chain reaction (PCR) tubes by FACS, equipped with an automated cell deposition unit and analysed by PCR to detect proviral DNA. Based on Poisson distribution, the expected level of infection was calculated. Viral replication was determined by amplifying double-spliced, single-spliced, and full-length transcripts of HIV using serially diluted cDNA of the FACS-sorted cells. RESULTS: An up to 10-fold higher frequency of infected cells was found in the CD57+CD4+ germinal centre T cells compared with CD57-CD4+ T cells. Furthermore, active viral replication was detected almost exclusively in the CD57+CD4+ T cells. CONCLUSIONS: The CD57+CD4+ germinal centre T cells are one of the sites of HIV infection and replication that may play a pivotal role in the pathogenesis of HIV infection.

Human Kupffer cells infected with HIV-1 in vivo.

Blood monocytes as well as cells of the macrophage-phagocytic system in several tissues are targets for HIV-1 in vivo and in vitro. However, the data on HIV-1 infection of liver macrophages/Kupffer cells (KCs), which make up the main pool of fixed-tissue macrophages, is controversial. We therefore studied HIV-1 infection of KCs in vivo. Blood and liver tissue was obtained from seven AIDS patients shortly after death. Liver tissue was minced before processing. Cell suspensions were further purified by density gradient centrifugation, stained with anti-CD14 (blood monocytes) or anti-macrophage 25F9 (KCs), and separated by fluorescence-activated cell sorting (FACS). These highly purified cell populations were then analyzed for HIV-1 proviral DNA by the polymerase chain reaction (PCR). By performing PCR on FACS-purified cell populations, we could show that both KCs and peripheral blood monocytes were HIV-1-infected in 3 of 7 patients. KCs harbored HIV-1 proviral DNA only if peripheral blood monocytes were infected. These data show that KCs of the liver are infected with HIV-1 in vivo.

Kinetics of productive and latent HIV infection in lymphatic tissue and peripheral blood during triple-drug combination therapy with or without additional interleukin-2.

OBJECTIVE: To study decay rates of productively and latently infected cells in peripheral blood and lymph nodes during triple antiretroviral therapy and the possible impact of interleukin-2 (IL-2) on viral kinetics. METHODS: In this non-randomized study, nine antiretroviral-naive HIV-positive patients received either saquinavir hard gel capsules 2400 mg three times daily (group I; four patients) or saquinavir soft gel capsules 1200 mg three times daily and IL-2 (group II), in both cases together with two nucleoside analogues. Plasma viraemia and lymphocyte subsets were analysed. Axillary lymph nodes were excised before and after 12 weeks of therapy. Lymph node sections were examined by in situ hybridization for HIV RNA, and productively infected cells were counted. Infection rates of FACS-sorted CD3, CD4 lymph node and peripheral blood mononuclear cells were determined by nested DNA PCR. RESULTS: Baseline plasma HIV RNA levels ranged from < 25 to > 1 x 10(6) copies/ml and remained undetectable throughout the study in one patient in group I. Plasma viraemia became undetectable after 3 months in four patients (three in group I). Productively infected cells were markedly reduced in the follow-up lymph node specimens. HIV DNA-positive CD4 T cells were reduced in lymphoid tissue and peripheral blood in all six evaluable patients. There were no significant differences between the groups in the clearance rates of plasma virus and of HIV DNA-positive cells. CONCLUSIONS: Combined antiretroviral therapy rapidly suppressed active HIV replication in plasma and lymphoid tissue. Latently infected cells were cleared at a slower rate. Viral clearance did not appear to be markedly affected by additional IL-2 therapy.

Antibodies to p24 antigen do not specifically detect HIV-infected lymphocytes in AIDS patients.

A flow cytometric assay (FCA), which detects the p24 antigen in HIV-infected cell lines and in peripheral blood mononuclear cells (PBMC) of AIDS patients, has been described in several studies. However, the results presented here clearly show that this p24-FCA, although useful for the analysis of HIV infection of cells in vitro, does not specifically detect HIV-infected PBMC from patients. Isotype control antibodies also stained PBMC from HIV-infected patients to a greater degree than the PBMC from healthy controls. Furthermore, the CD4-negative lymphocytes, which are generally not infected with HIV, were also found to stain with anti-p24. Finally, no enrichment of HIV-infected cells was found in the FACS-purified CD4+p24+ lymphocytes, compared to the CD4+p24- cell fraction. The p24-FCA, therefore, was not useful for determining the percentage of infected PBMCs from HIV-infected individuals.

Cytomegalovirus pp65 antigen-guided preemptive therapy with ganciclovir in solid organ transplant recipients: a prospective, double-blind, placebo-controlled study.

BACKGROUND: The aim of this study was to evaluate pp65 antigen-guided antiviral therapy in preventing human cytomegalovirus (HCMV) infection in solid organ transplant recipients. METHODS: Ten kidney and two liver transplant recipients with asymptomatic HCMV infection were randomized either for i.v. ganciclovir or placebo treatment in a prospective, double-blind study. All patients were positive by HCMV pp65 antigen test at levels >5 positive cells/2 x 10(5) investigated cells. RESULTS: No cases of HCMV end-organ disease occurred. In contrast to patients on placebo (5/7), none of the patients on ganciclovir (0/5) developed HCMV-associated symptoms (P=0.01). However, because of the small number of patients, all three high-risk patients (donor seropositive, recipient seronegative) were randomized to placebo and all three developed symptoms. CONCLUSIONS: Preemptive antiviral therapy guided by the pp65 antigen test seems to have a beneficial effect on preventing HCMV-associated symptoms in kidney and liver transplant recipients.

Detection of DNA in single cells using an automated cell deposition unit and PCR.

A highly effective single-cell PCR method using a fluorescence-activated cell sorting (FACS)-based automated cell deposition unit (ACDU) that sorts single cells directly into PCR tubes was developed. To evaluate the sensitivity of this method, single ACH-2 cells (containing one HIV-1 genome per cell) were sorted, and 220 out of 228 samples (96.5%) were HIV DNA-positive by PCR. Furthermore, the number of samples accidentally containing more than one cell was determined by sorting single cells from a mixture of human cytomegalovirus (HCMV)-infected fibroblasts and ACH-2 cells. Multiplex nested PCR (nPCR) was then performed, detecting HCMV and HIV DNA simultaneously. From 66 sorted cells, 2 (3%) were double-positive for HIV and HCMV, 31 (47%) for HCMV alone, 30 (45.5%) for HIV alone and 3 (4.5%) were PCR-negative. The ACDU was then programmed to sort defined numbers of cells into PCR tubes. This is similar to classic dilution assays in that it allows the determination of the percentage of cells that was positive for a specific DNA. The accuracy of multiple cell deposition by the ACDU was evaluated by determining the percentage of HIV-positive cells in defined mixtures of ACH-2 and uninfected H9 cells. Infection rates determined by the ACDU correlated well with the rates expected from the given dilutions.

Late cytomegalovirus polyradiculopathy following haploidentical CD34+-selected hematopoietic stem cell transplantation.

A 55-year-old man with acute myeloid leukemia in second relapse presented 4 months after haploidentical CD34+-selected hematopoietic stem cell transplantation (HSCT) with symmetric, progressive neurological deficits of the lower extremities. Although there was no molecular evidence for drug resistance in the cerebral-spinal fluid, antiviral combination therapy failed to control the rapidly progressing CMV polyradiculopathy (PRP) and encephalitis, which were confirmed by autopsy studies. Late CMV PRP as an unusual manifestation of CMV disease should be kept in mind in patients with suggestive neurological symptoms after HSCT.

Infection of blood and bone marrow cells with the human cytomegalovirus in vivo.

The human cytomegalovirus (HCMV) is a major pathogen in immunocompromised patients. Both, primary infection and reactivation of latent virus can cause disease. Peripheral blood leukocytes (PBL) most likely play an important role in viral persistence and dissemination of infection. However, an open question has been whether HCMV actively replicates in PBL in vivo and whether the progenitor cells in the bone marrow are also infected. Previous studies on this issue are controversial. Here we summarize data on the tropism of HCMV for mature leukocyte populations as well as bone marrow progenitor cells during HCMV viremia. All cell populations were highly purified by a fluorescence activated cell sorter (FACS) and analyzed by PCR for the presence of viral genomic DNA. Moreover, mature leukocyte populations were investigated for mRNA expression of regulatory and viral structural proteins. We could show, that HCMV DNA was detected most frequently in granulocytes and monocytes as well as in CD34+ progenitor cells of immunosuppressed patients. Viral mRNA expression was found in granulocytes, monocytes, and lymphocyte fractions. In contrast, no HCMV DNA was found in healthy, seropositive individuals.

Clinical utility of cytomegalovirus urine cultures for ophthalmic care in patients with HIV.

BACKGROUND: The utility of cytomegalovirus (CMV) urine cultures was checked in patients with HIV (a) to identify those at risk for CMV retinitis and (b) to guide clinical decisions on treatment and prophylaxis of CMV retinitis. METHODS: HIV infected patients were tested for CMVuria by shell vial cell cultures. The prevalence of CMVuria was related to CD4 count, HIV risk group, and time before and after diagnosis of CMV retinitis. RESULTS: A total of 639 shell vial cell cultures were obtained from 266 HIV infected ophthalmic patients. Only 4% of all patients with a CD4 count > 400 x 10(6)/l shed CMV in their urine compared with 42% with a CD4 count < or = 50 x 10(6)/l. Twenty three of 25 patients with CMV retinitis had a CD4 count < or = 50 x 10(6)/l. Among 130 patients with a CD4 count < or = 50 x 10(6)/l (a) those who were CMVuric had a nearly sevenfold risk (p < 0.0001) of developing CMV retinitis (35%) compared with those who did not shed CMV in their urine (5%), and (b) CMVuria and CMV retinitis were more frequent in homosexuals (58%/25%) than in intravenous drug users (23%/15%). More than 1 year before diagnosis of CMV retinitis 18% of patients were CMVuric compared with 83% of patients who were CMV culture positive in the last 3 months. CMVuria under virustatic maintenance therapy is associated with worsening of retinitis in two thirds of cases. CONCLUSION: Ophthalmic screening of patients with HIV should include those with a CD4 count < or = 50 x 10(6)/l and focus on the subgroup with additional CMVuria. Screening of other patients can be dropped without undue risk in order to spare AIDS patients unnecessary hospital visits. CMVuria as a single finding, however, does not justify antiviral prophylaxis of CMV retinitis.

[Virus diseases in patients returning from the tropics]. / Viruskrankheiten bei Tropenrückkehrern.

The high density of populations and insufficient sanitary conditions increases the risk to acquire viral diseases in tropical areas. This holds true for ubiquitous as well as for regional viral infections. Hepatitis and AIDS are found worldwide, but play a dominant role in tropical areas. Classical tropical viral infections are zoonoses. They are primarily infections of nonhuman vertebrates (e.g. rodents) and of arthropod vectors and can be transmitted to man. According to the clinical outcome these viral infections can be divided into three groups: influenza-like disease with arthralgia, encephalitis and hemorrhagic fevers. The majority of infections belong to the first group, followed by encephalitis cases. Viral hemorrhagic fevers are rare in visitors of tropical areas. Antibody detection is the method of choice in the diagnosis of tropical viral infections. In special situations (e.g. Lassa fever) the direct detection of the virus by PCR can be helpful. Tests for the detection of arboviruses, filoviruses and arenaviruses are only performed at a few centers worldwide.

A new approach for diagnosis of bovine coronavirus using a reverse transcription recombinase polymerase amplification assay.

Bovine coronavirus (BCoV) is an economically significant cause of calf scours and winter dysentery of adult cattle, and may induce respiratory tract infections in cattle of all ages. Early diagnosis of BCoV helps to diminish its burden on the dairy and beef industry. Real-time RT-PCR assay for the detection of BCoV has been described, but it is relatively expensive, requires well-equipped laboratories and is not suitable for on-site screening. A novel assay, using reverse transcription recombinase polymerase amplification (RT-RPA), for the detection of BCoV is developed. The BCoV RT-RPA was rapid (10-20 min) and has an analytical sensitivity of 19 molecules. No cross-reactivity with other viruses causing bovine gastrointestinal and/or respiratory infections was observed. The assay performance on clinical samples was validated by testing 16 fecal and 14 nasal swab specimens and compared to real-time RT-PCR. Both assays provided comparable results. The RT-RPA assay was significantly more rapid than the real-time RT-PCR assay. The BCoV RT-RPA constitutes a suitable accurate, sensitive and rapid alternative to the common measures used for BCoV diagnosis. In addition, the use of a portable fluorescence reading device extends its application potential to use in the field and point-of-care diagnosis.

Identification of genetic evidence for dobrava virus spillover in rodents by nested reverse transcription (RT)-PCR and TaqMan RT-PCR.

A survey of 158 rodents caught in the Czech Republic identified Dobrava virus sequences closely related to that of the Dobrava virus type strain in Apodemus sylvaticus and Mus musculus rodents. The identity of A. sylvaticus was unequivocally confirmed by random amplified polymorphic DNA analysis. The data seem to indicate hantavirus spillover from Apodemus flavicollis to other rodents.