West Nile Virus Surveillance, Guadeloupe, 2003–2004

We conducted extensive surveillance for West Nile virus infection in equines and chickens in Guadeloupe in 2003–2004. We showed a high seroprevalence in equines in 2003 related to biome, followed by a major decrease in virus circulation in 2004. No human or equine cases were reported during the study

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

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

Serologic Evidence of West Nile Virus Infection in Horses, Coahuila State, Mexico

Serum samples were obtained from 24 horses in the State of Coahuila, Mexico, in December 2002. Antibodies to West Nile virus were detected by epitope-blocking enzyme-linked immunosorbent assay and confirmed by plaque reduction neutralization test in 15 (62.5%) horses. We report the first West Nile virus activity in northern Mexico

Culex tarsalis is a competent vector species for Cache Valley virus

Background: Cache Valley virus (CVV) is a mosquito-borne orthobunyavirus endemic in North America. The virus is an important agricultural pathogen leading to abortion and embryonic lethality in ruminant species, especially sheep. The importance of CVV in human public health has recently increased because of the report of severe neurotropic diseases. However, mosquito species responsible for transmission of the virus to humans remain to be determined. In this study, vector competence of three Culex species mosquitoes of public health importance, Culex pipiens, Cx. tarsalis and Cx. quinquefasciatus, was determined in order to identify potential bridge vector species responsible for the transmission of CVV from viremic vertebrate hosts to humans. Results: Variation of susceptibility to CVV was observed among selected Culex species mosquitoes tested in this study. Per os infection resulted in the establishment of infection and dissemination in Culex tarsalis, whereas Cx. pipiens and Cx. quinquefasciatus were highly refractory to CVV. Detection of viral RNA in saliva collected from infected Cx. tarsalis provided evidence supporting its role as a competent vector. Conclusions: Our study provided further understanding of the transmission cycles of CVV and identifies Cx. tarsalis as a competent vector

Characterization of Newly Revealed Sequences in the Infectious Myonecrosis Virus Genome in \u3ci\u3eLitopenaeus vannamei\u3c/i\u3e

Infectious myonecrosis virus (IMNV) causes significant economic losses in farmed shrimp, where associated mortality in ponds can reach 70%. To explore host/pathogen interactions, a next-generation sequencing approach using lymphoid organ tissue from IMNV-infected Litopenaeus vannamei shrimp was conducted. Preliminary sequence assembly of just the virus showed that there were at least an additional 639 bp at the 5′ terminus and 23 nt at the 3′ terminus as compared with the original description of the IMNV genome (7561 nt). Northern blot and reverse transcription-PCR analysis confirmed the presence of novel sequence at both ends of the genome. Using 5′ RACE, an additional 4 nt were discovered; 3′ RACE confirmed the presence of 22 bp rather than 23 bp of sequence. Based on these data, the IMNV genome is 8226 bp in length. dsRNA was used to trigger RNA interference (RNAi) and suppress expression of the newly revealed genome sections at the 5′ end of the IMNV genome in IMNV-infected L. vannamei. An RNAi trigger targeting a 376 bp length of the 5′ UTR did not improve survival of infected shrimp. In contrast, an RNAi trigger targeting a 381 bp sequence in ORF1 improved survival to 82.2% as compared with 2.2% survival in positive control animals. These studies revealed the importance of the new genome sections to produce high-titre infection, and associated disease and mortality, in infected shrimp

Characterization of Newly Revealed Sequences in the Infectious Myonecrosis Virus Genome in \u3ci\u3eLitopenaeus vannamei\u3c/i\u3e

Infectious myonecrosis virus (IMNV) causes significant economic losses in farmed shrimp, where associated mortality in ponds can reach 70%. To explore host/pathogen interactions, a next-generation sequencing approach using lymphoid organ tissue from IMNV-infected Litopenaeus vannamei shrimp was conducted. Preliminary sequence assembly of just the virus showed that there were at least an additional 639 bp at the 5′ terminus and 23 nt at the 3′ terminus as compared with the original description of the IMNV genome (7561 nt). Northern blot and reverse transcription-PCR analysis confirmed the presence of novel sequence at both ends of the genome. Using 5′ RACE, an additional 4 nt were discovered; 3′ RACE confirmed the presence of 22 bp rather than 23 bp of sequence. Based on these data, the IMNV genome is 8226 bp in length. dsRNA was used to trigger RNA interference (RNAi) and suppress expression of the newly revealed genome sections at the 5′ end of the IMNV genome in IMNV-infected L. vannamei. An RNAi trigger targeting a 376 bp length of the 5′ UTR did not improve survival of infected shrimp. In contrast, an RNAi trigger targeting a 381 bp sequence in ORF1 improved survival to 82.2% as compared with 2.2% survival in positive control animals. These studies revealed the importance of the new genome sections to produce high-titre infection, and associated disease and mortality, in infected shrimp

Monitoring Sheep and Culicoides Midges in Montana for Evidence of Bunyamwera Serogroup Virus Infection

A serological and entomological investigation was performed to monitor for potential Bunyamwera (BUN) serogroup virus activity in Montana. To facilitate the serological investigation, sera were collected from 104 sheep in 2013 and 2014 and assayed by plaque reduction neutralization test using all six BUN serogroup viruses known to occur in the United States: Cache Valley virus (CVV), Lokern virus (LOKV), Main Drain virus (MDV), Northway virus, Potosi virus and Tensaw virus. BUN serogroup virus-specific antibodies were detected in 41 (39%) sheep. Of these, three were seropositive for MDV, one was seropositive for CVV, one was seropositive for LOKV and 36 had antibodies to an undetermined BUN serogroup virus. Additionally, 30,606 Culicoides sonorensis were collected in 2013 using Centers for Disease Control and Prevention (CDC) light traps and assayed for cytopathic virus by virus isolation in African Green Monkey kidney (Vero) cells. All midges were negative. Almost one-third of the midges were further tested by reverse transcription-polymerase chain reaction using BUN serogroup virus-reactive primers and all were negative. We provide evidence of BUN serogroup virus infection in sheep but not C. sonorensis in Montana in 2013-2014. This study also provides the first evidence of CVV, MDV and LOKV activity in Montana