Rift Valley Fever Virus Non-Structural Protein S Is Associated with Nuclear Translocation of Active Caspase-3 and Inclusion Body Formation.

Rift Valley fever phlebovirus (RVFV) causes Rift Valley fever (RVF), an emerging zoonotic disease that causes abortion storms and high mortality rates in young ruminants as well as severe or even lethal complications in a subset of human patients. This study investigates the pathomechanism of intranuclear inclusion body formation in severe RVF in a mouse model. Liver samples from immunocompetent mice infected with virulent RVFV 35/74, and immunodeficient knockout mice that lack interferon type I receptor expression and were infected with attenuated RVFV MP12 were compared to livers from uninfected controls using histopathology and immunohistochemistry for RVFV nucleoprotein, non-structural protein S (NSs) and pro-apoptotic active caspase-3. Histopathology of the livers showed virus-induced, severe hepatic necrosis in both mouse strains. However, immunohistochemistry and immunofluorescence revealed eosinophilic, comma-shaped, intranuclear inclusions and an intranuclear (co-)localization of RVFV NSs and active caspase-3 only in 35/74-infected immunocompetent mice, but not in MP12-infected immunodeficient mice. These results suggest that intranuclear accumulation of RVFV 35/74 NSs is involved in nuclear translocation of active caspase-3, and that nuclear NSs and active caspase-3 are involved in the formation of the light microscopically visible inclusion bodies.

NSG-Mice Reveal the Importance of a Functional Innate and Adaptive Immune Response to Overcome RVFV Infection.

Rift Valley fever (RVF) is a zoonotic disease caused by RVF Phlebovirus (RVFV). The RVFV MP-12 vaccine strain is known to exhibit residual virulence in the case of a deficient interferon type 1 response. The hypothesis of this study is that virus replication and severity of lesions induced by the MP-12 strain in immunocompromised mice depend on the specific function of the disturbed pathway. Therefore, 10 strains of mice with deficient innate immunity (B6-IFNARtmAgt, C.129S7(B6)-Ifngtm1Ts/J, B6-TLR3tm1Flv, B6-TLR7tm1Aki, NOD/ShiLtJ), helper T-cell- (CD4tm1Mak), cytotoxic T-cell- (CD8atm1Mak), B-cell- (Igh-Jtm1DhuN?+N2), combined T- and B-cell- (NU/J) and combined T-, B-, natural killer (NK) cell- and macrophage-mediated immunity (NOD.Cg-PrkdcscidIl2rgtm1WjI/SzJ (NSG) mice) were subcutaneously infected with RVFV MP-12. B6-IFNARtmAgt mice were the only strain to develop fatal disease due to RVFV-induced severe hepatocellular necrosis and apoptosis. Notably, no clinical disease and only mild multifocal hepatocellular necrosis and apoptosis were observed in NSG mice, while immunohistochemistry detected the RVFV antigen in the liver and the brain. No or low virus expression and no lesions were observed in the other mouse strains. Conclusively, the interferon type 1 response is essential for early control of RVFV replication and disease, whereas functional NK cells, macrophages and lymphocytes are essential for virus clearance.

A Putative Novel Hepatitis E Virus Genotype 3 Subtype Identified in Rabbit, Germany 2016.

Hepatitis E is an emerging viral disease that is the leading cause of viral hepatitis in the world. The vast majority of hepatitis E cases in developed countries are caused by zoonotic genotypes 3 and 4 of hepatitis E virus (HEV) for which pig and wild boar and to lesser extent rabbits are the main reservoir. According to recent reports rabbits are a source of human HEV infection and highlight the risk of zoonotic foodborne transmission. Here we report the molecular analysis of a novel HEV strain identified in a rabbit during a countrywide surveillance of rabbits and hares in Germany, 2016. The analysis of the complete genome reveals characteristics of a putative novel recombinant subtype of the species Orthohepevirus A within the clade of genotype 3 but not closely related to any known subtypes. Importantly, the genome of this strain possesses a nucleotide exchange in the overlapping region of open reading frames ORF2/ORF3 interfering with a broadly applied diagnostic real-time RT-PCR. In conclusion, a new type of HEV strain was identified in a German rabbit with atypical and novel sequence characteristics.

Vaccine Efficacy of Self-Assembled Multimeric Protein Scaffold Particles Displaying the Glycoprotein Gn Head Domain of Rift Valley Fever Virus.

Compared to free antigens, antigens immobilized on scaffolds, such as nanoparticles, generally show improved immunogenicity. Conventionally, antigens are conjugated to scaffolds through genetic fusion or chemical conjugation, which may result in impaired assembly or heterogeneous binding and orientation of the antigens. By combining two emerging technologies-i.e., self-assembling multimeric protein scaffold particles (MPSPs) and bacterial superglue-these shortcomings can be overcome and antigens can be bound on particles in their native conformation. In the present work, we assessed whether this technology could improve the immunogenicity of a candidate subunit vaccine against the zoonotic Rift Valley fever virus (RVFV). For this, the head domain of glycoprotein Gn, a known target of neutralizing antibodies, was coupled on various MPSPs to further assess immunogenicity and efficacy in vivo. The results showed that the Gn head domain, when bound to the lumazine synthase-based MPSP, reduced mortality in a lethal mouse model and protected lambs, the most susceptible RVFV target animals, from viremia and clinical signs after immunization. Furthermore, the same subunit coupled to two other MPSPs (Geobacillus stearothermophilus E2 or a modified KDPG Aldolase) provided full protection in lambs as well.

Experimental Challenge of Sheep and Cattle with Dugbe Orthonairovirus, a Neglected African Arbovirus Distantly Related to CCHFV.

Dugbe orthonairovirus (DUGV) is a tick-borne arbovirus within the order Bunyavirales. DUGV was first isolated in Nigeria, but virus isolations in ten further African countries indicate that DUGV is widespread throughout Africa. Humans can suffer from a mild febrile illness, hence, DUGV is classified as a biosafety level (BSL) 3 agent. In contrast, no disease has been described in animals, albeit serological evidence exists that ruminants are common hosts and may play an important role in the transmission cycle of this neglected arbovirus. In this study, young sheep and calves were experimentally inoculated with DUGV in order to determine their susceptibility and to study the course of infection. Moreover, potential antibody cross-reactivities in currently available diagnostic assays for Crimean-Congo hemorrhagic fever orthonairovirus (CCHFV) were assessed as DUGV is distantly related to CCHFV. Following subcutaneous inoculation, none of the animals developed clinical signs or viremia. However, all ruminants seroconverted, as demonstrated by two DUGV neutralization test formats (micro-virus neutralization test (mVNT), plaque reduction (PRNT)), by indirect immunofluorescence assays and in bovines by a newly developed DUGV recombinant N protein ELISA. Sera did not react in commercial CCHFV ELISAs, whereas cross-reactivities were observed by immunofluorescence and immunoblot assays.

Sheep and Cattle Are Not Susceptible to Experimental Inoculation with Hazara Orthonairovirus, a Tick-Borne Arbovirus Closely Related to CCHFV.

Hazara orthonairovirus (HAZV) is a tick-borne arbovirus closely related to Crimean-Congo hemorrhagic fever orthonairovirus (CCHFV). Whereas CCHFV is a biosafety level (BSL) 4 agent, HAZV is classified as BSL 2, as it is not known to cause any disease in humans. Belonging to the same serogroup as CCHFV, HAZV might act as a model which can provide a better understanding of this important zoonosis. Furthermore, the serological relatedness may cause diagnostic problems if antibodies against HAZV interfere with current CCHFV serological assays. Therefore, sheep and cattle-important natural hosts for CCHFV-were experimentally infected with HAZV to prove their susceptibility and evaluate potential antibody cross-reactivities. According to this study, neither sheep nor cattle are susceptible to experimental HAZV infections. Consequently, the HAZV infection in ruminants is clearly distinct from CCHFV infections. Sera of immunized animals weakly cross-reacted between HAZV and CCHFV in immunofluorescence and immunoblot assays, but not in commercial CCHFV ELISAs commonly used for field studies.

Two monoclonal antibodies against glycoprotein Gn protect mice from Rift Valley Fever challenge by cooperative effects.

Rift Valley fever virus (RVFV) is a zoonotic arbovirus that causes severe disease in humans and ruminants. The infection is characterized by abortions in pregnant animals, high mortality in neonates as well as febrile illness in humans that develop in 1% of cases encephalitis or hemorrhagic fever. There is presently no specific antiviral treatment for RVFV infection available. In this study, two monoclonal antibodies (mAbs), raised against glycoprotein Gn, were applied in a therapeutic study. Treatment of RVFV infected mice with neutralizing mAb Gn3 alone at two different time points (30 minutes before or 30 minutes after virus challenge) showed only moderate efficacy of about 58.3% survival in both applications. However, a combination therapy together with non-neutralizing mAb Gn32 demonstrated complete protection (100% survival) when applied 30 minutes after the lethal challenge dose. The increase of mAb efficacy is probably based on cooperative neutralization effects. These data suggest that a combination therapy with mAbs Gn3 and Gn32 could be an effective treatment option against RVFV infection.

Crimean-Congo haemorrhagic fever virus in ticks collected from livestock in Balochistan, Pakistan.

BACKGROUND: Crimean-Congo haemorrhagic fever virus (CCHFV) is a tick-borne zoonotic pathogen. It causes a fatal haemorrhagic disease in humans. Hard ticks, in particular Hyalomma spp., are considered to function as reservoir as well as vector for CCHFV. METHODS: A cross-sectional study was conducted in the province of Balochistan, Pakistan, from September to November 2017. Ticks were collected from cattle, sheep and goats in livestock farms. The ticks were morphologically identified, followed by confirmation with molecular methods (PCR and sequencing). Furthermore, ticks were examined for CCHFV genomes (S segment) by a one-step multiplex real-time RT-qPCR and positive samples were sequenced to determine the CCHFV genotype. RESULTS: In total, 525 of 529 livestock infesting adult ticks belonged to the genus Hyalomma, and 4 ticks to the genus Rhipicephalus (R. microplus 3×, R. turanicus 1×). In the genus Hyalomma, H. marginatum (28%), H. excavatum (26%), H. dromedarii (22%), H. anatolicum (16%) and H. scupense (8%) ticks were identified. Tick infestations were as follows: sheep 58%, goats 28% and cattle 14%. Four per cent (20/525) of ticks were CCHFV genome-positive, and all genomes clustered in CCHFV genotype Asia 1. Among CCHFV-positive ticks, 75% (15/20) were female and 25% (5/20) male. CCHFV genomes were most frequently detected in H. marginatum (30%, 6/20), followed by H. dromedarii (25%, 5/20), H. excavatum (20%, 4/20), H. anatolicum (20%, 4/20) and H. scupense (5%, 1/20). All CCHFV-positive ticks were found on sheep. The largest number of CCHFV-positive ticks were detected in the district of Kalat (60%, 12/20), followed by the districts of Quetta (30%, 6/20) and Killa Abdullah (10%, 2/20). CONCLUSIONS: This study confirms the circulation of CCHFV in ticks in Balochistan, south-western Pakistan. It is imperative to take effective tick control measures in this area, especially to control livestock tick infestations to prevent CCHF infections in humans.

Epidemiological investigations of Crimean-Congo haemorrhagic fever virus infection in sheep and goats in Balochistan, Pakistan.

Crimean-Congo haemorrhagic fever (CCHF) is a tick-borne zoonotic disease caused by the arbovirus Crimean-Congo haemorrhagic fever virus (CCHFV). Livestock serve as a transient reservoir for CCHFV, but do not show clinical signs. In this cross-sectional study, sheep and goats in Balochistan, Pakistan, were examined to determine the CCHFV seroprevalence, spatial distribution of seropositive sheep and goats, and to identify potential risk factors for seropositivity to CCHFV in these animals. To this end, farms and animals were selected by systematic sampling, blood samples from 800 sheep and 800 goats were collected and information regarding farm management and the kept animals were retrieved using a standard questionnaire. Sera were tested for antibodies against CCHFV in two independent ELISA formats and an immunofluorescence assay (IFA) following a hierarchical diagnostic decision tree. By these assays 149 (19 %, 95 %-CI: 16-21 %) out of 800 sheep serum samples and 37 (5 %, 95 %-CI: 3-6 %) out of 800 goat serum samples were positive for CCHFV-specific IgG antibodies. Interestingly, at least 8 (5 %, 95 %-CI: 2-10 %) out of 160 sera pools were from CCHFV viraemic sheep, as sera (in pools of 5) tested positive for CCHFV genome by real-time PCR (RT-qPCR). Risk factor analysis revealed that the open type of housing (OR = 3.76, 95 %-CI:1.57-9.56, p-value = 0.003), grazing (OR = 4.18, 95 %-CI:1.79-10.37, p-value = 0.001), presence of vegetation in or around the farm (OR = 3.13, 95 %-CI: 1.07-10.15, p-value = 0.043), lack of treatment against ticks (OR = 3.31, 95 %-CI: 1.16-10.21, p-value = 0.029), absence of rural poultry (OR = 2.93, 95 %-CI: 1.41-6.29, p-value = 0.004), animals with age ≥ 2 years (OR = 4.15, 95 %-CI: 2.84-6.19, p-value<0.001), animals infested with ticks (OR = 2.35, 95 %-CI: 1.59-3.52, p-value<0.001), and sheep species (OR = 4.72, 95 %-CI:3.24-6.86, p-value<0.001) represented statistically significant risk factors associated with seropositivity to CCHFV. Taken together this study confirms the circulation of CCHFV in livestock in Balochistan, Pakistan. The identification of risk factors might help to reduce the risk of infection in sheep and goats, which may also mitigate the risk for human infection. An interesting option for reducing the risk of CCHFV infection in small ruminants is keeping also chickens, since they pick ticks that transmit CCHFV.