Characterization of the transovarial transmission potential, tissue tropisms and genetic determinants of host specificity of single-host flaviviruses

Most known flaviviruses, including West Nile virus (WNV), are maintained in natural transmission cycles between hematophagous arthropods and vertebrate hosts; thus, they are dual-host viruses. Other flaviviruses such as Modoc virus (MODV) and Culex flavivirus (CxFV) are single-host viruses because they have host ranges restricted to vertebrates and insects, respectively. Numerous insect-specific flaviviruses (ISFs) including CxFV have been discovered in the last decade and most are widely spread in nature. However, little is known about the mechanism(s) by which ISFs are maintained in nature. In a previous study, CxFV was detected in both female and male mosquitoes collected in the field suggesting that this virus is maintained in nature by vertical transmission. The experiments outlined in chapter 2 were designed to test the hypothesis that efficient transovarial transmission (TOT) of CxFV occurs in the mosquito host. CxFV RNA was detected in 526 of 540 Culex pipiens progeny derived from CxFV-infected females and thus, the filial infection rate was 97.4%. Because all positive females produced infected offspring, the TOT prevalence was 100%. These data indicated that extremely efficient TOT of CxFV occurs in mosquitoes in nature. Tissue tropisms of CxFV were also defined. CxFV RNA was detected in all tissues tested: salivary glands, ovaries, testes, head, fat bodies and midguts. Time course experiments demonstrated that CxFV disseminates to the ovaries as early as 4 days post-inoculation. In chapter 3, the host range and genetic diversity of CxFV was investigated. Previously, a high prevalence of CxFV was reported in Cx. quinquefasciatus in the Yucatan Peninsula of Mexico. To determine whether other Culex spp. mosquitoes in this region are susceptible to natural CxFV infection, five other Culex spp. mosquitoes were tested for evidence of CxFV infection. Two pools of Cx. interrogator were positive. The envelope protein genes of these isolates and 16 isolates from Cx. quinquefasciatus were sequenced and shown to have \u3e99.2% nucleotide identity. These data suggest that there is limited genetic diversity among CxFV isolates in Yucatan Peninsula of Mexico. In chapter 4, studies were performed to increase our knowledge of the genetic elements that condition the differential host ranges of flaviviruses. Although flaviviruses possess a similar genomic organization, they differ in terms of their host specificity; some flaviviruses infect both vertebrates and arthropods whereas others have a vertebrate-specific or arthropod-specific phenotype. The genetic elements that condition these differential host ranges and transmission cycles have not been identified. Therefore, chimeric viruses were constructed by replacing the capsid (C), premembrane (prM) and envelope (E) genes or the prM-E genes of MODV with the corresponding regions of WNV and CxFV. Chimeric virus was recovered in cells transfected with the fusion product containing the prM-E genes of WNV in a MODV backbone. The virus could infect vertebrate but not mosquito cells, indicating that genetic elements outside of the prM-E gene region of MODV condition its vertebrate-specific phenotype. The three other chimeras did not produce detectable virus. Comparative studies between flaviviruses that possess differential host range profiles will help us understand why some flaviviruses can infect only vertebrate or only invertebrate organisms while other flaviviruses can infect both insect and vertebrate hosts and cause devastating disease in humans and animals

Evidence of Efficient Transovarial Transmission of Culex Flavivirus by Culex pipiens (Diptera: Culicidae)

This study determined the transovarial transmission (TOT) potential and tissue tropisms of Culex flavivirus (CxFV), an insect-specific flavivirus, in Culex pipiens (L.). Several hundred mosquito egg rafts were collected in the field, transferred to the insectaries, reared to the fourth larval instar, and identified using morphological characteristics. Cx. pipiens were reared to adults, allowed to oviposit in individual containers, and tested for CxFV RNA by reverse transcription-polymerase chain reaction (RT-PCR) and nucleotide sequencing. Eighteen CxFV RNA-positive females were identified from 26 females that oviposited viable egg rafts. Thirty F1 adults from each positive female were individually tested by RT-PCR for CxFV RNA. Viral RNA was detected in 526 of 540 progeny, and thus, the filial infection rate was 97.4%. Because all 18 positive females produced infected offspring, the TOT prevalence was 100%. These data indicated that efficient TOT of CxFV occurs in nature. To define the tissue tropisms of CxFV, different tissues (salivary glands, ovaries, testes, head, fat bodies, and midguts) were removed from the remainder of the F1 and tested by RT-PCR for CxFV RNA. Viral RNA was detected in all tissues. Additionally, uninfected laboratory-colonized Cx. pipiens were infected with CxFV by needle inoculation, and ovaries were collected at 4, 6, 8, and 12 d postinoculation and tested for CxFV RNA by RT-PCR. Viral RNA was detected at all time points, demonstrating that CxFV infects the ovaries as early as 4 d postinoculation. Surprisingly, however, we were unable to demonstrate transovarial transmission despite the presence of viral RNA in the ovaries. Nevertheless, the experiments performed with field-infected Cx. pipiens demonstrate that TOT is an efficient mechanism by which CxFV is maintained in mosquitoes in nature

Substitution of the premembrane and envelope protein genes of Modoc virus with the homologous sequences of West Nile virus generates a chimeric virus that replicates in vertebrate but not mosquito cells

Background: Most known flaviviruses, including West Nile virus (WNV), are maintained in natural transmission cycles between hematophagous arthropods and vertebrate hosts. Other flaviviruses such as Modoc virus (MODV) and Culex flavivirus (CxFV) have host ranges restricted to vertebrates and insects, respectively. The genetic elements that modulate the differential host ranges and transmission cycles of these viruses have not been identified. Methods: Fusion polymerase chain reaction (PCR) was used to replace the capsid (C), premembrane (prM) and envelope (E) genes and the prM-E genes of a full-length MODV infectious cDNA clone with the corresponding regions of WNV and CxFV. Fusion products were directly transfected into baby hamster kidney-derived cells that stably express T7 RNA polymerase. At 4 days post-transfection, aliquots of each supernatant were inoculated onto vertebrate (BHK-21 and Vero) and mosquito (C6/36) cells which were then assayed for evidence of viral infection by reverse transcription-PCR, Western blot and plaque assay. Results: Chimeric virus was recovered in cells transfected with the fusion product containing the prM-E genes of WNV. The virus could infect vertebrate but not mosquito cells. The in vitro replication kinetics and yields of the chimeric virus were similar to MODV but the chimeric virus produced larger plaques. Chimeric virus was not recovered in cells transfected with any of the other fusion products. Conclusions: Our data indicate that genetic elements outside of the prM-E gene region of MODV condition its vertebrate-specific phenotype

Management Factors Associated with Operation-Level Prevalence of Antibodies to Cache Valley Virus and Other Bunyamwera Serogroup Viruses in Sheep in the United States

A cross-sectional study was performed to identify operation-level risk factors associated with prevalence of antibody to Bunyamwera (BUN) serogroup viruses in sheep in the United States. Sera were obtained from 5150 sheep in 270 operations located in 22 states (three in the west, nine central states, and 10 in the east) and tested at a dilution of 1:20 by a plaque reduction neutralization test (PRNT) using Cache Valley virus (CVV). Antibodies that neutralized CVV were identified in 1455 (28%) sheep. Animal-level seroprevalence was higher in the east (49%) than the central (17%) and western (10%) states. A convenient subset (n = 509) of sera with antibodies that neutralized CVV was titrated and further analyzed by PRNT using all six BUN serogroup viruses that occur in the United States: CVV, Lokern virus (LOKV), Main Drain virus (MDV), Northway virus (NORV), Potosi virus (POTV), and Tensaw virus (TENV). Antibodies to CVV and LOKV were identified in sheep in all three geographic regions; MDV and POTV activity was detected in the central and eastern states, NORV activity was restricted to the west, and antibodies to TENV were not detected in any sheep. Several management factors were significantly associated with the presence of antibodies to BUN serogroup viruses. For instance, sheep housed during the lambing season inside structures that contained four walls and a roof and a door closed most of the time were more likely to be seropositive than other sheep. In contrast, herded/open-range sheep were less likely to be seropositive than their counterparts. These data can be used by producers to implement strategies to reduce the likelihood of BUN serogroup virus infection and improve the health and management practices of sheep

Characterization of the transovarial transmission potential, tissue tropisms and genetic determinants of host specificity of single-host flaviviruses

Most known flaviviruses, including West Nile virus (WNV), are maintained in natural transmission cycles between hematophagous arthropods and vertebrate hosts; thus, they are dual-host viruses. Other flaviviruses such as Modoc virus (MODV) and Culex flavivirus (CxFV) are single-host viruses because they have host ranges restricted to vertebrates and insects, respectively. Numerous insect-specific flaviviruses (ISFs) including CxFV have been discovered in the last decade and most are widely spread in nature. However, little is known about the mechanism(s) by which ISFs are maintained in nature. In a previous study, CxFV was detected in both female and male mosquitoes collected in the field suggesting that this virus is maintained in nature by vertical transmission. The experiments outlined in chapter 2 were designed to test the hypothesis that efficient transovarial transmission (TOT) of CxFV occurs in the mosquito host. CxFV RNA was detected in 526 of 540 Culex pipiens progeny derived from CxFV-infected females and thus, the filial infection rate was 97.4%. Because all positive females produced infected offspring, the TOT prevalence was 100%. These data indicated that extremely efficient TOT of CxFV occurs in mosquitoes in nature. Tissue tropisms of CxFV were also defined. CxFV RNA was detected in all tissues tested: salivary glands, ovaries, testes, head, fat bodies and midguts. Time course experiments demonstrated that CxFV disseminates to the ovaries as early as 4 days post-inoculation. In chapter 3, the host range and genetic diversity of CxFV was investigated. Previously, a high prevalence of CxFV was reported in Cx. quinquefasciatus in the Yucatan Peninsula of Mexico. To determine whether other Culex spp. mosquitoes in this region are susceptible to natural CxFV infection, five other Culex spp. mosquitoes were tested for evidence of CxFV infection. Two pools of Cx. interrogator were positive. The envelope protein genes of these isolates and 16 isolates from Cx. quinquefasciatus were sequenced and shown to have >99.2% nucleotide identity. These data suggest that there is limited genetic diversity among CxFV isolates in Yucatan Peninsula of Mexico. In chapter 4, studies were performed to increase our knowledge of the genetic elements that condition the differential host ranges of flaviviruses. Although flaviviruses possess a similar genomic organization, they differ in terms of their host specificity; some flaviviruses infect both vertebrates and arthropods whereas others have a vertebrate-specific or arthropod-specific phenotype. The genetic elements that condition these differential host ranges and transmission cycles have not been identified. Therefore, chimeric viruses were constructed by replacing the capsid (C), premembrane (prM) and envelope (E) genes or the prM-E genes of MODV with the corresponding regions of WNV and CxFV. Chimeric virus was recovered in cells transfected with the fusion product containing the prM-E genes of WNV in a MODV backbone. The virus could infect vertebrate but not mosquito cells, indicating that genetic elements outside of the prM-E gene region of MODV condition its vertebrate-specific phenotype. The three other chimeras did not produce detectable virus. Comparative studies between flaviviruses that possess differential host range profiles will help us understand why some flaviviruses can infect only vertebrate or only invertebrate organisms while other flaviviruses can infect both insect and vertebrate hosts and cause devastating disease in humans and animals.</p

Evidence of Efficient Transovarial Transmission of Culex Flavivirus by Culex pipiens (Diptera: Culicidae)

This study determined the transovarial transmission (TOT) potential and tissue tropisms of Culex flavivirus (CxFV), an insect-specific flavivirus, in Culex pipiens (L.). Several hundred mosquito egg rafts were collected in the field, transferred to the insectaries, reared to the fourth larval instar, and identified using morphological characteristics. Cx. pipiens were reared to adults, allowed to oviposit in individual containers, and tested for CxFV RNA by reverse transcription-polymerase chain reaction (RT-PCR) and nucleotide sequencing. Eighteen CxFV RNA-positive females were identified from 26 females that oviposited viable egg rafts. Thirty F1 adults from each positive female were individually tested by RT-PCR for CxFV RNA. Viral RNA was detected in 526 of 540 progeny, and thus, the filial infection rate was 97.4%. Because all 18 positive females produced infected offspring, the TOT prevalence was 100%. These data indicated that efficient TOT of CxFV occurs in nature. To define the tissue tropisms of CxFV, different tissues (salivary glands, ovaries, testes, head, fat bodies, and midguts) were removed from the remainder of the F1 and tested by RT-PCR for CxFV RNA. Viral RNA was detected in all tissues. Additionally, uninfected laboratory-colonized Cx. pipiens were infected with CxFV by needle inoculation, and ovaries were collected at 4, 6, 8, and 12 d postinoculation and tested for CxFV RNA by RT-PCR. Viral RNA was detected at all time points, demonstrating that CxFV infects the ovaries as early as 4 d postinoculation. Surprisingly, however, we were unable to demonstrate transovarial transmission despite the presence of viral RNA in the ovaries. Nevertheless, the experiments performed with field-infected Cx. pipiens demonstrate that TOT is an efficient mechanism by which CxFV is maintained in mosquitoes in nature.This article is from Journal of Medical Entomology 48 (2011): 1031–1038, doi:10.1603/ME11043.</p

Substitution of the premembrane and envelope protein genes of Modoc virus with the homologous sequences of West Nile virus generates a chimeric virus that replicates in vertebrate but not mosquito cells

Background: Most known flaviviruses, including West Nile virus (WNV), are maintained in natural transmission cycles between hematophagous arthropods and vertebrate hosts. Other flaviviruses such as Modoc virus (MODV) and Culex flavivirus (CxFV) have host ranges restricted to vertebrates and insects, respectively. The genetic elements that modulate the differential host ranges and transmission cycles of these viruses have not been identified. Methods: Fusion polymerase chain reaction (PCR) was used to replace the capsid (C), premembrane (prM) and envelope (E) genes and the prM-E genes of a full-length MODV infectious cDNA clone with the corresponding regions of WNV and CxFV. Fusion products were directly transfected into baby hamster kidney-derived cells that stably express T7 RNA polymerase. At 4 days post-transfection, aliquots of each supernatant were inoculated onto vertebrate (BHK-21 and Vero) and mosquito (C6/36) cells which were then assayed for evidence of viral infection by reverse transcription-PCR, Western blot and plaque assay. Results: Chimeric virus was recovered in cells transfected with the fusion product containing the prM-E genes of WNV. The virus could infect vertebrate but not mosquito cells. The in vitro replication kinetics and yields of the chimeric virus were similar to MODV but the chimeric virus produced larger plaques. Chimeric virus was not recovered in cells transfected with any of the other fusion products. Conclusions: Our data indicate that genetic elements outside of the prM-E gene region of MODV condition its vertebrate-specific phenotype.This article is from Virology Journal 11 (2014): 150, doi: 10.1186/1743-422X-11-150. Posted with permission.</p

Orthobunyavirus Antibodies in Humans, Yucatan Peninsula, Mexico

We performed a serologic investigation to determine whether orthobunyaviruses commonly infect humans in the Yucatan Peninsula of Mexico. Orthobunyavirus-specific antibodies were detected by plaque reduction neutralization test in 146 (18%) of 823 persons tested. Further studies are needed to determine health risks for humans from this potentially deadly group of viruses

Management Factors Associated with Operation-Level Prevalence of Antibodies to Cache Valley Virus and Other Bunyamwera Serogroup Viruses in Sheep in the United States

A cross-sectional study was performed to identify operation-level risk factors associated with prevalence of antibody to Bunyamwera (BUN) serogroup viruses in sheep in the United States. Sera were obtained from 5150 sheep in 270 operations located in 22 states (three in the west, nine central states, and 10 in the east) and tested at a dilution of 1:20 by a plaque reduction neutralization test (PRNT) using Cache Valley virus (CVV). Antibodies that neutralized CVV were identified in 1455 (28%) sheep. Animal-level seroprevalence was higher in the east (49%) than the central (17%) and western (10%) states. A convenient subset (n = 509) of sera with antibodies that neutralized CVV was titrated and further analyzed by PRNT using all six BUN serogroup viruses that occur in the United States: CVV, Lokern virus (LOKV), Main Drain virus (MDV), Northway virus (NORV), Potosi virus (POTV), and Tensaw virus (TENV). Antibodies to CVV and LOKV were identified in sheep in all three geographic regions; MDV and POTV activity was detected in the central and eastern states, NORV activity was restricted to the west, and antibodies to TENV were not detected in any sheep. Several management factors were significantly associated with the presence of antibodies to BUN serogroup viruses. For instance, sheep housed during the lambing season inside structures that contained four walls and a roof and a door closed most of the time were more likely to be seropositive than other sheep. In contrast, herded/open-range sheep were less likely to be seropositive than their counterparts. These data can be used by producers to implement strategies to reduce the likelihood of BUN serogroup virus infection and improve the health and management practices of sheep.This article is published as Meyers, Matthew T., Charlie S. Bahnson, Michael Hanlon, Christine Kopral, Saengchan Srisinlapaudom, Zachary N. Cochrane, Carlene E. Sabas et al. "Management Factors Associated with Operation-Level Prevalence of Antibodies to Cache Valley Virus and Other Bunyamwera Serogroup Viruses in Sheep in the United States." Vector-Borne and Zoonotic Diseases 15, no. 11 (2015): 683-693. Doi: 10.1089/vbz.2015.1810. </p