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West Nile virus (WNV) RNA was demonstrated in 5 of 25 (20%) urine samples collected from convalescent patients 573 to 2,452 days (1.6 to 6.7 years) after WNV infection. Four of the 5 amplicons sequenced showed >99% homology to the WNV NY99 strain. These findings show that individuals with chronic symptoms following WNV infection may have persistent renal infection over several years.
West Nile virus (WNV) is an important flavivirus in North America . Since WNV was first detected in the US in 1999 , approximately 25,000 human clinically evident infections have been reported, with more than 1,000 deaths [3,4]. Less than 1% of WNV infected people develop acute neuroinvasive disease, including meningitis, encephalitis, flaccid paralysis, and death .
Evidence of persistent WNV infection has been demonstrated in experimentally infected monkeys and hamsters with virus or viral RNA found in brain tissue [6,7]. In addition, hamsters experimentally infected with WNV developed chronic renal infection and shed virus in the urine for up to 8 months [8,9]. Serial urine specimens from these animals contained 102 to 104 plaque-forming units of infectious virus/ml. Immunohistochemical staining of tissues showed no evidence of WNV antigen in brain, liver, spleen, lungs or bladder, but kidney tissue showed moderate to strong antigen staining. Infectious virus was recovered by co-cultivation of trypsinized fresh kidney cells on Vero cells . These experimental data raise the possibility that persistent renal infection may occur in humans. To date, however, WNV has been demonstrated by reverse transcriptase polymerase chain reaction (RT-PCR) only in the urine of a patient with acute WNV infection .
When WNV reached Houston in 2002, we established a longitudinal study to follow hospitalized WNV patients to determine risk factors for encephalitis and to understand long-term clinical sequelae. Methods for WNV confirmation and study inclusion criteria were previously described . Informed consent was obtained upon recruitment. This cohort now numbers 112 patients who are evaluated every 6 months, at which time blood is collected and a questionnaire ascertaining subjective and objective measurements of clinical sequelae is completed. At one year post-infection, approximately 60% of patients remain symptomatic, particularly those who were encephalitic. Resolution of symptoms plateaued around two years post-infection, and after 5 years, 60% of patients who presented with encephalitis continued to report clinical symptoms. Chronic symptoms were significantly associated with the persistence of detectable anti-WNV serum IgM (p=0.026) and also with a history of hypertension. Cytokine studies showed that many of the chronically symptomatic patients also had significantly elevated plasma levels of interferon gamma inducing protein (IP-10), a marker of active viral infection (K. Murray, unpublished data). There was also evidence of suppression of the Th2 pathway, which might be an indicator of immunosilencing processes facilitating viral persistence. Finally, the deaths of five participants were attributed to chronic renal failure. Collectively, these observations led us to hypothesize that some members of the cohort were persistently infected with WNV and that the kidney could be a preferred site of continued replication and source of shedding. Accordingly, we developed protocols for the collection and processing of urine for the detection of viral nucleic acid using RT-PCR.
Fresh urine was collected from a group of cohort participants into RNAse-free tubes that contained 3.3 units/microliter of Protector RNAse inhibitor (Roche Diagnostics, Indianapolis, IN). An aliquot was immediately extracted and RNA was isolated from convalescent urine according to the manufacturer’s protocol for the Qiagen MinElute Virus Spin Kit (Qiagen, Valencia, CA, USA). The urine was not concentrated or pre-treated prior to testing. In addition to standard positive, negative and reagent controls, we included urine from known WNV antibody-negative healthy volunteers to exclude the possibility of amplicon or cross-over contamination during the extraction procedure. Extraction procedures, reactions, and electrophoresis were performed in separate laboratories for the same reason.
Oligonucleotides used for the RT-PCR have been described [12–14]. First-stage primer sequences were 1401: 5'-ACCAACTACTGTGGAGTC-3', and 1845: 5'-TTCCATCTTCACTCTACACT-3', amplifying a 445-bp region of the WNV envelope protein . The amplification reaction was performed according to manufacturer’s protocol for the One-Step RT-PCR kit (Qiagen). An aliquot from the first round of PCR was used for the nested PCR with the Taq PCR Core kit. Nested primers used were 1485: 5'-GCCTTCATACACACTAAAG-3' and 1732: 5'-CCAATGCTATCACAGACT-3' for amplifying a 248-bp region . For both the first round and nested PCR, ten µl of each reaction was resolved on an agarose gel (1% w/v), stained with ethidium bromide and visualized with the Gel Bio-Doc-It system for the presence of amplicons. PCR cleanup (Qiagen Quickspin) was used on the first round of PCR product of those specimens which had a positive band detected, and the amplicons were sent to Lone Star Laboratories (Houston, TX) for nucleic acid sequencing.
We tested urine specimens from 25 convalescent patients. RT-PCR was positive in both the first round and nested reactions in five of 25 (20%) urine specimens collected between 573 and 2,452 days post-onset acute clinical disease (see table). Four of the five amplicons from the PCR products from the primary RT-PCR reaction could be sequenced and were found to be >99% homologous to the WNV NY99 strain.
Of the five positive patients, four reported chronic symptoms, including weakness, fatigue, memory loss, and ataxia. All had a clinical presentation of encephalitis, were male, and had a prior history of hypertension. One patient developed kidney failure following his illness. Three patients were more than six years past their initial infection.
Although we suspected viral persistence in some patients with chronic symptoms, finding one in five urine specimens to be positive was unanticipated. For further assurance we repeated the entire RT-PCR procedure in a different location to further exclude the possibility of amplicon contamination. Urine specimens that had been flash frozen in dry ice and ethanol at the time of collection were sent to the University of Texas Medical Branch in Galveston, TX. RNA was extracted using the QIAamp viral RNA mini kit (Qiagen) and then screened by non-nested RT-PCR using oligonucleotide primers targeting a different region of the WNV E gene. Nucleotide sequencing of amplified products confirmed the presence of WNV RNA in samples from two of the patients (2002-42 and 2002-43; Genbank accession numbers GQ495619 and GQ495620, respectively). We are attempting to isolate virus from urine, but thus far have not succeeded. We expected to have difficulty obtaining as isolate since the previous study that identified WNV RNA in urine from an acutely infected human patient was unable to do so . We have also found that the viral RNA degrades quickly in urine, with RT-PCR becoming negative after two or more freeze/thaw cycles. This is a concern; therefore, we are working towards optimizing the testing urine for the presence of viral RNA.
We report here for the first time that WNV is capable of long-term persistence in patients, particularly in the presence of chronic clinical symptoms. The finding of viral RNA in the urine of these patients is suggestive of ongoing viral replication in renal tissue, consistent with the hamster model. The public health impact of these findings is considerable. It will be important to explore and understand the underlying mechanisms related to the shedding of viral RNA in the urine, whether shedding is constant or intermittent, and whether or not this represents true infection resulting in clinically evident disease. Additionally, all five of our positive individuals were older males, and we are unsure at this point if this is a significant finding or simply related to chance. We are currently establishing means to clinically evaluate all cohort participants, particularly with regard to renal function and other abnormalities possibly related to persistent infection with a focus on developing treatment options.
We thank the cohort participants for their contribution to this study, as well as Jennifer Bigbee, Rebecca Bryson, Liliana Rodriguez, Blanca Restrepo, Diana Gomez, and Shaper Mirza for their assistance. This study was funded in part by the NIH (NIH/NIAID/DAIT N01 AI 50027-03 and NIH/NIAID K23 AI057341). Development of the RT-PCR was supported in part by a grant from the United States Department of Defense, Army (grant #W81XWH-04-2-0035). R. Tesh and D. Beasley were supported by NIH contract N01-A125489. This study was approved by the University of Texas Health Science Center at Houston Committee for the Protection of Human Subjects (HSC-SPH-03-039).
Competing Interests Statement: All authors declare that they have no competing financial interests.
Kristy Murray—No conflict of interest
Christopher Walker—No conflict of interest
Emily Herrington—No conflict of interest
Jessica A. Lewis—No conflict of interest
Joseph McCormick—No conflict of interest
David Beasley—No conflict of interest
Robert B. Tesh—No conflict of interest
Susan Fisher-Hoch—No conflict of interest