SARS-CoV-2, a novelBetacoronavirus, was first reported circulating in human populations in Decemb... more SARS-CoV-2, a novelBetacoronavirus, was first reported circulating in human populations in December 2019 and has since become a global pandemic. Recent history involving SARS-like coronavirus outbreaks (SARS-CoV and MERS-CoV) have demonstrated the significant role of intermediate and reservoir hosts in viral maintenance and transmission cycles. Evidence of SARS-CoV-2 natural infection and experimental infections of a wide variety of animal species has been demonstrated, andin silicoandin vitrostudies have indicated that deer are susceptible to SARS-CoV-2 infection. White-tailed deer (Odocoileus virginianus) are amongst the most abundant, densely populated, and geographically widespread wild ruminant species in the United States. Human interaction with white-tailed deer has resulted in the occurrence of disease in human populations in the past. Recently, white-tailed deer fawns were shown to be susceptible to SARS-CoV-2. In the present study, we investigated the susceptibility and tr...
Background: The purpose of this study was the development of multiplex fluorescence microsphere i... more Background: The purpose of this study was the development of multiplex fluorescence microsphere immunoassay (FMIA) for the detection of Rift Valley fever virus (RVFV) IgG and IgM antibodies by incorporation of antigens from whole cell lysates and purified virions from MP12. Methods and Findings: Viral antigens were derived from whole cell material prepared from BHK cells infected with MP-12 (C-MP12). Virions from cell-free culture supernatant were purified on a sucrose cushion (V-MP12). The antigen material was treated with Triton X-100 or NP-40 prior to coating onto Luminex beads. V-MP12 and C-MP12 were assembled into a multiplex and tested in sheep and cattle infected with wild-type RVFV and the vaccine strain MP12. In these studies, V-MP12, C-MP12, and recombinant antigens (Gn, NSs, NSm, N) conjugated beads used in multiplex FMIA and the maximum level of IgG response to sheep in V-MP12 was recorded. IgG reactivity for the antigen targets varied with V-MP12>C-MP12. The positivi...
Journal of Veterinary Diagnostic Investigation, 1997
A reversible target capture viral RNA extraction procedure was combined with a reverse-transcript... more A reversible target capture viral RNA extraction procedure was combined with a reverse-transcriptase nested polymerase chain reaction (PCR) to develop a capture PCR assay providing a rapid and safe prediction method for circulating bluetongue virus in infected ruminants. This new assay was compared with virus isolation and a recently developed antigen-capture enzyme-linked immunosorbent assay (ELISA) for the detection of bluetongue virus. Eight Warhill crossbred sheep were inoculated subcutaneously with bluetongue virus serotype 10, and blood samples were taken sequentially over a period of 28 days. The capture PCR detected the peak of viremia, as determined by virus isolation and antigen-capture ELISA, from day 5 to day 14 after challenge. The results indicate that the rapid-capture bluetongue virus PCR provides a rapid indicator of samples in which virus can be isolated. In addition, this capture bluetongue virus PCR procedure does not require a lengthy phenol extraction or the us...
This communication summarizes the presentations given at the 1st international conference of the ... more This communication summarizes the presentations given at the 1st international conference of the World Society for Virology (WSV) held virtually during 16–18 June 2021, under the theme of tackling global viral epidemics. The purpose of this biennial meeting is to foster international collaborations and address important viral epidemics in different hosts. The first day included two sessions exclusively on SARS-CoV-2 and COVID-19. The other two days included one plenary and three parallel sessions each. Last not least, 16 sessions covered on-demand submitted talks. In total, 270 scientists from 49 countries attended the meeting, including 40 invited keynote speakers.
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is responsible for a global pandemic... more Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is responsible for a global pandemic that has had significant impacts on human health and economies worldwide. SARS-CoV-2 is highly transmissible and the cause of coronavirus disease 2019 (COVID-19) in humans. A wide range of animal species have also been shown to be susceptible to SARS-CoV-2 infection by experimental and/or natural infections. Domestic and large cats, mink, ferrets, hamsters, deer mice, white-tailed deer, and non-human primates have been shown to be highly susceptible, whereas other species such as mice, dogs, pigs, and cattle appear to be refractory to infection or have very limited susceptibility. Sheep (Ovis aries) are a commonly farmed domestic ruminant that have not previously been thoroughly investigated for their susceptibility to SARS-CoV-2. Therefore, we performed in vitro and in vivo studies which consisted of infection of ruminant-derived cell cultures and experimental challenge of sheep to inv...
SARS-CoV-2 is a recently emerged, highly contagious virus and the cause of the current COVID-19 p... more SARS-CoV-2 is a recently emerged, highly contagious virus and the cause of the current COVID-19 pandemic. It is a zoonotic virus, although its animal origin is not clear yet. Person-to-person transmission occurs by inhalation of infected droplets and aerosols, or by direct contact with contaminated fomites. Arthropods transmit numerous viral, parasitic, and bacterial diseases; however, the potential role of arthropods in SARS-CoV-2 transmission is not fully understood. Thus far, a few studies have demonstrated that SARS-CoV-2 replication is not supported in cells from certain insect species nor in certain species of mosquitoes after intrathoracic inoculation. In this study, we expanded the work of SARS-CoV-2 susceptibility to biting insects after ingesting a SARS-CoV-2-infected bloodmeal. Species tested included Culicoides sonorensis (Wirth & Jones) (Diptera: Ceratopogonidae) biting midges, as well as Culex tarsalis (Coquillett) and Culex quinquefasciatus (Say) mosquitoes (Diptera: ...
Background Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a recently emerged cor... more Background Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a recently emerged coronavirus that is the causative agent of the coronavirus disease 2019 (COVID-19) pandemic. COVID-19 in humans is characterized by a wide range of symptoms that range from asymptomatic to mild or severe illness including death. SARS-CoV-2 is highly contagious and is transmitted via the oral–nasal route through droplets and aerosols, or through contact with contaminated fomites. House flies are known to transmit bacterial, parasitic and viral diseases to humans and animals as mechanical vectors. Previous studies have shown that house flies can mechanically transmit coronaviruses, such as turkey coronavirus; however, the house fly’s role in SARS-CoV-2 transmission has not yet been explored. The goal of this work was to investigate the potential of house flies to mechanically transmit SARS-CoV-2. For this purpose, it was determined whether house flies can acquire SARS-CoV-2, harbor live virus...
Rift Valley fever virus (RVFV), a Category A pathogen and select agent, is the causative agent of... more Rift Valley fever virus (RVFV), a Category A pathogen and select agent, is the causative agent of Rift Valley fever. To date, no fully licensed vaccine is available in the U.S. for human or animal use and effective antiviral drugs have not been identified. The RVFV MP12 strain is conditionally licensed for use for veterinary purposes in the U.S. which was excluded from the select agent rule of Health and Human Services and the U.S. Department of Agriculture. The MP12 vaccine strain is commonly used in BSL-2 laboratories that is generally not virulent in mice. To establish a small animal model that can be used in a BSL-2 facility for antiviral drug development, we investigated susceptibility of six mouse strains (129S6/SvEv, STAT-1 KO, 129S1/SvlmJ, C57BL/6J, NZW/LacJ, BALB/c) to the MP12 virus infection via an intranasal inoculation route. Severe weight loss, obvious clinical and neurologic signs, and 50% mortality was observed in the STAT-1 KO mice, whereas the other 5 mouse strains did not display obvious and/or severe disease. Virus replication and histopathological lesions were detected in brain and liver of MP12-infected STAT-1 KO mice that developed the acute-onset hepatitis and delayed-onset encephalitis. In conclusion, the STAT-1 KO mouse strain is susceptible to MP12 virus infection, indicating that it can be used to investigate RVFV antivirals in a BSL-2 environment.
Rift Valley fever (RVF) is a zoonotic disease that causes severe epizootics in ruminants, charact... more Rift Valley fever (RVF) is a zoonotic disease that causes severe epizootics in ruminants, characterized by mass abortion and high mortality rates in younger animals. The development of a reliable challenge model is an important prerequisite for evaluation of existing and novel vaccines. A study aimed at comparing the pathogenesis of RVF virus infection in US sheep using two genetically different wild type strains of the virus (SA01-1322 and Kenya-128B-15) was performed. A group of sheep was inoculated with both strains and all infected sheep manifested early-onset viremia accompanied by a transient increase in temperatures. The Kenya-128B-15 strain manifested higher virulence compared to SA01-1322 by inducing more severe liver damage, and longer and higher viremia. Genome sequence analysis revealed sequence variations between the two isolates, which potentially could account for the observed phenotypic differences. We conclude that Kenya-128B-15 sheep infection represents a good and virulent challenge model for RVF.
Rift Valley fever virus (RVFV), a member of the Bunyaviridae family, is a mosquito-borne zoonotic... more Rift Valley fever virus (RVFV), a member of the Bunyaviridae family, is a mosquito-borne zoonotic pathogen that causes serious morbidity and mortality in livestock and humans. The recent spread of the virus beyond its traditional endemic boundaries in Africa to the Arabian Peninsula coupled with the presence of susceptible vectors in nonendemic countries has created increased interest in RVF vaccines. Subunit vaccines composed of specific virus proteins expressed in eukaryotic or prokaryotic expression systems are shown to elicit neutralizing antibodies in susceptible hosts. RVFV structural proteins, amino-terminus glycoprotein (Gn), and carboxylterminus glycoprotein (Gc), were expressed using a recombinant baculovirus expression system. The recombinant proteins were reconstituted as a GnGc subunit vaccine formulation and evaluated for immunogenicity in a target species, sheep. Six sheep were each immunized with a primary dose of 50 lg of each vaccine immunogen with the adjuvant montanide ISA25; at day 21, postvaccination, each animal received a second dose of the same vaccine. The vaccine induced a strong antibody response in all animals as determined by indirect enzyme-linked immunosorbent assay (ELISA). A plaque reduction neutralization test (PRNT 80) showed the primary dose of the vaccine was sufficient to elicit potentially protective virus neutralizing antibody titers ranging from 40 to 160, and the second vaccine dose boosted the titer to more than 1280. Furthermore, all animals tested positive for neutralizing antibodies at day 328 postvaccination. ELISA analysis using the recombinant nucleocapsid protein as a negative marker antigen indicated that the vaccine candidate is DIVA (differentiating infected from vaccinated animals) compatible and represents a promising vaccine platform for RVFV infection in susceptible species.
Previous phylogenetic analyses based on bluetongue virus (BTV) gene segment L3, which encodes the... more Previous phylogenetic analyses based on bluetongue virus (BTV) gene segment L3, which encodes the inner core protein, VP3, indicated a geographical distribution of different genotypes. The inner core protein, VP7, of BTV has been identified as a viral attachment protein for insect cell infection. Because the inner core proteins are involved with infectivity of insect cells, we hypothesized that certain VP7 protein sequences are preferred by the insect vector species present in specific geographic locations. We compared the gene segment S7, which encodes VP7, from 39 strains of BTV isolated from Central America, the Caribbean Basin, the United States, South Africa and Australia. For comparison, the S7 sequences from strains of the related orbiviruses, epizootic hemorrhagic disease virus (EHDV) and African horse sickness virus (AHSV) were included. The S7 gene was highly conserved among BTV strains and fairly conserved among the other orbiviruses examined. VP7 sequence alignment suggests that the BTV receptor-binding site in the insect is also conserved. Phylogenetic analyses revealed that the BTV S7 nucleotide sequences do not unequivocally display geographic distribution. The BTV strains can be separated into five clades based on the deduced VP7 amino acid sequence alignment and phylogeny but evidence for preferential selection by available gnat species for a particular VP7 clade is inconclusive. Differences between clades indicate allowable variation of the VP7 binding protein.
The Rift Valley fever virus (RVFV) encodes the structural proteins nucleoprotein (N), aminotermin... more The Rift Valley fever virus (RVFV) encodes the structural proteins nucleoprotein (N), aminoterminal glycoprotein (Gn), carboxyterminal glycoprotein (Gc), and L protein, 78-kD, and the nonstructural proteins NSm and NSs. Using the baculovirus system, we expressed the full-length coding sequence of N, NSs, NSm, Gc, and the ectodomain of the coding sequence of the Gn glycoprotein derived from the virulent strain of RVFV ZH548. Western blot analysis using anti-His antibodies and monoclonal antibodies against Gn and N confirmed expression of the recombinant proteins, and in vitro biochemical analysis showed that the two glycoproteins, Gn and Gc, were expressed in glycosylated form. Immunoreactivity profiles of the recombinant proteins in western blot and in indirect enzyme-linked immunosorbent assay against a panel of antisera obtained from vaccinated or wild type (RVFV)-challenged sheep confirmed the results obtained with anti-His antibodies and demonstrated the suitability of the baculo-expressed antigens for diagnostic assays. In addition, these recombinant proteins could be valuable for the development of diagnostic methods that differentiate infected from vaccinated animals (DIVA).
Epizootic hemorrhagic disease virus (EHDV) is a Culicoides-transmitted orbivirus that infects dom... more Epizootic hemorrhagic disease virus (EHDV) is a Culicoides-transmitted orbivirus that infects domestic and wild ruminants and is provisionally thought to be distributed throughout Africa, North America, Australia, East Asia and the Middle East. Historically, of the seven proposed serotypes of EHDV, only EHDV-1 and EHDV-2 have been reported from North America. In 2006, EHDV isolates were recovered from moribund or dead white-tailed deer (Odocoileus virginianus) in Indiana and Illinois that could not be identified as either EHDV-1 or EHDV-2 by virus neutralization tests or by serotype-specific RT-PCR. Additional serological and genetic testing identified the isolates as EHDV-6, a serotype that, although originally described from Australia, has recently been recognized as an emerging pathogen of cattle in Morocco, Algeria and Turkey. In 2007 and 2008, EHDV-6 was isolated again from white-tailed deer, this time in Missouri, Kansas and Texas, suggesting that the virus is capable of overwintering and that it may become, or already is, endemic in a geographically widespread region of the USA. Genetic characterization of the virus indicates that it is a reassortant, such that the outer capsid proteins determining serotype specificity (VP2 and VP5) are derived from exotic EHDV-6, whilst the remaining structural and non-structural proteins are apparently obtained from indigenous EHDV-2 (Alberta).
Bluetongue (BT) is an insect-transmitted, economically important disease of domestic and wild rum... more Bluetongue (BT) is an insect-transmitted, economically important disease of domestic and wild ruminants. Although only five of the 26 reported bluetongue virus (BTV) serotypes are considered endemic to the USA, 10 exotic serotypes have been isolated primarily in the southeastern region of the country since 1999. For an exotic BTV serotype to become endemic there must be susceptible animal species and competent vectors. In the USA, sheep and white-tailed deer (WTD) are the primary sentinel livestock and wildlife species, respectively. In 2006, BTV-8 was introduced into Northern Europe and subsequently overwintered, causing unprecedented livestock disease and mortality during the 2006– 2007 vector seasons. To assess the risk of the European strain of BTV-8 to North American WTD, and understand the role they could play after a similar introduction, eight bluetongue-seronegative WTD were inoculated with BTV-8. Body temperatures and clinical signs were recorded daily. Blood samples were ...
A cell line was established from Culex tarsalis Coquillett embryonated eggs and designated as CxT... more A cell line was established from Culex tarsalis Coquillett embryonated eggs and designated as CxTr. The cell line is heterogeneous, composed predominantly of small, round cells, and spindle-shaped cells with a doubling time of approximately 52–60 h. The identity of the cell line was verified as Cx. tarsalis by sequencing of cytochrome oxidase I and the cells were found to be free of contaminating cells, bacteria, fungi, and mycoplasma. The permissiveness of CxTr cells to arbovirus infection was investigated with vaccine and wildtype arboviruses from four viral families: Flaviviridae (Japanese encephalitis virus), Phenuiviridae (Rift Valley fever phlebovirus), Rhabdoviridae (vesicular stomatitis virus), and Togaviridae (Mayaro virus). All viruses were able to infect and replicate within CxTr cells.
SARS-CoV-2, a novelBetacoronavirus, was first reported circulating in human populations in Decemb... more SARS-CoV-2, a novelBetacoronavirus, was first reported circulating in human populations in December 2019 and has since become a global pandemic. Recent history involving SARS-like coronavirus outbreaks (SARS-CoV and MERS-CoV) have demonstrated the significant role of intermediate and reservoir hosts in viral maintenance and transmission cycles. Evidence of SARS-CoV-2 natural infection and experimental infections of a wide variety of animal species has been demonstrated, andin silicoandin vitrostudies have indicated that deer are susceptible to SARS-CoV-2 infection. White-tailed deer (Odocoileus virginianus) are amongst the most abundant, densely populated, and geographically widespread wild ruminant species in the United States. Human interaction with white-tailed deer has resulted in the occurrence of disease in human populations in the past. Recently, white-tailed deer fawns were shown to be susceptible to SARS-CoV-2. In the present study, we investigated the susceptibility and tr...
Background: The purpose of this study was the development of multiplex fluorescence microsphere i... more Background: The purpose of this study was the development of multiplex fluorescence microsphere immunoassay (FMIA) for the detection of Rift Valley fever virus (RVFV) IgG and IgM antibodies by incorporation of antigens from whole cell lysates and purified virions from MP12. Methods and Findings: Viral antigens were derived from whole cell material prepared from BHK cells infected with MP-12 (C-MP12). Virions from cell-free culture supernatant were purified on a sucrose cushion (V-MP12). The antigen material was treated with Triton X-100 or NP-40 prior to coating onto Luminex beads. V-MP12 and C-MP12 were assembled into a multiplex and tested in sheep and cattle infected with wild-type RVFV and the vaccine strain MP12. In these studies, V-MP12, C-MP12, and recombinant antigens (Gn, NSs, NSm, N) conjugated beads used in multiplex FMIA and the maximum level of IgG response to sheep in V-MP12 was recorded. IgG reactivity for the antigen targets varied with V-MP12>C-MP12. The positivi...
Journal of Veterinary Diagnostic Investigation, 1997
A reversible target capture viral RNA extraction procedure was combined with a reverse-transcript... more A reversible target capture viral RNA extraction procedure was combined with a reverse-transcriptase nested polymerase chain reaction (PCR) to develop a capture PCR assay providing a rapid and safe prediction method for circulating bluetongue virus in infected ruminants. This new assay was compared with virus isolation and a recently developed antigen-capture enzyme-linked immunosorbent assay (ELISA) for the detection of bluetongue virus. Eight Warhill crossbred sheep were inoculated subcutaneously with bluetongue virus serotype 10, and blood samples were taken sequentially over a period of 28 days. The capture PCR detected the peak of viremia, as determined by virus isolation and antigen-capture ELISA, from day 5 to day 14 after challenge. The results indicate that the rapid-capture bluetongue virus PCR provides a rapid indicator of samples in which virus can be isolated. In addition, this capture bluetongue virus PCR procedure does not require a lengthy phenol extraction or the us...
This communication summarizes the presentations given at the 1st international conference of the ... more This communication summarizes the presentations given at the 1st international conference of the World Society for Virology (WSV) held virtually during 16–18 June 2021, under the theme of tackling global viral epidemics. The purpose of this biennial meeting is to foster international collaborations and address important viral epidemics in different hosts. The first day included two sessions exclusively on SARS-CoV-2 and COVID-19. The other two days included one plenary and three parallel sessions each. Last not least, 16 sessions covered on-demand submitted talks. In total, 270 scientists from 49 countries attended the meeting, including 40 invited keynote speakers.
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is responsible for a global pandemic... more Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is responsible for a global pandemic that has had significant impacts on human health and economies worldwide. SARS-CoV-2 is highly transmissible and the cause of coronavirus disease 2019 (COVID-19) in humans. A wide range of animal species have also been shown to be susceptible to SARS-CoV-2 infection by experimental and/or natural infections. Domestic and large cats, mink, ferrets, hamsters, deer mice, white-tailed deer, and non-human primates have been shown to be highly susceptible, whereas other species such as mice, dogs, pigs, and cattle appear to be refractory to infection or have very limited susceptibility. Sheep (Ovis aries) are a commonly farmed domestic ruminant that have not previously been thoroughly investigated for their susceptibility to SARS-CoV-2. Therefore, we performed in vitro and in vivo studies which consisted of infection of ruminant-derived cell cultures and experimental challenge of sheep to inv...
SARS-CoV-2 is a recently emerged, highly contagious virus and the cause of the current COVID-19 p... more SARS-CoV-2 is a recently emerged, highly contagious virus and the cause of the current COVID-19 pandemic. It is a zoonotic virus, although its animal origin is not clear yet. Person-to-person transmission occurs by inhalation of infected droplets and aerosols, or by direct contact with contaminated fomites. Arthropods transmit numerous viral, parasitic, and bacterial diseases; however, the potential role of arthropods in SARS-CoV-2 transmission is not fully understood. Thus far, a few studies have demonstrated that SARS-CoV-2 replication is not supported in cells from certain insect species nor in certain species of mosquitoes after intrathoracic inoculation. In this study, we expanded the work of SARS-CoV-2 susceptibility to biting insects after ingesting a SARS-CoV-2-infected bloodmeal. Species tested included Culicoides sonorensis (Wirth & Jones) (Diptera: Ceratopogonidae) biting midges, as well as Culex tarsalis (Coquillett) and Culex quinquefasciatus (Say) mosquitoes (Diptera: ...
Background Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a recently emerged cor... more Background Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a recently emerged coronavirus that is the causative agent of the coronavirus disease 2019 (COVID-19) pandemic. COVID-19 in humans is characterized by a wide range of symptoms that range from asymptomatic to mild or severe illness including death. SARS-CoV-2 is highly contagious and is transmitted via the oral–nasal route through droplets and aerosols, or through contact with contaminated fomites. House flies are known to transmit bacterial, parasitic and viral diseases to humans and animals as mechanical vectors. Previous studies have shown that house flies can mechanically transmit coronaviruses, such as turkey coronavirus; however, the house fly’s role in SARS-CoV-2 transmission has not yet been explored. The goal of this work was to investigate the potential of house flies to mechanically transmit SARS-CoV-2. For this purpose, it was determined whether house flies can acquire SARS-CoV-2, harbor live virus...
Rift Valley fever virus (RVFV), a Category A pathogen and select agent, is the causative agent of... more Rift Valley fever virus (RVFV), a Category A pathogen and select agent, is the causative agent of Rift Valley fever. To date, no fully licensed vaccine is available in the U.S. for human or animal use and effective antiviral drugs have not been identified. The RVFV MP12 strain is conditionally licensed for use for veterinary purposes in the U.S. which was excluded from the select agent rule of Health and Human Services and the U.S. Department of Agriculture. The MP12 vaccine strain is commonly used in BSL-2 laboratories that is generally not virulent in mice. To establish a small animal model that can be used in a BSL-2 facility for antiviral drug development, we investigated susceptibility of six mouse strains (129S6/SvEv, STAT-1 KO, 129S1/SvlmJ, C57BL/6J, NZW/LacJ, BALB/c) to the MP12 virus infection via an intranasal inoculation route. Severe weight loss, obvious clinical and neurologic signs, and 50% mortality was observed in the STAT-1 KO mice, whereas the other 5 mouse strains did not display obvious and/or severe disease. Virus replication and histopathological lesions were detected in brain and liver of MP12-infected STAT-1 KO mice that developed the acute-onset hepatitis and delayed-onset encephalitis. In conclusion, the STAT-1 KO mouse strain is susceptible to MP12 virus infection, indicating that it can be used to investigate RVFV antivirals in a BSL-2 environment.
Rift Valley fever (RVF) is a zoonotic disease that causes severe epizootics in ruminants, charact... more Rift Valley fever (RVF) is a zoonotic disease that causes severe epizootics in ruminants, characterized by mass abortion and high mortality rates in younger animals. The development of a reliable challenge model is an important prerequisite for evaluation of existing and novel vaccines. A study aimed at comparing the pathogenesis of RVF virus infection in US sheep using two genetically different wild type strains of the virus (SA01-1322 and Kenya-128B-15) was performed. A group of sheep was inoculated with both strains and all infected sheep manifested early-onset viremia accompanied by a transient increase in temperatures. The Kenya-128B-15 strain manifested higher virulence compared to SA01-1322 by inducing more severe liver damage, and longer and higher viremia. Genome sequence analysis revealed sequence variations between the two isolates, which potentially could account for the observed phenotypic differences. We conclude that Kenya-128B-15 sheep infection represents a good and virulent challenge model for RVF.
Rift Valley fever virus (RVFV), a member of the Bunyaviridae family, is a mosquito-borne zoonotic... more Rift Valley fever virus (RVFV), a member of the Bunyaviridae family, is a mosquito-borne zoonotic pathogen that causes serious morbidity and mortality in livestock and humans. The recent spread of the virus beyond its traditional endemic boundaries in Africa to the Arabian Peninsula coupled with the presence of susceptible vectors in nonendemic countries has created increased interest in RVF vaccines. Subunit vaccines composed of specific virus proteins expressed in eukaryotic or prokaryotic expression systems are shown to elicit neutralizing antibodies in susceptible hosts. RVFV structural proteins, amino-terminus glycoprotein (Gn), and carboxylterminus glycoprotein (Gc), were expressed using a recombinant baculovirus expression system. The recombinant proteins were reconstituted as a GnGc subunit vaccine formulation and evaluated for immunogenicity in a target species, sheep. Six sheep were each immunized with a primary dose of 50 lg of each vaccine immunogen with the adjuvant montanide ISA25; at day 21, postvaccination, each animal received a second dose of the same vaccine. The vaccine induced a strong antibody response in all animals as determined by indirect enzyme-linked immunosorbent assay (ELISA). A plaque reduction neutralization test (PRNT 80) showed the primary dose of the vaccine was sufficient to elicit potentially protective virus neutralizing antibody titers ranging from 40 to 160, and the second vaccine dose boosted the titer to more than 1280. Furthermore, all animals tested positive for neutralizing antibodies at day 328 postvaccination. ELISA analysis using the recombinant nucleocapsid protein as a negative marker antigen indicated that the vaccine candidate is DIVA (differentiating infected from vaccinated animals) compatible and represents a promising vaccine platform for RVFV infection in susceptible species.
Previous phylogenetic analyses based on bluetongue virus (BTV) gene segment L3, which encodes the... more Previous phylogenetic analyses based on bluetongue virus (BTV) gene segment L3, which encodes the inner core protein, VP3, indicated a geographical distribution of different genotypes. The inner core protein, VP7, of BTV has been identified as a viral attachment protein for insect cell infection. Because the inner core proteins are involved with infectivity of insect cells, we hypothesized that certain VP7 protein sequences are preferred by the insect vector species present in specific geographic locations. We compared the gene segment S7, which encodes VP7, from 39 strains of BTV isolated from Central America, the Caribbean Basin, the United States, South Africa and Australia. For comparison, the S7 sequences from strains of the related orbiviruses, epizootic hemorrhagic disease virus (EHDV) and African horse sickness virus (AHSV) were included. The S7 gene was highly conserved among BTV strains and fairly conserved among the other orbiviruses examined. VP7 sequence alignment suggests that the BTV receptor-binding site in the insect is also conserved. Phylogenetic analyses revealed that the BTV S7 nucleotide sequences do not unequivocally display geographic distribution. The BTV strains can be separated into five clades based on the deduced VP7 amino acid sequence alignment and phylogeny but evidence for preferential selection by available gnat species for a particular VP7 clade is inconclusive. Differences between clades indicate allowable variation of the VP7 binding protein.
The Rift Valley fever virus (RVFV) encodes the structural proteins nucleoprotein (N), aminotermin... more The Rift Valley fever virus (RVFV) encodes the structural proteins nucleoprotein (N), aminoterminal glycoprotein (Gn), carboxyterminal glycoprotein (Gc), and L protein, 78-kD, and the nonstructural proteins NSm and NSs. Using the baculovirus system, we expressed the full-length coding sequence of N, NSs, NSm, Gc, and the ectodomain of the coding sequence of the Gn glycoprotein derived from the virulent strain of RVFV ZH548. Western blot analysis using anti-His antibodies and monoclonal antibodies against Gn and N confirmed expression of the recombinant proteins, and in vitro biochemical analysis showed that the two glycoproteins, Gn and Gc, were expressed in glycosylated form. Immunoreactivity profiles of the recombinant proteins in western blot and in indirect enzyme-linked immunosorbent assay against a panel of antisera obtained from vaccinated or wild type (RVFV)-challenged sheep confirmed the results obtained with anti-His antibodies and demonstrated the suitability of the baculo-expressed antigens for diagnostic assays. In addition, these recombinant proteins could be valuable for the development of diagnostic methods that differentiate infected from vaccinated animals (DIVA).
Epizootic hemorrhagic disease virus (EHDV) is a Culicoides-transmitted orbivirus that infects dom... more Epizootic hemorrhagic disease virus (EHDV) is a Culicoides-transmitted orbivirus that infects domestic and wild ruminants and is provisionally thought to be distributed throughout Africa, North America, Australia, East Asia and the Middle East. Historically, of the seven proposed serotypes of EHDV, only EHDV-1 and EHDV-2 have been reported from North America. In 2006, EHDV isolates were recovered from moribund or dead white-tailed deer (Odocoileus virginianus) in Indiana and Illinois that could not be identified as either EHDV-1 or EHDV-2 by virus neutralization tests or by serotype-specific RT-PCR. Additional serological and genetic testing identified the isolates as EHDV-6, a serotype that, although originally described from Australia, has recently been recognized as an emerging pathogen of cattle in Morocco, Algeria and Turkey. In 2007 and 2008, EHDV-6 was isolated again from white-tailed deer, this time in Missouri, Kansas and Texas, suggesting that the virus is capable of overwintering and that it may become, or already is, endemic in a geographically widespread region of the USA. Genetic characterization of the virus indicates that it is a reassortant, such that the outer capsid proteins determining serotype specificity (VP2 and VP5) are derived from exotic EHDV-6, whilst the remaining structural and non-structural proteins are apparently obtained from indigenous EHDV-2 (Alberta).
Bluetongue (BT) is an insect-transmitted, economically important disease of domestic and wild rum... more Bluetongue (BT) is an insect-transmitted, economically important disease of domestic and wild ruminants. Although only five of the 26 reported bluetongue virus (BTV) serotypes are considered endemic to the USA, 10 exotic serotypes have been isolated primarily in the southeastern region of the country since 1999. For an exotic BTV serotype to become endemic there must be susceptible animal species and competent vectors. In the USA, sheep and white-tailed deer (WTD) are the primary sentinel livestock and wildlife species, respectively. In 2006, BTV-8 was introduced into Northern Europe and subsequently overwintered, causing unprecedented livestock disease and mortality during the 2006– 2007 vector seasons. To assess the risk of the European strain of BTV-8 to North American WTD, and understand the role they could play after a similar introduction, eight bluetongue-seronegative WTD were inoculated with BTV-8. Body temperatures and clinical signs were recorded daily. Blood samples were ...
A cell line was established from Culex tarsalis Coquillett embryonated eggs and designated as CxT... more A cell line was established from Culex tarsalis Coquillett embryonated eggs and designated as CxTr. The cell line is heterogeneous, composed predominantly of small, round cells, and spindle-shaped cells with a doubling time of approximately 52–60 h. The identity of the cell line was verified as Cx. tarsalis by sequencing of cytochrome oxidase I and the cells were found to be free of contaminating cells, bacteria, fungi, and mycoplasma. The permissiveness of CxTr cells to arbovirus infection was investigated with vaccine and wildtype arboviruses from four viral families: Flaviviridae (Japanese encephalitis virus), Phenuiviridae (Rift Valley fever phlebovirus), Rhabdoviridae (vesicular stomatitis virus), and Togaviridae (Mayaro virus). All viruses were able to infect and replicate within CxTr cells.
Recent Orbivirus occurrences in the Americas have been investigated using whole genome amplificat... more Recent Orbivirus occurrences in the Americas have been investigated using whole genome amplification and sequencing followed by phylogenetic analysis. The bluetongue virus (BTV) and epizootic haemorrhagic disease virus (EHDV) whole genomes were amplified without prior sequence knowledge and deep sequenced. This technology was applied to evaluate BTV‑3 isolates spanning 4 decades from Florida, Arkansas, Mississippi, South Dakota, Central America, and the Caribbean basin. The results of the dataset analysis are consistent with the hypothesis that these viruses were introduced into the United States from Central America and the Caribbean basin. A similar analysis has been performed on a recent BTV‑2 isolate from California. It indicates that the BTV‑2 strain was likely introduced into Florida and then moved South to the Caribbean and West to California. A historical (1955‑2012) molecular characterisation of EHDV strains was also completed, and subsequently used as reference sequence for comparison of genomes from recent 2012 cattle isolates associated with clinical disease. Finally, this analysis was performed on BTV‑11 isolated from 2 canine cases and demonstrated that the genome sequences of the virus isolates from these cases were almost identical. These studies indicate the value of this technology in understanding virus epidemiology and ecology.
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Papers by William Wilson