Coxsackievirus B5 Strains Research Articles

Characterisation of new animal cell cultures’ sensitivity to <i>Coxsackievirus B5</i> and <i>Herpes simplex virus‑1</i>

The increase in the number of cell cultures for virology and biotechnology enhances the chances of a successful response to threats related to outbreaks of well-known and new human infectious diseases. It is a vital task to search for cell cultures sensitive to a wide spectrum of viruses.The aim of the study was to investigate the sensitivity of new diploid animal cell cultures (fibroblasts of a foetal pig’s kidneys and larynx) to Coxsackievirus B5 (CVB5) and Herpes simplex virus-1 (HSV-1).Materials and methods. The cultures of porcine foetal kidney fibroblasts (PFKF) and porcine foetal larynx fibroblasts (PFLF) were derived from a foetus of a healthy pig by mild trypsinisation. The study determined the sensitivity of these new PFKF and PFLF cultures to the above-mentioned viruses by the cytopathic effect (CPE) expressed as a percentage. The infectious activity of CVB5 was studied using real-time polymerase chain reaction (PCR) with the determination of amplification cycle threshold values (Ct); that of HSV-1 was studied using quantitative titration of the virus-containing liquid (VCL). Infectious activity values were expressed as tissue culture 50% infective doses (TCID50).Results. The authors developed diploid PFKF and PFLF cell cultures. PFKF cells demonstrated high sensitivity to CVB5, with a CPE of 87.5±3.3% after passage 3 and a satisfactory concentration of enterovirus RNA in the VCL of 22–24 Ct . The sensitivity of PFKF cells to HSV-1 corresponded to a CPE of 92.1±5.5%. In these cells, the infectious activity of HSV-1 corresponded to 104.25 TCID50/0.2 mL. The experiments with PFLF cells showed low CPE and infectious activity values for both viruses.Conclusions. The study demonstrated high CPE values with the CVB5 (CB5-8100) and HSV-1 (HSV-1/L-2) strains as examples and confirmed the sensitivity of the new diploid PFKF cell culture to these test viruses. Thus, the PFKF cell culture offers potential applications in virology and biotechnology and may be a candidate for testing other strains of CVB5 and HSV-1.

Epidemiological and etiological characteristics of viral meningitis for hospitalized pediatric patients in Yunnan, China.

Background:Viral infection is the most common cause of aseptic meningitis. The purpose of this study was to identify the viruses responsible for aseptic meningitis to better understand the clinical presentations of this disease.Method:Between March 2009 and February 2010, we collected 297 cerebrospinal fluid specimens from children with aseptic meningitis admitted to a pediatric hospital in Yunnan (China). Viruses were detected by using “in house” real-time quantitative polymerase chain reaction or reverse-transcription real-time quantitative polymerase chain reaction from these samples. Phylogenetic analyses were conducted using the Molecular Evolutionary Genetic Analysis version 7.0 software, with the neighbor-joining method.Results:Viral infection was diagnosed in 35 of the 297 children (11.8%). The causative viruses were identified to be enteroviruses in 25 cases (71.4%), varicella-zoster virus in 5 cases (14.3%), herpes simplex virus 1 in 2 cases (5.7%), and herpes simplex virus 2, Epstein–Barr virus, and human herpesvirus 6 in 1 case each (2.9% each). Of the enteroviruses, coxsackievirus B5 was the most frequently detected serotype (10/25 cases; 40.0%) and all coxsackievirus B5 strains belonged to C group.Conclusions:In the study, a causative virus was only found in the minority of cases, of them, enteroviruses were the most frequently detected viruses in patients with viral meningitis, followed by varicella-zoster virus and herpes simplex virus. Our findings underscore the need for enhanced surveillance and etiological study of aseptic meningitis.

Etiology and clinical analysis of central nervous system infection caused by Coxsackievirus B5 in severe hand, foot and mouth disease in Qingdao City, 2013-2014

Objective: To illuminate the gene characteristics and clinical characterization of Coxsackievirus B5 (CV-B5) strains isolated from patients with sevre hand, foot and mouth disease (HFMD) in Qingdao city. Methods: A total of 1 844 patients of HFMD were consecutively admitted to Qingdao Women and Children's Hospital from 2013 to 2014. Information of the study population described above was collected retrospectively. The samples were collected from at least 1 site (throat swab, cerebrospinal fluid), which viral nucleic acid extracted and the entire VP1 gene sequences of CV-B5 isolates were amplified and sequenced, then the homology and phylogeny analysis were conducted by MEGA7.0. The prototype Faulkner strain and other VP1 amino acid sequences were derived from the GenBank database. Results: A total of 8 CV-B5 positive cases were obtained, including 4 males and 4 females; 6 severe hospitalized cases and 2 outpatients. The age of 6 hospitalized patients ranged from 3 to 48 months, with a median of 26 months. For the six inpatients, fever, convulsions vomiting, diarrhea and rash were the main clinical manifestation, and all combined with viral encephalitis. Compared with the prototype strain Faulkner, in the VP1 region,the nucleotide and the amino acid homologies was 77.3%-78.8% and 95.5%-97.0% respectively. Five out of the six severe cases with substitution of serine (S) to asparagine (N) at amino acid site 95 in the VP1 region. The sequences of 8 CV-B5 strains were classified into genogroup D. Conclusion: Hand, foot and mouth disease associated with CV-B5 virus infection can result in nervous system involvement and the main complication was viral encephalitis. The CV-B5 strains associated with severe hand, foot and mouth disease had high nucleotide homology and present a certain regional aggregation.

Full-length genome analysis of a coxsackievirus B5 strain isolated from Shandong Province in 2014

Full-length genome analysis of a coxsackievirus B5 strain isolated from Shandong Province in 2014

Antiviral Activity of Synthetic Peptides Derived from Physiological Proteins

Background/Aims: New antivirals are needed to supplement or replace currently used drugs. The aim of this study was to evaluate the antiviral activity of synthetic peptides derived from physiological proteins. Methods: Vero cell monolayers were infected with herpes simplex virus 1, vesicular stomatitis virus, adenovirus, and coxsackievirus B5 strains in the presence of different concentrations of the selected peptides and viral yield was determined by plaque reduction assays to evaluate the antiviral activity of the peptides. Virucidal activity was evaluated by determining the residual infectivity of viral suspensions treated for 1 h with the peptides at the same concentrations as in the viral yield assays. Results: Among the investigated peptides, the killer peptide proved to exert a considerable antiviral activity against herpes simplex virus, attributable to a direct effect on virus particles, while its derivative K10S showed to be effective against the four investigated virus strains only at the highest concentration tested, yet, the inhibitory effects were only partial. Conclusion: Overall, initial evidence is provided on the antiviral activity of several peptides, as well as of their derivatives. Further investigation is warranted to ascertain the mechanism of action in order to develop new potential antiviral drugs.

Serotypes of HFMD-associated HEV-B species and genetic characteristics of the VP1 gene in coxsackievirus B2 and B5 strains isolated in Anyang area from 2011 to 2015

Objective To investigate the serotypes of human enterovirus B (HEV-B) species causing hand, foot and mouth disease (HFMD) and to analyze the genetic characteristics of VP1 region in coxsackievirus B2 (CVB2) and coxsackievirus B5 (CVB5) strains circulating in Anyang area during 2011 to 2015. Methods Real-time RT-PCR and semi-nested RT-PCR were performed to identify coxsackievirus A16 (CVA16), enterovirus 71 (EV71) and other serotypes of enterovirus in order to obtain the complete etiologic composition of HFMD. The numbers of HEV-B serotypes and the percentages of specimens positive for every serotype in all enterovirus-positive specimens were calculated. As CVB2 and CVB5 were the predominant serotypes of HEV-B species, five pairs of primers targeting the VP1 regions of CVB2 and CVB5 were designed to obtain the complete nucleotide sequences of CVB2 and CVB5 VP1 regions. The phylogenetic trees were constructed based on the VP1 sequences obtained in this study and those submitted to GenBank by using MEGA7.0 and BioEdit7.2. The selection pressures on VP1 regions of CVB2 and CVB5 strains circulating in China in recent years were evaluated with the online program of DataMonkey. Results A total of 57 specimens that belonged to 14 serotypes of HEV-B species were detected in Anyang area from 2011 to 2015. The 14 serotypes of HEV-B species accounted for 56% of all serotypes of enterovirus and the specimens positive for HEV-B species accounted for 3.06% of all enterovirus-positive specimens. The HFMD cases caused by most of the HEV-B serotypes were sporadic cases. Small outbreaks of HFMD could also be caused by some serotypes of HEV-B such as CVB2 and CVB5. The complete sequences of VP1 region were obtained from 8 CVB2 strains and 9 CVB5 strains. The phylogenetic trees based on the VP1 sequences demonstrated that the CVB2 strains were classified into four genotypes (A-D). The mean evolutionary distances between different genotypes ranged from 0.191 to 0.208 and the similarities in nucleotide sequences ranged from 79.7% to 85.8%. The CVB5 strains were classified into 6 genotypes (A-F). The mean evolutionary distances and the similarities in nucleotide sequences between different genotypes of CVB5 strains ranged from 0.170 to 0.285 and 76.0% to 86.8%, respectively. Strains of different genotypes varied significantly in the residues on positons 157 and 263 in the VP1 region of CVB2 strains and on positions 75, 90 and 95 in the VP1 region of CVB5 strains. All of the CVB2 strains isolated in Anyang area belonged to D genotype and located intensively in one lineage. The CVB5 strains circulated in Anyang area belonged to F genotype and located in two lineages. The selection pressures on CVB2 strains of D genotype and CVB5 strains of F genotype circulating in China in recent years were 0.037 and 0.036, respectively. Six positively selected amino acid sites were found in the VP1 region of CVB5 strains, but no positively selected amino acid site was found in the VP1 region of CVB2 strains. Conclusion HEV-B species was an essential component of the etiologic spectrum of HFMD in Anyang area during 2011 to 2015, of which CVB5 and CVB2 were the predominant serotypes. The VP1 region of CVB5 was more complex and active than that of CVB2 over the course of evolution. Key words: Hand, foot and mouth disease; Enterovirus; Coxsackievirus B2; Coxsackievirus B5; Phylogenetic tree; Positive selection pressure

Epidemiological survey and rapid etiological identification of an clustered viral encephalitis in Jinan, 2014

Objective To rapidly identify the pathogen of a clustered viral encephalitis in Jinan, 2014, and to analyze the epidemiological and etiological characteristics. Methods Uniform questionnaire was used to investigate each case.Both stool and cerebrospinal fluid specimens of 4 children were collected.At first, real-time fluorescence quantitative(RT) PCR was performed to detect the viral nucleic acid of pan-enterovirus(PE), enterovirus 71(EV71), coxsackievirus A16(CVA16), coxsackievirus A10(CVA10)and coxsackievirus A6(CVA6). Then nested reverse transcription-PCR based on 5'-UTR gene of enterovirus was conducted for the PE positive specimens.The PCR products were sequenced and homologous analysis were performed. Results The initial case appeared on the April 8, 2014 in the from of clustered viral encephalitis and the transmission mode was person-to-person.The RT-PCR results showed that PE was positive for all the specimens, while others including EV71, CVA16, CVA10 and CVA6 were negative.Analysis based on 5'-UTR through Basic Local Alignment Search Tool(http: //blast.ncbi.nlm.nih.gov/Blast.cgi) sequences indicated that coxsackievirus B5(CVB5) was the main pathogen and 99.2%-100.0% homology from each other.Data from homologous comparisons showed that these sequences had homology of 96.7%-97.4% with Henan CVB5 strain(HQ998851). These 4 children all recovered after the treatment with antiviral medications, anti-infective agents, and supportive therapy. Conclusions CVB5 was responsible for the clustered viral encephalitis in Jinan, 2014 and it had high homology with Henan CVB5 strain.RT-PCR and nested reverse transcription-PCR are rapid and effective methods to identify viral encephalitis caused by enterovirus. Key words: Viral encephalitis; Coxsackievirus B5; Real-time fluorescence quantitative polymerase chain reaction; Nested reverse transcription-polymersae chain reaction

Genome sequences of coxsackievirus B5 isolated from viral encephalitis patients in Henan province, 2011

To analyze the complete genome sequences of two coxsackievirus B5 (CVB5) isolated in Henan province, 2011. Specimens were collected from viral encephalitis patients and followed by viral isolation on them. RNA were extracted from positive isolates and the amplified products were sequenced. The full-length genomes of them were acquired by assembling the fragments, using DNAStar 5.01 software while phylogenetic analysis were performed with Mega 5.05 and other software. The genomes RNA of 03001N and 17Y showed 7408 bp and 7404 bp long, and the 5'- and 3'-untranslated regions were 747 bp, 743 bp and 103 bp, 103 bp, respectively. BLAST analysis of these two isolates, based on the complete genome, showed 97% identity, with both of them having the highest similarity(98%, 99%)to the CVB5 strain isolated from Henan in 2010 rather than other CVB5 strains. Coding regions of both isolates were 6558 bp, code for a polyprotein of 2185 amino acids (aa) and both of them showed 99% amino acid identity. Phylogenetic tree in VP1 region showed that the two isolates belonged to the same clade with other strains isolated from all over the country in the past years, except for some CVB5 strains isolated from Henan and Shandong province in 2009 that formed the other cluster. It seemed that more than one group of CVB5 were circulating in Henan province and these two isolates appeared the main epidemic strains circulating in the past years.

Recombination in human coxsackievirus B5 strains that caused an outbreak of viral encephalitis in Henan, China

In 2011, human coxsackievirus B5 (CVB5) caused an outbreak of viral encephalitis in Henan, China. Complete genome sequence analysis based on two isolates showed that the 5' half of the genome (nt 1-4540) had high similarity (>97%) to that of CVB5 strain GU376747, and the 3' half (nt 4700-7402) showed high similarity (>96%) to that of human coxsackievirus B3 (CVB3) strain GQ141875. These isolates had the highest similarity (97.7%) to the Changchun strain, based on the complete genome, rather than to other CVB5 strains isolated from Henan in recent years. There were therefore at least two groups of CVB5 circulating in Henan Province, which evolved at different rates.

Full Genome Sequence of a Novel Coxsackievirus B5 Strain Isolated from Neurological Hand, Foot, and Mouth Disease Patients in China

Coxsackievirus B5 (CVB5) belongs to the human enterovirus B species within the family Picornaviridae. We report the complete genome sequence of a novel CVB5 strain, CVB5/SD/09, that is associated with neurological hand, foot, and mouth disease in China. The complete genome consists of 7,399 nucleotides, excluding the 3' poly(A) tail, and has an open reading frame that maps between nucleotide positions 744 and 7301 and encodes a 2,185-amino-acid polyprotein. Phylogenetic analysis based on different genome region regions reveals that CVB5/SD/09 belongs to a novel CVB5 lineage, and similarity plotting and bootscanning analysis based on the whole genome of CVB5 in the present study and those available in GenBank indicate that the genome of CVB5/SD/09 has a mosaic-like structure, suggesting that recombination between different CVB5 strains may occur.

Epidemics of enterovirus infection in Chungnam Korea, 2008 and 2009.

Previously, we explored the epidemic pattern and molecular characterization of enteroviruses isolated in Chungnam, Korea from 2005 to 2006. The present study extended these observations to 2008 and 2009. In this study, enteroviruses showed similar seasonal prevalent pattern from summer to fall and age distribution to previous investigation. The most prevalent month was July: 42.9% in 2008 and 31.9% in 2009. The highest rate of enterovirus-positive samples occurred in children < 1-year-old-age. Enterovirus-positive samples were subjected to sequence determination of the VP1 region, which resolved the isolated enteroviruses into 10 types in 2008 (coxsackievirus A4, A16, B1, B3, echovirus 6, 7, 9, 11, 16, and 30) and 8 types in 2009 (coxsackievirus A2, A4, A5, A16, B1, B5, echovirus 11, and enterovirus 71). The most prevalent enterovirus serotype in 2008 and 2009 was echovirus 30 and coxsackievirus B1, respectively, whereas echovirus 18 and echovirus 5 were the most prevalent types in 2005 and 2006, respectively. Comparison of coxsackievirus B1 and B5 of prevalent enterovirus type in Korea in 2009 with reference strains of each same serotype were conducted to genetic analysis by a phylogenetic tree. The sequences of coxsackievirus B1 strains segregated into four distinct clusters (A, B, C, and D) with some temporal and regional sub-clustering. Most of Korean coxsackievirus B1 strains in 2008 and 2009 were in cluster D, while only "Kor08-CVB1-001CN" was cluster C. The coxsackievirus B5 strains segregated in five distinct genetic groups (clusters A-E) were supported by high bootstrap values. The Korean strains isolated in 2001 belonged to cluster D, whereas Korean strains isolated in 2005 and 2009 belonged to cluster E. Comparison of the VP1 amino acid sequences of the Korean coxsackievirus B5 isolates with reference strains revealed amino acid sequence substitutions at nine amino acid sequences (532, 562, 570, 571, 576-578, 582, 583, and 585).

Update on molecular characterization of coxsackievirus B5 strains

Among Coxsackie B viruses, Coxsckievirus B5 is one of the most predominant serotypes in human, it is frequently associated with cases of neurological diseases, epidemics of meningitis and is a common cause of cardiomyopathy and diabetes. In the present study 27 isolates of Coxsackievirus B5 from North Africa, obtained from cerebrospinal fluid and stool samples of healthy individuals, patients with acute flaccid paralysis or aseptic meningitis were investigated by partial sequencing in the 5' half of the VP1 region and compared to the up-to-date published Coxsackievirus B5 sequences in the same genomic region. Four distinct genomic groups and ten different clusters were individualized. Most of the isolates from Algeria and Tunisia belonged to two clusters. For both, the sequences from North Africa clustered mainly with sequences from European countries, the majority isolated recently during the 2000s. The analysis of the alignment of amino-acids sequences in the VP1 gene revealed four major substitutions in strains from different clusters, we also noticed changes in the BC-loop region; this region is associated with viral antigenicity. This study permit to better identify circulating Coxsackievirus B5 strains throughout the world and their genetic relationship. The protein analysis showed changes that could imply some antigenic significance. J. Med. Virol. 83:1247-1254, 2011. © 2011 Wiley-Liss, Inc.

Genetic variation of coxsackie virus B5 strains associated with aseptic meningitis in Greece

In order to explore the genetic relationships among coxsackie virus B5 strains in Greece, the nucleotide sequences of the partial VP1 gene in strains isolated from aseptic cases of meningitis were determined and compared with those of strains isolated from other countries. Phylogenetic analysis showed a high degree of divergence (25%) among Greek strains isolated in different years, which clustered with high bootstrap values in a different subgroup of viruses, suggesting that enterovirus types vary with time rather than geographical distribution. A non-synonymous mutation present in the strains of this study was not observed in other coxsackie virus B5 strains.