Peter Marszal

Immunization with Modified Vaccinia Virus Ankara-Based Recombinant Vaccine against Severe Acute Respiratory Syndrome Is Associated with Enhanced Hepatitis in Ferrets

ABSTRACT Severe acute respiratory syndrome (SARS) caused by a newly identified coronavirus (SARS-CoV) is a serious emerging human infectious disease. In this report, we immunized ferrets (Mustela putorius furo) with recombinant modified vaccinia virus Ankara (rMVA) expressing the SARS-CoV spike (S) protein. Immunized ferrets developed a more rapid and vigorous neutralizing antibody response than control animals after challenge with SARS-CoV; however, they also exhibited strong inflammatory responses in liver tissue. Inflammation in control animals exposed to SARS-CoV was relatively mild. Thus, our data suggest that vaccination with rMVA expressing SARS-CoV S protein is associated with enhanced hepatitis.

Invasion of the Central Nervous System in a Porcine Host by Nipah Virus

ABSTRACT Nipah virus, a newly emerged zoonotic paramyxovirus, infects a number of species. Human infections were linked to direct contact with pigs, specifically with their body fluids. Clinical signs in human cases indicated primarily involvement of the central nervous system, while in pigs the respiratory system was considered the primary virus target, with only rare involvement of the central nervous system. Eleven 5-week-old piglets were infected intranasally, orally, and ocularly with 2.5 × 105 PFU of Nipah virus per animal and euthanized between 3 and 8 days postinoculation. Nipah virus caused neurological signs in two out of eleven inoculated pigs. The rest of the pigs remained clinically healthy. Virus was detected in the respiratory system (turbinates, nasopharynx, trachea, bronchus, and lung in titers up to 105.3 PFU/g) and in the lymphoreticular system (endothelial cells of blood and lymphatic vessels, submandibular and bronchiolar lymph nodes, tonsil, and spleen with titers up to 106 PFU/g). Virus presence was confirmed in the nervous system of both sick and apparently healthy animals (cranial nerves, trigeminal ganglion, brain, and cerebrospinal fluid, with titers up to 107.7 PFU/g of tissue). Nipah virus distribution was confirmed by immunohistochemistry. The study presents novel findings indicating that Nipah virus invaded the central nervous system of the porcine host via cranial nerves as well as by crossing the blood-brain barrier after initial virus replication in the upper respiratory tract.

Development and characterisation of neutralising monoclonal antibody to the SARS-coronavirus

Abstract There is a global need to elucidate protective antigens expressed by the SARS-coronavirus (SARS-CoV). Monoclonal antibody reagents that recognise specific antigens on SARS-CoV are needed urgently. In this report, the development and immunochemical characterisation of a panel of murine monoclonal antibodies (mAbs) against the SARS-CoV is presented, based upon their specificity, binding requirements, and biological activity. Initial screening by ELISA, using highly purified virus as the coating antigen, resulted in the selection of 103mAbs to the SARS virus. Subsequent screening steps reduced this panel to seventeen IgG mAbs. A single mAb, F26G15, is specific for the nucleoprotein as seen in Western immunoblot while five other mAbs react with the Spike protein. Two of these Spike-specific mAbs demonstrate the ability to neutralise SARS-CoV in vitro while another four Western immunoblot-negative mAbs also neutralise the virus. The utility of these mAbs for diagnostic development is demonstrated. Antibody from convalescent SARS patients, but not normal human serum, is also shown to specifically compete off binding of mAbs to whole SARS-CoV. These studies highlight the importance of using standardised assays and reagents. These mAbs will be useful for the development of diagnostic tests, studies of SARS-CoV pathogenesis and vaccine development.

Experimental Infection of Pigs with the Human 1918 Pandemic Influenza Virus

ABSTRACT Swine influenza was first recognized as a disease entity during the 1918 “Spanish flu” pandemic. The aim of this work was to determine the virulence of a plasmid-derived human 1918 pandemic H1N1 influenza virus (reconstructed 1918, or 1918/rec, virus) in swine using a plasmid-derived A/swine/Iowa/15/1930 H1N1 virus (1930/rec virus), representing the first isolated influenza virus, as a reference. Four-week-old piglets were inoculated intratracheally with either the 1930/rec or the 1918/rec virus or intranasally with the 1918/rec virus. A transient increase in temperature and mild respiratory signs developed postinoculation in all virus-inoculated groups. In contrast to other mammalian hosts (mice, ferrets, and macaques) where infection with the 1918/rec virus was lethal, the pigs did not develop severe respiratory distress or become moribund. Virus titers in the lower respiratory tract as well as macro- and microscopic lesions at 3 and 5 days postinfection (dpi) were comparable between the 1930/rec and 1918/rec virus-inoculated animals. In contrast to the 1930/rec virus-infected animals, at 7 dpi prominent lung lesions were present in only the 1918/rec virus-infected animals, and all the piglets developed antibodies at 7 dpi. Presented data support the hypothesis that the 1918 pandemic influenza virus was able to infect and replicate in swine, causing a respiratory disease, and that the virus was likely introduced into the pig population during the 1918 pandemic, resulting in the current lineage of the classical H1N1 swine influenza viruses.

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.

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.

Development and evaluation of one-step rRT-PCR and immunohistochemical methods for detection of Rift Valley fever virus in biosafety level 2 diagnostic laboratories

Rift Valley fever virus (RVFV) is a zoonotic insect transmitted virus endemic to Africa and the Arabian Peninsula. Infection causes abortions and high mortality in newborn ruminants. The overall human infection rate is <1%; however, fatality rates in those with severe clinical disease have been reported as high as 29%. The potential of RVFV as a bioterrorism agent and/or being accidentally introduced into North America is widely recognized. Currently, regional veterinary biosafety level 2 (BSL-2) diagnostic laboratories lack safe, modern, validated diagnostic tests to detect RVFV. An existing one-step real-time RT-PCR (rRT-PCR) assay was modified for quick virus inactivation for use in BSL-2 laboratories, evaluated on serum and tissue samples from experimentally infected lambs and calves, and compared to virus isolation. Viremia was detected in all inoculated sheep with titers reaching 10(6.5) plaque forming units/ml, or up to 10(10) viral RNA copies/ml. Viremia in calves was lower and not detected in all inoculated animals; however, all animals became transiently febrile and were infected as determined by rRT-PCR of tissues. Virus was isolated from rRT-PCR-positive liver and/or spleen in 33% of lamb and 41% of calf samples between 2 and 7 days post inoculation. For RVFV antigen detection, reagents are typically produced at BSL-3Ag or BSL-4 conditions and require inactivation and safety testing for use outside of containment. In this study, antiserum against recombinant RVFV-nucleocapsid (N) was produced to develop an immunohistochemical (IHC) assay which was subsequently evaluated on formalin fixed lamb and calf tissues at BSL-2 laboratory conditions. Antigen was detected by IHC in 79% of rRT-PCR-positive sheep and 70% of rRT-PCR-positive calf tissues tested. Once validated and approved by national regulatory agencies, these assays can be safely produced and distributed to regional diagnostic laboratories, providing capacity for early detection of RVFV in suspected ruminant samples.

Efficacy of a recombinant Rift Valley fever virus MP-12 with NSm deletion as a vaccine candidate in sheep

Rift Valley fever virus (RVFV), a mosquito-borne virus in the Bunyaviridae family and Phlebovirus genus, causes RVF, a disease of ruminants and man, endemic in Sub-Saharan African countries. However, outbreaks in Yemen and Saudi Arabia demonstrate the ability for RVFV to spread into virgin territory and thus the need exists to develop safe and efficacious vaccines that can be used outside the endemic zones. Commercial RVFV vaccines are available but have limitations that prevent their use in disease-free countries. Consequently, there are ongoing efforts to develop and/or improve RVFV vaccines with global acceptability. In this study a previously developed MP-12-derived vaccine candidate with a large deletion of the NSm gene in the pre Gn region of the M segment (arMP-12-ΔNSm21/384) developed by T. Ikegami, that was already shown to be safe in pregnant sheep causing neither abortion nor fetal malformation was further evaluated. This vaccine was tested for protection of sheep from viremia and fever following challenge with virulent RVFV ZH501 strain. A single vaccination with arMP-12-ΔNSm21/384 fully protected sheep when challenged four weeks post vaccination, thereby demonstrating that this vaccine is efficacious in protecting these animals from RVFV infection.

1918 and 2009 H1N1 influenza viruses are not pathogenic in birds

The susceptibility of chickens to both 1918 and 2009 H1N1 influenza virus was evaluated. The intravenous pathogenicity index of 1918 and 2009 H1N1 viruses in chickens was 0. Chickens did not develop clinical signs following experimental inoculation simulating natural infection. No gross pathological changes were observed in any tissues of chickens between 2 and 18 days post-infection (p.i.) and viral RNA was not detected by real-time RT-PCR in mucosal secretions or tissues. Seroconversion was not detected in any of the chickens following inoculation with H1N1 2009 virus, whereas half the chickens developed influenza-specific antibodies at 28 days p.i. with 1918 influenza, suggesting limited infection. Viral RNA was detected by real-time RT-PCR in mallard ducks following inoculation with 1918 influenza virus at 3 days p.i. in cloacal swabs, but not in tissues, and all ducks seroconverted by 28 days p.i. Both 1918 and 2009 H1N1 influenza viruses behave as LPAI in gallinaceous poultry.