Detection of the mosquito-borne flaviviruses, West Nile, Dengue, Saint Louis Encephalitis, Ilheus, Bussuquara, and Yellow Fever in free-ranging black howlers (Alouatta caraya) of Northeastern Argentina.

Several medically important mosquito-borne flaviviruses have been detected in Argentina in recent years: Dengue (DENV), St. Louis encephalitis (SLEV), West Nile (WNV) and Yellow Fever (YFV) viruses. Evidence of Bussuquara virus (BSQV) and Ilheus virus (ILHV) activity were found, but they have not been associated with human disease. Non-human primates can act as important hosts in the natural cycle of flaviviruses and serological studies can lead to improved understanding of virus circulation dynamics and host susceptibility. From July-August 2010, we conducted serological and molecular surveys in free-ranging black howlers (Alouatta caraya) captured in northeastern Argentina. We used 90% plaque-reduction neutralization tests (PRNT90) to analyze 108 serum samples for antibodies to WNV, SLEV, YFV, DENV (serotypes 1and 3), ILHV, and BSQV. Virus genome detection was performed using generic reverse transcription (RT)-nested PCR to identify flaviviruses in 51 antibody-negative animals. Seventy animals had antibodies for one or more flaviviruses for a total antibody prevalence of 64.8% (70/108). Monotypic (13/70, 19%) and heterotypic (27/70, 39%) patterns were differentiated. Specific neutralizing antibodies against WNV, SLEV, DENV-1, DENV-3, ILHV, and BSQV were found. Unexpectedly, the highest flavivirus antibody prevalence detected was to WNV with 9 (8.33%) monotypic responses. All samples tested by (RT)-nested PCR were negative for viral genome. This is the first detection of WNV-specific antibodies in black howlers from Argentina and the first report in free-ranging non-human primates from Latin-American countries. Given that no animals had specific neutralizing antibodies to YFV, our results suggest that the study population remains susceptible to YFV. Monitoring of these agents should be strengthened to detect the establishment of sylvatic cycles of flaviviruses in America and evaluate risks to wildlife and human health.

Evolutionary relationships of West Nile virus detected in mosquitoes from a migratory bird zone of Colombian Caribbean.

BACKGROUND: West Nile virus (WNV) is a member of the genus Flavivirus, and it is transmitted between Culex sp. mosquitoes and avian hosts. Equids and humans are commonly infected with WNV as dead-end hosts, and the signs and symptoms of infection range from mild illness to neurologic symptoms as encephalitis, meningitis and sometimes death. Previous phylogenetic studies have classified WNV into six genetically distinct lineages and provided valuable insight on WNV dispersal patterns within the Americas and its emergence in different geographic areas. In this study, we isolated, sequenced and genetically characterized the NS5 and envelope genes for two WNV strains detected from Northern of Colombia. Herein we describe the evolutionary relationships with representative WNV-strains isolated in a variety of epidemic outbreaks and countries, to define the phylogeographic origin and possible implications in the epidemiology of this emergent virus in Colombia. FINDINGS: Fragments of the NS5 and Envelope genes were amplified with RT-PCR and sequenced to obtain 1186-nt and 1504-nt portions, respectively. Our sequences were aligned with 46 sequences from WNV-strains collected in the U.S., Mexico and Argentina for phylogenetic reconstruction using Bayesian methods. Sequence analyses identified unique non-synonymous substitutions in the envelope gene of the WNV strains we detected, and our sequences clustered together with those from the attenuated Texas - 2002 genotype. CONCLUSIONS: A new strain closely related to attenuated strains collected in Texas during 2002 was identified from Colombia by phylogenetic analysis. This finding may explain the absence of human/equine cases of WNV-encephalitis or severe disease in Colombia and possibly other regions of South America. Follow-up studies are needed in ecosystems used by migratory birds areas and virological/entomological surveillance.

Complete genome sequences and phylogenetic analysis of two West Nile virus strains isolated from equines in Argentina in 2006 could indicate an early introduction of the virus in the Southern Cone.

The complete nucleotide sequences of two West Nile virus (WNV) strains isolated in Argentina were determined. Phylogenetic trees were constructed from the aligned nucleic acid sequences of these two strains along with other previously published complete WNV genome sequences. Phylogenetic data showed that both strains belonged to clade 1a of lineage 1 and clustered in a subclade with American strains isolated during 1999-2002. These results suggest two independent routes of introduction of WNV in Argentina and that the virus could have been circulating in Argentina for some time before being isolated.

Characterization of West Nile viruses isolated from captive American Flamingoes (Phoenicopterus ruber) in Medellin, Colombia.

Serum samples from a total of 71 healthy captive birds belonging to 18 species were collected in July of 2008 in Medellin (Colombia) and tested for flaviviruses. Eighteen of 29 samples from American Flamingoes (Phoenicopterus ruber) were positive for West Nile virus (WNV) by reverse transcription-polymerase chain reaction. Selected positive samples were serially passaged and WNV was confirmed by immunofluorescence. Two isolates (524/08, 9835/08) were characterized in vitro and in vivo. Sequence analysis revealed WNV with 16 nucleotide substitutions resulting in six amino acid changes when compared with the NY99 strain. Colombian (COL) viruses were more closely related to Louisiana isolates from 2001. When compared with attenuated strains isolated from Texas, COL isolates differed in their plaque size and temperature sensitivity phenotype. The COL viruses were pathogenic in embryonated chicken eggs and Balb/c mice.

West Nile virus from blood donors, vertebrates, and mosquitoes, Puerto Rico, 2007.

West Nile virus (WNV) was isolated from a human blood donor, a dead falcon, and mosquitoes in Puerto Rico in 2007. Phylogenetic analysis of the 4 isolates suggests a recent introduction of lineage I WNV that is closely related to WNV currently circulating in North America.

Detection of RNA from a novel West Nile-like virus and high prevalence of an insect-specific flavivirus in mosquitoes in the Yucatan Peninsula of Mexico.

As part of our ongoing surveillance efforts for West Nile virus (WNV) in the Yucatan Peninsula of Mexico, 96,687 mosquitoes collected from January through December 2007 were assayed by virus isolation in mammalian cells. Three mosquito pools caused cytopathic effect. Two isolates were orthobunyaviruses (Cache Valley virus and Kairi virus) and the identity of the third infectious agent was not determined. A subset of mosquitoes was also tested by reverse transcription-polymerase chain reaction (RT-PCR) using WNV-, flavivirus-, alphavirus-, and orthobunyavirus-specific primers. A total of 7,009 Culex quinquefasciatus in 210 pools were analyzed. Flavivirus RNA was detected in 146 (70%) pools, and all PCR products were sequenced. The nucleotide sequence of one PCR product was most closely related (71-73% identity) with homologous regions of several other flaviviruses, including WNV, St. Louis encephalitis virus, and Ilheus virus. These data suggest that a novel flavivirus (tentatively named T'Ho virus) is present in Mexico. The other 145 PCR products correspond to Culex flavivirus, an insect-specific flavivirus first isolated in Japan in 2003. Culex flavivirus was isolated in mosquito cells from approximately one in four homogenates tested. The genomic sequence of one isolate was determined. Surprisingly, heterogeneous sequences were identified at the distal end of the 5' untranslated region.

Introductions of West Nile virus strains to Mexico.

Complete genome sequencing of 22 West Nile virus isolates suggested 2 independent introductions into Mexico. A previously identified mouse-attenuated glycosylation variant was introduced into southern Mexico through the southeastern United States, while a common US genotype appears to have been introduced incrementally into northern Mexico through the southwestern United States.

Phylogenetic analysis of West Nile virus, Nuevo Leon State, Mexico.

West Nile virus RNA was detected in brain tissue from a horse that died in June 2003 in Nuevo Leon State, Mexico. Nucleotide sequencing and phylogenetic analysis of the premembrane and envelope genes showed that the virus was most closely related to West Nile virus isolates collected in Texas in 2002.