The World Health Organization declared that a new strain of novel swine-origin influenza A (H1N1) virus was responsible for the pandemic infection in June 2009. We report a case of encephalitis diagnosed as the H1N1 virus infection. We describe a 17-year-old patient who had a seizure attack, diagnosed with a H1N1 virus infection via real time reverse-transcriptase polymerase chain reaction (RT-PCR). The H1N1 virus infection can be causative of the encephalitis, as with other influenza virus infections. Careful monitoring is essential for reducing complications.
Adolescent ; Animals ; Encephalitis, Viral/*diagnosis/*virology ; Humans ; Influenza A Virus, H1N1 Subtype/*pathogenicity ; Male ; Swine/*virology

OBJECTIVETo develop a rapid, cost effective RT-PCR method for the mass scale diagnosis of such diseases at the viremia stage to find out the actual disease burden in that area.

METHODSFor this purpose, cases with the history of only short febrile illness were considered. Thus 157 samples with the history of dengue/chikungunya like illness and only 58 samples with a history of acute encephalitis syndrome (AES) were selected.

RESULTSOut of 157 samples, 42 and 74 were detected as dengue and chikungunya, respectively and out of 58 AES cases only 23 could be detected as Japanese encephalitis by this RT-PCR method.

CONCLUSIONSThis cost effective RT-PCR method can detect the total positive cases that remain undetected by ELISA method. Moreover, this method is capable to detect the viral RNA from patients' sera even after the appearance of IgM antibody at one fifth costs as compared with the other commercially available kits.

Antibodies, Viral ; blood ; Arbovirus Infections ; diagnosis ; virology ; Arboviruses ; genetics ; Chikungunya Fever ; diagnosis ; virology ; Dengue ; diagnosis ; virology ; Encephalitis Virus, Japanese ; genetics ; Encephalitis, Japanese ; diagnosis ; virology ; Fever ; diagnosis ; virology ; Humans ; Immunoglobulin M ; blood ; Mass Screening ; RNA, Viral ; blood ; Reverse Transcriptase Polymerase Chain Reaction ; economics ; methods ; Sensitivity and Specificity ; Viremia ; diagnosis ; virology

OBJECTIVETo ascertain the pathogen of aseptic encephalitis epidemic in Long-Yan city in Fujian, and to find out the genetic characteristics of the virus.

METHODSRapid detection of enteroviral RNA by reverse transcription polymerasechain reaction (RT-PCR) was directly carried out in cerebrospinal fluid(CSF) to isolate and identify the viruses from CSF at the same time, and to detect the neutralization antibody in two serum specimens collected in acute and convalescence phase. Nucleotides of VP1 region was also analyzed by constructing phylogenetic tree.

RESULTSECHO 19 infection was rapidly diagnosed and sequence analysed by RT-PCR, and then echovirus type 19 from 16 of 30 CSF samples (53.33%) was isolated and detected using RD and Hep-2 cells simultaneity. The titer of ECHO 19 neutralization antibody became positive or increased by 4 times from acute to convalescence phase in 4 of the 5 patients. Phylogenetic analyses of the VP1 genes of these isolates showed that their nucleotides identity were 98.9% -100.0% which were different from those ECHO 19 from GeneBank database by 13.0%-22.4%.

CONCLUSIONThe etiology of the epidemic of aseptic encephalitis was attributed to ECHO 19. The method of molecular identification not only provided rapid diagnosis of enterovirus infections, but also information about the genetic character of the viruses.

Antibodies, Neutralizing ; analysis ; China ; epidemiology ; Echovirus Infections ; diagnosis ; immunology ; virology ; Encephalitis, Viral ; epidemiology ; virology ; Enterovirus B, Human ; genetics ; isolation & purification ; Humans ; Phylogeny ; Reverse Transcriptase Polymerase Chain Reaction

One step TaqMan reverse transcription polymerase chain reaction (RT-PCR) using TaqMan probe was developed for detection of Japanese encephalitis virus (JEV). Real-time RT-PCR was optimized to quantify JEV using the detection system (Rotor Gene 2000 detector) and dual-labeled fluorogenic probes. The gene specific labeled fluorogenic probe for the 3' non-translated region (3' NTR) was used to detect JEV. When the specificity of the assay using specific JEV primers was evaluated by testing three different JEV strains, other swine viruses and bovine viral diarrhea virus, no cross-reactions were detected with non-JE reference viruses. A single tube TaqMan assay was shown to be 10-fold more sensitive than the conventional two-step RT-PCR method. Detection limits of two step and real-time RT-PCR for JEV were 112 TCID50 /ml and 11.2 TCID50 /ml, respectively. Quantification of JEV was accomplished by a standard curve plotting cycle threshold values (Ct ) versus infectivity titer. Real-time RT-PCR assay using single tube method could be used as a sensitive diagnostic test, and supplied the results in real time for detection and quantification of JEV. We could detect JEV RNA genome in plasma samples of pigs inoculated with KV1899 strain at 2 days post inoculation, but couldn't in 41 fetus samples. This assay was sensitive, specific, rapid and quantitative for the detection of JEV from laboratory and field samples.
Animals ; DNA Primers/chemistry ; DNA Probes/chemistry ; Encephalitis Virus, Japanese/genetics/*isolation&purification ; Encephalitis, Japanese/diagnosis/*veterinary/virology ; RNA, Viral/analysis ; Reproducibility of Results ; Reverse Transcriptase Polymerase Chain Reaction/methods/*veterinary ; Sensitivity and Specificity ; Swine ; Swine Diseases/*diagnosis/virology ; *Taq Polymerase

OBJECTIVEHuman herpesvirus 6 (HHV-6) isolates are classified into two variants, HHV-6A and HHV-6B, based on distinct genetic, antigenic and biological characteristics. HHV-6 has been associated with encephalitis in children recently. This study aimed to establish a real time PCR assay for simultaneous detection of the two subtypes of HHV-6, and apply this new assay to children with suspected encephalitis, then analyze the relationship between the infection with HHV-6 and encephalitis in children.

METHODThe universal primers and variant-specific TaqMan probes were designed based on the highly conserved sequences of the DNA polymerase gene (U38) of HHV-6. The 5' end of the probes for HHV-6A and HHV-6B was labeled with the fluorescein reporter tetrachloro-6-carboxyfluorescein and 6-carboxyfluorescein (6-FAM), separately, while the 3' end were quenched with 6-carboxy-tetramethylrhodamine. The real time PCR assay for simultaneous detection of HHV-6A and HHV-6B was established. Then, the plasmids of HHV-6A and -6B which were diluted by a 10-fold series from 10(9) to 10(0) copies/microl, together with controls were used for testing both sensitivity and specificity of the real time PCR assay. The cerebrospinal fluid (CSF) specimens from 445 cases of suspected encephalitis were tested with this real time PCR and positive samples were then sequenced.

RESULTBoth HHV-6A (strain ZJ-159) and HHV-6B (strain GS) were positive on the real time PCR assay. There were no cross-reaction with herpes simplex virus type 1, type 2 (HSV-1, HSV-2), varicella-zoster virus (VZV), cytomegalovirus (CMV), Epstein-Barr virus (EBV), hepatitis B virus, Staphylococcus aureus, Mycoplasma pneumoniae and human DNA. A linear regression curve was obtained when plotting Ct values against the log10 of the viral DNA input for both subtypes of HHV-6. The sensitivity threshold was 10 copies/microl for the real time PCR. HHV-6 positive rate by the real time PCR assay was 4.72% (21/445), including 4 cases with HHV-6A infection, 16 cases of HHV-6B infection and 1 case with mixed HHV-6A and HHV-6B infection. The new PCR assay usually took 2 to 3 hours to provide results.

CONCLUSIONThis new real time PCR assay can simultaneously detect both subtypes of HHV-6, and have high specificity and sensitivity. It will provide an early and sensitive diagnosis of HHV-6 encephalitis in children.

Adolescent ; Child ; Child, Preschool ; DNA Fingerprinting ; DNA Primers ; DNA, Viral ; Encephalitis, Viral ; cerebrospinal fluid ; diagnosis ; virology ; Female ; Fluorometry ; Genotype ; Herpesvirus 6, Human ; genetics ; isolation & purification ; Humans ; Infant ; Infant, Newborn ; Polymerase Chain Reaction ; methods ; Sensitivity and Specificity

OBJECTIVETo understand the epidemiological characteristics and the main clinical symptoms of viral encephalitis in Gansu.

METHODSA total of 322 viral encephalitis patients were recruited from province sentinel hospitals in Gansu province from 2009 to 2011, and their basic information were collected as well as their serum samples and cerebrospinal fluid samples. 296 out of the 322 cases were qualified for our study. Based on the patients' epidemiological characteristics and clinical features, we determined the detection of the virus types (at least one kind of virus detection was carried out for each case). ELISA was applied to test the IgM antibody of Japanese encephalitis (JE) virus (JEV), enterovirus (EV: including Coxsackie virus, echovirus, enterovirus 71), mumps virus and herpes simplex virus (HSV) in cerebrospinal fluid and serum specimen. The difference of positive detected rate between types of virus, among patients from different regions, time, or at different ages, as well as the different clinical symptoms between JE patients and other viral encephalitis patients, were analyzed and compared.

RESULTSThe positive detected rate of virus in the 296 patients was 27.03% (80/296); the positive rate of JEV, EV, mumps virus, HSV detected was separately 7.53% (22/292), 8.75% (23/263), 13.84% (22/159) and 15.09% (40/265), and the difference was statistically significant (χ(2) = 10.849, P < 0.05). 90.91% (20/22) of the JEV positive cases were distributed in Tianshui, Longnan and Pingliang, and 95.45% (21/22) patients were infected from July to September. All the 23 EV detected positive patients were infected from April to December, while the ages of patients ranged from 1 to 44 years old. Mumps virus, HSV testing positive cases had onset every month. Logistic regression analysis showed that the patients who had the symptoms as disturbance of consciousness (OR = 15.487, 95%CI: 2.266 - 105.852), somnolence (OR = 11.659, 95%CI: 1.783 - 76.242), convulsions (OR = 11.062, 95%CI: 1.687 - 72.530) were more likely to infect JEV.

CONCLUSIONHSV was the principal pathogen of viral encephalitis in Gansu. An obvious central tendency in the regional and time distribution was found in JEV infection; and the clinical symptoms of JE patients were more severe.

Adolescent ; Adult ; Antibodies, Viral ; blood ; cerebrospinal fluid ; Child ; Child, Preschool ; China ; epidemiology ; Encephalitis Virus, Japanese ; Encephalitis, Viral ; diagnosis ; epidemiology ; virology ; Enterovirus ; Enzyme-Linked Immunosorbent Assay ; Female ; Humans ; Immunoglobulin M ; blood ; cerebrospinal fluid ; Infant ; Male ; Mumps virus ; RNA, Viral ; blood ; cerebrospinal fluid ; Simplexvirus ; Young Adult