Canine distemper virus epithelial cell infection is required for clinical disease but not for immunosuppression

To characterize the importance of infection of epithelial cells for morbillivirus pathogenesis, we took advantage of the severe disease caused by canine distemper virus (CDV) in ferrets. To obtain a CDV that was unable to enter epithelial cells but retained the ability to enter immune cells, we transferred to its attachment (H) protein two mutations shown to interfere with the interaction of measles virus H with its epithelial receptor, human nectin-4. As expected for an epithelial receptor (EpR)-blind CDV, this virus infected dog and ferret epithelial cells inefficiently and did not cause cell fusion or syncytium formation. On the other hand, the EpR-blind CDV replicated in cells expressing canine signaling lymphocyte activation molecule (SLAM), the morbillivirus immune cell receptor, with similar kinetics to those of wild-type CDV. While ferrets infected with wild-type CDV died within 12 days after infection, after developing severe rash and fever, animals infected with the EpR-blind virus showed no clinical signs of disease. Nevertheless, both viruses spread rapidly and efficiently in immune cells, causing similar levels of leukopenia and inhibition of lymphocyte proliferation activity, two indicators of morbillivirus immunosuppression. Infection was documented for airway epithelia of ferrets infected with wild-type CDV but not for those of animals infected with the EpR-blind virus, and only animals infected with wild-type CDV shed virus. Thus, epithelial cell infection is necessary for clinical disease and efficient virus shedding but not for immunosuppression.     

Canine Distemper Viruses Expressing a Hemagglutinin without N-Glycans Lose Virulence but Retain Immunosuppression

Paramyxovirus glycoproteins are posttranslationally modified by the addition of N-linked glycans, which are often necessary for correct folding, processing, and cell surface expression. To establish the contribution of N glycosylation to morbillivirus attachment (H) protein function and overall virulence, we first determined the use of the potential N-glycosylation sites in the canine distemper virus (CDV) H proteins. Biochemical characterization revealed that the three sites conserved in all strains were N glycosylated, whereas only two of the up to five additional sites present in wild-type strains are used. A wild-type virus with an H protein reproducing the vaccine strain N-glycosylation pattern remained lethal in ferrets but with a prolonged course of disease. In contrast, introduction of the vaccine H protein in the wild-type context resulted in complete attenuation. To further characterize the role of N glycosylation in CDV pathogenesis, the N-glycosylation sites of wild-type H proteins were successively deleted, including a nonstandard site, to ultimately generate a nonglycosylated H protein. Despite reduced expression levels, this protein remained fully functional. Recombinant viruses expressing N-glycan-deficient H proteins no longer caused disease, even though their immunosuppressive capacities were retained, indicating that reduced N glycosylation contributes to attenuation without affecting immunosuppression.Measles virus (MeV) and Canine distemper virus (CDV) are members of the genus Morbillivirus in the family Paramyxoviridae that are highly contagious and can cause severe disease. MeV is known to infect only humans and certain nonhuman primates and is rarely fatal, while CDV infects a broad range of carnivores and can result in up to 100% mortality (13). The signaling lymphocyte activation molecule (SLAM; CD150), which is expressed on activated T and B cells (10), is the primary receptor for morbilliviruses (3, 15, 42). Morbillivirus attachment to susceptible cells occurs via interaction between SLAM and the viral hemagglutinin (H) protein, one of two glycoproteins that are inserted into the viral membrane and subsequently expressed on the surfaces of infected cells. Upon binding, the conformational change of the H protein conducts a signal to the fusion (F) protein, which then mediates fusion between the viral and host cell membranes. However, the extent and efficiency of cell-cell fusion are H protein dependent (21, 52).Morbillivirus vaccine strains were developed during the 1960s by serial passages in eggs and different cell lines (11, 35). Consequently, there are significant molecular differences between the glycoproteins of wild-type and vaccine CDV strains. The H protein of the large-plaque Onderstepoort strain (OL) differs from the wild-type 5804P at 60 amino acid residues, introducing several additional N-glycosylation sites. Based on the observation that the vaccine strain H proteins have a lower apparent molecular weight than the wild-type proteins (9, 52), it has been hypothesized that reduced N glycosylation is an important attenuating factor and that an increase in N glycosylation may eventually result in vaccine failure (19).Posttranslational modifications, including N-linked glycosylation, are essential for the functions of many integral membrane and secreted proteins. N-glycan chains are added to asparagine (N) residues in the endoplasmic reticulum (ER) at the consensus sequence N-X-S/T, where “X” can be any amino acid except proline. As the nascent protein chain is inserted into the ER and moves through the Golgi apparatus and finally to the plasma membrane, N-glycans are trimmed and modified into elaborate structures that can account for a substantial proportion of the overall molecular weight of a given glycoprotein (43). N glycosylation has been shown to be important for the correct folding, transport, and function of other paramyxovirus fusion and attachment glycoproteins, including MeV H (17), Sendai virus hemagglutinin-neuraminidase (HN) (39), simian virus 5 HN (31), and Newcastle disease virus HN (27). In most cases, one or two N-glycans at specific locations are essential for proper folding and transport to the cell surface, and at least one or two N-glycans are required for the F or H/HN protein to function correctly (30, 47). Thus, the total deletion of N-glycans is generally not tolerated.To characterize the contribution of H protein N glycosylation to morbillivirus pathogenesis, we took advantage of the CDV ferret model (48, 50). We first determined the role of decreased natural N glycosylation of the vaccine strain H protein in protein function and virulence. Based on our finding that reduced N glycosylation results in prolonged disease, we performed a comprehensive mutational analysis to determine the minimal complement of H protein N glycosylation needed for proper intracellular transport and function. Using viruses bearing increasingly deglycosylated H proteins, we demonstrate that a CDV H protein without N-glycans remains completely functional. However, minimal N glycosylation is required for virulence.     

Importance of soil-water relations in assessing the endpoint of bioremediated soils

This study was conducted to investigate water movement in hydrocarbon contaminated soils. Three soils were studied, a hydrocarbon contaminated soil, the same soil after 3 years of bioremediation, and a control soil from the same site. There was a critical soil water content around 18% (bioremediated soil) and 20% (contaminated soil), above which the sorptivity of the contaminated soil was near that of the control soil. For soils with water contents below this value, there was a strong divergence in sorptivity between contaminated and control or bioremediated soils. Results suggest that water availability in contaminated soils will be highly dependent on soil water properties as water potential approaches the permanent wilting point (-1.5 MPa matrix potential).Infiltration of water into air dry (2% m.c. w/w) hydrocarbon contaminated soils was up to three orders of magnitude slower than for the control soil. For air dried soils, the infiltration rate of the contaminated and bioremediated soils was constant with time. This was in contrast to the control soil where infiltration rate was a function of the reciprocal of the square root of time.     

Safe and effective two-in-one replicon-and-VLP minispike vaccine for COVID-19: Protection of mice after a single immunization

Vaccines of outstanding efficiency, safety, and public acceptance are needed to halt the current SARS-CoV-2 pandemic. Concerns include potential side effects caused by the antigen itself and safety of viral DNA and RNA delivery vectors. The large SARS-CoV-2 spike (S) protein is the main target of current COVID-19 vaccine candidates but can induce non-neutralizing antibodies, which might cause vaccination-induced complications or enhancement of COVID-19 disease. Besides, encoding of a functional S in replication-competent virus vector vaccines may result in the emergence of viruses with altered or expanded tropism. Here, we have developed a safe single round rhabdovirus replicon vaccine platform for enhanced presentation of the S receptor-binding domain (RBD). Structure-guided design was employed to build a chimeric minispike comprising the globular RBD linked to a transmembrane stem-anchor sequence derived from rabies virus (RABV) glycoprotein (G). Vesicular stomatitis virus (VSV) and RABV replicons encoding the minispike not only allowed expression of the antigen at the cell surface but also incorporation into the envelope of secreted non-infectious particles, thus combining classic vector-driven antigen expression and particulate virus-like particle (VLP) presentation. A single dose of a prototype replicon vaccine complemented with VSV G, VSVΔG-minispike-eGFP (G), stimulated high titers of SARS-CoV-2 neutralizing antibodies in mice, equivalent to those found in COVID-19 patients, and protected transgenic K18-hACE2 mice from COVID-19-like disease. Homologous boost immunization further enhanced virus neutralizing activity. The results demonstrate that non-spreading rhabdovirus RNA replicons expressing minispike proteins represent effective and safe alternatives to vaccination approaches using replication-competent viruses and/or the entire S antigen.     

Importance of soil-water relations in assessing the endpoint of bioremediated soils

This study was conducted during 1992–1994 under semi-arid conditions in Burkina Faso. Our aim was to assess the influence of different mulch materials on soil variables affecting crop growth; i.e. water content, nutrient contents and temperature. The grain yield of Sorghum bicolor was used as a bioindicator, i.e. as an integrated measure of mulch effects.Six treatments were tested, two of which were leaf mulches of special interest for agroforestry. The treatments were chosen to represent mulch materials differing in nutrient content and decomposability (assumed to influence the duration of the impact on soil water content and temperature). The treatments were as follows: (1) control (no addition), (2) Azadirachta indica (neem) leaves, (3) neem leaves + aerobic compost of sorghum straw, (4) aerobic compost, (5) Acacia holocericea (acacia) phyllodes, and (6) wild grass. The mulching rate was 5 t dry matter ha^-1, and base mineral fertilizers were applied to all plots.It was shown that the neem leaves, neem leaves + compost, wild grass and acacia phyllodes treatments all significantly influenced the soil by conserving water and reducing temperatures compared with the control or the treatment with compost alone. Plots treated with either neem leaves, neem leaves + compost or compost alone gave higher yields than the three other treatments, generally poorer in nutrients, between which there was little difference. Neem leaves gave the numerically highest response: 1.54 × control, corresponding to a grain yield increase of 554 kg dry mass ha^-1 yr^-1 when averaged over the three years of study.Thus, yields did not always increase in spite of increased soil moisture and decreased soil temperatures. It was concluded that nutrients were more limiting than water or high soil temperatures under the conditions studied. The highest yields were achieved with a mulch that combines high nutrient delivery with water conservation and temperature reduction, namely mulch from neem leaves.     

Comparative Pathogenesis of Alkhumra Hemorrhagic Fever and Kyasanur Forest Disease Viruses in a Mouse Model

Kyasanur Forest disease virus (KFDV) and Alkhumra hemorrhagic fever virus (AHFV) are genetically closely-related, tick-borne flaviviruses that cause severe, often fatal disease in humans. Flaviviruses in the tick-borne encephalitis (TBE) complex typically cause neurological disease in humans whereas patients infected with KFDV and AHFV predominately present with hemorrhagic fever. A small animal model for KFDV and AHFV to study the pathogenesis and evaluate countermeasures has been lacking mostly due to the need of a high biocontainment laboratory to work with the viruses. To evaluate the utility of an existing mouse model for tick-borne flavivirus pathogenesis, we performed serial sacrifice studies in BALB/c mice infected with either KFDV strain P9605 or AHFV strain Zaki-1. Strikingly, infection with KFDV was completely lethal in mice, while AHFV caused no clinical signs of disease and no animals succumbed to infection. KFDV and high levels of pro-inflammatory cytokines were detected in the brain at later time points, but no virus was found in visceral organs; conversely, AHFV Zaki-1 and elevated levels of cytokines were found in the visceral organs at earlier time points, but were not detected in the brain. While infection with either virus caused a generalized leukopenia, only AHFV Zaki-1 induced hematologic abnormalities in infected animals. Our data suggest that KFDV P9605 may have lost its ability to cause hemorrhagic disease as the result of multiple passages in suckling mouse brains. However, likely by virtue of fewer mouse passages, AHFV Zaki-1 has retained the ability to replicate in visceral organs, cause hematologic abnormalities, and induce pro-inflammatory cytokines without causing overt disease. Given these striking differences, the use of inbred mice and the virus passage history need to be carefully considered in the interpretation of animal studies using these viruses.