Microbiological and physicochemical quality enhancement of treated wastewater using raw and chemically modified clays from Sidi Bouzid region, Tunisia.

Environmental discharge of wastewater represents a source of chemical and biological pollutants. This study firstly evaluates the microbiological and physicochemical quality of treated wastewaters collected from two wastewater treatment plants (WWTPs) located in two different Tunisian cities namely Sidi Bouzid (SB) and Gafsa (G). Then, the capacity of three raw and acid/base-activated local clays to enhance the quality of wastewaters was assessed. The results indicate that the quantities of enteric bacteria (oscillating from 1.381 × 103 to 1.4 × 108 CFU/100 mL), fungi (between 1.331 × 103 and 1.781 × 104 CFU/100 mL), as well as SARS-CoV-2 (between 4.25 × 103 and 5.05 × 105 CFU/100 mL) and Hepatitis A virus RNA (form 4.25 × 103 to 7.4 × 104 CFU/100 mL) detected in effluent wastewaters were not in compliance with the Tunisian standards for both studied WWTPs. Likewise for other indicators such as electrical conductivity (ranging 4.9-5.4 mS/cm), suspended matter (145-160 g l-1), chemical oxygen demand (123-160 mg l-1), biological oxygen demand 5 (172-195 mg l-1), chloride, Total Kjeldahl nitrogen (TKN) and phosphorus contents (710, 58-66 and 9.47-10.83 mg l-1 respectively), the registered values do not agree with the set standards established for wastewater treatment. On the other hand, the pH values fitted (oscillating from 6.86 (at G) to 7.24 (at SB) with the Tunisian standards for both WWTPs. After treatment, wastewaters showed better values for the microbiological parameters, especially for the clays designed as AM and HJ1, which eliminated 100% of viruses. In addition, when acid-activated AM clays were applied, a marked improvement in the quality of physicochemical parameters was obtained, especially for suspended matter (2 and 4 g l-1 for SB and G, respectively), TKN (5.2 (SB) and 6.40 (G) mg/l), phosphorus (1.01 (SB) and 0.81 (G) mg/l). Our results open perspectives for the possibility of efficiently using these specific clays in the enhancement of the quality of treated wastewaters.

Type B coxsackieviruses and central nervous system disorders: critical review of reported associations.

Type B coxsackieviruses (CV-B) frequently infect the central nervous system (CNS) causing neurological diseases notably meningitis and encephalitis. These infections occur principally among newborns and children. Epidemiological studies of patients with nervous system disorders demonstrate the presence of infectious virus, its components, or anti-CV-B antibodies. Some experimental studies conducted in vitro and in vivo support the potential association between CV-B and idiopathic neurodegenerative diseases such as amyotrophic lateral sclerosis and psychiatric illness such as schizophrenia. However, mechanisms explaining how CV-B infections may contribute to the genesis of CNS disorders remain unclear. The proposed mechanisms focus on the immune response following the viral infection as a contributor to pathogenesis. This review describes these epidemiological and experimental studies, the modes of transmission of CV-B with an emphasis on congenital transmission, the routes used by CV-B to reach the brain parenchyma, and plausible mechanisms by which CV-B may induce CNS diseases, with a focus on potential immunopathogenesis.

Immunopathology in the brain of mice following vertical transmission of Coxsackievirus B4.

Coxsackie B viruses (CV-B) are associated with several central nervous system (CNS) disorders. These viruses are predominantly transmitted by fecal-oral route but vertical transmission can also occur. This work attempted to study the immune response ensuing vertical transmission of CV-B to the brain, and its eventual implementation in the brain pathogenesis. To this end, pregnant Swiss albino mice were inoculated with CV-B4 E2 or with sterile medium for control animals. At different ages after birth, brains were collected and analyzed for virus infection, histopathological changes and immune response. Infectious particles were detected in offspring's brain which demonstrates vertical transmission of the virus. This infection is persistent since the long lasting detection of viral RNA in offspring's brain. Some pathological signs including meningitis, edema and accumulation of inflammatory cells within and surrounding the inflammatory areas were observed. Immunoflorescence staining unveiled the presence of T lymphocytes and microgliosis in the sites of lesion for a long period after birth. Multiplex cytokines measurement upon supernatants of in vitro mixed brain cells and extracted mononuclear cells from offspring's brain has demonstrated an elevated secretion of the pro-inflammatory cytokines TNFα, IL-6 and IFNα and the chemokines RANTES and MCP-1. Hence, vertical transmission of CV-B4 and its persistence within offspring's brain can lead to pathological features linked to increased and sustained immune response.

Coxsackievirus B4 infection and interneuronal spread in primary cultured neurons.

Coxsackie B viruses (CV-B) are usually transmitted via the fecal-oral route and the virus gains the central nervous system (CNS) via the bloodstream. Nevertheless, other routes of spread of the virus to the CNS cannot be excluded, including the neuronal route. Neuronal cells, as well as non-neuronal cells (fibroblasts), were isolated from mice and inoculated with CV-B4 in the absence and presence of neutralizing serum. In the absence of neutralizing serum, virus titers recorded in neuron cultures and rates of infected neurons were non-significantly different compared to those recorded in fibroblast cultures. Higher cell mortality was noted among neurons than fibroblasts. The addition of neutralizing serum to neurons did not reduce significantly virus titers or rates of infected cells and cell viability was not significantly augmented, while virus titers and rates of infected fibroblasts were significantly reduced and their viability was significantly enhanced as well. Our results demonstrate the ineffectiveness of neutralizing serum to prevent neurons infection with CV-B4 which suggests a trans-synaptic transmission of CV-B4 between neurons.

Coxsackievirus B4 vertical transmission in a murine model.

BACKGROUND: Life-threatening infections with type B Coxsackieviruses (CV-B) are frequently encountered among newborns and are partly attributed to vertically-transmitted virus. Our current study investigates this alternative way of contamination by CV-B, using a mouse model. METHODS: Pregnant Swiss mice were intraperitoneally inoculated with CV-B4 E2 at gestational day 10(G) or 17G. Dams and offspring were monitored for mortality and morbidity, and sampled at different time-points to document the infection and explore eventual vertical transmission. RESULTS: Inoculation at day 10G induced an important rate of abortion and a decrease in the number of delivered pups per litter, whereas inoculation at day 17G was marked by preterm delivery and significant behavioral changes in dams. Only one case of spastic paralysis and one case of pancreatitis were recorded among surviving pups. Seroneutralization revealed anti-CV-B4 neutralizing antibodies in infected dams and their partial transfer to offspring. Viral genome detection by RT-PCR and viral progeny titration in several tissues (dams' uteri, amniotic sac, amniotic fluid, placenta, umbilical cord, pancreas and heart) attested and documented CV-B4 vertical transmission to the majority of analyzed offspring. Virus detection in fetuses suggests transplacental transmission, but perinatal transmission during delivery could be also suggested. Vertically transmitted CV-B might even persist since prolonged viral RNA detection was noticed in the pancreas and heart from offspring born to dams inoculated at day 17G. CONCLUSION: This model of CV-B4 vertical transmission in mice, in addition to allow a better understanding of CV-B infections in fetuses and newborns, constitutes a useful tool to investigate the pathogenesis of CV-B associated chronic diseases.

Essential Oil Composition, Antioxidant, Cytotoxic and Antiviral Activities of Teucrium pseudochamaepitys Growing Spontaneously in Tunisia.

The chemical composition, antioxidant, cytotoxic and antiviral activities of the essential oil obtained by hydrodistillation from the aerial parts of Teucrium pseudochamaepitys (Lamiaceae) collected from Zaghouan province of Tunisia are reported. The essential oil was analyzed by gas chromatography equipped with a flame ionization detector (GC-FID) and gas chromatography coupled with mass spectrometry (GC/MS). Thirty-one compounds were identified representing 88.6% of the total essential oil. Hexadecanoic acid was found to be the most abundant component (26.1%) followed by caryophyllene oxide (6.3%), myristicin (4.9%) and α-cubebene (3.9%). The antioxidant capacity of the oil was measured on the basis of the scavenging activity to the stable 2,2-diphenyl-1-picrylhydrazyl (DPPH). The IC50 value of the oil was evaluated as 0.77 mg·mL(-1). In addition, the essential oil was found to possess moderate cytotoxic effects on the HEp-2 cell line (50% cytotoxic concentration (CC50)=653.6 µg·mL(-1)). The potential antiviral effect was tested against Coxsackievirus B (CV-B), a significant human and mouse pathogen that causes pediatric central nervous system disease, commonly with acute syndromes. The reduction of viral infectivity by the essential oil was measured using a cytopathic (CPE) reduction assay.

Detection of SARS-CoV-2 in the sewerage system in Tunisia: a promising tool to confront COVID-19 pandemic.

Aim: The current study undertaken in Tunisia examines the use of wastewaters to monitor SARS-CoV-2 circulation. Materials & methods: Viral genetic materials collected in wastewaters during two different periods (September-October 2020 and February-April 2021) were concentrated using the adsorption-elution method. SARS-CoV-2 genes were researched by real-time PCR. Results: During the first period of the study, viral RNA was detected in 61.11% of the analyzed samples collected from Monastir city with a rate of 88.88% for raw wastewaters and 33.33% for treated wastewaters. Then, during the second period of the study, the quantitative analysis of wastewaters collected from seven governorates showed the presence of viral RNA among around 25% of them with variable RNA loads. The increased amounts of viral RNA detected in wastewaters were accompanied by an increase in the number of COVID-19 patients in Tunisia. Conclusion: Our results emphasize the importance of sewage survey in SARS-CoV-2 tracking.

Coxsackievirus B4 Transplacental Infection Severely Disturbs Central Tolerogenic Mechanisms in the Fetal Thymus.

Thymus plays a fundamental role in central tolerance establishment, especially during fetal life, through the generation of self-tolerant T cells. This process consists in T cells education by presenting them tissue-restricted autoantigens promiscuously expressed by thymic epithelial cells (TECs), thus preventing autoimmunity. Thymus infection by Coxsackievirus B (CV-B) during fetal life is supposed to disturb thymic functions and, hence, to be an inducing or accelerating factor in the genesis of autoimmunity. To further investigate this hypothesis, in our current study, we analyzed thymic expression of autoantigens, at the transcriptional and protein level, following in utero infection by CV-B4. mRNA expression levels of Igf2 and Myo7, major autoantigens of pancreas and heart, respectively, were analyzed in whole thymus and in enriched TECs together along with both transcription factors, Aire and Fezf2, involved in autoantigens expression in the thymus. Results show that in utero infection by CV-B4 induces a significant decrease in Igf2 and Myo7 expression at both mRNA and protein level in whole thymus and in enriched TECs as well. Moreover, a correlation between viral load and autoantigens expression can be observed in the whole thymus, indicating a direct effect of in utero infection by CV-B4 on autoantigens expression. Together, these results indicate that an in utero infection of the thymus by CV-B4 may interfere with self-tolerance establishment in TECs by decreasing autoantigen expression at both mRNA and protein level and thereby increase the risk of autoimmunity onset.

Assessment of Thymic Output Dynamics After in utero Infection of Mice With Coxsackievirus B4.

The thymus is the main organ of the lymphatic system, in which T cells undergo a rigorous selection to ensure that their receptors (TCRs) will be functional and will not react against the self. Genes encoding for TCR chains are fragmented and must be rearranged by a process of somatic recombination generating TCR rearrangement excision circles (TRECs). We recently documented coxsackievirus B4 (CV-B4) infection of Swiss albino mouse thymus in the course of in utero transmission. In the current study, we intended to evaluate thymic output in this experimental model. For this purpose, pregnant Swiss albino mice were inoculated with CV-B4 at day 10 or 17 of gestation, and thymus and spleen were sampled from offspring at different time points and then subjected to quantification of TREC molecules and Ptk7 gene expression. Results showed a pronounced effect of in utero CV-B4 infection on the thymus with an increase in the cellularity and, consequently, the weight of the organ. sj and DßTREC analysis, by real-time PCR, revealed a significant decrease following CV-B4 infection compared to controls, a decrease which gets worse as time goes by, both in the thymus and in the periphery. Those observations reflect a disturbance in the export of T cells to the periphery and their accumulation within the thymus. The evaluation of Ptk7 transcripts in the thymus, for its part, showed a decrease in expression, especially following an infection at day 10 of gestation, which supports the hypothesis of T cell accumulation in a mature stage in the thymus. The various effects observed correlate either negatively or positively with the viral load in the thymus and spleen. Disruption in thymic export may indeed interfere with T cell maturation. We speculate that this may lead to a premature release of T cells and the possibility of circulating autoreactive or proliferation-impaired T cell clones.

Housekeeping Gene Expression in the Fetal and Neonatal Murine Thymus Following Coxsackievirus B4 Infection.

The thymus fulfills the role of T-cell production and differentiation. Studying transcription factors and genes involved in T-cell differentiation and maturation during the fetal and neonatal periods is very important. Nevertheless, no studies to date have been interested in evaluating the expressions of housekeeping genes as internal controls to assess the varying expressions of different genes inside this tissue during that period or in the context of viral infection. Thus, we evaluated by real-time quantitative polymerase chain reaction (qPCR) the expression of the most common internal control genes in the thymus of Swiss albino mice during the fetal and neonatal period, and following in utero infection with Coxsackievirus B4. The stability of expression of these reference genes in different samples was investigated using the geNorm application. Results demonstrated that the expression stability varied greatly between genes. Oaz1 was found to have the highest stability in different stages of development, as well as following Coxsackievirus B4 infection. The current study clearly demonstrated that Oaz1, with very stable expression levels that outperformed other tested housekeeping genes, could be used as a reference gene in the thymus and thymic epithelial cells during development and following Coxsackievirus B4 infection.

Central nervous system infection following vertical transmission of Coxsackievirus B4 in mice.

Coxsackie B viruses (CV-B) are important pathogens associated with several central nervous system (CNS) disorders. CV-B are mainly transmitted by the faecal-oral route, but there is also evidence for vertical transmission. The outcome of in utero CV-B infections on offspring's CNS is poorly explored. The aim of this study was to investigate vertical transmission of CV-B to the CNS. For this purpose, pregnant Swiss albino mice were intraperitoneally inoculated with CV-B4 E2 at gestational days 10G or 17G. Different CNS compartments were collected and analyzed for virus infection and histopathological changes. Using plaque assays, we demonstrated CV-B4 E2 vertical transmission to offspring's CNS. Viral RNA persisted in the CNS up to 60 days after birth, as evidenced by a sensitive semi-nested(sn) reverse transcripton(RT)-PCR method. This was despite infectious particles becoming undetectable at later time points. Persistence was associated with inflammatory lesions, lymphocyte infiltration and viral dsRNA detected by immunohistochemistry. Offspring born to dams mock- or virus-infected at day 17G were challenged by the same virus at day 21 after birth (-+ and ++ groups, respectively). Sn-RT-PCR and histology results compared between both ++ and -+ groups, show that in utero infection did not enhance CNS infection during challenge of the offspring with the same virus.