Progress 02/01/15 to 01/31/20
Outputs
Target Audience:
Nothing Reported
Changes/Problems:2015 The tragic death of the original UK PI, Dr. Richard Elliott, inevitably had an impact on the collaborative progress of the project. The well-qualified Dr. Alain Kohl has been appointed as the new UK PI. The USDA permitting process to ship viruses from the UK to the US was obtained relatively quickly but amendments to the permit to enable research in specific rooms at the Biosecurity Research Institute are taking much longer than expected and we are awaiting inspections of two rooms. Permits related to research in the UK are also taking longer than anticipated. 2016 United States In March 2016, the Higgs/Vanlandingham laboratories at Kansas State University's Biosecurity Research Institute successful passed the USDA/APHIS inspection. Researcher access to previuosly held viruses was granted in April 2016 after approval of permit amendments enabling objective 1 work to begin. United Kingdom Dr. James Dunlop joined the project in early 2016. A short transition time occurred while Dr. Dunlop interpreted the previous results generated by Dr. Aitor Navaro. One technicial challenge was the inability to amplify and sequence from viral RNA the large genomic segment of Cache Valley virus. The solution employed deep sequencing and DNA synthesis to generate a DNA copy of this genomic segment in order to progress with the reverse genetics development. 2017 Originally it was proposed to carry out Objective 2 with Schmallenberg virus but a NSs/NSm-deletant vaccine has recently been published by others (Kraatz et al, J Virol 2015) and we decided to switch to CVV instead given its importance as human and animal pathogens in North and Central America. The United States PIs have recently obtained a no-cost extension of the grant until January 31, 2019 due to administrative delays to the project that held back the start of experiments. 2018 United States The U.S. research team received a second no-cost extension for the project in October 2018 due to delays with permits. The new end date is 01/31/2020. United Kingdom Mosquito work could not be undertaken due to time constraints. UK portion of the project ended 06/30/2018. What opportunities for training and professional development has the project provided?2015 United Kingdom Virologist post doc Aitor Navarro has a solid background in viruses but until now arboviruses were not his field of study. He is being mentored by UK PI Dr. Alain Kohl and every laboratory meeting and seminar have been a source of knowledge that helps him in the development of the project. Dr. Navarro attended the 2015 International Meeting on Arboviruses and their Vectors held at the University of Glasgow (September 7-8, 2015). 2016 United Kingdom James Dunlop started working of the project in early 2016. Dr. Dunlop has a BSc (Hons) degree in Biochemistry from Glasgow and a PhD in molecular immunology from Dundee. His last post-doctoral position was for 2 years researching the Hepatitis C virus; and previous Post-doctoral research topics also include molecular immunology and neuroscience. Dr. Dunlop attended a small focused meeting at St. Andrews University titled "Within host RNA virus persistence: mechanisms and consequences". He learned a great deal of background knowledge and novel techniques from this informal international meeting in a series of presentations over 3 days by principal investigators. 2017 United States Yan-Jang Scott Huang, Research Assistant Professor at Kansas State University, attended and presented at the American Society for Virology - 2017 Annual Meeting in June. Victoria Ayers, PhD Graduate Student at Kansas State University, attended and presented a poster at both the American Society for Virology - 2017 Annual Meeting in June and the 16th Annual Great Plains Infectious Disease Meeting in November. Amy Lyons, MS Graduate Student at Kansas State University, attended and presented a poster at the 16th Annual Great Plains Infectious Disease Meeting in November. Christopher Clarkston, Early Admission Student to the College of Veterinary Medicine at Kansas State University, is involved in rearing mosquitoes for this project. 2018 United States Five DVM and graduate students have been involved in this project. They have learned techniques and procedures related to virology, entomology, and livestock research. Victoria Ayers, PhD Graduate Student at Kansas State University, presented a poster at the College of Veterinary Medicine Phi Zeta Research Day titled "Culicoides sonorensis Susceptible to Schmallenberg Virus." March 2018. 2019 United States Four DVM and graduate students have been involved in this project. They have learned techniques and procedures related to virology, entomology, and livestock research. How have the results been disseminated to communities of interest?2017 "Identification of Multiple Susceptible Vector Species for Cache Valley Virus" was presented at the 2nd International Meeting on Arboviruses and their Vectors in Glasgow, United Kingdom, September 7-8, 2017. 2018 Higgs, S. Presentation. NIFA's US-UK Collaboration to Control Viral Threats to Livestock. 122nd USAHA Annual Meeting, USDA Research Reviews, New Horizons in Zoonotic and Emerging Diseases. October 21, 2018. Sheraton Crown Center Hotel, Kansas City, Missouri. Ayers, V. B., Huang, Y-J. S., Lyons, A. C., Holderman, C. J., Unlu, I., Alto, B. W., Kohl, A., Higgs, S., Vanlandingham. D. L. Presentation. Determining the susceptibility of Culex Species Mosquitoes to Cache Valley Virus. Regional Infectious Disease Workshop. October 23-24, 2018. Lied Lodge & Conference Center, Nebraska City, Nebraska. Higgs, S., Kohl, A., Dunlop, J.I., Huang, Y-J. S., and Vanlandingham, D. L. Poster. US-UK Collaborative Control of Emerging Bunyaviruses. USDA AFRI Animal Health and Well-being Annual Project Director Meeting at the "Conference of Research Workers in Animal Diseases (CRWAD)" December 2-4, 2018. Chicago, Illinois. 2019 United States Vanlandingham, D.L. US-UK Collaborative Control of Emerging Bunyaviruses. Center of Excellence for Emerging and Zoonotic Animal Disease (CEEZAD) 2019 Biological Safety Level 3 (BSL-3) Training Program. June 13, 2019. Manhattan, Kansas. Higgs, S., Kohl, A., Dunlop, J.I., Huang, Y-J. S., and Vanlandingham, D. L. US-UK Collaborative Control of Emerging Bunyaviruses. USDA AFRI Animal Health and Well-being Annual Project Director Meeting at the "Conference of Research Workers in Animal Diseases (CRWAD)." November 2-5, 2019. Chicago, Illinois. Ayers, V. B., et al, Huang, Y-J. S., Vanlandingham, D. L., Higgs, S. Safety and Immunogenicity of a Recombinant Rift Valley fever virus Vaccine Candidate Containing a Modified Two-Segment Genome. American Society of Tropical Medicine and Hygiene 68th Annual Meeting. November 20-24, 2019. Gaylord National Resort and Convention Center. National Harbor, Maryland. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
Current - 2019 Objective 2 United States Evaluation of the Rift Valley fever virus (RVFV) recombinant vaccine candidate focused on safety and immunogenicity testing by immunization of mice and sheep. The protective efficacy was demonstrated by comparative pathology studies in immunologically naïve and immunized sheep experimentally challenged with the virulent ZH501 strain. The RVFV vaccine was produced by deleting the two virulence factors, NSm and NSs genes, and by combining the coding regions for Gn, Gc and nucleocapsid (N) proteins in a single genomic segment. The recombinant virus (rbRVFV) contains a bi-segmented genome and retained the attenuated phenotypes in vitro. To optimize dosage to elicit neutralizing antibody responses in vertebrate hosts, a pilot immunization study consisting of two immunization regimens was conducted in CD-1 mice. The quantity of neutralizing antibodies in serum samples collected between 20 and 42 days post-immunization was determined using plaque reduction neutralization test (PRNT) in Vero76 cells. The first group of CD-1 mice received a single immunization at 103, 104, or 105 plaque forming unit (p.f.u.) of rbRVFV vaccine through subcutaneous injection. A two-dosage immunization regimen was evaluated in the second group, which received 103, 104, or 105 p.f.u. of rbRVFV followed by a booster dose of 105 p.f.u. All vaccinated mice produced neutralizing antibodies without demonstrable adverse effects. The two-dose regimen delivering 105 p.f.u. at the initial immunization and 105 p.f.u. booster induced serum neutralizing responses consistently above the immune protection threshold of 1:10 PRNT50 titer in CD1 mice and so was selected as the regimen for further study. To evaluate the safety and immunogenicity of rbRVFV vaccine in large animals, fourteen adult sheep were enrolled in the immunization and challenge experiment. Seven adult sheep were vaccinated twice at 105 p.f.u. while the other seven control group animals received sterile cell media. No adverse events were observed in vaccinated and control animals. At 35 days, animals were challenged with 106 p.f.u. of the virulent RVFV ZH501 strain. The immunization with rbRVFV vaccine induced neutralizing antibody responses in all seven sheep. The geometric mean of PRNT50 titer among vaccinated sheep was 44.2 and 44.2 at 14 and 21 days post-immunization. The comparison of viremic titers and tissue viral loads between immunologically naïve and immunized animals, which received the experimental challenge with 106 p.f.u. of ZH501 strain, demonstrated the protective efficacy of the rbRVFV vaccine. In vaccinated animals, the onset of viremia was delayed and the maximal titer was reduced in comparison with control animals. The viremic titer reached the highest level among all naïve sheep at one day post infection (geometric mean titer at 4.5*103 p.f.u./ml) while all but one vaccinated animal develop viremia (1.0*101 p.f.u./ml). At three days post infection, infectious viruses were detected in three vaccinated animals (geometric mean titer at 1.3*101 p.f.u./ml) but remained lower than the viremic titer among naïve animals (geometric mean titer at 4.3*101 p.f.u./ml). Infectious viruses were detected in the liver (1.2*103 p.f.u./ml), kidney (3.0*102 p.f.u.), and spleen (8.0*101 p.f.u./ml) of one naïve animal at seven days post infection, consistent with the viscerotropism reported in RVFV infections in vertebrate hosts. None of the immunized animals had detected viral load in any tissues. In conclusion, the rbRVFV vaccine candidate is safe and immunogenic in both mice and sheep. A two-dose immunization regimen elicited neutralizing antibody responses and protected sheep against the experimental challenge against the virulent ZH501 strain. Previous In 2015, a material transfer agreement was finalized and the following viruses were shipped from the U.K. to the U.S.: Akabane virus (AKAV), Cache Valley virus (CVV), Kairi virus (KRIV), Schmallenberg virus (SBV), and Rift Valley fever virus (RVFV) (MP12). Also, several mosquito species were colonized in the laboratory. In the U.K., the first step of the project characterized several arboviruses, beginning with CVV and KRIV, and a reverse genetics system for these viruses was developed that allowed production of attenuated viruses for the vaccine platform. Cloning of a plasmid vector containing a mutated version of the non-structural protein (NSs protein) involved in counteracting the immune system, was completed. This approach was used to produce an attenuated virus candidate for the vaccine platform. In 2016, CVV was propagated in Vero76 cells and used for oral infection of Culex tarsalis and Culex pipiens mosquitoes. Our results demonstrated that CVV infected Cx. tarsalis, but Cx. pipiens was completely refractory to CVV indicating it is less likely to be involved in the transmission of CVV in nature. The research group received approval for IACUC protocol titled "Evaluation of an experimental live-attenuated vaccine candidate for Schmallenberg virus in sheep". In the U.K., the first step of the project involved the characterization of several arboviruses, beginning with CVV and KRIV. Sequencing for all genomic segments of both viruses was completed (deposited in Genbank) which enabled us to design specific antibodies to both viruses. Initial testing suggested that these antibodies could have useful diagnostic and laboratory applications. Reverse genetics for KRIV and CVV was completed and growth characteristics in a large variety of cell lines was performed. Between June and November of 2017, our group conducted multiple oral challenge experiments of Culicoides sonorensis with SBV. The results demonstrated that Cs. sonorensis in the United States are susceptible to the oral infection of SBV and could potentially be a competent vector in the event of SBV introduction. In the U.K., while reverse genetics were available for AKAV and SBV, they had to be produced de novo for CVV and KRIV. Both CVV and KRIV delNSs viruses were shown to be potent inducers of type I interferon responses and thus potential vaccine candidates. Reassortment viruses produced by reverse genetics were characterized in vitro. Chimeric viruses in which parts of the glycoproteins were exchanged were designed. Work carried out by the Kohl group and colleagues laid the groundwork for RNAi studies in insects with RVFV. A two segmented RVFV had already been produced and they developed an NSs and NSm deletant CVV. Initial infections of sheep with CVV (delNSs, delNSm) vaccine candidate were therefore planned in the U.S. for 2018 with determination of anti-CVV antibody responses (ELISA, neutralization assays). Challenge experiments were planned using needle and vector transmission infection. In 2018, further mosquito infection studies were carried out with CVV in preparation for infections of sheep by vector feeding. Transmission of CVV by Ae. albopictus under laboratory conditions demonstrated the feasibility of using infected mosquitoes for transmission studies. Evaluation of a vaccine candidate for CVV was conducted in sheep from July to September. Animals received the immunization of 103 tissue culture infectious dose (TCID50) of the CVV-delNSm-delNSs mutant. Challenge was performed through needle inoculation or by allowing the feeding of infected Cx. tarsalis mosquitoes on individual animals. Both IgM and IgG antibodies reactive to CVV were detected by immunofluorescence in almost all vaccinated sheep, compared to the absence of antibodies in the sheep not vaccinated. Due to the weak presence of antibodies, further studies will be done in order to determine if the titer of the vaccine used was not strong enough to show significant results.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2018
Citation:
United States
Ayers, V. B., Huang, Y-J.S., Lyons, A.C., Park, S.L., Higgs, S., Dunlop, J.I., Kohl, A., Alto, B.W., Unlu, I., Blitvich, B.J., and
Vanlandingham, D.L. 2018. Culex tarsalis is a competent vector species for Cache Valley virus. Parasit Vectors. Sep
20;11(1):519. PMCID: PMC6149065. DOI: 10.1186/s13071-018-3103-2.
- Type:
Journal Articles
Status:
Published
Year Published:
2018
Citation:
United Kingdom
Dunlop, J.I., Szemiel, A.M., Navarro, A., Wilkie, G.S., Tong, L., Modha, S., Mair, D., Sreenu, V.B., Da Silva Filipe, A., Li, P., Huang, Y-J.S., Brennan, B., Hughes, J., Vanlandingham, D.L., Higgs, S., Elliott, R.M., and Kohl, A. 2018. Development of reverse genetics systems and investigation of host response antagonism and reassortment potential for Cache Valley and Kairi viruses, two emerging orthobunyaviruses of the Americas. PLoS Negl Trop Dis. 12(10): e0006884. https://doi.org/10.1371/journal.pntd.0006884.
- Type:
Journal Articles
Status:
Published
Year Published:
2019
Citation:
United States
Ayers, V. B., Huang, Y-J.S., Lyons, A.C., Park, S.L., Dunlop, J.I., Unlu, I., Kohl, A., Higgs, S., Blitvich, B.J., and Vanlandingham, D.L.** 2019. Infection and transmission of Cache Valley virus by Aedes albopictus and Aedes aegypti mosquitoes. Parasit Vectors. DOI: 10.1186/s13071-019-3643-0.
|
Progress 02/01/15 to 01/27/20
Outputs
Target Audience:
Nothing Reported
Changes/Problems:2015 The tragic death of the original UK PI, Dr. Richard Elliott, inevitably had an impact on the collaborative progress of the project. The well-qualified Dr. Alain Kohl has been appointed as the new UK PI. The USDA permitting process to ship viruses from the UK to the US was obtained relatively quickly but amendments to the permit to enable research in specific rooms at the Biosecurity Research Institute are taking much longer than expected and we are awaiting inspections of two rooms. Permits related to research in the UK are also taking longer than anticipated. 2016 United States In March 2016, the Higgs/Vanlandingham laboratories at Kansas State University's Biosecurity Research Institute successful passed the USDA/APHIS inspection. Researcher access to previuosly held viruses was granted in April 2016 after approval of permit amendments enabling objective 1 work to begin. United Kingdom Dr. James Dunlop joined the project in early 2016. A short transition time occurred while Dr. Dunlop interpreted the previous results generated by Dr. Aitor Navaro. One technicial challenge was the inability to amplify and sequence from viral RNA the large genomic segment of Cache Valley virus. The solution employed deep sequencing and DNA synthesis to generate a DNA copy of this genomic segment in order to progress with the reverse genetics development. 2017 Originally it was proposed to carry out Objective 2 with Schmallenberg virus but a NSs/NSm-deletant vaccine has recently been published by others (Kraatz et al, J Virol 2015) and we decided to switch to CVV instead given its importance as human and animal pathogens in North and Central America. The United States PIs have recently obtained a no-cost extension of the grant until January 31, 2019 due to administrative delays to the project that held back the start of experiments. 2018 United States The U.S. research team received a second no-cost extension for the project in October 2018 due to delays with permits. The new end date is 01/31/2020. United Kingdom Mosquito work could not be undertaken due to time constraints. UK portion of the project ended 06/30/2018. What opportunities for training and professional development has the project provided?2015 United Kingdom Virologist post doc Aitor Navarro has a solid background in viruses but until now arboviruses were not his field of study. He is being mentored by UK PI Dr. Alain Kohl and every laboratory meeting and seminar have been a source of knowledge that helps him in the development of the project. Dr. Navarro attended the 2015 International Meeting on Arboviruses and their Vectors held at the University of Glasgow (September 7-8, 2015). 2016 United Kingdom James Dunlop started working of the project in early 2016. Dr. Dunlop has a BSc (Hons) degree in Biochemistry from Glasgow and a PhD in molecular immunology from Dundee. His last post-doctoral position was for 2 years researching the Hepatitis C virus; and previous Post-doctoral research topics also include molecular immunology and neuroscience. Dr. Dunlop attended a small focused meeting at St. Andrews University titled "Within host RNA virus persistence: mechanisms and consequences". He learned a great deal of background knowledge and novel techniques from this informal international meeting in a series of presentations over 3 days by principal investigators. 2017 United States Yan-Jang Scott Huang, Research Assistant Professor at Kansas State University, attended and presented at the American Society for Virology - 2017 Annual Meeting in June. Victoria Ayers, PhD Graduate Student at Kansas State University, attended and presented a poster at both the American Society for Virology - 2017 Annual Meeting in June and the 16th Annual Great Plains Infectious Disease Meeting in November. Amy Lyons, MS Graduate Student at Kansas State University, attended and presented a poster at the 16th Annual Great Plains Infectious Disease Meeting in November. Christopher Clarkston, Early Admission Student to the College of Veterinary Medicine at Kansas State University, is involved in rearing mosquitoes for this project. 2018 United States Five DVM and graduate students have been involved in this project. They have learned techniques and procedures related to virology, entomology, and livestock research. Victoria Ayers, PhD Graduate Student at Kansas State University, presented a poster at the College of Veterinary Medicine Phi Zeta Research Day titled "Culicoides sonorensis Susceptible to Schmallenberg Virus." March 2018. 2019 United States Four DVM and graduate students have been involved in this project. They have learned techniques and procedures related to virology, entomology, and livestock research. How have the results been disseminated to communities of interest?2017 "Identification of Multiple Susceptible Vector Species for Cache Valley Virus" was presented at the 2nd International Meeting on Arboviruses and their Vectors in Glasgow, United Kingdom, September 7-8, 2017. 2018 Higgs, S. Presentation. NIFA's US-UK Collaboration to Control Viral Threats to Livestock. 122nd USAHA Annual Meeting, USDA Research Reviews, New Horizons in Zoonotic and Emerging Diseases. October 21, 2018. Sheraton Crown Center Hotel, Kansas City, Missouri. Ayers, V. B., Huang, Y-J. S., Lyons, A. C., Holderman, C. J., Unlu, I., Alto, B. W., Kohl, A., Higgs, S., Vanlandingham. D. L. Presentation. Determining the susceptibility of Culex Species Mosquitoes to Cache Valley Virus. Regional Infectious Disease Workshop. October 23-24, 2018. Lied Lodge & Conference Center, Nebraska City, Nebraska. Higgs, S., Kohl, A., Dunlop, J.I., Huang, Y-J. S., and Vanlandingham, D. L. Poster. US-UK Collaborative Control of Emerging Bunyaviruses. USDA AFRI Animal Health and Well-being Annual Project Director Meeting at the "Conference of Research Workers in Animal Diseases (CRWAD)" December 2-4, 2018. Chicago, Illinois. 2019 United States Vanlandingham, D.L. US-UK Collaborative Control of Emerging Bunyaviruses. Center of Excellence for Emerging and Zoonotic Animal Disease (CEEZAD) 2019 Biological Safety Level 3 (BSL-3) Training Program. June 13, 2019. Manhattan, Kansas. Higgs, S., Kohl, A., Dunlop, J.I., Huang, Y-J. S., and Vanlandingham, D. L. US-UK Collaborative Control of Emerging Bunyaviruses. USDA AFRI Animal Health and Well-being Annual Project Director Meeting at the "Conference of Research Workers in Animal Diseases (CRWAD)." November 2-5, 2019. Chicago, Illinois. Ayers, V. B., et al, Huang, Y-J. S., Vanlandingham, D. L., Higgs, S. Safety and Immunogenicity of a Recombinant Rift Valley fever virus Vaccine Candidate Containing a Modified Two-Segment Genome. American Society of Tropical Medicine and Hygiene 68th Annual Meeting. November 20-24, 2019. Gaylord National Resort and Convention Center. National Harbor, Maryland. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
Current - 2019 Objective 2 United States Evaluation of the Rift Valley fever virus (RVFV) recombinant vaccine candidate focused on safety and immunogenicity testing by immunization of mice and sheep. The protective efficacy was demonstrated by comparative pathology studies in immunologically naïve and immunized sheep experimentally challenged with the virulent ZH501 strain. The RVFV vaccine was produced by deleting the two virulence factors, NSm and NSs genes, and by combining the coding regions for Gn, Gc and nucleocapsid (N) proteins in a single genomic segment. The recombinant virus (rbRVFV) contains a bi-segmented genome and retained the attenuated phenotypes in vitro. To optimize dosage to elicit neutralizing antibody responses in vertebrate hosts, a pilot immunization study consisting of two immunization regimens was conducted in CD-1 mice. The quantity of neutralizing antibodies in serum samples collected between 20 and 42 days post-immunization was determined using plaque reduction neutralization test (PRNT) in Vero76 cells. The first group of CD-1 mice received a single immunization at 103, 104, or 105 plaque forming unit (p.f.u.) of rbRVFV vaccine through subcutaneous injection. A two-dosage immunization regimen was evaluated in the second group, which received 103, 104, or 105 p.f.u. of rbRVFV followed by a booster dose of 105 p.f.u. All vaccinated mice produced neutralizing antibodies without demonstrable adverse effects. The two-dose regimen delivering 105 p.f.u. at the initial immunization and 105 p.f.u. booster induced serum neutralizing responses consistently above the immune protection threshold of 1:10 PRNT50 titer in CD1 mice and so was selected as the regimen for further study. To evaluate the safety and immunogenicity of rbRVFV vaccine in large animals, fourteen adult sheep were enrolled in the immunization and challenge experiment. Seven adult sheep were vaccinated twice at 105 p.f.u. while the other seven control group animals received sterile cell media. No adverse events were observed in vaccinated and control animals. At 35 days, animals were challenged with 106 p.f.u. of the virulent RVFV ZH501 strain. The immunization with rbRVFV vaccine induced neutralizing antibody responses in all seven sheep. The geometric mean of PRNT50 titer among vaccinated sheep was 44.2 and 44.2 at 14 and 21 days post-immunization. The comparison of viremic titers and tissue viral loads between immunologically naïve and immunized animals, which received the experimental challenge with 106 p.f.u. of ZH501 strain, demonstrated the protective efficacy of the rbRVFV vaccine. In vaccinated animals, the onset of viremia was delayed and the maximal titer was reduced in comparison with control animals. The viremic titer reached the highest level among all naïve sheep at one day post infection (geometric mean titer at 4.5*103 p.f.u./ml) while all but one vaccinated animal develop viremia (1.0*101 p.f.u./ml). At three days post infection, infectious viruses were detected in three vaccinated animals (geometric mean titer at 1.3*101 p.f.u./ml) but remained lower than the viremic titer among naïve animals (geometric mean titer at 4.3*101 p.f.u./ml). Infectious viruses were detected in the liver (1.2*103 p.f.u./ml), kidney (3.0*102 p.f.u.), and spleen (8.0*101 p.f.u./ml) of one naïve animal at seven days post infection, consistent with the viscerotropism reported in RVFV infections in vertebrate hosts. None of the immunized animals had detected viral load in any tissues. In conclusion, the rbRVFV vaccine candidate is safe and immunogenic in both mice and sheep. A two-dose immunization regimen elicited neutralizing antibody responses and protected sheep against the experimental challenge against the virulent ZH501 strain. Previous In 2015, a material transfer agreement was finalized and the following viruses were shipped from the U.K. to the U.S.: Akabane virus (AKAV), Cache Valley virus (CVV), Kairi virus (KRIV), Schmallenberg virus (SBV), and Rift Valley fever virus (RVFV) (MP12). Also, several mosquito species were colonized in the laboratory. In the U.K., the first step of the project characterized several arboviruses, beginning with CVV and KRIV, and a reverse genetics system for these viruses was developed that allowed production of attenuated viruses for the vaccine platform. Cloning of a plasmid vector containing a mutated version of the non-structural protein (NSs protein) involved in counteracting the immune system, was completed. This approach was used to produce an attenuated virus candidate for the vaccine platform. In 2016, CVV was propagated in Vero76 cells and used for oral infection of Culex tarsalis and Culex pipiens mosquitoes. Our results demonstrated that CVV infected Cx. tarsalis, but Cx. pipiens was completely refractory to CVV indicating it is less likely to be involved in the transmission of CVV in nature. The research group received approval for IACUC protocol titled "Evaluation of an experimental live-attenuated vaccine candidate for Schmallenberg virus in sheep". In the U.K., the first step of the project involved the characterization of several arboviruses, beginning with CVV and KRIV. Sequencing for all genomic segments of both viruses was completed (deposited in Genbank) which enabled us to design specific antibodies to both viruses. Initial testing suggested that these antibodies could have useful diagnostic and laboratory applications. Reverse genetics for KRIV and CVV was completed and growth characteristics in a large variety of cell lines was performed. Between June and November of 2017, our group conducted multiple oral challenge experiments of Culicoides sonorensis with SBV. The results demonstrated that Cs. sonorensis in the United States are susceptible to the oral infection of SBV and could potentially be a competent vector in the event of SBV introduction. In the U.K., while reverse genetics were available for AKAV and SBV, they had to be produced de novo for CVV and KRIV. Both CVV and KRIV delNSs viruses were shown to be potent inducers of type I interferon responses and thus potential vaccine candidates. Reassortment viruses produced by reverse genetics were characterized in vitro. Chimeric viruses in which parts of the glycoproteins were exchanged were designed. Work carried out by the Kohl group and colleagues laid the groundwork for RNAi studies in insects with RVFV. A two segmented RVFV had already been produced and they developed an NSs and NSm deletant CVV. Initial infections of sheep with CVV (delNSs, delNSm) vaccine candidate were therefore planned in the U.S. for 2018 with determination of anti-CVV antibody responses (ELISA, neutralization assays). Challenge experiments were planned using needle and vector transmission infection. In 2018, further mosquito infection studies were carried out with CVV in preparation for infections of sheep by vector feeding. Transmission of CVV by Ae. albopictus under laboratory conditions demonstrated the feasibility of using infected mosquitoes for transmission studies. Evaluation of a vaccine candidate for CVV was conducted in sheep from July to September. Animals received the immunization of 103 tissue culture infectious dose (TCID50) of the CVV-delNSm-delNSs mutant. Challenge was performed through needle inoculation or by allowing the feeding of infected Cx. tarsalis mosquitoes on individual animals. Both IgM and IgG antibodies reactive to CVV were detected by immunofluorescence in almost all vaccinated sheep, compared to the absence of antibodies in the sheep not vaccinated. Due to the weak presence of antibodies, further studies will be done in order to determine if the titer of the vaccine used was not strong enough to show significant results.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2018
Citation:
United States
Ayers, V. B., Huang, Y-J.S., Lyons, A.C., Park, S.L., Higgs, S., Dunlop, J.I., Kohl, A., Alto, B.W., Unlu, I., Blitvich, B.J., and
Vanlandingham, D.L. 2018. Culex tarsalis is a competent vector species for Cache Valley virus. Parasit Vectors. Sep
20;11(1):519. PMCID: PMC6149065. DOI: 10.1186/s13071-018-3103-2.
- Type:
Journal Articles
Status:
Published
Year Published:
2018
Citation:
United Kingdom
Dunlop, J.I., Szemiel, A.M., Navarro, A., Wilkie, G.S., Tong, L., Modha, S., Mair, D., Sreenu, V.B., Da Silva Filipe, A., Li, P., Huang, Y-J.S., Brennan, B., Hughes, J., Vanlandingham, D.L., Higgs, S., Elliott, R.M., and Kohl, A. 2018. Development of reverse genetics systems and investigation of host response antagonism and reassortment potential for Cache Valley and Kairi viruses, two emerging orthobunyaviruses of the Americas. PLoS Negl Trop Dis. 12(10): e0006884. https://doi.org/10.1371/journal.pntd.0006884.
- Type:
Journal Articles
Status:
Published
Year Published:
2019
Citation:
United States
Ayers, V. B., Huang, Y-J.S., Lyons, A.C., Park, S.L., Dunlop, J.I., Unlu, I., Kohl, A., Higgs, S., Blitvich, B.J., and Vanlandingham, D.L.** 2019. Infection and transmission of Cache Valley virus by Aedes albopictus and Aedes aegypti mosquitoes. Parasit Vectors. DOI: 10.1186/s13071-019-3643-0.
|
Progress 02/01/18 to 01/31/19
Outputs
Target Audience:
Nothing Reported
Changes/Problems: United States The U.S. research team received a second no-cost extension for the project in October 2018 due to delays with permits. The new end date is 01/31/2020. United Kingdom Mosquito work could not be undertaken due to time constraints. UK portion of the project ended 06/30/2018. What opportunities for training and professional development has the project provided? Five DVM and graduate students have been involved in this project. They have learned techniques and procedures related to virology, entomology, and livestock research. Victoria Ayers, PhD Graduate Student at Kansas State University, presented a poster at the College of Veterinary Medicine Phi Zeta Research Day titled "Culicoides sonorensis Susceptible to Schmallenberg Virus." March 2018. How have the results been disseminated to communities of interest? Higgs, S. Presentation. NIFA's US-UK Collaboration to Control Viral Threats to Livestock. 122nd USAHA Annual Meeting, USDA Research Reviews, New Horizons in Zoonotic and Emerging Diseases. October 21, 2018. Sheraton Crown Center Hotel, Kansas City, Missouri. Ayers, V. B., Huang, Y-J. S., Lyons, A. C., Holderman, C. J., Unlu, I., Alto, B. W., Kohl, A., Higgs, S., Vanlandingham. D. L. Presentation. Determining the susceptibility of Culex Species Mosquitoes to Cache Valley Virus. Regional Infectious Disease Workshop. October 23-24, 2018. Lied Lodge & Conference Center, Nebraska City, Nebraska. Higgs, S., Kohl, A., Dunlop, J.I., Huang, Y-J. S., and Vanlandingham, D. L. Poster. US-UK Collaborative Control of Emerging Bunyaviruses. USDA AFRI Animal Health and Well-being Annual Project Director Meeting at the "Conference of Research Workers in Animal Diseases (CRWAD)" December 2-4, 2018. Chicago, Illinois. What do you plan to do during the next reporting period to accomplish the goals? To determine the safety and immunogenicity of a modified live-attenuated Rift Valley fever virus (RVFV) MP12 vaccine strain lacking the NSm and NSs genes (MP12-delNSm-delNSs), ICR mice will be immunized with serially diluted virus under two different vaccination regimens in a pilot study. The first group of animals will be vaccinated with one single immunization. The second group will receive two vaccinations. Animals will be maintained for up to 42 days. Blood will be collected weekly after the initial immunization and used for the determination of neutralizing antibody titers. A cut-off of neutralizing antibody titers above 1:10 will be used as an indicator for protective immunity. The results will be used to determine the vaccination regimen required for eliciting protective immune responses against RVFV in outbred animals. The efficacy of MP12-delNSm-delNSs vaccine strain will be evaluated using a challenge protection study in sheep. The immunization will be performed based on the results of the pilot study. Three to four-month-old sheep will be immunized followed by the challenge with the virulent ZH501 strain. Protective efficacy will be evaluated based on the difference in mortality rate, viremia, and tissue viral loads between vaccinated and immunologically naïve animals. Vector competence of Culicoides sonorensis for two orthobunyaviruses, Schmallenberg and Akabane viruses, may be performed through oral challenge. Infectious viruses in arthropod tissues will be quantified using cell culture followed by the detection of viruses or viral RNA in saliva.
Impacts
What was accomplished under these goals?
Objective 1 United States Competence studies were carried out with Cache Valley virus (CVV) in Culex tarsalis, Cx. pipiens, and Cx. quinquefasciatus. These species are relevant to North America, and importantly the data show that Cx. tarsalis can be a competent vector for CVV, with viral dissemination taking place and viral RNA detected in saliva after blood meal infection. Intriguingly, the other two mosquito species were highly refractory to CVV. This is an important result and establishes the use of these in vivo infection systems for further studies on other viruses. Vector competence of Aedes aegypti and Ae. albopictus for CVV has also been determined. The species of Ae. albopictus is of great importance for human and animal health because multiple isolates of CVV have been isolated from the species including a newly emerging genetic lineage in North America. Our preliminary results demonstrated that orally infected Ae. albopictus develop a disseminated form of infection followed by the detection of viral RNA in the saliva of infected mosquitoes. Transmission of CVV by Ae. albopictus under laboratory conditions demonstrated the feasibility of using infected mosquitoes for transmission studies. Objective 2 United States Evaluation of a vaccine candidate for CVV was conducted in sheep during July to September. Animals received the immunization of 10^3 tissue culture infectious dose (TCID50) of the CVV-delNSm-delNSs mutant. Challenge was performed through needle inoculation or by allowing the feeding of infected mosquitoes on individual animals. Serum and plasma samples were collected weekly after immunization and daily post challenge. Nasal swabs were also taken on days 0-7, 14, 21, and 28-35 to determine the shedding kinetics of CVV in vaccinated or challenged animals. During necropsy, tissue samples from various organs were collected for virus titration and isolation, and RNA extractions. Processing of serum samples from the study has been completed for neutralization test and reverse transcriptase quantitative polymerase chain reaction.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2018
Citation:
United States
Ayers, V. B., Huang, Y-J.S., Lyons, A.C., Park, S.L., Higgs, S., Dunlop, J.I., Kohl, A., Alto, B.W., Unlu, I., Blitvich, B.J., and Vanlandingham, D.L. 2018. Culex tarsalis is a competent vector species for Cache Valley virus. Parasit Vectors. Sep 20;11(1):519. PMCID: PMC6149065. DOI: 10.1186/s13071-018-3103-2.
- Type:
Journal Articles
Status:
Published
Year Published:
2018
Citation:
United Kingdom
Dunlop, J.I., Szemiel, A.M., Navarro, A., Wilkie, G.S., Tong, L., Modha, S., Mair, D., Sreenu, V.B., Da Silva Filipe, A., Li, P., Huang, Y-J.S., Brennan, B., Hughes, J., Vanlandingham, D.L., Higgs, S., Elliott, R.M., and Kohl, A. 2018. Development of reverse genetics systems and investigation of host response antagonism and reassortment potential for Cache Valley and Kairi viruses, two emerging orthobunyaviruses of the Americas. PLoS Negl Trop Dis 12(10): e0006884. https://doi.org/10.1371/journal.pntd.0006884.
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Progress 02/01/17 to 01/31/18
Outputs
Target Audience:
Nothing Reported
Changes/Problems:Originally it was proposed to carry out Objective 2 with Schmallenberg virus but a NSs/NSm-deletant vaccine has recently been published by others (Kraatz et al, J Virol 2015) and we decided to switch to CVV instead given its importance as human and animal pathogens in North and Central America. The United States PIs have recently obtained a no-cost extension of the grant until January 31, 2019 due to administrative delays to the project that held back the start of experiments. What opportunities for training and professional development has the project provided?Yan-Jang Scott Huang, Research Assistant Professor at Kansas State University, attended and presented at the American Society for Virology - 2017 Annual Meeting in June. Victoria Ayers, PhD Graduate Student at Kansas State University, attended and presented a poster at both the American Society for Virology - 2017 Annual Meeting in June and the 16th Annual Great Plains Infectious Disease Meeting in November. Amy Lyons, MS Graduate Student at Kansas State University, attended and presented a poster at the 16th Annual Great Plains Infectious Disease Meeting in November. Christopher Clarkston, Early Admission Student to the College of Veterinary Medicine at Kansas State University, is involved in rearing mosqutioes for this project. How have the results been disseminated to communities of interest?"Identification of Multiple Susceptible Vector Species for Cache Valley Virus" was presented at the 2nd International Meeting on Arboviruses and their Vectors in Glasgow, United Kingdom, September 7-8, 2017. What do you plan to do during the next reporting period to accomplish the goals?Objective 1: Determine Competence of US Vectors for Imported Bunyaviruses The vector competence of Culicoides sonorensis for Schmallenberg virus and Akabane virus will continue to be determined by isolating and detecting the presence of infectious viruses and viral genome in the saliva of infected arthropods.Chimeric viruses in which parts of glycoproteins are exchanged are currently in process of being designed. RNAi experiments in insects with mutant RVFV or 2-segment RVFV are now being planned. Objective 2: Develop a Novel Bunyavirus Vaccine Platform Preparations for CVV vaccination studies in sheep at Kansas State University are now under way and scheduled for Spring 2018.
Impacts
What was accomplished under these goals?
Objective 1: Determine Competence of US Vectors for Imported Bunyaviruses United States Between June and November of 2017, our group has conducted multiple oral challenge experiments of Culicoides sonorensis with Schamllenberg virus (SBV). Infection was performed with virus stocks generated from baby hamster kidney BHK-21 cells. Orally challenged arthropods were maintained at 28oC for 7 days under 16 hour:8 hour light:dark photoregimen and analyzed for the presence of infectious viruses with 50% tissue culture infectious dose (TCID50) based titration assay. Infection was observed in greater than 50% of arthropods in three independent experiments followed by the development of disseminated form of infection in greater than 60% of infected arthropods. The results demostrated Culicoides sonorensis in the United States are susceptible to the oral infection of SBV and can potentially become a competent vector in the event of its introduction. United Kingdom The main aim here was to assess whether US midges and mosquitoes are susceptible to bunayviruses that could be imported. These include orthobunyaviruses such as Akabane virus (AKAV), Schmallenberg virus (SBV), Kairi virus (KRIV), Cache Valley virus (CVV) as well the phlebovirus Rift Valley fever (RVFV). This includes susceptibility/transmission studies in insects. The orthobunyavirus genome consists of three RNA segments of negative polarity: L (encoding RNA dependent RNA polymerase L), M (encoding glycoprotein Gn and Gc as well as the non-structural protein NSm), and S (encoding nucleocapsid protein N and non-structural protein NSs). The non-structural proteins, especially NSs, are well characterized as a type I interferon antagonist and may be necessary for infections in vivo in mosquitoes; the role of NSm in this bunyavirus genus is less clear. 1.1 Viruses, reverse genetics, and production of virus mutants While reverse genetics were available for AKAV and SBV, they had to be produced de novo for CVV and KRIV. For this purpose, resequencing of viral RNA from infected cells was combined with available information for isolates of these viruses. L, M and S segments were cloned for both viruses, as well as expression plasmids for L and N proteins to support viral replication which can be necessary to recover recombinant virus through reverse genetics. In addition, a panel of minigenome systems, in which reporter genes such as Renilla luciferase are flanked by segment-derived non-coding 3' and 5' terminal sequences (necessary to direct replication) were developed. These are useful to study replicative processes including complementation studies between L and N proteins (both required for replication) of different viruses to see if these can function together to support replicative processes which may be important for re-assortment. Importantly, we successfully rescued both CVV and KRIV viruses by reverse genetics, as well as delNSs viruses (and also CVV delNSm). More rescue combinations are underway. Both delNSs viruses were shown to be potent inducers of type I interferon responses and thus potential vaccine candidates. Reassortment experiments by reverse genetics, in which cDNAs encoding L, M or S segments from different orthobunyaviruses are mixed to give viruses of different parental genetic backgrounds, are about to be completed, with some combinations, as expected, not producing virus and some reassortant viruses obtained. These are currently being characterized in cell culture. Chimeric viruses in which parts of the glycoproteins are exchanged are currently in the process of being designed. 1.2 Characterize innate immune responses of insect vectors to selected bunyaviruses Recent work carried out by the Kohl group and colleagues (Dietrich et al, PLoS Neglected Tropical Diseases 2017; Dietrich et al., mSphere 2017) has laid the groundwork for RNAi studies in insects with RVFV. The experiments suggested here for mutant RVFV or 2-segment RVFV are now in planning. Objective 2: Develop a Novel Bunyavirus Vaccine Platform United Kingdom With vaccines being a key tool in fighting virus infections, this aim proposes to assess attenuated orthobunyaviruses -NSs or NSm or double deletant viruses- as vaccine candidates. Important challenge experiments are to be conducted with infected arthropods to deliver viruses and thus reproduce natural arbovirus infections. 2.1 Generate attenuated bunyavirus candidates Two segmented RVFV has already been produced (Brennan et al, J Virol 2011); and as described under 1.1 we have developed NSs and NSm deletant CVV. Originally we proposed to carry out this objective with SBV but a NSs/NSm-deletant vaccine has recently been published by others (Kraatz et al, J Virol 2015) and we decided to switch to CVV instead given its importance as a human and animal pathogen in North and Central America. Preparations for vaccination studies at KSU are now under way and scheduled for Spring 2018 (see 2.3). 2.2 Reassortment potential of R2segMP12 Reassortment experiments to verify the two-segmented RVFV can reassort with the original RVFV MP12 (vaccine strain) are being planned for the end of 2017. 2.3 Evaluate immunogenicity of selected vaccine candidates in large animals and test selected vaccine candidates using needle-inoculated and vector-transmitted challenge virus As described above (2.1), initial infections of sheep with CVV (delNSs, delNSm) vaccine candidates are now being prepared for spring 2018. This will include determination of anti-CVV antibody responses (ELISA, neutralisation assays). Challenge experiments will be conducted by needle and vector transmission infection.
Publications
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Progress 02/01/16 to 01/31/17
Outputs
Target Audience:
Nothing Reported
Changes/Problems:United States In March 2016, the Higgs/Vanlandingham laboratories at Kansas State University's Biosecurity Research Institute successful passed the USDA/APHIS inspection. Researcher access to previuosly held viruses was granted in April 2016 after approval of permit amendments enabling objective 1 work to begin. United Kingdom Dr. James Dunlop joined the project in early 2016. A short transition time occurred while Dr. Dunlop interpreted the previous results generated by Dr. Aitor Navaro. One technicial challenge was the inability to amplify and sequence from viral RNA the large genomic segment of Cache Valley virus. The solution employed deep sequencing and DNA synthesis to generate a DNA copy of this genomic segment in order to progress with the reverse genetics development. What opportunities for training and professional development has the project provided?United Kingdom James Dunlop started working of the project in early 2016. Dr. Dunlop has a BSc (Hons) degree in Biochemistry from Glasgow and a PhD in molecular immunology from Dundee. His last post-doctoral position was for 2 years researching the Hepatitis C virus; and previous Post-doctoral research topics also include molecular immunology and neuroscience. Dr. Dunlop attended a small focused meeting at St. Andrews University titled "Withinhost RNA virus persistence: mechanisms and consequences". He learned a great deal of background knowledge and novel techniques from this informal international meeting in a series of presentations over 3 days by principal investigators. How have the results been disseminated to communities of interest?
Nothing Reported
What do you plan to do during the next reporting period to accomplish the goals?United States Objective 1 research will focus on testing various lines of Aedes species mosquitoes and begin Culicoides work in early 2017. Objective 2 will involve continued scheduling and planning of upcoming vaccine evaluation study. United Kingdom Objective 2 studies on re-assorted and chimeric glycoprotein viruses in 2017 will provide the foundation work for the development of the novel vaccine platform.
Impacts
What was accomplished under these goals?
United States Objective 1 - Oral infection of Cache Valley virus in medically important Culex species mosquitoes Cache valley virus (CVV) was propagated in Vero76 cells and used in the oral infection of Culex tarsalis and Culex pipiens mosquitoes. Culex tarsalis is derived from an existing colony in our collection. Culex pipiens was collected from the Ewing township in New Jersey in the summer of 2015. 8-10-day-old female mosquitoes were deprived of sugar and water for up to 48 and 24 hours prior to the feeding, respectively. Viremic blood meals were orally administered through the Hemotek feeder apparatus and cotton pledgets. Mosquitoes were collected at 7 and 14 days post-infection (d.p.i.) to determine the status of infection and dissemination based on the isolation of infectious viruses in homogenized mosquito tissues with 50% tissue culture dose infection (TCID50). Characterization of replication kinetics was performed by titration in whole mosquitoes. Oral challenge of Cx. tarsalis leads to the infection and dissemination observed at 7 and 14 d.p.i. The infection rates were comparable at 7 (81.8%, 18/22) and 14 (82.6%, 19/23) d.p.i. Dissemination into secondary tissues occurred at both time points. Whilst an increase in dissemination rate was observed between 7 (72.7%, 8/11) and 14 (100.0%, 12/12) d.p.i. there was no demonstrable difference identified through statistical analysis. Titers of homogenized whole mosquitoes were comparable at 7 (5.41 ±2.06 logTCID50/ml) and 14 (5.47± 1.07 logTCID50/ml) d.p.i. Cx. pipiens exposure to Cache Valley virus through per os infection did not lead to any established infection during the length of the experiment. There were 28 and 27 mosquitoes analyzed at 7 and 14 days post infection, respectively. No infection or dissemination was observed in Cx. pipiens. In addition to Anopheles quadrimaculatus and Culiseta inornata, a known competent vector species for CVV, our results demonstrate that CVV is able to infect Cx. tarsalis. As other reports have demonstrated their feeding behaviors do involve feeding on deer and other ruminants (Washino et al., 1983), CVV may potentially utilize it as a competent vector. Interestingly, the other medically important species, Cx. pipiens, is completely refractory to CVV indicating it is less likely to be involved in the transmission of CVV in nature. Objective 2 - Evaluate immunogenicity of selected vaccine candidates in large animals The research group has received approval for IACUC protocol titled "Evaluation of an experimental live-attenudated vaccine candidate for Schmallenberg virus in sheep". This will be the first of two vaccine candidate studies to be conducted in sheep at the Biosecurity Research Institute. References Washino RK, Tempelis CH. Mosquito host bloodmeal identification: methodology and data analysis. Annu Rev Entomol. 1983;28:179-201. United Kingdom Objective 2 - Genetic characterization and reverse genetics system development of Cache Valley and Kairi viruses The first step of the project involves the characterization of several arboviruses, beginning with Cache Valley virus (CVV) and Kairi virus (KRIV). Development of a reverse genetics system will allow us to design attenuated viruses for the vaccine platform. The reverse genetic system requires sequencing of these poorly characterized viruses since their genome sequence is not fully available in the databases. We have completed sequencing for all genomic segments of both viruses and this has enabled us to design specific antibodies to both viruses*. Initial testing of these antibodies, by western blotting, using a panel of orthobunyaviruses demonstrated that they specifically bind to a few members of the bunyamwera serogroup of which CVV and KRIV are both members. This suggests these antibodies could have useful diagnostic and laboratory applications and raises the possibility of licensing these valuable reagents. We have completed the reverse genetics for KRIV and are now studying the growth characteristics of this virus in a large variety of cell lines including mosquito, midge, and horse. This includes studies of interferon antagonism utilizing a recombinant virus containing mutations in the NSs protein. A reverse engineered virus construct containing an inactivated NSs protein can result in the formation of an attenuated virus that could form the basis of a vaccine platform. After overcoming technical difficulties with the molecular genetics of CVV, we have also recently completed the reverse genetics and are presently studying the growth characteristics of this virus in a variety of cell lines. *The previously unavailable viral genomic sequences will be deposited in Genbank
Publications
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Progress 02/01/15 to 01/31/16
Outputs
Target Audience:
Nothing Reported
Changes/Problems: The tragic death of the original UK PI, Dr. Richard Elliott, inevitably had an impact on the collaborative progress of the project. The well-qualified Dr. Alain Kohl has been appointed as the new UK PI. The USDA permitting process to ship viruses from the UK to the US was obtained relatively quickly but amendments to the permit to enable research in specific rooms at the Biosecurity Research Institute are taking much longer than expected and we are awaiting inspections of two rooms. Permits related to research in the UK are also taking longer than anticipated. What opportunities for training and professional development has the project provided?US None UK Virologist post doc Aitor Navarro has a solid background in viruses but until now arboviruses were not his field of study. He is being mentored by UK PI Dr. Alain Kohl and every laboratory meeting and seminar have been a source of knowledge that helps him in the development of the project. Dr. Navarro attended the 2015 International Meeting on Arboviruses and their Vectors held at the University of Glasgow (September 7-8, 2015). How have the results been disseminated to communities of interest?
Nothing Reported
What do you plan to do during the next reporting period to accomplish the goals?US Once we have access to the viruses, stocks will be produced and titers determined. Viruses will be presented via blood meals to established mosquito colonies to determine competence for infection and transmission. We will also assist in competence studies on the mutants and reassortant viruses developed by the UK collaborators. UK To continue with the project goals, the first step will be to fully develop a reverse genetic system for CVV and KRIV. We already have the "tools" for KRIV and we will attempt the rescue of recombinant KRIV as soon as possible. We will follow the same steps with CVV. The samples are ready to complete the sequence of the L segment using deep-sequencing; and we hope to have the results soon. Once we have developed the reverse genetic system for CVV and KRIV, we will start generating recombinant viruses with mutations in the protein (NSs) responsible for counteracting the immune system. The recombinant viruses will be characterize in mammalian and insect cells (interferon response, viral growth, etc). In the meantime, we will use wild type CVV and KRIV to characterize the immune response and we will use the minigenome system, previously developed in the laboratory for other bunyaviruses, to answer questions regarding the swapping of domains of membrane glycoproteins Gn and Gc. Regarding personal development and communication of the work, I plan to send an abstract to attend either the 35th Annual Meeting of the American Society of Virology Meeting (ASV 2016) that will be held at Virginia Tech, Blacksburg, Virginia or the 6th European Congress of Virology (ECV 2016) that will be held in Hamburg, Germany. I will apply for travel grants for both meetings.
Impacts
What was accomplished under these goals?
US Receipt of Project Viruses - A material transfer agreement has been signed by the collaborating laboratories in the UK and the USA, and the following viruses have been received from the UK to the US: Akabane virus (AKAV), Cache Valley virus (CVV), Kairi virus (KRIV), Schmallenberg virus (SBV), and Rift Valley fever virus (RVFV) (MP12). Colonization of US Arthropod Species - Several mosquito species have been colonized in the laboratory that are currently available for studies: (Species, Origin, Generations) Aedes aegypti, Puerto Rico, F>20; Culex pipiens, Anderson, California, F12; Culex pipiens, Trenton, New Jersey, F4; Culex quinquefasciatus, Valdosta, Georgia, F14; Culex quinquefasciatus, Vero Beach, Florida, F3; and Culex tarsalis, Kern, California, F>20. UK The first step of the project involves the characterization of several arboviruses, beginning with Cache Valley virus (CVV) and Kairi virus (KRIV), and the development of a reverse genetics system for these viruses that allows us to develop attenuated viruses for the vaccine platform. These viruses are poorly characterized and their genome sequence is not fully available in the databases. These sequences are needed for the development of the reverse genetic system. During this period (less than 6 months), the 3' and 5' end sequences have been determined, this has allowed us to clone the whole genome in plasmid vectors prepared to develop a reverse genetic system in mammalian cells. We have determined the complete sequence of two of the three segments of CVV and KRIV (segments S and M) and we are working towards the complete sequencing of the L segment. We have characterized the growth of CVV and KRIV in mammalian cells resulting in the expected viral growth curve with the viruses reaching the expected viral titers. As part of the development of the attenuated viruses for the vaccine platform we have designed and started the cloning of a plasmid vector containing a mutated version of the non-structural protein (NSs protein) involved in counteracting the immune system. This approach will be used to produce an attenuated virus that will be a candidate for the vaccine platform.
Publications
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