Detection and genome characterization of Middelburg virus strains isolated from CSF and whole blood samples of humans with neurological manifestations in South Africa.

BACKGROUND: The Old world Alphavirus, Middelburg virus (MIDV), is not well known and although a few cases associated with animal illness have previously been described from Southern Africa, there has been no investigation into the association of the virus with human illness. The current study aimed to investigate possible association of MIDV infection with febrile or neurological manifestations in hospitalized or symptomatic patients fromGauteng, South Africa. METHODS: This study is a descriptive retrospective and prospective laboratory based study. Archived cerebrospinal fluid (CSF) samples submitted to the National Health Laboratory Service (NHLS), Tshwane Academic division for viral investigation from public sector hospitals in Gauteng as well as EDTA (ethylenediaminetetraacetic acid) whole blood samples from ad hoc cases of veterinary students, presenting with neurological and febrile illness, were selected and screened for the presence of alphaviruses using real-time reverse transcription(rtRT) PCR.Virus isolations from rtRT-PCR positive samples were conducted in Vero cell culture and used to obtain full genome sequences. Basic descriptive statistical analysis was conducted using EpiInfo. RESULTS: MIDV was detected by rtRT-PCR in 3/187 retrospective CSF specimens obtained from the NHLS from hospitalised patients in the Tshwane region of Gauteng and 1/2 EDTA samples submitted in the same year (2017) from ad hoc query arbovirus cases from veterinary students from the Faculty of Veterinary Science University of Pretoria.Full genome sequences were obtained for virus isolates from two cases; one from an EDTA whole blood sample (ad hoc case) and another from a CSF sample (NHLS sample).Two of the four Middelburg virus positive cases,for which clinical information was available, had other comorbidities or infections at the time of infection. CONCLUSION: Detection of MIDV in CSF of patients with neurological manifestations suggests that the virus should be investigated as a human pathogen with the potential of causing or contributing to neurological signs in children and adults.

Development & evaluation of biotinylated DNA probe for clinical diagnosis of chikungunya infection in patients' acute phase serum & CSF samples.

BACKGROUND & OBJECTIVES: The resurgence of chikungunya virus (CHIKV) in the Indian Ocean Islands and India has drawn worldwide attention due to its explosive nature, high morbidity and complex clinico-pathological manifestations. The early confirmatory diagnosis of CHIKV is essential for management as well as control of unprecedented epidemics. The present study describes the development and evaluation of a highly sensitive and specific E1 structural gene specific biotinylated DNA probe for detection of chikungunya virus in clinical samples using a dot blot format. METHODS: The complementary DNA (cDNA) of CHIKV was spotted on to nylon membrane. The membrane was subjected to prehybridization and hybridization and developed using a colour development solution containing DAB chromogen. RESULTS: The CHIKV E1 specific DNA probe was highly sensitive detecting picogram levels of target nucleic acid. The comparative evaluation with SYBR Green I based real-time RT-PCR revealed 99 per cent accordance with a sensitivity and specificity of 99 and 98 per cent, respectively. The specificity of this assay was further confirmed through cross-reaction studies with confirmed dengue and Japanese encephalitis (JE) patient serum samples along with infected culture supernatant of Ross River and Saint Louis encephalitis and plasmid DNA of O'Nyong Nyong, Semlinki forest and Sindbis viruses. INTERPRETATION & CONCLUSION: The DNA probe reported in this study may be useful for specific, sensitive and confirmatory clinical diagnosis of chikungunya infection in acute phase human patient serum and CSF samples. This assay can also be used in the laboratory for quantification of viral antigen in cell culture supernatant for research purpose.

Case reports of neuro-Chikungunya in southern Thailand.

There has been a recent increase in reports of neurologic complications as major causes of morbidity and mortality in chikungunya virus infection. As a part of 2004-2009 global outbreaks, an unprecedented large chikungunya epidemic occurred in Southern Thailand during 2008-2009 in which 49,069 cases were reported. During this period, we encountered two patients with meningoencephalitis and another patient with myeloneuropathy among 1,018 cases diagnosed as chikungunya in our hospital. The clinical pictures are presented and the key points are used to recognize and differentiate chikungunya from Japanese encephalitis virus, dengue virus, and herpesvirus infections, which are more common causes of meningoencephalitis and myelitis in this region.

Systemic involvements and fatalities during Chikungunya epidemic in India, 2006.

BACKGROUND: In addition to classical manifestations of Chikungunya infection, severe infections requiring hospitalization were reported during outbreaks in India in 2006. OBJECTIVES: To describe the systemic syndromes and risk groups of severe Chikungunya infections. STUDY DESIGN: We prospectively investigated suspected Chikungunya cases hospitalized in Ahmedabad, Gujarat during September-October 2006, and retrospectively investigated laboratory-confirmed Chikungunya cases hospitalized with neurologic syndromes in Pune, Maharashtra. Hospital records were reviewed for demographic, comorbidity, clinical and laboratory information. Sera and/or cerebrospinal fluid were screened by one or more methods, including virus-specific IgM antibodies, viral RNA and virus isolation. RESULTS: Among 90 laboratory-confirmed Chikungunya cases hospitalized in Ahmedabad, classical Chikungunya was noted in 25 cases and severe Chikungunya was noted in 65 cases, including non-neurologic (25) and neurologic (40) manifestations. Non-neurologic systemic syndromes in the 65 severe Chikungunya cases included renal (45), hepatic (23), respiratory (21), cardiac (10), and hematologic manifestations (8). Males (50) and those aged >or=60 years (50) were commonly affected with severe Chikungunya, and age >or=60 years represented a significant risk. Comorbidities were seen in 21 cases with multiple comorbidities in 7 cases. Among 18 deaths, 14 were males, 15 were aged >or=60 years and 5 had comorbidities. In Pune, 59 laboratory-confirmed Chikungunya cases with neurologic syndromes were investigated. Neurologic syndromes in 99 cases from Ahmedabad and Pune included encephalitis (57), encephalopathy (42), and myelopathy (14) or myeloneuropathy (12). CONCLUSIONS: Chikungunya infection can cause systemic complications and probably deaths, especially in elderly adults.

Chikungunya virus and central nervous system infections in children, India.

Chikungunya virus (CHIKV) is a mosquito-borne alphavirus best known for causing fever, rash, arthralgia, and occasional neurologic disease. By using real-time reverse transcription-PCR, we detected CHIKV in plasma samples of 8 (14%) of 58 children with suspected central nervous system infection in Bellary, India. CHIKV was also detected in the cerebrospinal fluid of 3 children.

[Neurological manifestations in Chikungunya: about 23 cases collected in Reunion Island]. / Manifestations neurologiques du chikungunya: à propos de 23 cas colligés à la Réunion.

INTRODUCTION: We are reporting 23 cases of patients presenting neurological symptoms in the setting of a chikungunya outbreak that occurred in the Indian Ocean from March 2005 to April 2006. These symptoms were the cause of admission in our ward, mainly via the emergency room. CASE REPORTS: In the acute phase of their illness, 23 patients presented neurological symptoms associated with positive CSF tests (specific IgM or RT- PCR). Clinical manifestations included disrupted behavior or altered mental status in 95% of patients, headache in 30.4%, seizure in 26%, motor dysfunction in 4.3% and sensorial disorders in 8.7%. Outcome was fatal in two patients during their hospitalisation and several months after discharge in three other elderly bedridden patients with altered general status. CSF analysis was sometimes but not always inflammatory. CT or MRI, when done, showed no recent abnormality. EEG disclosed most often of a diffuse moderately slowed activity with no pseudo-periodic or unusual pattern. A few epileptic aspects were seen in known epileptic patients. The outcome of the neurological symptoms was generally good over a few days, contrasting with persisting impairment of general status and severe joint pains leading to a bedridden state and death in three patients. CONCLUSION: Nervous system involvement was not uncommon during the chikungunya outbreak in Reunion Island in 2005 and 2006. The most frequent expression was moderate confusion occurring during the acute phase of infection. Peripheral nerve involvement in the form of a typical Guillain Barré syndrome was also observed. In general, the neurological outcome was very good. Fatal issues occurring in the early stages or later on (five out of 23 patients) were related to altered general condition in debilitated bedridden elderly patients.

Increased blood-brain barrier permeability is not a primary determinant for lethality of West Nile virus infection in rodents.

Blood-brain barrier (BBB) permeability was evaluated in mice and hamsters infected with West Nile virus (WNV, flavivirus) as compared to those infected with Semliki Forest (alphavirus) and Banzi (flavivirus) viruses. BBB permeability was determined by measurement of fluorescence in brain homogenates or cerebrospinal fluid (CSF) after intraperitoneal (i.p.) injection of sodium fluorescein, by macroscopic examination of brains after i.p. injection of Evans blue, or by measurement of total protein in CSF compared to serum. Lethal infection of BALB/c mice with Semliki Forest virus and Banzi virus caused the brain : serum fluorescence ratios to increase from a baseline of 2-4% to as high as 11 and 15%, respectively. Lethal infection of BALB/c mice with WNV did not increase BBB permeability. When C57BL/6 mice were used, BBB permeability was increased in some, but not all, of the WNV-infected animals. A procedure was developed to measure BBB permeability in live WNV-infected hamsters by comparing the fluorescence in the CSF, aspirated from the cisterna magnum, with the fluorescence in the serum. Despite a time-dependent tendency towards increased BBB permeability in some WNV-infected hamsters, the highest BBB permeability values did not correlate with mortality. These data indicated that a measurable increase in BBB permeability was not a primary determinant for lethality of WNV infection in rodents. The lack of a consistent increase in BBB permeability in WNV-infected rodents has implications for the understanding of viral entry, viral pathogenesis and accessibility of the CNS of rodents to drugs or effector molecules.