Constructed wetlands for combined sewer overflow treatment: A state-of-the-art review.

Combined sewer overflows (CSOs) are a major source of surface water pollution and degradation. This is particularly visible where sewage collection with combined sewer and centralized treatment are well established, such as in Europe and North America: an overwhelming number of surface water bodies are in insufficient status of ecology, hydrology and physico-chemical parameters. Therefore, several countries have started implementing constructed wetlands (CWs) as mainstream on-spot treatment. This paper summarizes the main design approaches that can be adopted. We identified eight different schemes for the implementation of CSO-CWs, based on our international experience and documented by a literature analysis. The performance review includes conventional water quality parameters, as well as pathogen and emergent contaminant removal. Furthermore, modelling tools for advanced design and for understanding a wide applicability of these green infrastructures are presented. This paper also provides a review on other side benefits offered by the adoption of Nature-Based Solutions for CSO treatment, such as ecosystem services, and the most common issues related to their operation and maintenance. Our analysis has produced a list of key factors for design and operation, all derived from full-scale installations in operation up to more than ten years.

A patient with severe respiratory failure caused by novel human coronavirus.

We report a case of a 45-year-old patient who developed severe acute respiratory distress syndrome accompanied by renal failure. An infection with a novel human coronavirus was confirmed and found to be the reason for rapidly progressive respiratory failure of our patient.

Specific serology for emerging human coronaviruses by protein microarray.

We present a serological assay for the specific detection of IgM and IgG antibodies against the emerging human coronavirus hCoV-EMC and the SARS-CoV based on protein microarray technology. The assay uses the S1 receptor-binding subunit of the spike protein of hCoV-EMC and SARS-CoV as antigens. The assay has been validated extensively using putative cross-reacting sera of patient cohorts exposed to the four common hCoVs and sera from convalescent patients infected with hCoV-EMC or SARS-CoV.

IFN-α subtypes: distinct biological activities in anti-viral therapy.

During most viral infections, the immediate host response is characterized by an induction of type I IFN. These cytokines have various biological activities, including anti-viral, anti-proliferative and immunomodulatory effects. After induction, they bind to their IFN-α/ß receptor, which leads to downstream signalling resulting in the expression of numerous different IFN-stimulated genes. These genes encode anti-viral proteins that directly inhibit viral replication as well as modulate immune function. Thus, the induction of type I IFN is a very powerful tool for the host to fight virus infections. Many viruses evade this response by various strategies like the direct suppression of IFN induction or inhibition of the IFN signalling pathway. Therefore, the therapeutic application of exogenous type I IFN or molecules that induce strong IFN responses should be of great potential for future immunotherapies against viral infections. Type I IFN is currently used as a treatment in chronic hepatitis B and C virus infection, but as yet is not widely utilized for other viral infections. One reason for this restricted clinical use is that type I IFN belongs to a multigene family that includes 13 different IFN-α subtypes and IFN-ß, whose individual anti-viral and immunomodulatory properties have so far not been investigated in detail to improve IFN therapy against viral infections in humans. In this review, we summarize the recent achievements in defining the distinct biological functions of type I IFN subtypes in cell culture and in animal models of viral infection as well as their clinical usage in chronic hepatitis virus infections.

Assays for laboratory confirmation of novel human coronavirus (hCoV-EMC) infections.

We present a rigorously validated and highly sensitive confirmatory real-time RT-PCR assay (1A assay) that can be used in combination with the previously reported upE assay. Two additional RT-PCR assays for sequencing are described, targeting the RdRp gene (RdRpSeq assay) and N gene (NSeq assay), where an insertion/deletion polymorphism might exist among different hCoV-EMC strains. Finally, a simplified and biologically safe protocol for detection of antibody response by immunofluorescence microscopy was developed using convalescent patient serum.

Interaction of oxygen concentration and retention of pollutants in vertical flow constructed wetlands for CSO treatment.

The EU Water Framework Directive (WFD) calls for a good quality of all water bodies. Retention soil filters (RSF) have been developed to treat discharges from combined sewers systems. RSF have proved over the past 15 years to be the most effective measure to meet the EU WFD standards, especially for small or particularly sensitive receiving waters, which require an enhanced reduction of emissions from combined sewer overflows (CSOs). The paper presents results from laboratory-scale experiments, in which the oxygen measurement in the filter plays a main role. The results show remarkable differences in oxygen concentrations in different filter depths. The highest oxygen consumption takes place in the upper part of the filter. In the lower part the re-aeration of sewage from the soil air dominates. This indicates that the biological activity is limited to the upper part of the filter. The availability of oxygen in the filter is a sign for degradation of wastewater compounds (ammonium, COD) under certain conditions and already takes place during the filter operation. The removal of ammonium especially cannot be strictly divided into phases of sorption during the loading and oxidation during the dry period any more.

Constructed wetlands for CSO treatment: an overview of practice and research in Germany.

Vertical flow treatment wetlands have been developed as very useful tools for treatment of combined sewage overflow. Several systems have been in operation for over 15 years. Based on recent research work, new technical guidelines now recommend systems with a drained filter of sand 0/2 mm and a throttled outflow. COD, NH4-N and SS removal rates of 85-99% can be expected from this type of filter. SS loadings that are too high and very long or frequent inundation affect the performance adversely. Information for successful long-term operation were derived from various existing plants.

Simulation of a subsurface vertical flow constructed wetland for CSO treatment.

Constructed wetlands (CWs) have proved to be a highly effective measure to reduce the ecological impact of combined sewer overflows (CSOs) on receiving waters. Due to the stochastic nature of the loading regime and the multitude of environmental influences, assessment of the performance of such plants requires detailed mathematical modelling. A multi-component reactive transport module (CW2D) was applied to simulate the flow, transport and degradation processes occurring in a CW for CSO treatment. CW2D was originally developed to simulate the treatment of municipal wastewater in subsurface flow CWs. Loading and operational conditions in CSO treatment differ fundamentally from the conditions occurring for wastewater treatment. Despite these differences, first results from the simulation of lab-scale experiments show, that the model is generally applicable to this type of plant. Modelling of adsorption, degradation processes, and influent fractionation, however, require further research.

Role of interleukin-4 (IL-4), IL-12, and gamma interferon in primary and vaccine-primed immune responses to Friend retrovirus infection.

The immunological resistance of a host to viral infections may be strongly influenced by cytokines such as interleukin-12 (IL-12) and gamma interferon (IFN-gamma), which promote T helper type 1 responses, and IL-4, which promotes T helper type 2 responses. We studied the role of these cytokines during primary and secondary immune responses against Friend retrovirus infections in mice. IL-4- and IL-12-deficient mice were comparable to wild-type B6 mice in the ability to control acute and persistent Friend virus infections. In contrast, more than one-third of the IFN-gamma-deficient mice were unable to maintain long-term control of Friend virus and developed gross splenomegaly with high virus loads. Immunization with a live attenuated vaccine virus prior to challenge protected all three types of cytokine-deficient mice from viremia and high levels of spleen virus despite the finding that the vaccinated IFN-gamma-deficient mice were unable to class switch from immunoglobulin M (IgM) to IgG virus-neutralizing antibodies. The results indicate that IFN-gamma plays an important role during primary immune responses against Friend virus but is dispensable during vaccine-primed secondary responses.

Enhanced cellular immune response and reduced CD8(+) lymphocyte apoptosis in acutely SIV-infected Rhesus macaques after short-term antiretroviral treatment.

Losing the decisive virus-specific functions of both CD4(+) and CD8(+) T lymphocytes in the first weeks after immunodeficiency virus infection ultimately leads to AIDS. The SIV/rhesus monkey model for AIDS was used to demonstrate that a 4-week chemotherapeutic reduction of viral load during acute SIV infection of macaques allowed the development of a competent immune response able to control virus replication after discontinuation of treatment in two of five monkeys. Increasing SIV-specific CD4(+) T-helper-cell proliferation was found in all macaques several weeks after treatment, independent of their viral load. However, only macaques with low viral loads showed persistent T-cell reactivity of lymph node cells. In contrast to animals with higher viral loads, T-helper-cell counts and memory T-helper cells did not decline in the two macaques controlling viral replication. Lymphocyte apoptosis was consistently low in all treated macaques. In contrast, high CD8(+) lymphocyte death but only slightly increased CD4(+) lymphocyte apoptosis were observed during the first weeks after infection in untreated control animals, indicating that early apoptotic death of virus-specific CTL could be an important factor for disease development. Antiretroviral treatment early after infection obviously retained virus-specific and competent T lymphocytes, whereby a virus-specific immune response could develop in two animals able to control the viral replication after cessation of treatment.

Cellular and molecular mechanisms of vaccine-induced protection against retroviral infections.

More than 15 years after the discovery of human immunodeficiency virus (HIV), researchers are still struggling to design a protective AIDS vaccine. A remaining problem is a lack of basic knowledge about the immunological requirements for protection against retroviruses. Infection of macaque monkeys with simian immunodeficiency virus is still the best model for HIV vaccine research. However, in this model it remains difficult to determine protective immunological mechanisms because of limited numbers of experimental animals and their genetic heterogeneity. Thus, fundamental concepts in retroviral immunology have to be defined in other ways such as mouse models. This minireview summarizes new findings on cellular and molecular mechanisms in protection of mice against Friend murine retrovirus infection. It has been shown that complex immune responses, including B and T cell responses, are required for efficient protection in this model. Multiple viral antigens are necessary to elicit such broad immune reactivity. Efficacious vaccines must protect not only against acute disease, but also against the establishment of persistent infections or the host is at serious risk of virus reactivation. The minireview closes with a discussion on the relevance of findings from the mouse model on the design of a protective vaccine against HIV.

Different immunological requirements for protection against acute versus persistent Friend retrovirus infections.

The propensity of retroviruses to rapidly establish persistent infections poses a formidable problem in vaccination strategies. In the current study, we use a live attenuated vaccine to study protection against acute and persistent Friend virus infections in mice. Adoptive transfers of immune CD8(+) T cells combined with passive immunizations with virus-neutralizing antibodies increased protection against acute disease compared with either treatment alone, but there was no protection against the establishment of persistent infection. In addition, the protection against acute disease elicited by the combination treatment was dependent on endogenous CD4(+) T cells as no protection was achieved in CD4(+) T-cell-depleted mice. Quantitative studies showed that doubling the numbers of immune lymphocytes used in adoptive transfer experiments increased protection against acute disease depending on the type of lymphocyte subset used in the transfer. CD8(+) T cells were the most potent subset for the transfer of such protection. However, even high numbers of immune CD8(+) T cells gave no protection against the establishment of persistent infections. The data indicate that strengthening the numbers of specific immune cell subsets may have a beneficial effect on protection against acute disease, but protection from establishment of persistence requires complex immune responses involving multiple lymphocyte subsets.

Herpesvirus saimiri-transformed macaque T cells are tolerated and do not cause lymphoma after autologous reinfusion.

Human T cells are transformed in vitro to stable growth after infection with herpesvirus saimiri subgroup C strain C488, and they retain their antigen-specific reactivity and other important functional features of mature activated T lymphocytes. The virus persists as nonintegrating episomes in human T cells under restricted viral gene expression and without production of virus particles. This study analyzes the behavior of herpesvirus-transformed autologous T cells after reinfusion into the donor under close-to-human experimental conditions. T cells of 5 macaque monkeys were transformed to stable interleukin-2 dependent growth and were intravenously infused into the respective donor. The animals remained healthy, without occurrence of lymphoma or leukemia for an observation period of more than 1 year. Over several months virus genomes were detectable in peripheral blood cells and in cultured T cells by polymerase chain reaction. In naive control animals, a high-dose intravenous infection rapidly induced pleomorphic peripheral T-cell lymphoma. In contrast, monkeys were protected from lymphoma after challenge infection if they had previously received autologous T-cell transfusions. High levels of antibodies against virus antigens were detectable after challenge infection only. Taken together, herpesvirus-transformed T cells are well tolerated after autologous reinfusion. This may allow us to develop a novel concept for adoptive T-cell mediated immunotherapy.

Accelerated clearance of SHIV in rhesus monkeys by virus-like particle vaccines is dependent on induction of neutralizing antibodies.

Recombinant, insect cell derived SIV Pr56(gag) virus-like particles (VLPs) have been modified either by inserting HIV-1 Gp160 derived peptides into the Pr56(gag) precursor or by integrating the complete HIV-1 gp120 in the particle membrane. To investigate the protective efficacy of these particulate antigens, rhesus macaques were immunized with VLPs both adjuvant-free or adsorbed to alum. In addition, recombinant Semliki Forest viruses (SFV) expressing proteins corresponding to the VLP constructs were established and administered as live vaccines in combination with particulate antigens. Vaccination induced specific humoral responses irrespective of the immunization regimen. However, in contrast to Pr56(gag)-specific antibodies, Env-specific antibody titers could not be increased by booster immunizations in this study. Cell-mediated immune responses were detected following vaccination with VLP-preparations as well as recombinant SFVs. A tendency towards stimulating both enhanced cell mediated as well as humoral immune responses was observed following priming with recombinant SFVs. Upon challenge with SHIV-4 all vaccinated monkeys became infected. However, animals, that were vaccinated with VLPs presenting the complete gp120, managed to clear virus faster than nonimmunized controls. The observed virus elimination significantly correlated with an anamnestic antibody response and an accelerated appearance of neutralizing antibodies postchallenge.

Kinetics of the development of protective immunity in mice vaccinated with a live attenuated retrovirus.

Vaccination of mice with a live attenuated vaccine virus induces potent protection against subsequent challenge with pathogenic Friend retroviral complex. The kinetic studies presented here demonstrate protection from acute splenomegaly as early as 1 week postvaccination. At this time point virus-specific cytotoxic T lymphocytes (CTL) were demonstrable in direct chromium release assays. However, during the first 2 weeks after vaccination protection was incomplete since the mice were not protected against establishment of low-level persistent infections in the spleen. By 3 weeks postvaccination the animals were protected against the establishment of persistent virus as well as acute splenomegaly. The timing of this complete protection correlated with the presence of both virus-neutralizing antibodies and primed CTL in the immunized mice. Within 3 days of virus challenge, vaccinated mice showed high levels of activated B cells and CD4(+) and CD8(+) T cells, indicating an efficient priming of all lymphocyte subsets. Despite very limited replication of the vaccine virus, the protective effect was long lived and was still present 6 months after immunization.

Construction and characterization of recombinant VLPs and Semliki-Forest virus live vectors for comparative evaluation in the SHIV monkey model.

For testing of recombinant virus-like particles (VLPs) in the SHIV monkey model, SIVmac239 Pr56gag precursor-based pseudovirions were modified by HIV-1 gp160 derived peptides. First, well-characterized epitopes from the HIV-1 envelope glycoprotein were inserted into the Pr56gag precursor by replacing defined regions that were shown to be dispensable for virus particle formation. Expression of these chimeric proteins in a baculovirus expression system resulted in efficient assembly and release of non-infectious, hybrid VLPs. In a second approach the HIV-1IIIB external glycoprotein gp120 was covalently linked to an Epstein-Barr virus derived transmembrane domain. Coexpression of the hybrid envelope derivative with the Pr56gag precursor yielded recombinant SIV derived Pr56gag particles with the HIV-1 gp120 firmly anchored on the VLP surface. Immunization of rhesus monkeys with either naked VLPs or VLPs adsorbed to alum induced substantial serum antibody titers and promoted both T helper cell and cytotoxic T lymphocyte responses. Furthermore, priming macaques with the corresponding set of recombinant Semliki-Forest viruses tended to enhance the immunological outcome. Challenge of the immunized monkeys with chimeric SHIV resulted in a clearly accelerated reduction of the plasma viremia as compared to control animals.