Identification of a common conserved neutralizing linear B-cell epitope in the VP3 protein of waterfowl parvoviruses

ABSTRACT

Waterfowl parvoviruses (WPVs) including goose parvovirus (GPV), novel GPV-related virus (NGPV) and Muscovy duck parvovirus (MDPV) cause significant economic losses and epizootic threat to the waterfowl industries, and little is known about the B-cell epitopes of WPVs. In this study, a monoclonal antibody (mAb) 5B5 against the VP3 protein of NGPV was used to identify the possible epitope in the three kinds of WPVs. The mAb 5B5 had neutralizing activities to the three viruses, and reacted with the conserved linear B-cell epitopes of ⁴³⁸LHNPPP⁴⁴³ in VP3 protein of GPV, NGPV and MDPV. To the authors’ best knowledge, this is the first report on identification of the common conserved neutralizing linear B-cell epitope on VP3 protein of three different WPVs, which would facilitate the development of a novel immunodiagnostic assay for rapid detection of WPV infection.     

Isolation and molecular characterization of a new Muscovy duck parvovirus from Muscovy ducks in the USA.

Between 1997 and 1999 several cases of a new disease in Muscovy ducks were reported in Pennsylvania, USA. The cases were characterized by locomotor dysfunction, weakness, recumbency, 40 to 60% morbidity and 10 to 40% mortality. The most characteristic microscopic lesions were moderate to severe degenerative rhabodomyopathy. In order to characterize the aetiological agent, virus isolation was attempted from the spleen, liver, heart, skeletal muscle and intestine by inoculation of 14-day-old Muscovy duck embryos with tissue homogenates. Deaths occurred on the second egg passage and parvoviruses were isolated by serial passage of allantoic fluid from dead embryos and then in Muscovy duck embryo fibroblast (MDEF) cultures. Parvovirus particles were observed in allantoic fluids and supernatants of MDEF cultures by transmission electron microscopy. Two genomic fragments, comprising 1108 nucleotides of the right open reading frame that codes for the structural viral proteins 1, 2 and 3, were amplified by polymerase chain reaction from one of the isolates, Muscovy duck parvovirus (MDPV)/PSU-31010. Comparison of this fragment with available sequences of other MDPV and related goose parvovirus (GPV) isolates showed that it had only 84.5% sequence identity with other MDPV isolates and 84.6% identity with the GPV isolates. This region shares over 99% identity among previously sequenced MDPV isolates and 95% identity among the related GPV isolates. This suggests that MDPV/PSU-31010 is divergent from all other sequenced MDPV and GPV isolates, and may represent a new group of avian parvoviruses.     

120例儿科患儿微小病毒B19感染的近期研究

目的 探讨人类微小病毒B19（HPVB19）在儿科疾病中的感染状况.方法 应用巢式聚合酶链反应的方法（PCR）以及ELISA法,对120例患儿（观察组）及40例随机挑选本院门诊健康体检儿童（对照组）血浆中微小病毒B19-DNA以及B19-VP2-IgM检测.结果 120例患儿（观察组）B19-DNA检测总阳性检出率为31.7%（38/120）,40例健康儿童（对照组）B19-DNA检测均为阴性,两组比较有显著差别（P＜0.005）.其中特发性血小板减少性紫癜（ITP）和心肌炎阳性检出率最高,分别为40%（12/30）和37.1%（13/35）.120例患儿（观察组）B19-VP2- IgM总阳性检出率31.7% （38/120）,40例健康儿童（对照组）B19-DNA检测均为阴性,两组比较有显著差别（P＜0.005）,B19 DNA和B19 VP2 IgM一致率为100%.40例健康儿童（对照组）B19-VP2- IgM均为阴性,两组比较有显著差别（P＜0.005）.结论 儿童对B19有较高的感染率,尤其ITP和心肌炎患儿与B19感染关系更为密切.     

Dissecting the Structure and Mechanism of a Complex Duplication–Triplication Rearrangement in the DMD Gene

Pathogenic complex genomic rearrangements are being increasingly characterized at the nucleotide level, providing unprecedented opportunities to evaluate the complexities of mutational mechanisms. Here, we report the molecular characterization of a complex duplication–triplication rearrangement involving exons 45–60 of the DMD gene. Inverted repeats facilitated this complex rearrangement, which shares common genomic organization with the recently described duplication‐inverted triplication–duplication (DUP–TRP/INV‐DUP) events; specifically, a 690‐kb region comprising DMD exons from 45 to 60 was duplicated in tandem, and another 46‐kb segment containing exon 51 was inserted inversely in between them. Taking into consideration (1) the presence of a predicted PRDM9 binding site in the near vicinity of the junction involving two inverted L1 elements and (2) the inherent properties of X–Y chromosome recombination during male meiosis, we proposed an alternative two‐step model for the generation of this X‐linked DMD DUP–TRP/INV‐DUP event.     

The evolution and molecular characteristics of H9N2 avian influenza viruses in Jiangxi of China

To understand the evolution and molecular characteristics of Jiangxi H9N2 viruses, we isolated 17 viruses in 2011 and analyzed their characteristics. Phylogenetic analyses revealed that their hemagglutinin genes originate from JS/1/00-like sublineage, neuraminidase genes originate from BJ/94-like sublineage, PB1, PA, NP, and NS genes all come from SH/F/98-like sublineage, PB2 genes originate from ST/163/04-like sublineage, while M genes come from G1-like sublineage. Genotype analysis showed that our isolates were classified as genotype 57. Molecular analyses indicated that our strains contained specific sites characteristic of low-pathogenic viruses. The current study once again highlights the necessity for continued surveillance of novel H9N2 viruses.     

Conventional and molecular investigation of meningococcal isolates in relation to two outbreaks in the area of Athens, Greece

Two local outbreaks caused by serogroup B Neisseria meningitidis occurred in the Athens area of Greece during 2003. In total, 30 N. meningitidis isolates from patients and carriers, as well as sporadic cases, were investigated by conventional techniques (serogroup, serotype and serosubtype), multilocus sequence typing (MLST), analysis of variable number tandem repeats (VNTR) and random amplified polymorphic DNA (RAPD) analysis. Compared with the two other molecular techniques, VNTR analysis was a simple, reliable and highly discriminatory method for fine typing of meningococcal isolates, showing a good correlation with the epidemiological data for the two outbreaks analysed.     

Multiple clusters of A(H1N1)pdm09 virus circulating in severe cases of influenza during the 2010–2011 season: A phylogenetic and molecular analysis of the neuraminidase gene

The molecular characterization of circulating influenza A viruses is crucial to detect mutations potentially involved in increased virulence, drug resistance and immune escape. A molecular and phylogenetic analysis of A(H1N1)pdm09 neuraminidase (NA) gene sequences from different patient categories defined according to the severity of influenza infection were analyzed. A total of 126 influenza A(H1N1)pdm09 positive samples from patients with severe infections in comparison with those with moderate and mild infections was performed in Lombardy (Northern Italy, nearly 10 million inhabitants) during the 2010–2011 season. NA sequences included in this study segregated into five distinct clusters. Nineteen amino acid substitutions were detected exclusively in NA sequences of viruses identified in patients with severe or moderate influenza infection. Three of them (F74S, S79P, E287K) were observed in virus strains with the 222G/N hemagglutinin mutation. None of NA sequences under study had mutations related to the resistance to the NA inhibitors. Four out of 126 (3.2%) NA sequences from patients with severe infection lost a N‐linked glycosylation site due to the change from N to K at residue 386. Two additional N‐linked glycosylation sites in the NA stalk region (residues 42 and 44) were found in 12 (9.5%) NA sequences. Sporadic NA mutations were detected in NA viral sequences from critically ill patients, and no variants with reduced sensitivity to NA inhibitors were observed either in treated or untreated patients. J. Med. Virol. 85: 944–952, 2013. © 2013 Wiley Periodicals, Inc.     

Antibiotic resistance and molecular epidemiology of the beta‐lactamase‐producing Haemophilus influenzae isolated in Chongqing,China

This study aimed to determine the antibiotic resistance and molecular epidemiology of Haemophilus influenzae isolated from children with acute respiratory infection in Chongqing, China. To this end, 1967 H. influenzae isolates from 2006 to 2009 were analysed regarding β‐lactamase production and antibiotic resistance. Ninety‐nine β‐lactamase‐producing H. influenzae isolates from 2010 were analysed for antibiotic resistance and promoter regions of bla_TEM‐1. β‐lactamase production was found in 35.8% (705/1967) of the strains. All ninety‐nine β‐lactamase‐producing strains from 2010 were of the TEM‐1 type as determined by PCR but did not produce the predicted 1075 bp product. According to PCR‐SSCP and DNA sequencing, the promoter regions of bla_TEM‐1 were categorized into 6 genotypes as SSCP1 (Pdel), SSCP2 (Pa/Pb), SSCP3 (P4), SSCP4 (Prpt.b), SSCP5 (2Prpt) and SSCP6 (P3.b). The Pdel, Pa/Pb and Prpt.b were common promoters of bla_TEM‐1 for H. influenzae isolated from children in Chongqing. Strains with Prpt.b were more resistant to ampicillin (AMP) than strains with Pdel, Pa/Pb and P4 (p < 0.05). Therefore, bla_TEM‐1 β‐lactamase is the main mechanism for resistance of H. influenzae to ampicillin in Chongqing. Furthermore, the Prpt.b promoters may be related to the high resistance of H. influenzae to AMP.     

Forensic characteristics and phylogenetic analysis of 23 Y-STR loci in the Miao population from Guizhou province,southwest China

Background: Investigation of haplotype/allele frequency data of Y-STR loci in ethnically diverse populations is essential for forensic reference database construction and genetic application. However, the population genetic characteristics of the Chinese Miao minority from Guizhou Province remain uncharacterised.

Aim: To assess forensic characteristics for 23 Y-Chromosomal STR loci in Guizhou Miao and explore population genetic relationships with geographically neighbouring populations.

Subjects and methods: Twenty-three Y-Chromosomal STRs were genotyped using the Powerplex^® Y23 system in 103 unrelated Chinese Miao males from Guizhou Province, southwest China. Haplotypes and forensic parameters were obtained. Population relationships of Guizhou Miao with others were revealed using AMOVA and an MDS plot.

Results: A total of 96 haplotypes were identified with overall haplotype diversity (HD) and discrimination capacity (DC) of 0.9985 and 0.9320, respectively. Genetic differentiation was observed with most of the comparison populations, prominently for Guizhou Shui.

Conclusion: The 23 Y-STR loci were highly polymorphic and discriminating in the Guizhou Miao population and could be used for forensic practice and population genetic studies. Population relationship analysis revealed Guizhou Miao had a close genetic relationship with geographically close Guizhou Gelao, as well as Han majorities derived from different regions.     

Two novel types of contiguous gene deletion of the AVPR2 and ARHGAP4 genes in unrelated Japanese kindreds with nephrogenic diabetes insipidus

Study of two families containing individuals with nephrogenic diabetes insipidus (NDI) indicated different types of 21.3 kb and 26.3 kb deletions involving the AVPR2 and ARHGAP4 (RhoGAP C1) genes. In the case of the 21.3 kb deletion, the deletion consensus motif (5′‐TGAAGG‐3′) and polypurine runs, known as the arrest site of polymerase alpha, were detected in the vicinity of the deletion junction. Inverted repeats (7/8 matches), believed to potentiate DNA loop formation, flank the deletion breakpoint. We propose this deletion to be the result of slipped mispairing during DNA replication. In the case of the 26.3 kb deletion, the 12,945 bp inverted region with the 10,003 bp internal deletion was accompanied with the 2,509 bp deletion in the 5′‐side and the 13,785 bp deletion in the 3′‐side. We defined three deletion junctions in this rearrangement (DJ1, DJ2, and DJ3) from the 5′‐side. The surrounding sequence of DJ1 (5′‐CCC‐3′) closely resembled that of DJ3 (5′‐AGGG‐3′) (DJ1; 5′‐cCCCgaggg‐3′, DJ3; 5′‐ccccAGGG‐3′), and DJ1 was located in the 5′‐side of DJ3 without any overlapping in sequence. The immunoglobulin class switch (ICS) motif (5′‐TGGGG‐3′) was found around the complementary sequence of DJ3. There was a 10‐base palindrome (5′‐aGACAtgtct‐3′) in the alignment of the DJ2 (5′‐GACA‐3′) region. From these findings, we propose a novel mutation process with the rearrangement probably resulting from stem‐loop induced non‐homologous recombination in an ICS‐like fashion. Both patients, despite lacking ARHGAP4, had no morphological, clinical, or laboratory abnormalities except for those usually found in patients with NDI. Hum Mutat 19:23–29, 2002. © 2001 Wiley‐Liss, Inc.     

手足口病例中埃克病毒11型河南株分子流行病学研究

目的分析埃克病毒11型（ Echo11）河南分离株的分子流行病学特征。方法对2010-2012年河南手足口病监测系统中采集的粪便标本,采用RD、Hep2细胞进行病毒分离,对阳性分离物进行VP1区扩增、测序,生物学软件分析测序结果,BLAST比对后确定病毒基因型;扩增10株Echo11河南分离株VP1完整编码区序列,与国内外其他Echo11病毒株进行同源性分析并构建VP1区全基因亲缘进化树,分析其进化来源及不同基因型的流行范围。结果2010-2012年河南手足口病标本共分离出184株非EV71/CA16型肠道病毒,其中Echo1110株,占5．43％。 Echo11河南株VP1区全长共876个核苷酸,编码292个氨基酸;10株Echo11河南株VP1区之间核苷酸同源性为93．1％～100％,氨基酸同源性为97．3％～100％;与原型Gregory株之间核苷酸同源性为77．8％～78．8％,氨基酸同源性为90．8％～91．8％。结论 Echo11也是引起河南省手足口病的主要病原体,河南省存在Echo11病毒A基因型的流行。     

Clinical and molecular characteristics of the 2009 pandemic influenza H1N1 infection with severe or fatal disease from 2009 to 2011 in Shenzhen,China

In the past 3 years, the 2009 pandemic influenza virus H1N1 (pH1N1) has led to many severe or fatal cases. The virus‐related factors that cause severe or fatal disease are not clear. The clinical and molecular characteristics of pH1N1 infections with severe or fatal disease were examined to understand the correlation between pH1N1 infection and disease severity. Since 2009, three pH1N1 influenza epidemic outbreaks have occurred in Shenzhen, China. One hundred forty‐six severe cases were confirmed in the first wave in 2009. In severe cases, a high proportion (49.3%) of patents displayed high fever (>39.0°C), and 73.2% of patients had pneumonia and tracheobronchitis. Seven fatal cases were recorded: three with viral encephalitis and four with respiratory failure. The results of sequencing and phylogenetic analysis showed that the viruses from fatal or severe cases were scattered throughout the phylogenetic tree. Four substitutions (D222G, D222N, D222E, and Q223R) were observed on the 220‐loop of the receptor‐binding sites of the HA gene. Both D222G and D222N were associated statistically with severe disease. The 2011 viruses had evolved into two distinct branches. Ten specific point mutations occurred in the 2011 virus. In summary, high fever, lower respiratory tract infections and serious complications were the main features of severe cases. Gene variation seemed not to be the main reason for severe disease. Vaccination is the effective mean to prevent infection and severe disease. J. Med. Virol. 85:405–412, 2013. © 2012 Wiley Periodicals, Inc.