Susceptibility of neuroblastoma cells to rabies virus may be affected by passage number.

Maintaining a healthy, continuous immortalized cell line is essential for rabies laboratories that perform virus isolation assays and test for the presence of viral neutralizing antibodies. Individuals who routinely work with rabies virus, such as rabies laboratory employees, or those who may have a high potential for exposure to rabies virus, including veterinarians, should be tested for the presence of anti-rabies viral neutralizing antibodies (VNA) every 6-24 months, depending on potential exposure level. The gold standard for serum neutralization assays require the use of live rabies virus and cells that are sensitive to rabies virus infection. Additionally, virus isolation assays are routinely performed in rabies laboratories as a back-up for the direct fluorescent antibody test (dFAT). Currently there are no guidelines or publications recommending the use of low, intermediate, or high passage cell lines in rabies assays. In this study, we compared the sensitivity of intermediate, high, and extremely high passaged neuroblastomas to rabies virus using virus isolation, serum neutralization, and real time RT-PCR techniques. Additionally, cells were examined microscopically to determine changes in morphology and dissemination of rabies virus antigen between intermediate, high, and extremely high passage cells. No significant difference was found between cell passage numbers and viral susceptibility between intermediate and high passaged cells. However, extremely high passaged cells (≥1200 passages) were less susceptible to viral infection and/or produced less virus following inoculation. As a result, rabies laboratories that use viral isolation and serum neutralization assays should regularly assess cell susceptibility to ensure the integrity and repeatability of the test.

Overwintering of Rabies Virus in Silver Haired Bats (Lasionycteris noctivagans).

Silver-haired bats, (Lasionycteris noctivagans) are semi-colonial, migratory tree bats that have infrequent contact with humans. Despite the species rarity, the L. noctivagans rabies variant is the most commonly reported rabies virus variant (RABV) in domestically acquired human rabies cases in the US. Unlike big brown bats (Eptesicus fuscus) and little brown bats (Myotis lucifugus), L. noctivagans are not considered true hibernators. It is unknown if RABV can overwinter in hibernating L. noctivagans or is only maintained in members of this taxa that migrate to warmer climates. To better understand RABV overwintering in this species, L. noctivagans were inoculated intramuscularly with either a homologous RABV (L. noctivagans Virus 1) or one of two heterologous RABV (Eptesicus fuscus Virus 2 and Myotis lucifugus Virus 1). Five days following inoculation, L. noctivagans were placed in a hibernation chamber for 6 weeks. Our results demonstrate that rabies virus can overwinter in L. noctivagans yet the incubation period was extended 6 weeks when compared to bats maintained at ambient temperatures. Additionally, we found that the longer the incubation period, the greater the viral dissemination to the salivary glands. Similar to our previous studies, L. noctivagans were most susceptible to a homologous variant. In summary, we found that RABV incubation is extended following a subcutaneous exposure or maintenance in hibernation and longer incubation times increase dissemination and potential for transmission.

Comparison of Automated Quantitative Reverse Transcription-PCR and Direct Fluorescent-Antibody Detection for Routine Rabies Diagnosis in the United States.

Rabies virus found worldwide and prevalent throughout the United States continues to be a public health concern. Direct-fluorescent antibody (DFA) detection remains the gold standard for rabies virus diagnostics. Assessing the utility of a high-throughput molecular platform such as the QIAsymphony SP/AS, in conjunction with quantitative reverse transcription-PCR (qRT-PCR), to augment or potentially replace the DFA test, was the focus of this project. Here we describe a triplex qRT-PCR assay, including assembly and evaluation for sensitivity, specificity, and ability to detect variants. Additionally, we compared the qRT-PCR assay to the gold standard direct fluorescent-antibody test. More than 1,000 specimens submitted for routine rabies diagnosis were tested to directly compare the two methods. All results were in agreement between the two methods, with one additional specimen detected by qRT-PCR below the limits of the DFA sensitivity. With the proper continued validation for variant detection, molecular methods have a place in routine rabies diagnostics within the United States.

Extended incubation period of rabies virus in a captive big brown bat (Eptesicus fuscus).

Rabies virus incubation in bats is typically less than 180 days, yet longer incubation periods have been described. We report a 267-day incubation in a big brown bat (Eptesicus fuscus) exposed to rabies virus before entering our captive colony.

Molecular detection of viral causes of encephalitis and meningitis in New York State.

The etiology of encephalitis and meningitis, serious diseases of the central nervous system (CNS), in most cases remains unknown. The importance of establishing a diagnosis however, becomes even more important as advances are made in effective therapy. Molecular methods of detection, in particular, PCR, are being used routinely and have established a place in the arsenal of tools for diagnosis of CNS infections. In this study a viral etiological agent was detected by PCR in 340 of the total 2,357 specimens from patients who exhibited symptoms of encephalitis or meningitis. The detection rate increased from 8.9% during the first year of the study to 14.8% during the second year of the study with improved methodology and an expanded panel of viral agents. Methods were enhanced by developing real-time PCR assays (some multiplexed), using increased automation, superior nucleic acid extraction, and reverse transcription (RT) methods, and incorporation of an internal extraction control. Additionally, adenovirus and human herpes virus 6 (HHV-6) were added to the original panel of 10 viruses that included enteroviruses, herpesviruses, and arboviruses. The most common viruses detected were enteroviruses (129; 5.5%), Epstein-Barr virus (EBV) (85; 3.6%), herpes simplex viruses (HSVs) 1 and 2 (67; 2.8%), and varicella zoster virus (VZV) (44; 1.9%).

Fatal case of deer tick virus encephalitis.

Deer tick virus is related to Powassan virus, a tickborne encephalitis virus. A 62-year-old man presented with a meningoencephalitis syndrome and eventually died. Analyses of tissue samples obtained during surgery and at autopsy revealed a widespread necrotizing meningoencephalitis. Nucleic acid was extracted from formalin-fixed tissue, and the presence of deer tick virus was verified on a flavivirus-specific polymerase-chain-reaction (PCR) assay, followed by sequence confirmation. Immunohistochemical analysis with antisera specific for deer tick virus identified numerous immunoreactive neurons, with prominent involvement of large neurons in the brain stem, cerebellum, basal ganglia, thalamus, and spinal cord. This case demonstrates that deer tick virus can be a cause of fatal encephalitis.

Development of human patient simulation programs: achieving big results with a small budget.

The benefits of simulation in nursing education are well documented. Nursing students learn in a safe environment that enhances critical thinking and collaboration. Barriers to simulation include cost, resources, and fear of technology. This article describes how to design and implement a quality simulation program for less than $20,000.

Detection and typing of enteroviruses from CSF specimens from patients diagnosed with meningitis/encephalitis.

BACKGROUND: Human enteroviruses are the most common cause of viral meningitis. Rapid enterovirus detection and identification is important in order to ruleout other causes of disease, initiate appropriate patient management and to aid in epidemiological investigations. OBJECTIVES: A 2-year study (2005-2006) of patients with symptoms of meningitis/encephalitis was performed in New York State (NYS) to determine the underlying enteroviral etiology. STUDY DESIGN: Reverse-transcription, followed by a sequential PCR strategy targeting the 5'-nontranslated and VP1 regions, were used to first detect and then type, these RNA viruses. RESULT: From a total of 1374 specimens tested, enterovirus was detected in 67 specimens (4.9%); of these, 59 could subsequently be typed. Coxsackievirus B5 was found in 14 cases in 2005, but none in 2006. Overall, 14 enterovirus serotypes were detected. CONCLUSIONS: The most prevalent enteroviruses in this cohort were Coxsackievirus B5, and echoviruses 18, and 6 collectively accounting for 46%. 2005 was a period of high activity for Coxsackievirus B5 in NYS. A large majority of the enterovirus-positive patients suffered from headache and fever. In most cases, the cerebrospinal fluid profile was reported and generally showed elevated protein levels (>45mg/dl) and a higher than normal white blood cell count (>5mm(3)).

Detection and typing of human herpesvirus 6 by molecular methods in specimens from patients diagnosed with encephalitis or meningitis.

Human herpesvirus 6 (HHV-6) was detected in specimens from patients hospitalized with symptoms of encephalitis or meningitis. A real-time PCR assay was developed which has a linear dynamic range of 5 to 5 x 10(6) copies of HHV-6 and a sensitivity of five gene copies per reaction. While the assay detects both subtypes, HHV-6A and HHV-6B, it is specific and does not cross-react with a selected specificity panel. A total of 1,482 patient specimens, which were collected between 2003 and 2007, were tested; 26 specimens from 24 patients were found to be positive for HHV-6 by real-time PCR. The HHV-6 detection rate in this population was therefore 1.75%. The majority of the specimens tested (>95%) were cerebrospinal fluid (CSF) specimens. We were able to type 20 of the 26 positive specimens by conventional PCR and sequence analysis; all were HHV-6B. Forty-two percent of the patients were 3 years of age or younger, which may indicate a primary infection in these patients. Given the ages of the remaining patients (from 4 to 81 years), their infections were most probably due to virus reactivations. Where information was available, symptoms of patients included fever (71%), altered mental status (67%), and abnormal CSF profile (75%). Fifty percent of patients of 3 years of age or younger suffered from seizures. The detection of HHV-6 in specimens from patients diagnosed with encephalitis or meningitis, in the absence of a positive PCR result for other agents, strongly suggests a role for HHV-6 in the pathogenesis of these central nervous system diseases.

CCR2 genotype and disease progression in a treated population of HIV type 1-infected women.

Both antiretroviral therapy and the human coreceptor polymorphism CCR2-V64I slow progression of human immunodeficiency virus type 1 (HIV-1) disease. To examine the effect of V64I on disease progression in patients receiving therapy, we determined CCR2 genotypes in the Women's Interagency HIV Study cohort. We studied 2047 HIV-1-infected women, most of whom initiated treatment during the study. No association was seen between CCR2 genotype and either disease progression or therapeutic response, suggesting that the benefits of treatment most likely overshadow the salutary effects of the V64I polymorphism.

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 Prevention's 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.

CC chemokine receptor 5 genotype and susceptibility to transmission of human immunodeficiency virus type 1 in women.

The human gene for CC chemokine receptor 5, a coreceptor for human immunodeficiency virus type 1 (HIV-1), affects susceptibility to infection. Most studies of predominantly male cohorts found that individuals carrying a homozygous deleted form of the gene, Delta 32, were protected against transmission, but protection did not extend to Delta 32 heterozygotes. The role played by this mutation in HIV-1 transmission to women was studied in 2605 participants in the Women's Interagency HIV Study. The Delta 32 gene frequency was 0.026 for HIV-1-seropositive women and 0.040 for HIV-1-seronegative women, and statistical analyses showed that Delta 32 heterozygotes were significantly less likely to be infected (odds ratio, 0.63 [95% confidence interval, 0.44-0.90]). The CCR5 Delta 32 heterozygous genotype may confer partial protection against HIV-1 infection in women. Because Delta 32 is rare in Africans and Asians, it seems plausible that differential genetic susceptibility, in addition to social and behavioral factors, may contribute to the rapid heterosexual spread of HIV-1 in Africa and Asia.

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.