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.

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.

Sequence and phylogenetic data indicate that an orthobunyavirus recently detected in the Yucatan Peninsula of Mexico is a novel reassortant of Potosi and Cache Valley viruses.

We determined the complete nucleotide sequences of the small (S) and medium (M) RNA segments of an orthobunyavirus isolated from mosquitoes in the Yucatan Peninsula of Mexico. A 528-nt region of the large (L) RNA segment was also sequenced. The S RNA segment has greatest nucleotide identity to the homologous region of Cache Valley virus (CVV; 98%) followed by Potosi virus (POTV; 89%) and Northway virus (86%). The M RNA segment has 96% nucleotide identity to the homologous region of POTV, and less than 74% nucleotide identity to the homologous regions of all other orthobunyaviruses for which M segment sequence data are available. The L RNA segment has greatest nucleotide identity to the homologous region of POTV (98%) followed by CVV (82%) and Tensaw virus (77%). These data indicate that the virus, tentatively named Cholul virus (CHLV), is a novel reassortant that acquired its S RNA segment from CVV and its M and L RNA segments from POTV. Phylogenetic data support this conclusion.

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.

Isolation and sequence analysis of Culex flavivirus from Culex interrogator and Culex quinquefasciatus in the Yucatan Peninsula of Mexico.

Previously, we reported a high prevalence of Culex flavivirus (CxFV) in Culex quinquefasciatus (Say) in the Yucatan Peninsula of Mexico. To determine whether other Culex spp. mosquitoes in this region are susceptible to natural CxFV infection, Cx. bahamensis (Dyar and Knab), Cx. coronator (Dyar and Knab), Cx. interrogator (Dyar and Knab), Cx. nigripalpus (Theobald) and Cx. opisthopus (Komp) in the Yucatan Peninsula of Mexico were tested for CxFV. 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 > or =99.2% nucleotide identity. These data suggest that there is limited genetic diversity among CxFV isolates in the Yucatan Peninsula of Mexico.

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.

Immunohistochemical staining of IFN-gamma positive cells in porcine reproductive and respiratory syndrome virus-infected lungs.

Paraffin-embedded lungs were obtained from a previous porcine reproductive and respiratory syndrome virus (PRRSV)-challenged experiment involving three groups: an uninfected control group, a low virulence (LV, Resp PRRSV/Repro)-infected group, and a high virulence (HV, VR-2385)-infected group. Tissues were collected at 3, 7, 10, 14 or 28 days post-inoculation (DPI) (n=5). Lungs were examined to detect IFN-gamma positive cells by immunohistochemical staining using polyclonal antibodies to IFN-gamma. The microscopic lung lesions induced by the HV group were more severe than those in the LV group. A significant increase in number of lymphocytes in the HV group was observed at 10 DPI (24.90+/-9.79%), 14 DPI (22.00+/-11.47%) and 28 DPI (28.95+/-15.11%) (P<0.05). A relative decrease in macrophage numbers was observed and correlated well with the increase in lymphocyte numbers when the disease progressed. IFN-gamma positive cells were demonstrated in both lymphocytes and macrophages, particularly pulmonary alveolar macrophages. A significant increase in IFN-gamma positive cells was found at 7 DPI (15.90+/-13.65%), 10 DPI (46.95+/-13.79%), 14 DPI (10.90+/-5.13%) and 28 DPI (13.40+/-4.89%) in the HV group (P<0.05). The results suggested that the increase in IFN-gamma positive cells in the HV group correlated well with the severity of the lung lesions, which may be because of the presence of PRRSV in the lung.

Orthobunyaviruses, a common cause of infection of livestock in the Yucatan peninsula of Mexico.

To determine the seroprevalence of selected orthobunyaviruses in livestock in the Yucatan Peninsula of Mexico, a serologic investigation was performed using serum samples from 256 domestic animals (182 horses, 31 sheep, 1 dog, 37 chickens, and 5 turkeys). All serum samples were examined by plaque reduction neutralization test using Cache Valley virus (CVV), Cholul virus (CHLV), South River virus (SOURV), Kairi virus, Maguari virus, and Wyeomyia virus. Of the 182 horses, 60 (33.0%) were seropositive for CHLV, 48 (26.4%) were seropositive for CVV, 1 (0.5%) was seropositive for SOURV, 60 (33.0%) had antibodies to an undetermined orthobunyavirus, and 13 (7.1%) were negative for orthobunyavirus-specific antibody. Of the 31 sheep, 6 (19.3%) were seropositive for CHLV, 3 (9.7%) were seropositive for CVV, 4 (12.9%) were seropositive for SOURV, 16 (51.6%) had antibodies to an undetermined orthobunyavirus, and 2 (6.5%) were negative for orthobunyavirus-specific antibody. The single dog was seropositive for SOURV. Four (11%) chickens had antibodies to an undetermined orthobunyavirus, and 1 (20%) turkey was seropositive for CHLV. These data indicate that orthobunyaviruses commonly infect livestock in the Yucatan Peninsula.