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

Four types of scrapie in goats differentiated from each other and bovine spongiform encephalopathy by biochemical methods

Scrapie in goats has been known since 1942, the archetype of prion diseases in which only prion protein (PrP) in misfolded state (PrPSc) acts as infectious agent with fatal consequence. Emergence of bovine spongiform encephalopathy (BSE) with its zoonotic behaviour and detection in goats enhanced fears that its source was located in small ruminants. However, in goats knowledge on prion strain typing is limited. A European-wide study is presented concerning the biochemical phenotypes of the protease resistant fraction of PrPSc (PrPres) in over thirty brain isolates from transmissible spongiform encephalopathy (TSE) affected goats collected in seven countries. Three different scrapie forms were found: classical scrapie (CS), Nor98/atypical scrapie and one case of CH1641 scrapie. In addition, CS was found in two variants—CS-1 and CS-2 (mainly Italy)—which differed in proteolytic resistance of the PrPres N-terminus. Suitable PrPres markers for discriminating CH1641 from BSE (C-type) appeared to be glycoprofile pattern, presence of two triplets instead of one, and structural (in)stability of its core amino acid region. None of the samples exhibited BSE like features. BSE and these four scrapie types, of which CS-2 is new, can be recognized in goats with combinations of a set of nine biochemical parameters

Shuni virus replicates at the maternal-fetal interface of the ovine and human placenta

Shuni virus (SHUV) is a neglected teratogenic and neurotropic orthobunyavirus that was discovered in the 1960s in Nigeria and was subsequently detected in South Africa, Zimbabwe, and Israel. The virus was isolated from field-collected biting midges and mosquitoes and shown to disseminate efficiently in laboratory-reared biting midges, suggesting that members of the families Culicidae and Ceratopogonidae may function as vectors. SHUV infections have been associated with severe neurological disease in horses, a variety of wildlife species, and domesticated ruminants. SHUV infection of ruminants is additionally associated with abortion, stillbirth, and congenital malformations. The detection of antibodies in human sera also suggests that the virus may have zoonotic potential. To understand how SHUV crosses the ruminant placenta, we here infected pregnant ewes and subsequently performed detailed clinical-and histopathological examination of placental tissue. We found that SHUV targets both maternal epithelial cells and fetal trophoblasts, that together form the maternal-fetal interface of the ovine placenta. Experiments with human placental explants, furthermore, revealed replication of SHUV in syncytiotrophoblasts, which are generally highly resistant to virus infections. Our findings provide novel insights into vertical transmission of SHUV in sheep and call for research on the potential risk of SHUV infection during human pregnancies.</p

Humoral and cellular immune responses elicited by vaccination of mice with NSR or NSR-Gn.

<p>(A) VNT titers in sera collected from mice vaccinated with NSR or NSR-Gn before vaccination (DPV -1) and 13, 22 and 29 DPV. Bars represent average titers (n = 6) of each group with standard deviation. The detection limit of the assay is depicted by the interrupted line. (B) Detection of IFN-γ-producing splenocytes isolated from mice vaccinated with NSR or NSR-Gn. Splenocytes were isolated and seeded at a density of 5×10⁵ cells/well in triplicate and stimulated for 12 hours with the indicated peptides or the ectodomain of Gn. Bars represent an average number of IFN-γ producing cells (n = 4) per group with standard deviation. The non-parametric Mann-Whitney test was used for statistical analysis and statistical significance between the groups is depicted by asterisks (*p<0.05; **p<0.01).</p

Rectal temperatures of vaccinated and mock-vaccinated lambs before and after challenge with RVFV.

<p>Fever was defined as a rectal body temperature above 40.5°C (interrupted line). Body temperatures of mock-vaccinated lambs (C1–C8) and lambs vaccinated with a low dose (L1–L8), medium dose (M1–M8) or high dose (H1–H7) of NSR-Gn are depicted individually.</p

Detection of anti-N antibodies by ELISA.

<p>Sera were obtained 7, 14 and 21 days post vaccination (DPV) and at 0, 7, 14 and 21 days post challenge (DPC). Titers are expressed as percentage competition ratio of the optical densities (OD) of the sample and the OD of the negative control (%S/N). All values lower than 40% are considered positive, between 40–50% are considered doubtful and above 50% are considered negative. The 40% and 50% cut-offs are represented by solid and interrupted lines, respectively. Results obtained from analysis of each individual animal from the mock-group (C1–C8), low-dose group (L1–L8), medium-dose group (M1–M8) or high-dose group (H1–H7) are depicted.</p

Virus neutralization test (VNT).

<p>Sera were obtained from lambs of the mock group, low-dose group, medium-dose group and high-dose group. The white bars represent VNT titers determined 21 days post vaccination (DPV) and the black bars represent the VNT titers determined 21 days post challenge (DPC). Results obtained from analysis of each individual animal from the mock-group (C1–C8), low-dose group (L1–L8), medium-dose group (M1–M8) and high-dose group (H1–H7) are depicted. The detection limit of the assay is represented by an interrupted line. Lamb C3 died 7 days after challenge, therefore no serum sample was collected at 21 DPC.</p

Detection of viral RNA in plasma by qRT-PCR.

<p>Plasma samples were collected daily at the first 7 days post challenge (DPC) and subsequently on DPC 9, 11, 14 and 21. Viral RNA copy numbers detected in individual animals of the mock-vaccinated group (C1–C8), low-dose group (L1–L8), medium-dose group (M1–M8) and high-dose group (H1–H7) are depicted.</p

Construction of the S-Gn segment and expression of Gn.

<p>(A) Schematic representation of the S segment of RVFV (upper panel) and the S segment in which the NSs gene is replaced for the codon-optimized Gn gene (lower panel). Distribution of Gn in Rep-Gn cells (B and E) and in BHK cells infected with NSR-Gn at an MOI of 0.5 (C and F). Panels D and G represent BHK control cells. Upper panels represent permeabilized cells and lower panels represent nonpermeabilized cells. The cells were stained with an anti-Gn monoclonal antibody and a Texas Red-labeled secondary antibody. Nuclei were visualized by DAPI staining.</p

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