Highly efficient and sensitive membrane-based concentration process allows quantification, surveillance, and sequencing of viruses in large volumes of wastewater.

Wastewater-based epidemiology is experiencing exponential development. Despite undeniable advantages compared to patient-centered approaches (cost, anonymity, survey of large populations without bias, detection of asymptomatic infected peoples…), major technical limitations persist. Among them is the low sensitivity of the current methods used for quantifying and sequencing viral genomes from wastewater. In situations of low viral circulation, during initial stages of viral emergences, or in areas experiencing heavy rains, the extremely low concentrations of viruses in wastewater may fall below the limit of detection of the current methods. The availability during crisis and the cost of the commercial kits, as well as the requirement of expensive materials such as high-speed centrifuge, can also present major blocks to the development of wastewater-based epidemiological survey, specifically in low-income countries. Thereby, highly sensitive, low cost and standardized methods are still needed, to increase the predictability of the viral emergences, to survey low-circulating viruses and to make the results from different labs comparable. Here, we outline and characterize new protocols for concentrating and quantifying SARS-CoV-2 from large volumes (500 mL-1 L) of untreated wastewater. In addition, we report that the methods are applicable for monitoring and sequencing. Our nucleic acid extraction technique (the routine C: 5 mL method) does not require sophisticated equipment such as automatons and is not reliant on commercial kits, making it readily available to a broader range of laboratories for routine epidemiological survey. Furthermore, we demonstrate the efficiency, the repeatability, and the high sensitivity of a new membrane-based concentration method (MBC: 500 mL method) for enveloped (SARS-CoV-2) and non-enveloped (F-specific RNA phages of genogroup II / FRNAPH GGII) viruses. We show that the MBC method allows the quantification and the monitoring of viruses in wastewater with a significantly improved sensitivity compared to the routine C method. In contexts of low viral circulation, we report quantifications of SARS-CoV-2 in wastewater at concentrations as low as 40 genome copies per liter. In highly diluted samples collected in wastewater treatment plants of French Guiana, we confirmed the accuracy of the MBC method compared to the estimations done with the routine C method. Finally, we demonstrate that both the routine C method processing 5 mL and the MBC method processing 500 mL of untreated wastewater are both compatible with SARS-CoV-2 sequencing. We show that the quality of the sequence is correlated with the concentration of the extracted viral genome. Of note, the quality of the sequences obtained with some MBC processed wastewater was improved by dilutions or enzyme substitutions suggesting the presence of specific enzyme inhibitors in some wastewater. To the best of our knowledge, our MBC method is one of the first efficient, sensitive, and repeatable method characterized for SARS-CoV-2 quantification and sequencing from large volumes of wastewater.

SARS-CoV-2 genome quantification in wastewaters at regional and city scale allows precise monitoring of the whole outbreaks dynamics and variants spreading in the population.

SARS-CoV-2 is a coronavirus causing a globalized outbreak called COVID-19. SARS-CoV-2 transmission is associated with inhalation of contaminated respiratory droplets and could causes severe complications. Until today several "waves" of infections have been observed despite implementation of strict health policies. Decisions for such sanitary measures are based on population health monitoring. Unfortunately, for COVID-19, a significant proportion of individuals are asymptomatic but play a role in the virus transmission. To overcome these limitations, several strategies were developed including genome quantification in wastewater that could allow monitoring of the health status of population, since shedding of SARS-CoV-2 in patient stool is frequent. Wastewater-based epidemiology (WBE) was established and several countries implemented this approach to allow COVID-19 outbreak monitoring. In France, the OBEPINE project performed a quantitative analysis of SARS-CoV-2 in raw wastewater samples collected from major wastewater treatment plants (WWTP) since March 2020. In the greater Paris area 1101 samples (507 for five WWTP and 594 for sewer) were collected. This 16 months monitoring allows us to observe the outbreak dynamics. Comparison of WBE indicators with health data lead to several important observation; the good level of correlation with incidence rates, the average 3 days lead time, and the sensitivity (WBE change when incidence is > to 7/100000 inhabitants). We also compared the local monitoring (city level) with the regional monitoring, to help cluster identification. Moreover, variants of concern (VOC) emerged due to the selection pressure. We developed a specific RT-qPCR method targeting the deletion H69-V70 in the spike protein, using this deletion as a proxy of the B.1.1.7 presence in the wastewater. With this data we demonstrate the predominant role played by this strain in the third wave. All these results allow a better description and understanding of the pandemic and highlight the role of such WBE indicators.

[The French Armed Forces Biomedical Research Institute (IRBA) and wastewater-based epidemiology: Applicability and relevance in armed forces]. / L'Institut de recherche biomédicale des armées (IRBA) et l'épidémiologie des eaux usées : intérêt pour les forces armées.

The French Armed Forces Biomedical Research Institute (IRBA) deeply involved in research on SARS-COV-2, participated in the creation of the Obépine sentinel network in charge of detecting, qualifying and quantifying the virus genome in wastewater in France. During this pandemic, wastewater-based epidemiology has proven to be a first class public health tool for assessing viral dynamics in populations and environment. Obépine has also conducted research demonstrating the low infectivity of faeces and wastewater and allowed for early detection of epidemic waves linked to new variants. The IRBA has adapted this powerful tool to the monitoring of viral infections on board the aircraft carrier Charles-de-Gaulle in order to get an operational system for anticipation after the first local outbreak in 2020. The presence of this surveillance and anticipation tool has allowed a better management of SARS-CoV-2 contingent introductions on board during stopovers or crewmembers entries. The combination of a mandatory vaccination protocol and the surveillance of viral circulation in black waters has made it possible to identify and locate cases, and thus to continue the operational mission in the COVID-19 environment while limiting the spread and preserving the health of the crew. This innovative tool can easily be redirected to the search for any other pathogens in blackwater or even, in the long term, to ensure health surveillance of any military establishment, at sea or on land, in France or on overseas bases.

Several forms of SARS-CoV-2 RNA can be detected in wastewaters: Implication for wastewater-based epidemiology and risk assessment.

The ongoing global pandemic of coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been a public health emergency of international concern. Although SARS-CoV-2 is considered to be mainly transmitted by inhalation of contaminated droplets and aerosols, SARS-CoV-2 is also detected in human feces and to a less extent in urine, and in raw wastewaters (to date viral RNA only) suggesting that other routes of infection may exist. Monitoring SARS-CoV-2 genomes in wastewaters has been proposed as a complementary approach for tracing the dynamics of virus transmission within human population connected to wastewater network. The understanding on SARS-CoV-2 transmission through wastewater surveillance, the development of epidemic modeling and the evaluation of SARS-CoV-2 transmission from contaminated wastewater are largely limited by our knowledge on viral RNA genome persistence and virus infectivity preservation in such an environment. Using an integrity based RT-qPCR assay this study led to the discovery that SARS-CoV-2 RNA can persist under several forms in wastewaters, which provides important information on the presence of SARS-CoV-2 in raw wastewaters and associated risk assessment.

Evaluation of lockdown effect on SARS-CoV-2 dynamics through viral genome quantification in waste water, Greater Paris, France, 5 March to 23 April 2020.

IntroductionSevere acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the etiological agent of coronavirus disease (COVID-19). People infected with SARS-CoV-2 may exhibit no or mild non-specific symptoms; thus, they may contribute to silent circulation of the virus among humans. Since SARS-CoV-2 RNA can be detected in stool samples, monitoring SARS-CoV-2 RNA in waste water (WW) has been proposed as a complementary tool to investigate virus circulation in human populations.AimTo test if the quantification of SARS-CoV-2 genomes in WW correlates with the number of symptomatic or non-symptomatic carriers.MethodWe performed a time-course quantitative analysis of SARS-CoV-2 by RT-qPCR in raw WW samples collected from several major WW treatment plants in Greater Paris. The study period was 5 March to 23 April 2020, including the lockdown period in France (from 17 March).ResultsWe showed that the increase of genome units in raw WW accurately followed the increase of human COVID-19 cases observed at the regional level. Of note, the viral genome could be detected before the epidemic grew massively (around 8 March). Equally importantly, a marked decrease in the quantities of genome units was observed concomitantly with the reduction in the number of new COVID-19 cases, 29 days following the lockdown.ConclusionThis work suggests that a quantitative monitoring of SARS-CoV-2 genomes in WW could generate important additional information for improved monitoring of SARS-CoV-2 circulation at local or regional levels and emphasises the role of WW-based epidemiology.

[The psychiatrist and the Tako Tsubo]. / Le psychiatre de liaison et le Tako Tsubo

INTRODUCTION: Stress cardiopathy, also called "Tako Tsubo" is a cardiac pathology linked to an acute coronary syndrome with electrocardiographic signs and an increase in the level of cardiac enzymes, without any abnormality on coronarography. This syndrome is secondary to great physical or mental stress. Mortality and the risk of recurrence are low. However, there is no consensus for treatment or prevention. CASE REPORT: We report the case of 75-year-old woman presenting recurrent cardiogenic shocks. A symptomatic sinusal dysfunction motivated the introduction of a pacemaker in March 2008. One month later, she was hospitalized for a new cardiogenic shock with left ventricular dysfunction, a 40% ejection fraction, and a third degree mitral insufficiency. Cardiac enzymes were initially elevated. Electrocardiogram showed an ST elevation. The transthoracic echocardiogram revealed a left anterolateral ventriculogram dysfunction, and cardiac catheterization showed healthy coronary arteries. The cardiologist wondered about the existence of an anxiodepressive syndrome. No personal psychiatric background was known. The patient was widowed 3 years earlier. She described herself as a naturally anxious person. She hadn't experienced any recent stressing event. She was not depressed and wasn't taking any psychotropic drug. Her family was caring for her. The next day, the patient had another cardiogenic shock and died a few hours later. Maybe the introduction of the pacemaker occasioned one more stress for this patient… DISCUSSION: We know that people with a stressing job have probably more chance to suffer a myocardial infarction (the risks are 1.5 or two times greater for them). The prevalence of cardiomyopathy syndrome is 4.9% for women. These women have gone through the menopause, with a history of hypertension and anxiodepressive symptoms. However, we do not find any similar description (behavioural scheme type A) as is shown by the psychosomatic school in cases of patients who have gone through myocardial infarction. We also can question ourselves about the fact that some people can be predisposed to suffer from "Tako Tsubo" cardiomyopathy and about the existence of personality disorders. What then is the role of the psychiatrist with these patients?

[Pathophysiological mechanisms of dengue fever: critical review of current concepts]. / Mécanismes physiopathologiques de la dengue: revue critique des hypothèses.

In the 20th century dengue fever became one of the leading causes of morbidity and mortality throughout the tropics. The dengue virus is an arbovirus transmitted by Aedes mosquitoes. There are four distinct serotypes of dengue arbovirus (DENV-1, 2, 3, 4). According to the World Health Organization, a person infected by one of the dengue viruses can develop symptoms ranging from the classical self-limiting form characterized by high temperature, headache, myalgia, and arthralgia to the severe, potentially fatal, form known as dengue shock syndrome. For over 40 years the main explanation for the pathogenesis of dengue has been based on the "antibody-dependent enhancement" (ADE) concept stating that enhancing antibodies acquired during a primary infection increase the number of infected cells, and thus the level of viremia, during secondary infection. However the severity of dengue is not limited to dengue shock syndrome and there are many cases that do not conform to the ADE concept. A meta-analysis could provide crucial information for resolving this controversy and open the way for development of a monovalent vaccine against the dengue virus as for the closely related yellow fever virus.

Validation of single real-time TaqMan PCR assay for the detection and quantitation of four major genotypes of hepatitis E virus in clinical specimens.

Since the characterization of the genome of the hepatitis E virus (HEV) in 1990, a large genetic diversity has been described. A single real-time reverse transcription (RT)-PCR assay with TaqMan technology has been validated which uses only one set of primers and probe within the ORF2 HEV region (nt 5207-5292) for the detection and quantification of the four major genotypes of HEV. This assay proved to be as efficient as the conventional RT-PCR methodology for the detection of HEV in clinical samples testing positive previously. The real-time RT-PCR and conventional RT-PCR were performed comparatively on 60 pairs of sera and stools collected during a recent outbreak of hepatitis E in Darfur. The real-time RT-PCR assay was 10- to 100-fold sensitive than for conventional RT-PCR assays used in this study with a range quantitation from 1.8 x 10(1) to 7.2 x 10(3) RNA copies/microl in clinical samples (serum and stools).

Clinical characteristics and risk factors for Kraepelinian subtype of schizophrenia: replication of previous findings and relation to summer birth.

The aims of the study were: (1) to replicate findings that patients with Kraepelinian schizophrenia constitute a distinct subgroup and (2) to examine the relationship between season of birth and the Kraepelinian subtype. Thirty-one Kraepelinian patients, defined on the basis of a longitudinal criterion--at least 5 years of continuous and complete dependence on others to maintain the basic necessities of life, including food, clothing and shelter--were compared with 279 non-Kraepelinian schizophrenic patients. All patients met ICD-10 criteria for schizophrenia and were evaluated with the Positive and Negative Syndrome Scale. Kraepelinian schizophrenic patients had more negative symptoms and were more disorganized than non-Kraepelinian patients. Positive and anxious-depressive symptoms did not differ between the two groups. Among Kraepelinian patients, there was an excess number of births in the month of July. These findings are consistent with previous reports that Kraepelinian patients could have a disease with an etiopathophysiology separate from that of other schizophrenic patients.

Human immunodeficiency virus type 1 entry into macrophages mediated by macropinocytosis.

Whereas human immunodeficiency virus (HIV) infects various cell types by fusion at the plasma membrane, we observed a different entry route in human primary macrophages, in which macropinocytosis is active. Shortly after exposure of macrophages to HIV-1 and irrespective of viral envelope-receptor interactions, particles were visible in intracellular vesicles, which were identified as macropinosomes. Most virions appeared subsequently degraded. However, fusion leading to capsid release in the cytosol and productive infection could take place inside vesicles when particles were properly enveloped. These observations provide new insights into HIV-1 interactions with a cell target relevant to pathogenesis. They may have implications for the design of soluble inhibitors aimed at interfering with the fusion or entry processes.

Close but distinct regions of human herpesvirus 8 latency-associated nuclear antigen 1 are responsible for nuclear targeting and binding to human mitotic chromosomes.

Human herpesvirus 8 is associated with all forms of Kaposi's sarcoma, AIDS-associated body cavity-based lymphomas, and some forms of multicentric Castleman's disease. Herpesvirus 8, like other gammaherpesviruses, can establish a latent infection in which viral genomes are stably maintained as multiple episomes. The latent nuclear antigen (LANA or LNAI) may play an essential role in the stable maintenance of latent episomes, notably by interacting concomitantly with the viral genomes and the metaphase chromosomes, thus ensuring an efficient transmission of the neoduplicated episomes to the daughter cells. To identify the regions responsible for its nuclear and subnuclear localization in interphase and mitotic cells, LNAI and various truncated forms were fused to a variant of green fluorescent protein. This enabled their localization and chromosome binding activity to be studied by low-light-level fluorescence microscopy in living HeLa cells. The results demonstrate that nuclear localization of LNAI is due to a unique signal, which maps between amino acids 24 and 30. Interestingly, this nuclear localization signal closely resembles those identified in EBNA1 from Epstein-Barr virus and herpesvirus papio. A region encompassing amino acids 5 to 22 was further proved to mediate the specific interaction of LNA1 with chromatin during interphase and the chromosomes during mitosis. The presence of putative phosphorylation sites in the chromosome binding sites of LNA1 and EBNA1 suggests that their activity may be regulated by specific cellular kinases.

[Activated protein C resistance and cardiolipin antibodies in leg ulcers]. / Résistance à la protéine C activée et anticorps anti-cardiolipine dans les ulcères de jambe.

BACKGROUND: We conducted a prospective study to determine the prevalence of activated protein C resistance and anticardiolipin antibodies in leg ulcers, whatever venous, arterial or arteriovenous. PATIENTS AND METHODS: One hundred fifteen patients hospitalized for leg ulcers, without antiphospholipid syndrome were included. The vascular abnormalities were studied by clinical examination, Doppler, duplex Doppler and, when required, arteriography. Activated protein C resistance was isolated by a "classic" test (normalized APTT ratio in PCa presence or absence) and by a "second generation test" (by preliminary dilution with deficient factor V plasma). All patients with abnormal results on the second test were screened for the factor V Leiden (by PCR amplication with use of restriction enzymes). Anticardiolipin antibodies were investigated with an ELISA method with Harris standards as reference, in which the positive threshold was established at 20 units. RESULTS: Among these 115 patients, 50 venous (43.5 p. 100), 23 arterial (20 p. 100), 42 arteriovenous (36.5 p. 100) leg ulcers were identified. Activated protein C resistance was isolated in 12 cases (10.4 p. 100) (heterozygous carriers): 7 venous ulcers, 3 arteriovenous, 2 arterial. Anticardiolipin antibodies were measured at significant level in 49 cases (42.6 p. 100): 21 venous ulcers, 18 arteriovenous, 10 arterial. DISCUSSION: In this study, there was no statistical difference between the activated protein C resistance prevalence in leg ulcers when compared with Lorraine population (p=0.27). Factor V Leiden or anticardiolipin antibodies abnormalities were isolated in 56 cases (48.7 p. 100) without statistical difference between the 3 types of ulcers. Finally, the pathophysiology of venous, arterial and arteriovenous leg ulcers remains complex, suggesting several coagulation perturbations.

Quantification of enterovirus RNA in sludge samples using single tube real-time RT-PCR.

We have developed a quantitative RT-PCR method that can be used to determine the amount of enterovirus RNA in urban sludge samples. This method combines Taq-Man technology with the ABI Prism 7700 real-time sequence detection system. We optimized a one-step RT-PCR that uses a dual-labeled fluorogenic probe to quantify the 5' noncoding region of enteroviruses. For accurate quantification of the number of copies, a Mahoney type 1 poliovirus RNA standard was designed and produced using genetic engineering. This fragment, quantified using the Ribogreen method, was used in serial dilutions as an external standard. The method had a 7-log dynamic range (5 to 2 x 10(7)). PCR inhibitors were removed by extracting viral RNA (after virus concentration) using the RNeasy mini kit with added polyvinylpyrrolidone (PVP) and running the amplification reaction with a mixture containing PVP and T4 gene 32 protein. This real-time quantification of enterovirus RNA allows large numbers of samples to be screened. Its sensitivity, simplicity and reproducibility render it suitable as a screening method with which to characterize enteroviruses, the presence of infectious particles being subsequently confirmed by cell culture.

Lytic infection by double-strand DNA viruses and cell cycle alterations.

Polyomaviruses, papillomaviruses, adenoviruses and herpesviruses are double-stranded DNA viruses that replicate in the nucleus of the cells they infect and have evolved various strategies to create a cellular environment that is optimally conducive to their replication. One of these strategies consists of activating cellular genes, mostly S-phase genes that are required for the replication of the viral genome. Concomitantly, they encode one or several proteins that negatively regulate the response of the cell to viral infection, notably cell cycle arrest and/or apoptosis. As a result, these viruses profoundly alter the biochemical pathways that normally control cellular growth, and may thus promote uncontrolled cell proliferation. This review describes some well-known mechanisms of cell cycle alteration induced by these viruses.

Quantitative analysis of human herpesvirus 8 viral load using a real-time PCR assay.

We have developed a quantitative real-time PCR (TaqMan) assay aimed at measuring the cellular human herpesvirus 8 (HHV-8) DNA load in various clinical samples. Standard curves were obtained by serial dilutions of a control plasmid containing both HHV-8 (ORF73 gene) and the cellular target (human albumin gene). The assay appeared to be very sensitive (100% detection rate for at least 10 copies per well) and specific and was easily reproducible (less than 3% intra-assay variability, 5% interassay variability). This method allowed us to quantify precisely the average HHV-8 copy number per cell in various persistently HHV-8-infected cell lines (BBG-1 cells, n = 200; BC-1 cells, n = 59; BCBL-1 cells, n = 70). A retrospective study was also conducted to assess the HHV-8 DNA load in 12 human immunodeficiency virus-infected patients with either Kaposi's sarcoma (KS; seven patients monitored over a 3-month period) or multicentric Castleman's disease (MCD; five patients). The HHV-8 DNA load ranged from 0 to 9,171 copies/10(6) cells in low-risk KS patients (T0, I0, S0 according to the classification of the AIDS Clinical Trials group). We also measured the viral loads in MCD patients either during symptomatic periods or during remission. The results are in agreement with previously published data, with high viral loads correlating with clinical symptoms (1.3 x 10(6) copies/10(6) cells) and low viral loads correlating with asymptomatic periods (less than 5,000 copies/10(6) cells).

High expression of MDM2 protein and low rate of p21(WAF1/CIP1) expression in SCID mice Epstein Barr virus-induced lymphoproliferation.

To study the prevalence of p53 inactivation and MDM2/p21(WAFI/CIP1) expression in severe combined immunodeficient (SCID) mice Epstein-Barr virus (EBV)-induced lymphoproliferation, 19 samples obtained after ip injection of peripheral blood mononuclear cells (PBMCs) from EBV-seropositive donors or lymphoblastoid cell lines (LCL) were analyzed. In all samples tested, overexpression of Ki-67 antigen was shown by immunohistochemistry, indicating a high proliferative index of SCID mice EBV-induced lymphoproliferation. P53 mutations were screened by functional assay in yeast in 14 samples. With this test, a p53-inactivating mutation was found in only one case; the remaining cases exhibited a wild-type p53 pattern. However, an accumulation of p53 protein was detected by immunohistochemistry in six of 19 samples. P21 expression was found in seven of 19 samples but was not correlated with the rate of p53 protein in tumors. In contrast, high levels of nuclear accumulation of MDM2 were found in all samples by immunohistochemistry. These results suggest that a high Ki-67 proliferative index in SCID mice EBV-induced lymphoproliferation is not due to the inactivation of p53 by mutation, but could be associated with an overexpression of MDM2, which would act by a p53-independent mechanism.(J Histochem Cytochem 47:1315-1321, 1999)

Opposite effects of SDF-1 on human immunodeficiency virus type 1 replication.

The alpha-chemokine SDF-1 binds CXCR4, a coreceptor for human immunodeficiency virus type 1 (HIV-1), and inhibits viral entry mediated by this receptor. Since chemokines are potent chemoattractants and activators of leukocytes, we examined whether the stimulation of HIV target cells by SDF-1 affects the replication of virus with different tropisms. We observed that SDF-1 inhibited the entry of X4 strains and increased the infectivity of particles bearing either a CCR5-tropic HIV-1 envelope or a vesicular stomatitis virus G envelope. In contrast to the inhibitory effect of SDF-1 on X4 strains, which is at the level of entry, the stimulatory effect does not involve envelope-receptor interactions or proviral DNA synthesis. Rather, we observed an increased ability of Tat to transactivate the HIV-1 long terminal repeat in the presence of the chemokine. Therefore, the effects of SDF-1 on the HIV-1 life cycle can be multiple and opposite, including both an inhibition of viral entry and a stimulation of proviral gene expression.