Echovirus Research Articles

COPI Is Required for Enterovirus 71 Replication

Enterovirus 71 (EV71), a member of the Picornaviridae family, is found in Asian countries where it causes a wide range of human diseases. No effective therapy is available for the treatment of these infections. Picornaviruses undergo RNA replication in association with membranes of infected cells. COPI and COPII have been shown to be involved in the formation of picornavirus-induced vesicles. Replication of several picornaviruses, including poliovirus and Echovirus 11 (EV11), is dependent on COPI or COPII. Here, we report that COPI, but not COPII, is required for EV71 replication. Replication of EV71 was inhibited by brefeldin A and golgicide A, inhibitors of COPI activity. Furthermore, we found EV71 2C protein interacted with COPI subunits by co-immunoprecipitation and GST pull-down assay, indicating that COPI coatomer might be directed to the viral replication complex through viral 2C protein. Additionally, because the pathway is conserved among different species of enteroviruses, it may represent a novel target for antiviral therapies.

Echovirus 7 Entry into Polarized Intestinal Epithelial Cells Requires Clathrin and Rab7

ABSTRACTEnteroviruses invade the host by crossing the intestinal mucosa, which is lined by polarized epithelium. A number of enteroviruses, including echoviruses (EV) and group B coxsackieviruses (CVB), initiate infection by attaching to decay-accelerating factor (DAF), a molecule that is highly expressed on the apical surface of polarized epithelial cells. We previously observed that entry of DAF-binding CVB3 into polarized intestinal epithelial cells occurs by an unusual endocytic mechanism that requires caveolin but does not involve clathrin or dynamin. Here we examined the entry of a DAF-binding echovirus, EV7. We found that drugs, small interfering RNAs (siRNAs), and dominant negative mutants that target factors required for clathrin-mediated endocytosis, including clathrin and dynamin, inhibited both EV7 infection and internalization of virions from the cell surface. Once virus had entered the cell, it colocalized with markers of early endosomes (EEA1) and then late endosomes (LAMP-2). Inhibition of endosomal maturation—with siRNAs or dominant negative mutants targeting Rab5 and Rab7—inhibited infection and prevented release of viral RNA into the cell. These results indicate that EV7 is internalized by clathrin-mediated endocytosis and then moves to early and late endosomes before releasing its RNA. Trafficking through endosomes is known to be important for viruses that depend on low pH or endosomal cathepsin proteases to complete the entry process. However, we found that EV7 infection required neither low pH nor cathepsins.

A single amino acid substitution controls DAF-dependent phenotype of echovirus 11 in rhabdomyosarcoma cells

Decay accelerating factor (DAF, CD55) is used by DAF-dependent (Daf+) variants of echovirus 11 (EV11) as a primary cellular receptor. The interaction of EV11 with DAF is completely reversible, therefore DAF-dependent variants require an unidentified coreceptor to initiate uncoating. Daf− variants of EV11, which do not interact with DAF, use an alternative primary cellular receptor. The aim of this study was to test the hypothesis whether the coreceptor, which is necessary for the uncoating of DAF-dependent variants, may act as an alternative primary receptor for the Daf− variants of EV11. By using the model of the two closely related daf+ and daf− clones of EV11 in rhabdomyosarcoma (RD) cell line, it was shown that a single amino acid substitution in the capsid protein VP2 could control the expression of the DAF-dependent phenotype. Anti-DAF monoclonal antibody has blocked the infection of RD cells by the DAF-dependent daf+ clone, but not by the daf− clone of EV11. Since the structural proteins of the two clones differed only in the receptor binding site for DAF, the unidentified non-DAF primary receptor for the daf− clone might have the same conformation as the uncoating coreceptor required for the daf+ clone. Despite the difference in primary receptors, both daf+ and daf− clones were equally inhibited by a monoclonal antibody to beta2-microglobulin. The monoclonal antibody B9.12.1 to class I human leukocyte antigen molecules showed no inhibitory effect in regards to either clone. The hypothesis of convergent intracellular traffic of Daf+ and Daf− variants of EV11 is discussed.

Efficient synthesis and anti-enteroviral activity of 9-arylpurines

To further explore the anti-enteroviral activity of 9-aryl-6-chloropurines, three different series of compounds with a dialkylamino, (alkyl)amido, or oxazolidinone substituent at the aryl ring have been synthesized, in most cases with the aid of microwave-assisted synthesis. The resulting compounds efficiently inhibit Coxsackie virus type B3 (CVB3) replication with EC50 values varying from 3 to 15 μM, and with no significant toxicity in Vero cells. The most potent compounds also selectively inhibit the replication of other enteroviruses including Coxsackie virus B4 and Echo virus 11. The cross-resistance studies performed with different 9-aryl-6-chloropurines indicate that they all belong to the same pharmacological family and differ from other CVB3 drugs such as enviroxime.

A Patient with Mediastinal Mature Teratoma Presenting with Paraneoplastic Limbic Encephalitis

A 22-year-old woman with a convulsion and impaired consciousness was transferred from a local psychiatric clinic to Oita University Hospital. She had no symptoms until July 2004, when she had fever and convulsive seizures and was diagnosed encephalitis. She underwent continuous infusion of thiamylal sodium for status epilepticus. Autonomic changes, such as tachycardia and increased sweating, were also seen. On neurological examination, she was in coma and status epilepticus, showing spastic paralysis and central hypoventilation. Tracheal intubation was performed, and she was put on mechanical ventilation. There were no evident endocrine or metabolic abnormalities. Antinuclear antibodies were negative. Her cerebrospinal fluid (CSF) pressure was 270 mm H2O. The fluid was clear and contained 26 cells/ l, glucose 72 mg/dl, protein 21.6 mg/ml, immunoglobulin G 6 mg/dl, and adenosine deaminase 1.5 IU/L. Microscopic examinations of the CSF for tumor cells and microorganisms were negative, and cultures yielded no growth. There were no marked elevations of antiviral antibody titers, including Epstein-Barr virus, herpes simplex virus, echo virus, and cytomegalovirus. A subunit of anti-N-methyl-D-aspartate receptor antibodies, anti-NR2B (glutamate receptor [GluR] epsilon-2) antibody, and antiNR2A (glutamate receptors [GluR] epsilon-1) antibody were positive in both serum and CSF. An anterior mediastinal tumor, 4.5 cm in diameter, was found on chest computed tomography scan (Figure 1). These findings suggested paraneoplastic limbic encephalitis associated with a mediastinal teratoma. The patient received corticosteroid therapy but the neurological symptoms continued. Serological tests showed hypothyroidism. The tumor was resected without combined resection of the thymus 10 months after the neurological symptoms appeared. Pathologically, the tumor was a mature teratoma with various tissue components, including skin, thymus, striated muscle, cartilage, bronchial glands and mucosa, and pancreas (Figure 2). The neurological symptoms improved dramatically postoperatively. The seizures disappeared, and the thiamylal infusion was gradually tapered over 3 weeks after surgery and discontinued. The patient was weaned from ventilatory support within 4 weeks after tumor resection. The patient is currently well at 7 years after surgery with no evidence of tumor recurrence.

Evaluation of Reverse Transcription Loop-Mediated Isothermal Amplification assays for Rapid Detection of Human Enterovirus 71 and Coxsackievirus A16 in Clinical Samples

A sensitive reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay for human enterovirus 71 (EV71) and Coxsackievirus A16 (CVA16) infection was further evaluated. The one step reaction was performed in a single tube at 65?C for 45 min for EV71 and 35 min for CVA16. The detection limits of RT-LAMP assays for both EV71 and CVA16 were 0.1 of a 50% tissue culture infective dose (TCID50) per reaction, based on 10—Fold dilutions of a titrated EV71 or CVA16 strain. The specific assay showed there were no cross-reactions with Coxsackievirus A (CVA) viruses (CVA 2, 4, 5, 7, 9, 10, 14, and 25), Coxsackievirus B (CVB) viruses (CVB 1, 2, 3, 4, and 5) or ECHO viruses (ECHO 3, 6, 11, and 19). In parallel with commercial quantitative real-time polymerase chain reaction (qRT-PCR) diagnostic kits for EV71 and CVA16, the RT-LAMP assay was evaluated with 515 clinical specimens, the results showed the RT-LAMP assay and the qRT-PCR assay were in complete agreement for 513/515 (99.6%) of the specimens. Two samples with discrepant results from two methods were further verified by nested reverse transcription polymerase chain reaction (nRT-PCR) assay and sequencing to be true positives for CVA16. In conclusion, RT-LAMP assay is demonstrated to be a sensitive and specific assay and have a great potential for the rapid and visual screening of EV71 and CVA16 in China, especially in those resource-limited hospitals and rural clinics of provincial and municipal regions.

Genetic characteristic analysis of echovirus 11

Objective To study echovirus 11 ( ECHO11 ) sequences genetic variability of the VP1 gene of 82 isolates from 15 countries and 2 provinces in China.Methods The VP1 sequences of 82 strains of echovirus 11 were downloaded from the GenBank,and their nucleotide and amino acid diversity were calculated and phylogenetic tree was constructed using Mega software.Results 82 echovirus 11 strains were divided into 4 genotypes( A-D),Genotype A is further divided into 7 subgenotyps and genotype D into 7 subgenotyps.The prototype strain Gregory is the sole member of genotype B.Genotype C is divided into 3 genotypes.The sequence diversity between echovirus 11 strains is 21.7%-24.1% (AA diversity:6.0%-12.1% ).Conclusion Our results show that most genotypes contain isolates that have circulated over a wide geographical(several countries from different continents) and temporal range.Several genotypes were also shown to co-circulate in a region during the same period of time,showing that the epidemic of echovirus 11 has typical time and geographical characters. Key words: Echovirus 11 ; Genetic characteristics

The association of recombination events in the founding and emergence of subgenogroup evolutionary lineages of human enterovirus 71.

Enterovirus 71 (EV71) is responsible for frequent large-scale outbreaks of hand, foot, and mouth disease worldwide and represent a major etiological agent of severe, sometimes fatal neurological disease. EV71 variants have been classified into three genogroups (GgA, GgB, and GgC), and the latter two are further subdivided into subgenogroups B1 to B5 and C1 to C5. To investigate the dual roles of recombination and evolution in the epidemiology and transmission of EV71 worldwide, we performed a large-scale genetic analysis of isolates (n = 308) collected from 19 countries worldwide over a 40-year period. A series of recombination events occurred over this period, which have been identified through incongruities in sequence grouping between the VP1 and 3Dpol regions. Eleven 3Dpol clades were identified, each specific to EV71 and associated with specific subgenogroups but interspersed phylogenetically with clades of coxsackievirus A16 and other EV species A serotypes. The likelihood of recombination increased with VP1 sequence divergence; mean half-lives for EV71 recombinant forms (RFs) of 6 and 9 years for GgB and GgC overlapped with those observed for the EV-B serotypes, echovirus 9 (E9), E30, and E11, respectively (1.3 to 9.8 years). Furthermore, within genogroups, sporadic recombination events occurred, such as the linkage of two B4 variants to RF-W instead of RF-A and of two C4 variants to RF-H. Intriguingly, recombination events occurred as a founding event of most subgenogroups immediately preceding their lineage expansion and global emergence. The possibility that recombination contributed to their subsequent spread through improved fitness requires further biological and immunological characterization.

Genetic analysis of echovirus 30 isolated from acute meningitis/encephalitis syndrome cases in Linyi city of Shandong province, China

To investigate the pathogen spectrum constitution of HEV related to acute meningitis/encephalitis syndrome (AMES) and to analyze the genetic characterization of the echovirus 30 (Echo30) isolates. Cerebrospinal fluid, stool, and/or throat swab specimens from 101 AMES cases were collected for virus isolation with RD and HEp-2 cell lines in Linyi city from April to December, 2010. After typing by neutralization test, the entire VP1 gene of the isolates were amplified by RT-PCR and sequenced. Homologous comparison was carried out and phylogenetic analysis were performed. HEVs were isolated from 47 specimens of 34 patients (4 from cerebrospinal fluid, 18 from throut swab and 25 from stool), with 11 Echo30 isolates identified by microneutralization assay and molecular typing method. In alignment of VP1 sequences with global Echo30 isolates, relative high nucleotide homologies (91.2% - 95.4%) with Taian, Zhangqiu and Jiangsu isolates and high divergences (17.9% -19.6%) with prototype strain were observed. Nucleotide divergences among Linyi isolates were 0 - 10.2%, and 3 lineages were revealed via phylogenetic analysis, reflecting 3 transmission chains of Echo30 co-circulated in Linyi city, 2010. Echo30 was the predominant serotype of AMES cases in Linyi city, 2010. These isolates possessed considerable divergence with Echo30 from other countries.

Study on the pathological and molecular characteristics of viral meningitis outbreaks in Linhai county, Zhejiang province, 2004

In order to confirm the causes of viral meningitis outbreaks in Linhai county, Zhejiang province in 2004, and to analyze the relationship between hereditary variation and evolution of the pathogen. 60 cerebrospinal fluid (CSF) specimens were collected from the suspected patients. Virus strains from the specimens were isolated with RD and Hep-2 cell lines, and identified through neutralization test. VP1 and VP4/VP2 genes of the isolated viruses were sequenced. Both phylogenetic and homological trees were also constructed. 19 Echovirus type 30 (E30) strains were isolated from 60 CSFs, in which E30 accounted for 31.7%. All of the complete VP1 genes in 4 sequenced virus isolates of E30 were composed of 876 nt, encoding 292 amino acids (aa). The identity of nucleotide and amino acid in VP1 gene were 82.4% - 84.1% and 93.5% - 94.2% between the 4 Linhai strains and the prototype strain Bastianni of E30, were 87.1% - 99.9% and 97.9% - 100.0% among the 4 virus strains of E30 from Linhai, respectively. The 4 Linhai strains could be classified into two classes. The diversity of nt and aa was minimal in the same class but obvious between the two classes, with the range of diversities as 12.9% and 2.1%, respectively. The Linhai E30 strains had maximum similarity with the Zhejiang E30 strains in 2002 - 2003. The 4 Linhai strains of E30 in the phylogenetic tree of the VP1 gene were attributed into two branches of the G and H genotype, respectively. The G branch also included the E30 strains from Zhejiang, Jiangsu and Shangdong in 2003, while the H branch including E30 strains from Zhuji, Zhejiang in 2002. The phylogenetic tree of VP4/VP2 genes was similar to that of VP1 gene. The outbreak of viral meningitis in Linhai county in 2004 was caused by the two classes of E30 strains with G and H genotype existed simultaneously. The Linhai E30 strains had maximum genetic relations to the Zhejiang, Jiangsu and Shangdong strains of E30. The H genotype was inferred to be a new variant strain, which was first isolated in Zhejiang province in 2002.

Genome analysis of two type 6 echovirus (E6) strains recovered from sewage specimens in Greece in 2006

Echovirus 6 (E6) is one of the main enteroviral serotypes that was isolated from cases of aseptic meningitis and encephalitis during the last years in Greece. Two E6 (LR51A5 and LR61G3) were isolated from the sewage treatment plant unit in Larissa, Greece, in May 2006, 1 year before their characterization from aseptic meningitis cases. The two isolates were initially found to be intra-serotypic recombinants in the genomic region VP1, a finding that initiated a full genome sequence analysis. In the present study, nucleotide, amino acid, and phylogenetic analyses for all genomic regions were conducted. For the detection of recombination events, Simplot and bootscan analyses were carried out. The continuous phylogenetic relationship in 2C-3D genomic region of strains LR51A5 and LR61G3 with E30 isolated in France in 2002-2005 indicated that the two strains were recombinants. SimPlot and Bootscan analyses confirmed that LR51A5 and LR61G3 carry an inter-serotypic recombination in the 2C genomic region. The present study provide evidence that recombination events occurred in the regions VP1 (intraserotypic) and non-capsid (interserotypic) during the evolution of LR51A5 and LR61G3, supporting the statement that the genomes of circulating enteroviruses are a mosaic of genomic regions of viral strains of the same or different serotypes. In conclusion, full genome sequence analysis of circulating enteroviral strains is a prerequisite to understand the complexity of enterovirus evolution.

Molecular characterization and antiviral activity test of common drugs against echovirus 18 isolated in Korea

Genetic diversity and antiviral activity for five common antiviral drugs of echovirus (ECV) 5 isolated in Korea have been described. The present study extended these tests to a Korean ECV 18 isolate. An outbreak of aseptic meningitis caused by the ECV 18 isolate was reported in Korea in 2005, marking the first time this virus had been identified in the country since enterovirus surveillance began in 1993. Using a sample isolated from stool specimen of a 5-year-old male patient with aseptic meningitis, the complete genome sequence was obtained and was compared it with the Metcalf prototype strain. Unlike the ECV5 isolate, the 3' untranslated region had the highest identity value (94.2%) at the nucleotide level, while, at the amino acid level, the P2 region displayed the highest identity value (96.9%). These two strains shared all cleavage sites, with the exception of the 2B/2C site, which was RQ/NN in the Metcalf strain but RQ/NS in the Korean ECV 18 isolate. In Vero cells infected with the Korean ECV 18 isolate, no cytotoxicity was observed in the presence of azidothymidine, acyclovir, amantadine, lamivudine, or ribavirin, when the drugs were administered at a CC50 value >100 μg/mL. Of the five drugs, only amantadine (IC50: 4.97 ± 0.77 μg/mL, TI: 20.12) and ribavirin (IC50: 7.63 ± 0.87 μg/mL, TI: 13.11) had any antiviral activity against the Korean ECV 18 isolate in the five antiviral drugs. These antiviral activity effects were similar with results of the Korean ECV5 isolate.

Genetic analysis of a partial VP1 region and molecular identification of non-EV71, non-CA16 virus strains of hand, foot and mouth disease（HFMD） in Guangdong province between 2008 and 2009

Objective To discuss the prevalence of non-EV71, non-CA16 virus strains of hand, foot and mouth disease （ HFMD ） in Guangdong province between 2008 and 2009, and analyze the genetic evolution of these non-EVT1, non-CA16 virus strains. Methods Isolated viruses from stool samples collected from outpatient and in-patient cases of HFMD between 2008 and 2009 by human rhabdomyosarcoma（RD） cell and HEp-2 cell, cultures that exhibited a characteristic enterovirus cytopathic effect were evaluated by RT-PCR. Those strains which identified non-EVT1, non-CA16 were analyzed by VP1 sequencing and then were identified by BLAST program. A phylogenetic tree was constructed using the Neighor-Joinning method in the MEGA 4.0 software. Results Twenty-two virus strains of non-EVT1, non-CA16 were obtained, and nine of the twenty-two virus strains in 2008 were classified into CA2, CA4, and CB3 by BLAST ; thirteen of the twenty-two virus strains in 2009 were classified into EVS0, Echol3, Echo30, CBS, Echo24, CA10, CA6, and poliovirus 1 by BLAST. The homology of all strains was low, and all the strains belonged to CA, CB, Echoviruses, Enterovirus and poliovirus subgroup. Conclusion Except for EVT1 and CA16 was a major causative agent in prevail of HFMD in Guangdong province between 2008 and 2009, there also existed other subgroup Enterovirus. The other twenty-two strains respectively belonged to CA, CB, Echoviruses, Enterovirus and poliovirus subgroup, and none of those strains was predominant. Muti-species Enterovirus occurred concomitantly. Key words: Hand, foot and mouth disease; Enterovirus; VP1 gene; Type

Synthesis and in vitro Study of Antiviral and Virucidal Activity of Novel 2-[(4-Methyl-4H-1,2,4-triazol-3-yl)sulfanyl]acetamide Derivatives

2-[(4-Methyl-4H-1,2,4-triazol-3-yl)sulfanyl]acetamide derivatives were synthesized and their structures were confirmed by 1H NMR, IR, and elemental analysis. Cytotoxicity of the compounds towards HEK-293 and GMK cells was evaluated. Moreover, the antiviral and virucidal activities of these compounds against human adenovirus type 5 and ECHO-9 virus were assessed. Some of the newly synthesized derivatives have the potential to reduce the viral replication of both tested viruses.

Visual detection of human enterovirus 71 subgenotype C4 and Coxsackievirus A16 by reverse transcription loop-mediated isothermal amplification with the hydroxynaphthol blue dye

A sensitive reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay was developed for rapid visual detection of human enterovirus 71 subgenotype C4 (EV71-C4) and Coxsackievirus A16 (CVA16) infection, respectively. The reaction was performed in one step in a single tube at 65 °C for 60 min with the addition of the hydroxynaphthol blue (HNB) dye prior to amplification. The detection limits of the RT-LAMP assay were 0.33 and 1.58 of a 50% tissue culture infective dose (TCID 50) per reaction based on 10-fold dilutions of a titrated EV71 or CVA16 strain, respectively. No cross-reaction was observed with Coxsackievirus A (CVA) viruses (CVA2, 4, 5, 7, 9, 10, 14, and 24), Coxsackievirus B (CVB) viruses (CVB1,2,3,4, and 5) or ECHO viruses (ECHO3, 6, 11, and 19). The assay was further evaluated with 47 clinical stool specimens diagnosed previously with EV71, CVA16 or other human enterovirus infections. Virus isolates from stool samples were confirmed by virus neutralization testing and sequencing. RT-LAMP with HNB dye was demonstrated to be a sensitive and cost-effective assay for rapid visual detection of human EV71-C4 and CVA16.

Cocirculation of Two Transmission Lineages of Echovirus 6 in Jinan, China, as Revealed by Environmental Surveillance and Sequence Analysis

Enterovirus environmental surveillance on sewage from the city of Jinan, Shandong Province, China, was initiated in 2008. Thirty echovirus 6 (E6) strains-1 in 2008 and 29 in 2010-were isolated and identified. Most E6 isolates (n = 21) came from the sewage collected on August 2010, revealing high local E6 activity at that time. Interestingly, the VP1 sequences of most isolates, even from the same sewage, were not identical. Phylogenetic analysis of VP1 sequences revealed two lineages for these isolates, with 78.0 to 80.0% nucleotide identities with one another, 94.8 to 100.0% identity within the major lineage, and 92.7 to 98.5% identity within the minor one. The VP1 sequences of environmental isolates, clinical isolates from 1998 to 2010, and global E6 were subjected to evolutionary analysis using Bayesian phylodynamic methods. The inferred E6 VP1 ancestral sequence dated back to 1901 (range, 1873 to 1928) and evolved with 7.047 × 10(-3) substitutions per site per year. Shandong E6 segregated into three clusters, and the two environmental lineages belonged to clusters A and C, which originated in 2003 and 1992, respectively. The antigenicity analysis via neutralization assay confirmed great antigenic differences between Shandong isolates and a prototype strain. These findings underscore the value of continuous environmental surveillance and genetic analysis to monitor circulating enteroviruses in the population and give further insight into E6 evolution.

Phylogenetic analysis of complete VP1 sequences of echoviruses 11 and 6: high genetic diversity and circulation of genotypes with a wide geographical and temporal range

Echovirus 6 (E6) and echovirus 11 (E11) are common causes of meningitis and other human diseases; they are among the most frequently isolated enteroviruses worldwide. In the present work we have studied genetic variability over the entire VP1 gene of selected isolates representing a wide geographical and temporal range. Fifty new sequences from North Africa were included, together with previously published sequences from different countries. The sequence diversity between strains of the same type was high: 22 and 30 % for E6 and E11, respectively. Phylogenetic analysis revealed five genogroups within each type, the genetic diversity within a genogroup generally being <20 %. Some genogroups were further subdivided into genotypes, most containing isolates that had circulated over a wide geographical (several countries from different continents) and temporal (up to two decades) range. Several genotypes were also shown to co-circulate in a region during the same period of time. These features differ from other enteroviruses that divide into temporal or geographical clusters. This study reports new sequences from North Africa, updates the molecular epidemiology of E6 and E11, and proposes a new genogroup in each type.

Development of a recombinant CHO cell model for the investigation of CAR and DAF role during early steps of echovirus 6 infection

The early steps of echovirus 6 (E6) infection remain poorly understood and the only described receptor for haemagglutinating E6 strains is the decay accelerating factor (DAF). There is, however, accumulating evidence suggesting that E6 interaction with DAF is necessary but not sufficient for infection. In this report, we investigated the role of the coxsackie-adenovirus-receptor (CAR) as a potential DAF co-receptor during E6 infection. Using stably transfected Chinese Hamster Ovary (CHO) cells expressing CAR and DAF receptors, we found that DAF expression allowed attachment of both haemagglutinating and non-haemagglutinating E6 strains but was not sufficient for promoting E6 cell entry. Interestingly, the co-expression of DAF and CAR rendered 0.1–0.2% of cells permissive to some E6 strains’ infection. Although our results did not show a major role of the CAR/DAF cooperation for E6 infection, it nevertheless indicated the use of CAR in the cell entry step of some minor E6 quasispecies. Moreover, the present report validates the use of recombinant CHO cells as valuable cellular model for the further characterisation of E6 receptors.

Usefulness of various MRI sequences in the diagnosis of viral encephalitis

There is paucity of studies regarding the utility of various conventional MRI sequences in the diagnosis of viral encephalitis. The present study evaluates the usefulness of various MRI sequences in acute viral encephalitis. 88 consecutive viral encephalitis patients, aged 2–72 years were subjected to clinical evaluation. Consciousness was assessed by Glasgow Coma Scale (GCS). Serum or cerebrospinal fluid (CSF) was analyzed for dengue, Japanese encephalitis (JE), herpes, measles, echo, coxsackie and polio viruses using ELISA or PCR. Cranial MRI was done and T1, T2, FLAIR and DW images were obtained. The MRI changes were correlated with type of encephalitis and duration of illness. All the patients had altered sensorium and 37 had seizures. 22 patients had JE, 9 had dengue, 8 had herpes simplex encephalitis (HSE), 2 had Epstein-Barr virus encephalitis (EBVE) and 47 had non-specific encephalitis. The median duration of MRI study from onset was 10 days. In JE (20/22), HSE (8/8), and EBVE (2/2), MRI abnormalities were more common compared to dengue (2/9) and non-specific (20/47) encephalitis. The MRI abnormalities were more common in FLAIR (57.1%) compared to T2 (52.9%), DWI (38.1%) and T1 (19.3%) sequences. The mean ADC value in JE patients was lower (974.0 ± 110.85 ×10 −6 mm 2/s) than HSE (1024.33 ± 485.76 × 10 −6 mm 2/s). Additional MRI lesions were seen in 12.6% cases on FLAIR sequence. FLAIR and T2 sequences were more sensitive in revealing abnormalities in viral encephalitis.

Etiology analysis of 165 infant patients with viral pneumonia

Objective To comprehend etiology and clinical manifestation changes of infant pneumonia in this locality.Methods Indirect immunofluorescence (IIF) assay was applied in children with acute pneumonia to detect serum 11 kinds of viruses[respiratory syncytial virus (RSV),adenovirus (ADV),influenza virus (IFV-A+B),parain fluenza virus(PIV14) ,coxsackie B,virus(CB1V),Coxsackie A7 virus (CA7V) ,ECHO virus]specific antibody IgM,according to the serum virus-specific IgM positive,C-reactive protein(CRP)<8mg/L and no other pathogenic infection and laboratory evidence for the conditions of 436 cases detected in children with pneumonia.Results Detected a total 125 cases of antibody-positive,the positive detection rate is 37.99%.Of which 103 cases of single virus infection .accounting for 82.4% ,22 cases of mixed infection,accounting for 17.6%.RSV infection on top of the list followed by the rest of IFV,ADV and PIV.Infants of different ages,different seasons of the different types of virus susceptibility.Conclusion Pneumonia in infants were caused by pathogenic bacteria in addition to the virus of a wide range,and the incidence of age,the peak seasons and the clinical manifestations were vary.From an early stage of infection pathogen detection,clearing pathogen type,making the correct diagnosis of pneumonia in the treatment of infants had an important guiding significance. Key words: Infant; Pneumonia; Virulence; Analyses of viral etiology