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1.  Genetic Analysis of West Nile Virus Isolates from an Outbreak in Idaho, United States, 2006–2007 
West Nile virus (WNV) appeared in the U.S. in 1999 and has since become endemic, with yearly summer epidemics causing tens of thousands of cases of serious disease over the past 14 years. Analysis of WNV strains isolated during the 2006–2007 epidemic seasons demonstrates that a new genetic variant had emerged coincidentally with an intense outbreak in Idaho during 2006. The isolates belonging to the new variant carry a 13 nt deletion, termed ID-Δ13, located at the variable region of the 3′UTR, and are genetically related. The analysis of deletions and insertions in the 3′UTR of two major lineages of WNV revealed the presence of conserved repeats and two indel motifs in the variable region of the 3′UTR. One human and two bird isolates from the Idaho 2006–2007 outbreaks were sequenced using Illumina technology and within-host variability was analyzed. Continued monitoring of new genetic variants is important for public health as WNV continues to evolve.
PMCID: PMC3799518  PMID: 24065039
Flavivirus; West Nile virus; genetic variation; WNV evolution; indel motifs; next generation sequencing
2.  Evolutionary Dynamics of West Nile Virus in the United States, 1999–2011: Phylogeny, Selection Pressure and Evolutionary Time-Scale Analysis 
West Nile virus (WNV), an arbovirus maintained in a bird-mosquito enzootic cycle, can infect other vertebrates including humans. WNV was first reported in the US in 1999 where, to date, three genotypes belonging to WNV lineage I have been described (NY99, WN02, SW/WN03). We report here the WNV sequences obtained from two birds, one mosquito, and 29 selected human samples acquired during the US epidemics from 2006–2011 and our examination of the evolutionary dynamics in the open-reading frame of WNV isolates reported from 1999–2011. Maximum-likelihood and Bayesian methods were used to perform the phylogenetic analyses and selection pressure analyses were conducted with the HyPhy package. Phylogenetic analysis identified human WNV isolates within the main WNV genotypes that have circulated in the US. Within genotype SW/WN03, we have identified a cluster with strains derived from blood donors and birds from Idaho and North Dakota collected during 2006–2007, termed here MW/WN06. Using different codon-based and branch-site selection models, we detected a number of codons subjected to positive pressure in WNV genes. The mean nucleotide substitution rate for WNV isolates obtained from humans was calculated to be 5.06×10−4 substitutions/site/year (s/s/y). The Bayesian skyline plot shows that after a period of high genetic variability following the introduction of WNV into the US, the WNV population appears to have reached genetic stability. The establishment of WNV in the US represents a unique opportunity to understand how an arbovirus adapts and evolves in a naïve environment. We describe a novel, well-supported cluster of WNV formed by strains collected from humans and birds from Idaho and North Dakota. Adequate genetic surveillance is essential to public health since new mutants could potentially affect viral pathogenesis, decrease performance of diagnostic assays, and negatively impact the efficacy of vaccines and the development of specific therapies.
Author Summary
West Nile Virus (WNV) is a mosquito-borne virus of African origin that is widespread around the world. The WNV life-cycle involves mosquitoes and birds, but humans and other animals can be infected, although they are not considered to be important players in the transmission cycle. Clinically, most WNV infections are unapparent, but the virus can disseminate to the central nervous system causing a potentially fatal neurological disease, especially in susceptible populations including elderly and immunocompromised individuals. West Nile virus can also be transmitted by organ transplant and by transfusion of blood and blood components. Like other arboviruses, WNV has the extraordinary capacity of growing in the different microenvironments represented by the invertebrate vector and the vertebrate hosts. From an evolutionary standpoint, the arrival of WNV in the US in 1999 represents a unique opportunity to explore the processes involved in the adaptation and dissemination of an arbovirus in a naïve environment. From the study of WNV sequences, we can not only learn about the evolutionary mechanisms that govern arboviruses, but also update diagnostic tests that rely on the detection of the viral genome upon the occurrence of mutations and study the existence of genetic markers that may be responsible for increases in clinical cases and their severity.
PMCID: PMC3667762  PMID: 23738027
3.  Development and Application of a High-Throughput Micro-Neutralization Assay - Lack of XMRV/MLV Detection in Blood Donors 
Transfusion  2012;52(2):332-342.
Xenotropic murine leukemia virus (MLV)-related virus (XMRV) and other related MLVs have been described with chronic fatigue syndrome (CFS) and certain types of prostate cancer. In addition, prevalence rates as high as 7% have been reported in blood donors, raising the risk of transfusion-related transmission. Several laboratories have utilized micro-neutralization assays as a surrogate marker for detection of anti-MLV serological responses – with up to 25% of prostate cancer patients reported to harbor neutralizing antibody responses.
We developed a high-throughput micro-neutralization assay for research studies on blood donors using retroviral vectors pseudotyped with XMRV-specific envelopes. Infection with these pseudotypes was neutralized by sera from both macaques and mice challenged with XMRV, but not pre-immune serum. 354 plasma samples from blood donors in the Reno/Tahoe area were screened for neutralization.
6.5% of donor samples gave moderate neutralization of XMRV, but not control pseudotypes. However, further testing by Western blot revealed no evidence of antibodies against MLVs in any of these samples. Furthermore, no evidence of infectious virus or viral nucleic acid was observed.
A micro-neutralization assay was developed for detection of XMRV, and can be applied in a high-throughput format for large scale studies. Although a proportion of blood donors demonstrated the ability to block XMRV envelope-mediated infection, we found no evidence that this inhibition was mediated by specific antibodies elicited by exposure to XMRV/MLV. It is likely that this moderate neutralization is mediated through another, non-specific mechanism.
PMCID: PMC3299481  PMID: 22239212
High-throughput micro-neutralization assay; XMRV; MLV; Pseudoviruses; Donor screening
4.  Absence of reproducibly detectable low-level HIV viremia in highly exposed seronegative men and women. 
AIDS (London, England)  2011;25(5):619-623.
Transient HIV infections have been invoked to account for the cellular immune responses detected in highly virus-exposed individuals who have remained HIV seronegative. We tested for very low levels of HIV RNA in 524 seronegative plasma samples from 311 highly exposed women and men from 3 longitudinal HIV cohorts.
2073 transcription mediated amplification (TMA) HIV RNA tests were performed for an average of 3.95 TMA assays per plasma sample. Quadruplicate TMA assays, analyzing a total of 2 ml of plasma, provided an estimated sensitivity of 3.5 HIV RNA copies/ml.
Four samples from subjects who did not sero-convert within the following six months were positive for HIV RNA. For one sample, human polymorphism DNA analysis indicated a sample mix up. Borderline HIV RNA detection signals were detected for the other three positive samples and further replicate TMA testing yielded no positive results. Nested PCR assays (n=254) for HIV proviral DNA on PBMC from these 3 subjects were negative.
Transient viremia was not reproducibly detected in highly HIV exposed seronegative men and women. If transient infections do occur, plasma HIV RNA levels may remain below the detection limits of the sensitive assay used here, be of very short duration, or viral replication may be restricted to mucosal surfaces or their draining lymphoid tissues.
PMCID: PMC3458706  PMID: 21297421
5.  Neuroinvasive Disease and West Nile Virus Infection, North Dakota, USA, 1999–2008 
Emerging Infectious Diseases  2012;18(4):684-686.
To determine risk for West Nile virus (WNV) neuroinvasive disease in North Dakota, we tested plasma samples from blood donors for WNV IgG and compared infection rates with reported WNV neuroinvasive disease incidence. We estimate that 1 in 244 WNV infections leads to neuroinvasive disease; risk is substantially increased among men and older persons.
PMCID: PMC3309699  PMID: 22469465
West Nile virus; viruses; epidemiology; serology; incidence; blood donors; neuroinvasive disease; meningitis; encephalitis; flaccid paralysis; arbovirus; flavivirus; North Dakota; United States; vector-borne infections

Results 1-5 (5)