Cinnia Huang

Identification of a Kunjin/West Nile-like flavivirus in brains of patients with New York encephalitis

Molecular analysis of brains from patients of the recent New York City encephalitis outbreak reveals the presence of a flavivirus not previously described in the Americas.

First Isolation of West Nile virus from a patient with encephalitis in the United States.

West Nile virus (WNV) was isolated from a patient who developed encephalitis while undergoing treatment with CHOP (cyclophosphamide, hydroxydoxorubicin, vincristine [Oncovin], predisone) and rituximab for a non-Hodgkin B-cell lymphoma. Both standard reverse transcription–polymerase chain reaction (RT-PCR) and Taqman RT-PCR established the diagnosis of WNV infection from cerebrospinal fluid (CSF). Several whole blood samples and one serum sample underwent further testing. CSF and serum samples were negative for WNV antibody; however, all samples were positive by both RT-PCR assays. Infectious virus was recovered from a blood sample, and its identity was confirmed by using a WNV-specific immunofluorescence assay. The complete WNV genomes determined from CSF and from the virus isolate adapted from cell culture were the same. The results represent the first complete WNV genome sequence obtained directly from human CSF and the first time that infectious WNV has been recovered from a patient with encephalitis in North America.

Detection of arboviral RNA directly from mosquito homogenates by reverse-transcription - polymerase chain reaction.

Many arthropod-borne viruses (arboviruses) are important human pathogens medically. The development of an effective technique to detect the viruses by using nucleic acid amplification, such as polymerase chain reaction (PCR), improves not only clinical diagnosis but also virologic surveillance of mosquito vectors in the field. In this study, the development of an improved and simplified assay is described for detection of mosquitoes infected with eastern equine encephalitis (EEE) virus, Cache Valley (CV), and California (CAL) serogroup viruses from field-collected mosquito pools. As little as 5 microl of homogenate from mosquito pools was used in the reverse transcription (RT) reaction followed by the use of three sets of specific primers for the PCR. Positive pools were determined by finding PCR bands of the expected size for each arbovirus. The confirmation and identification of Bunyaviruses was done by sequencing the PCR product. In 1999, West Nile virus (WNV) was identified as the etiologic agent of an outbreak of human encephalitis in New York City. It is shown that this protocol is also able to detect West Nile viral RNA in a pool of 100 mosquitoes containing one infected mosquito.

Multiple-Year Experience in the Diagnosis of Viral Central Nervous System Infections with a Panel of Polymerase Chain Reaction Assays for Detection of 11 Viruses

BACKGROUND Polymerase chain reaction (PCR) is becoming more common in diagnostic laboratories. In some instances, its value has been established. In other cases, assays exist, but their beneficial use has not been determined. This article summarizes findings from 3485 patients who underwent testing over a 6-year period in our laboratory. METHODS A panel of PCR assays was used for the detection of a range of viruses associated with central nervous system (CNS) infections. PCR results were analyzed in conjunction with information about patient age and sex, the time between onset and specimen collection, and other variables. Medical chart review was conducted for 280 patients to gain diagnostic and epidemiologic insight with regard to cases of unresolved encephalitis. RESULTS A total of 498 PCR-positive samples (14.3%) were detected. Enteroviruses accounted for the largest number (360 [72.3%]) of positive PCR results, followed by herpes simplex virus (76 [15.3%]), varicella-zoster virus (29 [5.82%]), and West Nile virus (WNV) (18 [3.61%]). Of 360 patients who tested positive for enterovirus, only 46 met the Centers for Disease Control and Preventions encephalitis definition. It resulted in the greatest decrease (87.2%) in positive PCR results. Overall, the PCR positivity rate for specimens collected within 5 days after illness onset was 17.2%, compared with 8.6% for specimens collected > or =6 days after onset. CONCLUSIONS The value of PCR in the diagnosis of viral infections has been established. PCR is of lower value in the detection of WNV in CNS, compared with serological testing, but is of greater value in the detection of other arboviruses, particularly viruses in the California serogroup. Medical chart reviews indicated that apparent CNS infection resolves in approximately 50% of cases.

The S RNA genomic sequences of Inkoo, San Angelo, Serra do Navio, South River and Tahyna bunyaviruses

The complete nucleotide sequences of the small (S) genomic RNA segments of five California (CAL) serogroup bunyaviruses (two Inkoo virus strains, San Angelo virus, Serra do Navio virus, South River virus and Tahyna virus) were determined. In agreement with previously published data concerning CAL serogroup viruses, the nucleocapsid (N) and non-structural (NSs) proteins were encoded in over-lapping open reading frames (ORFs). All N protein ORFs were 708 nucleotides in length and encoded a 235 amino-acid gene product. The NSs ORFs were either 279 or 294 nucleotides in length, which would encode 92 or 97 amino-acid proteins, respectively. Comparative analysis of the nucleotide sequences and amino acids corresponding to the nucleocapsid protein resulted in a predicted relationship among these viruses that generally agreed with those determined by serology. The only exception was Inkoo virus, where comparisons based on the S RNA sequence and partial M RNA sequence suggest that this virus is more similar to Jamestown Canyon virus of the Melao complex than it is to viruses such as Tahyna and La Crosse viruses of the California encephalitis complex.

Sequence comparisons of medium RNA segment among 15 California serogroup viruses.

The complete nucleotide sequences have been determined for the M segment of 12 California (CAL) serogroup bunyaviruses. A method is described here of long reverse transcription-polymerase chain reaction (RT-PCR) that yields the full-length medium (M) RNA genomic segment. A phylogenetic tree was constructed by comparison of the open reading frames (ORFs) in the M RNA segment of 15 CAL serogroup viruses. Three distinct branches were identified and they are represented by the California encephalitis (CE), Melao (MEL), and Trivittatus (TVT) complexes. These groups correspond to those previously established by small (S) RNA genomic sequences. In addition, except for Inkoo virus, the predicted relationship among these viruses agreed with those found by serology.

Evidence that fatal human infections with La Crosse virus may be associated with a narrow range of genotypes

La Crosse (LAC) virus belongs to the California (CAL) serogroup of the genus Bunyavirus, family Bunyaviridae. It is considered one of the most important mosquito-borne pathogens in North America, especially in the upper Mid-West, where it is associated with encephalitis during the time of year when mosquitoes are active. Infections occur most frequently in children and young adults and, while most cases are resolved after a period of intense illness, a small fraction (< 1%) are fatal. At present there have only been three isolates of LAC virus from humans all made from brain tissue postmortem. The cases yielding viruses are separated chronologically by 33 years and geographically from Minnesota/Wisconsin (1960, 1978) to Missouri (1993). The M RNA sequence of the first two isolates was previously reported. The present study extends the observations to the isolate from the 1993 case and includes several mosquito isolates as well. A comparison of the M RNAs of these viruses shows that for the human isolates both nucleotide sequence and the deduced amino-acid sequence of the encoded proteins are highly conserved, showing a maximum variation of only 0.91% and 0.69%, respectively. This high degree of conservation over time and space leads to the hypothesis that human infections with this particular genotype of LAC virus are those most likely to have a fatal outcome. It is also shown that a virus with this genotype could be found circulating in mosquitoes in an area more or less intermediate between the locations of the first and second fatal cases.

Diagnosis of Jamestown Canyon Encephalitis by Polymerase Chain Reaction

In recent years, polymerase chain reaction (PCR) has been under study as a potential technique to improve the accuracy of diagnosis of suspected central nervous system viral infections. We describe a case of severe encephalitis in a previously healthy 20-year-old woman from New York who presented with headache, fever, and photophobia. Her illness was characterized by progressive worsening of her neurological status, leading to confusion, delirium, and status epilepticus. The diagnosis of Jamestown Canyon encephalitis was established by positive reverse transcriptase (RT)-PCR and nucleic acid sequencing of the band from both cerebrospinal fluid and brain tissue. The nucleotide sequence and the deduced amino acid sequence of the Jamestown Canyon virus from this patient were very similar to Jamestown Canyon virus isolates from mosquito pools in New York. This report suggests that RT-PCR assays could be important tools in the diagnostic workup of cases of encephalitis.

?Acute? West Nile Virus Encephalitis (Response to Krishnamoorthy et al.).

To the Editor: In a letter to the editor, Krishnamoorthy et al. question the diagnosis of “acute West Nile encephalitis” in our case report. We did not use the word “acute” in the paper, but the patient did in fact have an acute illness. We believe that this case report, in which West Nile virus (WNV) was isolated in cell culture, represents the best evidence for a WNV infection in a human in the United States. The diagnosis of West Nile encephalitis was based on clinical analysis (1); not everyone with the diagnosis undergoes an autopsy. In many instances, patients do recover. In our case, the patient had the clinical features of encephalitis consisting of unremitting fever associated with a rapid course of progressive confusion and lethargy followed by coma. In addition, increased depression of respiratory drive existed, pointing to brain stem involvement. We agree that the inflammatory changes in the brain were limited as compared to such changes in other reported cases of WNV; however, this limitation was attributable to the fact that the patient was both immunocompromized and neutropenic at the time of acute infection. Therefore, the usual inflammatory response cannot be expected. Even though the changes were limited, they were consistent with the histologic findings in previously published reports (1,2). The second point by Krishnamoorthy et al. represents their hypothesis about a human chronic carrier state for WNV. Although a chronic carrier state is possible, the viremic period associated with arboviral infections is typically short (3). While one cannot rule out persistent infection with WNV, until our report attempts to recover the virus by isolation in North America in humans have been uniformly unsuccessful. Also, previous reports of successful WNV isolations by Israeli investigators in immunocompetent hosts (4) have been from blood specimens before seroconversion. These considerations indicate that the virus is not routinely found in the blood in substantial amounts by the time clinical symptoms consistent with WNV infection occur. We do not know, nor have we speculated, about the timing of the infection as the patient had no recollection of a mosquito bite. Tests for both immunoglobulin (Ig) G and IgM antibodies to WNV were negative in our patient. Because the patient was immunocompromised, a humoral response was not expected; therefore, this information cannot be used as evidence that the patient had an acute infection. However, observations that the patient had no manifestation of encephalitis during a previous episode of neutropenia and that she had an acute febrile illness associated with neurologic signs of encephalitis point to an acute infection. The figure, in which WNV copy numbers are correlated with leukocyte count, is not intended to pinpoint the time of infection. However, as stated in the paper, this figure did show that the virus was rapidly cleared after resolution of neutropenia. A report by Camenga et al. (5) demonstrated that mice, infected with WNV develop only an inapparent infection. These mice will invariably die of fulminant encephalitis if only a single dose of cyclophosphamide is given. However, mice treated with one dose of cyclophosphamide demonstrate inflammatory changes in the brain. If a second dose of the drug is administered 5 days after infection, inflammation is completely suppressed in mice. Although mice are immunologically different from humans, this work, done almost 30 years ago, supports the argument for an acute infection in the current case report. If the patient in our study was a chronic carrier, she should have had manifestations of acute West Nile encephalitis immediately following the first course of combination chemotherapy, which was much more immunosuppressive than cyclophosphamide alone. This fact reemphasizes our major point in the article that patients who are immunocompromized and undergoing chemotherapy, which may cause neutropenia, should take extra precautions against being exposed to WNV.