Novel Flavivirus Attenuation Markers Identified in the Envelope Protein of Alfuy Virus.

Alfuy (ALFV) is an attenuated flavivirus related to the Murray Valley encephalitis virus (MVEV). We previously identified markers of attenuation in the envelope (E) protein of the prototype strain (ALFV3929), including the hinge region (E273-277) and lack of glycosylation at E154-156. To further determine the mechanisms of attenuation we assessed ALFV3929 binding to glycosaminoglycans (GAG), a known mechanism of flaviviruses attenuation. Indeed, ALFV3929 exhibited reduced binding to GAG-rich cells in the presence of heparin; however, low-passage ALFV isolates were relatively unaffected. Sequence comparisons between ALFV strains and structural modelling incriminated a positively-charged residue (K327) in ALFV3929 as a GAG-binding motif. Substitution of this residue to the corresponding uncharged residue in MVEV (L), using a previously described chimeric virus containing the prM & E genes of ALFV3929 in the backbone of MVEV (MVEV/ALFV-prME), confirmed a role for K327 in enhanced GAG binding. When the wild type residues at E327, E273-277 and E154-156 of ALFV3929 were replaced with the corresponding residues from virulent MVEV, it revealed each motif contributed to attenuation of ALFV3929, with the E327/E273-277 combination most dominant. These data demonstrate that attenuation of ALFV3929 is multifactorial and provide new insights for the rational design of attenuated flavivirus vaccines.

Deleterious effect of Usutu virus on human neural cells.

In the last decade, the number of emerging Flaviviruses described worldwide has increased considerably. Among them Zika virus (ZIKV) and Usutu virus (USUV) are African mosquito-borne viruses that recently emerged. Recently, ZIKV has been intensely studied due to major outbreaks associated with neonatal death and birth defects, as well as neurological symptoms. USUV pathogenesis remains largely unexplored, despite significant human and veterinary associated disorders. Circulation of USUV in Africa was documented more than 50 years ago, and it emerged in Europe two decades ago, causing massive bird mortality. More recently, USUV has been described to be associated with neurological disorders in humans such as encephalitis and meningoencephalitis, highlighting USUV as a potential health threat. The aim of this study was to evaluate the ability of USUV to infect neuronal cells. Our results indicate that USUV efficiently infects neurons, astrocytes, microglia and IPSc-derived human neuronal stem cells. When compared to ZIKV, USUV led to a higher infection rate, viral production, as well as stronger cell death and anti-viral response. Our results highlight the need to better characterize the physiopathology related to USUV infection in order to anticipate the potential threat of USUV emergence.

Usutu virus growth in human cell lines: induction of and sensitivity to type I and III interferons.

The mechanisms of Usutu virus (USUV) pathogenesis are largely unknown. The aim of this study was to evaluate the sensitivity of USUV to interferon (IFN) and the capacity of USUV to stimulate IFN production. Initial experiments were conducted to characterize the susceptibility of human cell lines to USUV infection and to evaluate the single-growth cycle replication curve of USUV. Results indicate that USUV is able to infect a variety of human cell lines, completing the replication cycle in Hep-2 and Vero cells within 48 h. Pre-treatment of cells with types I and III IFNs significantly inhibited the replication of USUV. However, the inhibitory effects of IFNs were considerably less if IFN was added after viral infection had been initiated. Also, USUV weakly induced types I and III IFNs.

Induction of indistinguishable gene expression patterns in rats by Vero cell-derived and mouse brain-derived Japanese encephalitis vaccines.

Transcriptomics is an objective index that reflects the overall condition of cells or tissues, and transcriptome technology, such as DNA microarray analysis, is now being introduced for the quality control of medical products. In this study, we applied DNA microarray analysis to evaluate the character of Japanese encephalitis (JE) vaccines. When administered into rat peritoneum, Vero cell-derived and mouse brain-derived JE vaccines induced similar gene expression patterns in liver and brain. Body weights and blood biochemical findings were also similar after administration of the two vaccines. Our results suggest that the two JE vaccines are likely to have equivalent characteristics with regard to reactivity in rats.

Simulation of climate-change scenarios to explain Usutu-virus dynamics in Austria.

The emergence and spread of infectious diseases in mid-latitudes, so far mainly observed in the tropics, considerably increase under the current situation of climate change. A recent example is the Usutu virus (USUV) outbreak in Austria. USUV is closely related to the West Nile virus in the U.S. and caused mass mortalities mainly of blackbirds (Turdus merula). The USUV flavivirus persists in a natural transmission cycle between vectors (mosquitoes) and host reservoirs (birds) and leads - once endemic in a population - to periodic outbreaks. In an epidemic model to explain the USUV dynamics in Austria 2001-2005, USUV dynamics were mainly determined by an interaction of bird immunity and environmental temperature. To investigate future scenarios, we entered temperature predictions from five global climate models into the USUV model and also considered four different climate-warming scenarios defined by the I ntergovernmental Panel on Climate Change, IPCC (20 different model-scenario combinations). We downscaled the 20 time series of predicted temperatures (through the year 2100) to represent the region around Vienna. Our simulations predict that USUV will persist in the host population after the epidemic peak observed in 2003. USUV-specific annual blackbird-mortality time series predict that the outbreak frequency increases successively from the beginning to the end of the century. Simulations of worst-case scenarios result in an endemic equilibrium with a decline of the blackbird population of about 24%. Additionally we calculated the annually averaged basic reproduction number for the period 1901-2100. The latter depict that undetected major outbreaks before 2000 were unlikely, whereas it is likely that the USUV becomes endemic after 2040.

Explaining Usutu virus dynamics in Austria: model development and calibration.

Usutu virus (USUV), a flavivirus of the Japanese encephalitis virus complex, was for the first time detected outside Africa in the region around Vienna (Austria) in 2001 by Weissenböck et al. [Weissenböck, H., Kolodziejek, J., Url, A., Lussy, H., Rebel-Bauder, B., Nowotny, N., 2002. Emergence of Usutu virus, an African mosquito-borne flavivirus of the Japanese encephalitis virus group, central Europe. Emerg. Infect. Dis. 8, 652-656]. USUV is an arthropod-borne virus (arbovirus) circulating between arthropod vectors (mainly mosquitoes of the Culex pipiens complex) and avian amplification hosts. Infections of mammalian hosts or humans, as observed for the related West Nile virus (WNV), are rare. However, USUV infection leads to a high mortality in birds, especially blackbirds (Turdus merula), and has similar dynamics with the WNV in North America, which, amongst others, caused mortality in American robins (Turdus migratorius). We hypothesized that the transmission of USUV is determined by an interaction of developing proportion of the avian hosts immune and climatic factors affecting the mosquito population. This mechanism is implemented into the present model that simulates the seasonal cycles of mosquito and bird populations as well as USUV cross-infections. Observed monthly climate data are specified for the temperature-dependent development rates of the mosquitoes as well as the temperature-dependent extrinsic-incubation period. Our model reproduced the observed number of dead birds in Austria between 2001 and 2005, including the peaks in the relevant years. The high number of USUV cases in 2003 seems to be a response to the early beginning of the extraordinary hot summer in that year. The predictions indicate that >70% of the bird population acquired immunity, but also that the percentage would drop rapidly within only a couple of years. We estimated annually averaged basic reproduction numbers between R (0)=0.54 (2004) and 1.35 (2003). Finally, extrapolation from our model suggests that only 0.2% of the blackbirds killed by USUV were detected by the Austrian USUV monitoring program [Chvala, S., Bakonyi, T., Bukovsky, C., Meister, T., Brugger, K., Rubel, F., Nowotny, N., Weissenböck, H., 2007. Monitoring of Usutu virus activity and spread by using dead bird surveillance in Austria, 2003-2005. Vet. Microbiol. 122, 237-245]. These results suggest that the model presented is able to quantitatively describe the process of USUV dynamics.

Immunogenicity and protective efficacy in mice of a formaldehyde-inactivated Indian strain of Japanese encephalitis virus grown in Vero cells.

P20778, an Indian strain of Japanese encephalitis virus (JEV) obtained from Vellore in the Southern India, was grown in Vero cells cultured on microcarriers in a spinner flask. The virus was formalin-inactivated and its immunogenicity and protective efficacy in mice were tested in comparison with a commercially available vaccine. Our studies indicated that formalin-inactivated JEV P20778 induced high levels of protective immunity in mice. Virus inactivation with formalin at 22 degrees C, which required shorter incubation period, was found to be as good or better to virus inactivation at 4 degrees C for generating high titers of anti-JEV antibodies. Similarly, the 22 degrees C-inactivated virus generated JEV neutralizing antibody titers as good or higher than those induced by the 4 degrees C-inactivated virus. Thus, for the vaccine production, inactivation of JEV with formalin at 22 degrees C would be a preferred method as it is faster and does not require cold room storage.

Effects of bafilomycin A1 on Japanese encephalitis virus in C6/36 mosquito cells.

Involvement of intracellular acidic compartments in the early phase of Japanese encephalitis (JE) virus infection of C6/36 mosquito cells was examined by bafilomycin A1, a specific inhibitor of vacuolar type H(+)-ATPase (V-ATPase). Dose dependent reduction of viral envelope protein (E) produced into the infected culture fluid was observed by pretreating the cells with 0.25 to 1.0 microM bafilomycin A1. In synchronized infection, cell surface-bound virions were internalized immediately by heating at 31 degrees C, followed by the release of nucleocapsid into the cytosol within a short lag period. Subcellular distribution of infecting 3H-uridine-labeled viral RNA (V-RNA) and its RNase sensitivity were analyzed by fractionation in Percoll density gradient centrifugation. At a 10 min chasing period, an RNase resistant V-RNA peak was found in fractions with a mean density of 1.05 g/ml corresponding to the endosome, while an RNase sensitive V-RNA peak was detected at density range of 1.052-1.054 g/ml corresponding to the ribosome in C6/36 cell homogenate. The results indicate that JE virus infection in C6/36 cells proceeded through the endocytic pathway involving intracellular acidic compartments which was affected by bafilomycin A1.

Japanese encephalitis virus infection in Vero cells: the involvement of intracellular acidic vesicles in the early phase of viral infection was observed with the treatment of a specific vacuolar type H+-ATPase inhibitor, bafilomycin A1.

The involvement of intracellular acidic vesicles in the early phase of Japanese encephalitis (JE) virus infection in Vero cells was observed by adding a specific vacuolar type H+-ATPase (V-ATPase) inhibitor (bafilomycin A1) in the cell culture medium. Studies with the detection of viral envelope (E) protein suggested that bafilomycin A1 inhibited virus infection in the cells. Subcellular distribution of incoming biotinylated virions and 3H-uridine-labeled viral RNA were observed in fractions of a Percoll density gradient. At 10 min of the chasing period, virions and viral RNA were found mainly in fractions with a mean density of 1.04 g/ml corresponding to the endosome both in the control and bafilomycin A1-treated cells. At 60 min of the chasing period, the peak of biotin activity was detected in fractions with a mean density of 1.08 g/ml corresponding to the lysosome, whereas the peak of radioactivity did not run parallel with that of biotin and shifted to fractions with a mean density of 1.05 g/ml and higher than 1.084 g/ml, respectively. At 60 min of the chasing period in bafilomycin A1-treated cells, the peak of biotin and radioactivity were still found mainly in the fraction with a density of 1.04 g/ml, representing the endosome. Subcellular fractionation by a Percoll density gradient revealed that bafilomycin A1 treatment resulted in the accumulation of virions in the endosome fraction and suggested the prevention of intracellular translocation of the virions which occurs during the early entry process of an infecting virus to the cells.