The Orthobunyavirus Germiston Enters Host Cells from Late Endosomes.

With more than 80 members worldwide, the Orthobunyavirus genus in the Peribunyaviridae family is a large genus of enveloped RNA viruses, many of which are emerging pathogens in humans and livestock. How orthobunyaviruses (OBVs) penetrate and infect mammalian host cells remains poorly characterized. Here, we investigated the entry mechanisms of the OBV Germiston (GERV). Viral particles were visualized by cryo-electron microscopy and appeared roughly spherical with an average diameter of 98 nm. Labeling of the virus with fluorescent dyes did not adversely affect its infectivity and allowed the monitoring of single particles in fixed and live cells. Using this approach, we found that endocytic internalization of bound viruses was asynchronous and occurred within 30 to 40 min. The virus entered Rab5a-positive (Rab5a+) early endosomes and, subsequently, late endosomal vacuoles containing Rab7a but not LAMP-1. Infectious entry did not require proteolytic cleavage, and endosomal acidification was sufficient and necessary for viral fusion. Acid-activated penetration began 15 to 25 min after initiation of virus internalization and relied on maturation of early endosomes to late endosomes. The optimal pH for viral membrane fusion was slightly below 6.0, and penetration was hampered when the potassium influx was abolished. Overall, our study provides real-time visualization of GERV entry into host cells and demonstrates the importance of late endosomal maturation in facilitating OBV penetration. IMPORTANCE Orthobunyaviruses (OBVs), which include La Crosse, Oropouche, and Schmallenberg viruses, represent a growing threat to humans and domestic animals worldwide. Ideally, preventing OBV spread requires approaches that target early stages of infection, i.e., virus entry. However, little is known about the molecular and cellular mechanisms by which OBVs enter and infect host cells. Here, we developed accurate, sensitive tools and assays to investigate the penetration process of GERV. Our data emphasize the central role of late endosomal maturation in GERV entry, providing a comprehensive overview of the early stages of an OBV infection. Our study also brings a complete toolbox of innovative methods to study each step of the OBV entry program in fixed and living cells, from virus binding and endocytosis to fusion and penetration. The information gained herein lays the foundation for the development of antiviral strategies aiming to block OBV entry.

Enjeux méthodologiques associés à la réalisation d'une EIS : l'exemple de la reconversion d'une caserne à Limoges.

INTRODUCTION: With the goal of testing the health impact assessment process to promote health in its policies, the City of Limoges selected the urban planning project aiming to reconvert the Marceau barracks for this experiment. The aim of this HIA was to consider the potential impacts on health of the developed pre-project. The stages of screening, scoping, impact assessment and recommendations took place between 2017 and the end of 2019.Purpose of research: To describe the methods and the different stages of an HIA based on a concrete example, in order to share an experience of an HIA and to relate the issues and difficulties encountered. METHODS: An evaluation team was set up to carry out the HIA coordinated by the Regional Health Observatory of Nouvelle-Aquitaine (ORS NA) and the City of Limoges. The team followed the recommendations from the international guides while adapting the principles to specific timing issues and the overall context. RESULTS: The HIA led to the identification of 121 potential impacts and 54 recommendations. The presence of a multidisciplinary team was a strong asset throughout the whole process, while the timing of the HIA and the participation of the population were the two aspects that required the most flexibility. CONCLUSIONS: The HIA has enabled a concerted and intersectoral effort on health issues through an urban planning project. While the time-consuming aspect of the process was noted, the need for a more systematic integration of health issues into urban projects with HIA was emphasized.

Making Rift Valley Fever Viral Particles Fluorescent.

Rift Valley fever virus (RVFV) is a mosquito-borne pathogen that represents a significant threat to both human and veterinary public health. Since its discovery in the Great Rift Valley of Kenya in the 1930s, the virus has spread across Africa and beyond, now posing a risk of introduction into Southern Europe and Asia. Despite recent progresses, early RVFV-host cell interactions remain largely uncharacterized. In this method chapter, we delineate the procedure for labeling RVFV particles with fluorescent organic dyes. This approach makes it feasible to visualize single viral particles in both fixed and living cells and study RVFV entry into host cells. We provide additional examples with two viruses closely related to RVFV, namely, Toscana virus and Uukuniemi virus. Furthermore, we illustrate how to utilize fluorescent viral particles to examine and quantify each step of the cell entry program of RVFV, which includes state-of-the-art fluorescence-based detection techniques such as fluorescence microscopy, flow cytometry, and fluorimetry.

The reported incidence of campylobacteriosis modelled as a function of earlier temperatures and numbers of cases, Montreal, Canada, 1990-2006.

Previous studies have detected an effect of earlier temperatures on the incidence of campylobacteriosis in humans, but without adjustment for earlier numbers of cases of the disease. We estimated the effect of temperature on the number of cases notified by week in Montreal, Canada, from 1 January 1990 to 26 March 2006, simultaneously with the effect of the numbers of cases notified in the preceding weeks. The current campylobacteriosis count (week 0) was modelled by negative binomial regression, with earlier weekly average temperatures and earlier counts as predictors. Secular trends were accounted for by cubic spline functions and seasonal variations by sine-cosine functions. Indicator variables identified weeks with fewer than 5 working days. In the final statistical model, a 1°C increase in temperature above 10°C during any of weeks -1 to -6 was associated with a 0.8% (95% CI: 0.3% to 1.3%) increase in the current count. For each additional notified case during any of weeks -1 to -5 or -9 to -12, the increase in the current count was approximately 0.5% (95% CI: 0.2% to 1.0%). Thus, earlier temperatures and earlier counts have independent effects, that of temperatures being the larger one. The temperature effect is too small to require short term public health planning. However, in Montreal, an increase in average temperature of the order of 4.5°C, forecast by some for 2055, could produce a 23% increase in incidence, resulting in about 4,000 excess cases per year.

Toward the reduction of water consumption in the vegetable-processing industry through membrane technology: case study of a carrot-processing plant.

The food industry consumes large amounts of clean, potable water and in turn generates a significant amount of wastewater. In order to minimize water consumption, membrane technologies represent a suitable solution for the treatment of wastewater before it is recycled as process water. Many studies have shown the effectiveness of this technology in the dairy industry, but there are few studies in the fruit- and vegetable-processing sectors. A recently developed methodology for the reduction of water consumption was tested here. Compounds to be eliminated were identified through chemical analysis of several wastewater samples from a carrot-peeling process. Drinking-water quality was selected as our target. Total suspended solids (TSS), fructose, glucose and sucrose were identified as key parameters. Salts (particularly Ca2+ and Mg2+), pH and carbonate hardness (CH) were identified as indicators for evaluating the risk of scaling and corrosion. Based on these results, sieving followed by a 0.5-µm microfiltration (MF) was chosen as the process for pre-treatment. Four nanofiltration (NF) membranes (NFW from SYNDER, DK from GE, NF270 from DOW and SR3D from KOCH) and three reverse osmosis (RO) membranes (ESPA4 from Nitto Group Company, BW30 from DOW and HRX from KOCH) were then tested for the capacity to minimize chemical oxygen demand (COD) and to principally remove sugars. These membranes were then evaluated in terms of permeability and rejection rates. High-quality water could be obtained with RO membranes at low pressure (up to 15 bar) while limiting fouling risks. Rejection rates up to 98.3, 98.0, 99.2, 99.2 and 99.4% for conductivity, COD, fructose, glucose and sucrose, respectively, were achieved. These results are very encouraging for future reuse in vegetable processing before the blanching step, after an additional disinfection treatment.

Activation/proliferation and apoptosis of bystander goat lymphocytes induced by a macrophage-tropic chimeric caprine arthritis encephalitis virus expressing SIV Nef.

Caprine arthritis encephalitis virus (CAEV) is the natural lentivirus of goats, well known for its tropism for macrophages and its inability to cause infection in lymphocytes. The viral genome lacks nef, tat, vpu and vpx coding sequences. To test the hypothesis that when nef is expressed by the viral genome, the virus became toxic for lymphocytes during replication in macrophages, we inserted the SIVsmm PBj14 nef coding sequences into the genome of CAEV thereby generating CAEV-nef. This recombinant virus is not infectious for lymphocytes but is fully replication competent in goat macrophages in which it constitutively expresses the SIV Nef. We found that goat lymphocytes cocultured with CAEV-nef-infected macrophages became activated, showing increased expression of the interleukin-2 receptor (IL-2R). Activation correlated with increased proliferation of the cells. Interestingly, a dual effect in terms of apoptosis regulation was observed in exposed goat lymphocytes. Nef was found first to induce a protection of lymphocytes from apoptosis during the first few days following exposure to infected macrophages, but later it induced increased apoptosis in the activated lymphocytes. This new recombinant virus provides a model to study the functions of Nef in the context of infection of macrophages, but in absence of infection of T lymphocytes and brings new insights into the biological effects of Nef on lymphocytes.

Simian immunodeficiency virus Vpr/Vpx proteins kill bystander noninfected CD4+ T-lymphocytes by induction of apoptosis.

The depletion of CD4+ T-lymphocytes central to the immunodeficiency in acquired immunodeficiency syndrome (AIDS) is largely mediated by apoptosis of both infected and uninfected cells, but the mechanisms involved and the viral proteins responsible are still poorly characterized. It has recently been suggested that, in human and simian immunodeficiency virus (HIV) and SIV, Vpr is a major modulator of apoptosis in infected cells. Recently, we have reported on a chimera of caprine arthritis-encephalitis virus (CAEV) carrying vpr/vpx genes from SIVmac239, which is replication competent in goat macrophages but not in lymphocytes or human cells. Despite infection being restricted to macrophages, inoculation of primary goat peripheral blood mononuclear cells (PBMCs) with this chimera induced apoptosis in the lymphocyte population. In addition, when infected goat synovial membrane (GSM) cells were co-cultured with human CD4+ T lymphocyte SupT1 cell line, these CD4+ T cells showed increased apoptosis. The parental CAEV induced no significant apoptosis in goat PBMC cultures or in co-cultures with human SupT1 lymphocytes. This indicates that SIV Vpr/Vpx proteins indeed mediate apoptosis of T-lymphocytes and, moreover, do so without the need for active infection of these cells. Moreover, this apoptosis was observed when SupT1s were cocultured in direct contact, but not in absence of contact with CAEV-pBSCAvpxvpr-infected GSM cells. In view of these data, we propose that SIV Vpx/Vpr activate cell-to-cell contact-dependent extracellular signaling pathways to promote apoptotic death of uninfected bystander T-lymphocytes. Understanding this mechanism might bring insight for intervening in the loss of CD4+ T lymphocytes in the SIV infection model and in human AIDS.

Clearance of a productive lentivirus infection in calves experimentally inoculated with caprine arthritis-encephalitis virus.

Lentiviruses have long been considered host-specific pathogens, but several recent observations demonstrated their capacity to conquer new hosts from different species, genera, and families. From these cross-species infections emerged new animal and human infectious diseases. The successful colonization and adaptation of a lentivirus to a nonnatural host depends on unspecific and specific host barriers. Some of those barriers exert a relative control of viral replication (i.e., cytotoxic T-lymphocyte response, viral inhibitory factors), but none of them was found able to totally clear the infection once the retrovirus is fully adapted in its host. In this study we examined the evolution of the host-lentivirus interactions occurring in an experimental animal model of cross-species infection in order to analyze the efficiency of those barriers in preventing the establishment of a persistent infection. Five newborn calves were inoculated with caprine arthritis-encephalitis virus (CAEV), and the evolution of infection was studied for more than 12 months. All the animals seroconverted in the first 0.75 to 1 month following the inoculation and remained seropositive for the remaining time of the experiment. Viral infection was productive during 4 months with isolation of replication competent virus from the blood cells and organs of the early euthanized animals. After 4 months of infection, neither replication-competent virus nor virus genome could be detected in blood cells or in the classical target organs, even after an experimental immunosuppression. No evidence of in vitro restriction of CAEV replication was observed in cells from tissues explanted from organs of these calves. These data provide the demonstration of a natural clearance of lentivirus infection following experimental inoculation of a nonnatural host, enabling perspectives of development of new potential vaccine strategies to fight against lentivirus infections.

Specific G2 arrest of caprine cells infected with a caprine arthritis encephalitis virus expressing vpr and vpx genes from simian immunodeficiency virus.

Primate lentivirus (HIV and SIV) vpr accessory genes encode 12- to 14-kDa proteins which induce cell cycle arrest at the G2 phase of infected cells, preventing them from going through mitosis. Members of the HIV-2/SIVmac/SIVsmm group also encode a second closely related accessory protein called Vpx. Vpx and HIV Vpr are critical for virus replication in nondividing cells due to their participation in nuclear import of the preintegration complex. Caprine arthritis encephalitis virus (CAEV) and maedi visna virus are the natural lentiviruses of domestic goat and sheep, respectively, and their genomes do not carry vpr and vpx genes. In this study, we generated chimeric CAEV-based genomes carrying vpr and vpx genes from SIVmac239 and tested their ability to induce G2 cell cycle arrest in infected caprine cells. CAEV-pBSCAvpxvpr is the chimeric genome that was shown to be infectious and replication competent. Our data demonstrated that CAEV-pBSCAvpxvpr-infected goat synovial membrane cell monolayer developed more cytopathic effects and a high proportion of cells remained in the G2 phase of cell cycle. This G2 arrest was observed both at the early and at the late stages of infection, while minimal effect was observed with the parental CAEV-pBSCA. These results, described for the first time in mammalian cells other than those of primates, indicate that Vpr-induced G2 cell cycle arrest is not restricted to only primate cells. Thus, conservation of Vpx/Vpr protein functions in caprine cells suggests a possible role for these proteins in the virus life cycle and its ability to adapt to new hosts. The data presented here thus raise a pertinent question about the biological significance of the conservation of Vpr and Vpx functions in caprine cells despite the high phylogenic distance between primates and small ruminants.