Susceptibility of Pigs and Chickens to SARS Coronavirus

An outbreak of severe acute respiratory syndrome (SARS) in humans, associated with a new coronavirus, was reported in Southeast Asia, Europe, and North America in early 2003. To address speculations that the virus originated in domesticated animals, or that domestic species were susceptible to the virus, we inoculated 6-week-old pigs and chickens intravenously, intranasally, ocularly, and orally with 106 PFU of SARS-associated coronavirus (SARS-CoV). Clinical signs did not develop in any animal, nor were gross pathologic changes evident on postmortem examinations. Attempts at virus isolation were unsuccessful; however, viral RNA was detected by reverse transcriptase-polymerase chain reaction in blood of both species during the first week after inoculation, and in chicken organs at 2 weeks after inoculation. Virus-neutralizing antibodies developed in the pigs. Our results indicate that these animals do not play a role as amplifying hosts for SARS-CoV

Innate Immune Response to Rift Valley Fever Virus in Goats

Rift Valley fever (RVF), a re-emerging mosquito-borne disease of ruminants and man, was endemic in Africa but spread to Saudi Arabia and Yemen, meaning it could spread even further. Little is known about innate and cell-mediated immunity to RVF virus (RVFV) in ruminants, which is knowledge required for adequate vaccine trials. We therefore studied these aspects in experimentally infected goats. We also compared RVFV grown in an insect cell-line and that grown in a mammalian cell-line for differences in the course of infection. Goats developed viremia one day post infection (DPI), which lasted three to four days and some goats had transient fever coinciding with peak viremia. Up to 4% of peripheral blood mononuclear cells (PBMCs) were positive for RVFV. Monocytes and dendritic cells in PBMCs declined possibly from being directly infected with virus as suggested by in vitro exposure. Infected goats produced serum IFN-γ, IL-12 and other proinflammatory cytokines but not IFN-α. Despite the lack of IFN-α, innate immunity via the IL-12 to IFN-γ circuit possibly contributed to early protection against RVFV since neutralising antibodies were detected after viremia had cleared. The course of infection with insect cell-derived RVFV (IN-RVFV) appeared to be different from mammalian cell-derived RVFV (MAM-RVFV), with the former attaining peak viremia faster, inducing fever and profoundly affecting specific immune cell subpopulations. This indicated possible differences in infections of ruminants acquired from mosquito bites relative to those due to contact with infectious material from other animals. These differences need to be considered when testing RVF vaccines in laboratory settings

Comparative characterization of Crimean-Congo hemorrhagic fever virus cell culture systems with application to propagation and titration methods

Abstract Crimean-Congo hemorrhagic fever orthonairovirus (CCHFV) is a biosafety level 4 and World Health Organization top priority pathogen. Infection leads to an often fatal hemorrhagic fever disease in humans. The tick-borne virus is endemic in countries across Asia, Europe and Africa, with signs of spreading into new regions. Despite the severity of disease and the potential of CCHFV geographic expansion to cause widespread outbreaks, no approved vaccine or treatment is currently available. Critical for basic research and the development of diagnostics or medical countermeasures, CCHFV viral stocks are commonly produced in Vero E6 and SW-13 cell lines. While a variety of in-house methods are being used across different laboratories, there has been no clear, specific consensus on a standard, optimal system for CCHFV growth and titration. In this study, we perform a systematic, side-by-side characterization of Vero E6 and SW-13 cell lines concerning the replication kinetics of CCHFV under different culture conditions. SW-13 cells are typically cultured in a CO2-free condition (SW-13 CO2 −) according to the American Type Culture Collection. However, we identify a CO2-compatible culture condition (SW-13 CO2 +) that demonstrates the highest viral load (RNA concentration) and titer (infectious virus concentration) in the culture supernatants, in comparison to SW-13 CO2 − and Vero E6 cultures. This optimal viral propagation system also leads to the development of two titration methods: an immunostaining-based plaque assay using a commercial CCHFV antibody and a colorimetric readout, and an antibody staining-free, cytopathic effect-based median tissue culture infectious dose assay using a simple excel calculator. These are anticipated to serve as a basis for a reproducible, standardized and user-friendly platform for CCHFV propagation and titration

Rift Valley fever virus incorporates the 78 kDa glycoprotein into virions matured in mosquito C6/36 cells.

Rift Valley fever virus (RVFV), genus Phlebovirus, family Bunyaviridae is a zoonotic arthropod-borne virus able to transition between distant host species, causing potentially severe disease in humans and ruminants. Viral proteins are encoded by three genomic segments, with the medium M segment coding for four proteins: nonstructural NSm protein, two glycoproteins Gn and Gc and large 78 kDa glycoprotein (LGp) of unknown function. Goat anti-RVFV polyclonal antibody and mouse monoclonal antibody, generated against a polypeptide unique to the LGp within the RVFV proteome, detected this protein in gradient purified RVFV ZH501 virions harvested from mosquito C6/36 cells but not in virions harvested from the mammalian Vero E6 cells. The incorporation of LGp into the mosquito cell line - matured virions was confirmed by immune-electron microscopy. The LGp was incorporated into the virions immediately during the first passage in C6/36 cells of Vero E6 derived virus. Our data indicate that LGp is a structural protein in C6/36 mosquito cell generated virions. The protein may aid the transmission from the mosquitoes to the ruminant host, with a possible role in replication of RVFV in the mosquito host. To our knowledge, this is a first report of different protein composition between virions formed in insect C6/36 versus mammalian Vero E6 cells

Antibody response in RVFV-infected goats.

<p>Antibody response to RVFV was measured by plaque reduction neutralisation test (PRNT). The reciprocal of the serum dilution giving at least 70% plaque inhibition relative to the virus control was taken as the PRNT₇₀ titre for that sample. MAM-RVFV = mammalian cell-derived RVFV, IN-RVFV = insect cell-derived RVFV.</p

Adaptive cell mediated immunity in RVFV-infected goats.

<p>Means of PBMC IFN-γ response from IN-RVFV-infected goats are represented by the open histograms and MAM-RVFV infected goats by the filled histograms. Error bars represent standard deviation of means (n = 4 goats each). MAM-RVFV = RVFV produced in the mammalian cell line Vero E6, IN-RVFV = RVFV produced in the insect cell line C6/36.</p

Changes in cell population frequencies in peripheral blood mononuclear cells in RVFV-infected goats.

<p>Column A: PBMCs from IN-RVFV-infected goats; Column B: PBMCs from MAM-RVFV-infected goats; Column C: Cell frequencies expressed as a percentage of pre-infection value for —▪— IN-RVFV and —♦—MAM RVFV- infected goats (n = 4 goats each). Data points in column A and B represent individual animals and the line represents the means. In column C data points represent means + standard deviation (error bars). MAM-RVFV = RVFV produced in the mammalian cell line Vero E6, IN-RVFV = RVFV produced in the insect cell line C6/36.</p