Enterovirus isolation from children with acute respiratory infections and presumptive identification by a modified microplate method

AbstractObjective: To evaluate a modified microplate method, utilizing HEF, HEp-2, Vero, MDCK and newly introduced RD-18S and GMK cell lines, for virus isolation.Methods: From June to October 2001, 723 throat swab specimens taken from children with acute respiratory infections (ARIs) were inoculated onto these cells. To analyze cell sensitivity, we also inoculated 20 serotypes of stocked enteroviruses.Results: During the period, we isolated 40 Coxsackie A2 (CoxA2), 13 CoxA4, 16 CoxA16, 1 CoxB2, 11 CoxB3, 2 CoxB5, 54 echo16, 2 entero7l and 1 polio2. By observing a cell sensitivity pattern with HEF, HEp-2, Vero, RD-18S, and GMK, we could finally differentiate five enterovirus groups: CoxA except for CoxA16, CoxA16/entero7l, CoxB, echovirus, and poliovirus.Conclusions: With this system, the RD-18S cell line enabled us to isolate CoxA virus, except for CoxA16, for the first time. Differentiation of five enterovirus groups by cell sensitivity simplified the specific identification by neutralization test as a presumptive identification. A modified microplate method may be an appropriate cell combination for virus isolation, especially for enteroviruses, and is expected to be used routinely for virologic diagnosis and to clarify the epidemiology of ARI in children

20１１ネン カラ ２０１３ネン ノ ヤマガタケン ニ オケル ミッツ ノ コトナル イデンシガタ ノ ハイエン マイコプラズマ ノ チイキテキ ヒロガリ ト マクロライド タイセイ カブ シュツゲン ノ タイミング ニ カンスル ケントウ

Background: We previously revealed that several multiple-locus variable-number tandem-repeat analyses（MLVA）and P1 types of Mycoplasma pneumoniae（M. pneumoniae）cocirculated between 2011 and 2013 in Yamagata, Japan. However, the regional spread of M. pneumoniae infection by genotype is not reported yet. It remains unclear whether there is a difference in the spread of macrolide-resistant M. pneumoniae among genotypes. Methods: Genotypes were labeled according to 4-locus（Mpn 13, 14, 15, and 16）MLVA and P1 types. A total of 208 strains belonging to three major genotypes, i.e., type 4-5-7-2, 1; 4-5-7-3, 1; and 3-5-6-2, 2c, were analyzed by combining with the information of macrolide resistance-associated mutation and the patients’ information including residence. Results and Discussion: The three genotypes were widely distributed over more than four cities and towns in Yamagata Prefecture, cocirculating between late 2011 and early 2013, and there was little difference in the duration of their epidemics. Timing of macrolide-resistant strain appearance during the epidemic period differed between type 4-5-7-2, 1 and type 4-5-7-3, 1, and it did not appear throughout type 3-5-6-2, 2c epidemic. These genotypic differences can account for the variation in the prevalence of macrolide resistance-associated mutations in each of the studied areas

A hydroxypropyl methylcellulose plaque assay for human respiratory syncytial virus

ウイルスの研究において感染性のあるウイルス粒子を正確に定量することは，病原性の評価のみならずウイルスに対する新規治療薬の効果を客観的に評価するために極めて重要な手技である．プラークアッセイ法はウイルス粒子を定量するために不可欠な技術であるが，Respiratory syncytial virus（RSV）において，プラークアッセイ法を安定して行うことはしばしば困難である．今回我々は，overlay material（充填化合物）が細胞増殖に与える影響を中心にRSVおよびhuman metapneumovirus（hMPV）におけるプラークアッセイ法の最適化の検討を行った

Heparan sulfate attachment receptor is a major selection factor for attenuated enterovirus 71 mutants during cell culture adaptation.

Enterovirus 71 (EV71) is a causative agent of hand, foot, and mouth disease (HFMD). However, this infection is sometimes associated with severe neurological complications. Identification of neurovirulence determinants is important to understand the pathogenesis of EV71. One of the problems in evaluating EV71 virulence is that its genome sequence changes rapidly during replication in cultured cells. The factors that induce rapid mutations in the EV71 genome in cultured cells are unclear. Here, we illustrate the population dynamics during adaptation to RD-A cells using EV71 strains isolated from HFMD patients. We identified a reproducible amino acid substitution from glutamic acid (E) to glycine (G) or glutamine (Q) in residue 145 of the VP1 protein (VP1-145) after adaptation to RD-A cells, which was associated with attenuation in human scavenger receptor B2 transgenic (hSCARB2 tg) mice. Because previous reports demonstrated that VP1-145G and Q mutants efficiently infect cultured cells by binding to heparan sulfate (HS), we hypothesized that HS expressed on the cell surface is a major factor for this selection. Supporting this hypothesis, selection of the VP1-145 mutant was prevented by depletion of HS and overexpression of hSCARB2 in RD-A cells. In addition, this mutation promotes the acquisition of secondary amino acid substitutions at various positions of the EV71 capsid to increase its fitness in cultured cells. These results indicate that attachment receptors, especially HS, are important factors for selection of VP1-145 mutants and subsequent capsid mutations. Moreover, we offer an efficient method for isolation and propagation of EV71 virulent strains with minimal selection pressure for attenuation

Growth Kinetics of Influenza C Virus Antigenic Mutants That Escaped from Anti-Hemagglutinin Esterase Monoclonal Antibodies and Viral Antigenic Changes Found in Field Isolates

The antigenicity of the hemagglutinin esterase (HE) glycoprotein of influenza C virus is known to be stable; however, information about residues related to antigenic changes has not yet been fully acquired. Using selection with anti-HE monoclonal antibodies, we previously obtained some escape mutants and identified four antigenic sites, namely, A-1, A-2, A-3, and Y-1. To confirm whether the residues identified as the neutralizing epitope possibly relate to the antigenic drift, we analyzed the growth kinetics of these mutants. The results showed that some viruses with mutations in antigenic site A-1 were able to replicate to titers comparable to that of the wild-type, while others showed reduced titers. The mutants possessing substitutions in the A-2 or A-3 site replicated as efficiently as the wild-type virus. Although the mutant containing a deletion at positions 192 to 195 in the Y-1 site showed lower titers than the wild-type virus, it was confirmed that this region in the 190-loop on the top side of the HE protein is not essential for viral propagation. Then, we revealed that antigenic changes due to substitutions in the A-1, A-3, and/or Y-1 site had occurred in nature in Japan for the past 30 years. These results suggest that some residues (i.e., 125, 176, 192) in the A-1 site, residue 198 in the A-3 site, and residue 190 in the Y-1 site are likely to mediate antigenic drift while maintaining replicative ability

Anti-influenza virus activity of two extracts of the blackcurrant (ribes nigrum L.) from new zealand and poland

We investigated the inhibitory effect of extracts of blackcurrant (Ribes nigrum L.) from New Zealand and Poland on 4 strains of influenza virus (IFV) by the inhibition of virus adsorption; pandemic flu from 2009-2010 (IFV-AH1pdm), Hong Kong flu (IFV-AH3), oseltamivir phosphate-resistant Russian flu (IFV-AH1tam(r)) and influenza virus type B (IFV-B). The inhibitory effect of the extracts of blackcurrant or blueberry on the infectivity of the virion were evaluated by the inhibition of virus adsorption on the cell surface (adsorption-inhibitory assay). Three percent solutions of the blackcurrant extracts from New Zealand and Poland were enough to disinfect more than half of IFV-AH1pdm and IFV-B, and 10% solutions from both regions disinfected all IFV strains completely. Our previous study showed that the antiviral effect of the blackcurrant differed according to viral species. Here we showed that although the antiviral effect of Blackcurrant was slightly different within viral strains from one species, the extract of Blackcurrant could disinfect all of 4 IFV strains we examined. The extracts of blackcurrant showed definite potential for use as a disinfectant and antiseptic agent to prevent IFV infection

Neutralizing Epitopes and Residues Mediating the Potential Antigenic Drift of the Hemagglutinin-Esterase Protein of Influenza C Virus

We mapped the hemagglutinin-esterase (HE) antigenic epitopes of the influenza C virus on the three-dimensional (3D) structure of the HE glycoprotein using 246 escape mutants that were selected by a panel of nine anti-HE monoclonal antibodies (MAbs), including seven of the C/Ann Arbor/1/50 virus and two of the C/Yamagata/15/2004 virus. The frequency of variant selection in the presence of anti-HE MAbs was very low, with frequencies ranging from 10−4.62 to 10−7.58 for the C/Ann Arbor/1/50 virus and from 10−7.11 to 10−9.25 for the C/Yamagata/15/2004 virus. Sequencing of mutant HE genes revealed 25 amino acid substitutions at 16 positions in three antigenic sites: A-1, A-2, and A-3, and a newly designated Y-1 site. In the 3D structure, the A-1 site was widely located around the receptor-binding site, the A-2 site was near the receptor-destroying enzyme site, and the Y-1 site was located in the loop on the topside of HE. The hemagglutination inhibition reactions of the MAbs with influenza C viruses, circulating between 1947 and 2016, were consistent with the antigenic-site amino acid changes. We also found some amino acid variations in the antigenic site of recently circulating strains with antigenic changes, suggesting that viruses that have the potential to alter antigenicity continue to circulate in humans