Reverse genetics system for shuni virus, an emerging orthobunyavirus with zoonotic potential

The genus Orthobunyavirus (family Peribunyaviridae, order Bunyavirales) comprises over 170 named mosquito- and midge-borne viruses, several of which cause severe disease in animals or humans. Their three-segmented genomes enable reassortment with related viruses, which may result in novel viruses with altered host or tissue tropism and virulence. One such reassortant, Schmallenberg virus (SBV), emerged in north-western Europe in 2011. Shuni virus (SHUV) is an orthobunyavirus related to SBV that is associated with neurological disease in horses in southern Africa and recently caused an outbreak manifesting with neurological disease and birth defects among ruminants in Israel. The zoonotic potential of SHUV was recently underscored by its association with neurological disease in humans. We here report a reverse genetics system for SHUV and provide first evidence that the non-structural (NSs) protein of SHUV functions as an antagonist of host innate immune responses. We furthermore report the rescue of a reassortant containing the L and S segments of SBV and the M segment of SHUV. This novel reverse genetics system can now be used to study SHUV virulence and tropism, and to elucidate the molecular mechanisms that drive reassortment events.The Dutch Ministry of Agriculture, Nature and Food Quality of the Netherlands and the European Union’s Horizon 2020 research and innovation programme under LEAP-Agri grant agreement No 727715.http://www.mdpi.com/journal/viruseshj2020Medical Virolog

Quantifying Rift Valley fever virus transmission efficiency in a lamb-mosquito-lamb model

Rift Valley fever virus (RVFV) is a (re)emerging mosquito-borne pathogen impacting human and animal health. How RVFV spreads through a population depends on population-level and individual-level interactions between vector, host and pathogen. Here, we estimated the probability for RVFV to transmit to naive animals by experimentally exposing lambs to a bite of an infectious mosquito, and assessed if and how RVFV infection subsequently developed in the exposed animal. Aedes aegypti mosquitoes, previously infected via feeding on a viremic lamb, were used to expose naive lambs to the virus. Aedes aegypti colony mosquitoes were used as they are easy to maintain and readily feed in captivity. Other mosquito spp. could be examined with similar methodology. Lambs were exposed to either 1-3 (low exposure) or 7-9 (high exposure) infectious mosquitoes. All lambs in the high exposure group became viremic and showed characteristic signs of Rift Valley fever within 2-4 days post exposure. In contrast, 3 out of 12 lambs in the low exposure group developed viremia and disease, with similar peak-levels of viremia as the high exposure group but with some heterogeneity in the onset of viremia. These results suggest that the likelihood for successful infection of a ruminant host is affected by the number of infectious mosquitoes biting, but also highlights that a single bite of an infectious mosquito can result in disease. The per bite mosquito-to-host transmission efficiency was estimated at 28% (95% confidence interval: 15 - 47%). We subsequently combined this transmission efficiency with estimates for life traits of Aedes aegypti or related mosquitoes into a Ross-McDonald mathematical model to illustrate scenarios under which major RVFV outbreaks could occur in naïve populations (i.e., R0 >1). The model revealed that relatively high vector-to-host ratios as well as mosquitoes feeding preferably on competent hosts are required for R0 to exceed 1. Altogether, this study highlights the importance of experiments that mimic natural exposure to RVFV. The experiments facilitate a better understanding of the natural progression of disease and a direct way to obtain epidemiological parameters for mathematical models

Crimean-congo hemorrhagic fever virus subunit vaccines induce high levels of neutralizing antibodies but no protection in STAT1 knockout mice

Crimean-Congo hemorrhagic fever virus is a tick-borne bunyavirus of the Nairovirus genus that causes hemorrhagic fever in humans with high case fatality. Here, we report the development of subunit vaccines and their efficacy in signal transducer and activator of transcription 1 (STAT1) knockout mice. Ectodomains of the structural glycoproteins Gn and Gc were produced using a Drosophila insect cell-based expression system. A single vaccination of STAT129 mice with adjuvanted Gn or Gc ectodomains induced neutralizing antibody responses, which were boosted by a second vaccination. Despite these antibody responses, mice were not protected from a CCHFV challenge infection. These results suggest that neutralizing antibodies against CCHFV do not correlate with protection of STAT1 knockout mice.</p

Molecular Characterization of <i>Serratia marcescens</i> Strain Isolated from Yellow Mealworms, <i>Tenebrio molitor</i>, in The Netherlands

Insect culture has developed rapidly worldwide; it faces important security and safety control issues, including animal infections and disease development. In the Netherlands, in 2021, a ~30% mortality of mealworms, Tenebrio molitor, occurred at one farm, where over-humid sites in the substrate were observed. Bacterial cultures from both the external and internal partsof fry and larger mealworms were identified by MALDI-TOF to predominantly Serratia marcescens, Staphylococcus xylosus and Staphylococus saprofyticus. Due to the important role of S. marcescens as a potential zoonotic bacterium, we performed a molecular characterization of the isolated strain. Genomic analysis showed a multidrug-resistant S. marcescens isolate carrying a tet (41), aac (6′)-Ic, and blaSST-1 chromosomal class C beta-lactamase-resistantgenes, all located on the chromosome. Additionally, several virulence genes were identified. The phylogenetic tree revealed that the S. marcescens strain from this study was similar to other S. marcescens strains from different ecological niches. Although the entomopathogenic activity was not confirmed, this case demonstrates that T. molitor can act as a reservoir and as an alternative path for exposing clinically important antibiotic-resistant bacteria that can affect animals and humans. It underlines the need to keep management factors optimal, before insects and their products enter the feed and food chain

Transmission of Rift Valley fever virus from European-breed lambs to Culex pipiens mosquitoes

Background: Rift Valley fever virus (RVFV) is a mosquito-borne bunyavirus of the genus Phlebovirus that is highly pathogenic to ruminants and humans. The disease is currently confined to Africa and the Arabian Peninsula, but globalization and climate change may facilitate introductions of the virus into currently unaffected areas via infected animals or mosquitoes. The consequences of such an introduction will depend on environmental factors, the availability of susceptible ruminants and the capacity of local mosquitoes to transmit the virus. We have previously demonstrated that lambs native to the Netherlands are highly susceptible to RVFV and we here report the vector competence of Culex (Cx.) pipiens, the most abundant and widespread mosquito species in the country. Vector competence was first determined after artificial blood feeding of laboratory-reared mosquitoes using the attenuated Clone 13 strain. Subsequently, experiments with wild-type RVFV and mosquitoes hatched from field-collected eggs were performed. Finally, the transmission of RVFV from viremic lambs to mosquitoes was studied. Principal findings: Artificial feeding experiments using Clone 13 demonstrated that indigenous, laboratory-reared Cx. pipiens mosquitoes are susceptible to RVFV and that the virus can be transmitted via their saliva. Experiments with wild-type RVFV and mosquitoes hatched from field-collected eggs confirmed the vector competence of Cx. pipiens mosquitoes from the Netherlands. To subsequently investigate transmission of the virus under more natural conditions, mosquitoes were allowed to feed on RVFV-infected lambs during the viremic period. We found that RVFV is efficiently transmitted from lambs to mosquitoes, although transmission was restricted to peak viremia. Interestingly, in the mosquito-exposed skin samples, replication of RVFV was detected in previously unrecognized target cells. Significance: We here report the vector competence of Cx. pipiens mosquitoes from the Netherlands for RVFV. Both laboratory-reared mosquitoes and well as those hatched from field-collected eggs were found to be competent vectors. Moreover, RVFV was transmitted efficiently from indigenous lambs to mosquitoes, although the duration of host infectivity was found to be shorter than previously assumed. Interestingly, analysis of mosquito-exposed skin samples revealed previously unidentified target cells of the virus. Our findings underscore the value of including natural target species in vector competence experiments

Reproducing the Rift Valley fever virus mosquito-lamb-mosquito transmission cycle

Rift Valley fever virus (RVFV) is a mosquito-borne bunyavirus that is pathogenic to ruminants and humans. The virus is endemic to Africa and the Arabian Peninsula where outbreaks are characterized by abortion storms and mortality of newborns, particularly in sheep herds. Vector competence experiments in laboratory settings have suggested that over 50 mosquito species are capable of transmitting RVFV. Transmission of mosquito-borne viruses in the field is however influenced by numerous factors, including population densities, blood feeding behavior, extrinsic incubation period, longevity of vectors, and viremia levels in vertebrate hosts. Animal models to study these important aspects of RVFV transmission are currently lacking. In the present work, RVFV was transmitted to European (Texel-swifter cross-breed) lambs by laboratory-reared Aedes aegypti mosquitoes that were infected either by membrane feeding on a virus-spiked blood meal or by feeding on lambs that developed viremia after intravenous inoculation of RVFV. Feeding of mosquitoes on viremic lambs resulted in strikingly higher infection rates as compared to membrane feeding. Subsequent transmission of RVFV from lamb to lamb by infected mosquitoes was highly efficient in both models. The animal models described here can be used to study mosquito-mediated transmission of RVFV among the major natural target species and to evaluate the efficacy of vaccines against mosquito-mediated RVFV infection.</p

Effect of NSR infection on intracellular and extracellular CD83 levels.

<p>(A) The levels of soluble CD83 in supernatants from cells harvested 24 h after stimulation with LPS, infection with NSR, or from cells mock infected with NSRmock were determined by ELISA. Bars represent average CD83 concentrations ±SD. Results from one of two independently performed experiments with similar results are shown. (B) Detection of CD83 in cell lysates by Western blot at 24 hpi. The different treatments are shown above the top panel and the probed proteins are depicted at the right. The positions of molecular weight standard proteins are shown at the left. The top blot was stripped and re-probed with antibodies against GAPDH and GFP, which served as loading control and control to confirm NSR infection, respectively. Results from one of two independent experiments with cells from two donors are shown.</p

Analysis of CD83, CD80, GAPDH and PPIA mRNA levels.

<p>DC RNA samples, prepared 24 h after treatment (as indicated), were analysed by qRT-PCR. Bars represent average Ct values from triplicates ±SD with cells from one donor. The experiment is a representative of 3 independently performed experiments with cells from 3 different donors. Statistical significance is indicated with an asterisk.</p

Cytokine secretion by NSR-infected DCs.

<p>Supernatants of infected or control-treated DCs were harvested at 24 hpi and analysed with a luminex-based cytokine assay. Bars represent the mean cytokine concentrations ± SD of triplicates with cells from one donor. Statistical significance between infected (NSR) and mock-infected (NSRmock) conditions is indicated.</p