Diagnosis of influenza viruses with special reference to novel H1N1 2009 influenza virus

On 15 April and 17 April 2009, novel swineorigin influenza A (H1N1) virus was identifi ed in specimens obtained from two epidemiologically unlinked patients in the United States. The ongoing outbreak of novel H1N1 2009 influenza (swine influenza) has caused more than 3,99,232 laboratory confi rmed cases of pandemic influenza H1N1 and over 4735 deaths globally. This novel 2009 influenza virus designated as H1N1 A/swine/California/04/2009 virus is not zoonotic swine flu and is transmitted from person to person and has higher transmissibility then that of seasonal influenza viruses. In India the novel H1N1 virus infection has been reported from all over the country. A total of 68,919 samples from clinically suspected persons have been tested for influenza A H1N1 across the country and 13,330 (18.9%) of them have been found positive with 427 deaths. At the All India Institute of Medical Sciences, New Delhi India, we tested 1096 clinical samples for the presence of novel H1N1 influenza virus and seasonal influenza viruses. Of these 1096 samples, 194 samples (17.7%) were positive for novel H1N1 influenza virus and 197 samples (18%) were positive for seasonal influenza viruses. During outbreaks of emerging infectious diseases accurate and rapid diagnosis is critical for minimizing further spread through timely implementation of appropriate vaccines and antiviral treatment. Since the symptoms of novel H1N1 influenza infection are not specifi c, laboratory confi rmation of suspected cases is of prime importance.     

引发2009年流感暴发的H1N1新型流感病毒的研究进展

引起流感世界性大流行的主要原因与流感病毒表面抗原血凝素(HA)和神经氨酸酶(NA)频发的变异有很大关系,抗原的变异使得流感病毒可以逃逸机体的免疫防御,而且使许多应用中的疫苗失去防御效果。综述2009年世界暴发的H1N1新型流感病毒的结构在进化过程中发生的变异,有助于增加人们对流感病毒的了解,从而有效的治疗和预防流感大流行。     

A single-tube nested real-time polymerase chain reaction for sensitive contained detection of Cryptosporidium parvum

Aim:  A new real-time polymerase chain reaction (PCR) was developed for sensitive contained detection of Cryptosporidium parvum .
Methods and Results:  The method is a nested PCR targeting a specific region of rDNA of C. parvum , which takes place in one tube, using different annealing temperatures to control the first and the second rounds of PCR, with real-time fluorogenic probe-based detection of the second round of PCR. The DNA-based detection limit of the method was 2 fg, which corresponds to approx. one genome per reaction. The detection level determined using diluted samples of C. parvum oocysts was ten oocysts per millilitre.
Conclusions:  The method facilitates sensitive detection of C. parvum thanks to the nested format, while reducing the risk of laboratory contamination thanks to the single-tube, real-time fluorimetric format.
Significance and Impact of the Study:  The developed method may be useful for sensitive contained detection of C. parvum in environmental and food samples, after appropriate separation of oocysts.     

Group-specific primer and probe sets to detect methanogenic communities using quantitative real-time polymerase chain reaction

Real-time polymerase chain reaction (PCR) is a highly sensitive method that can be used for the detection and quantification of microbial populations without cultivating them in anaerobic processes and environmental samples. This work was conducted to design primer and probe sets for the detection of methanogens using a real-time PCR with the TaqMan system. Six group-specific methanogenic primer and probe sets were designed. These sets separately detect four orders (Methanococcales, Methanobacteriales, Methanomicrobiales, and Methanosarcinales) along with two families (Methanosarcinaceae and Methanosaetaceae) of the order Methanosarcinales. We also designed the universal primer and probe sets that specifically detect the 16S rDNA of prokaryotes and of the domain Bacteria and Archaea, and which are fully compatible with the TaqMan real-time PCR system. Target-group specificity of each primer and probe set was empirically verified by testing DNA isolated from 28 archaeal cultures and by analyzing potential false results. In general, each primer and probe set was very specific to the target group. The primer and probe sets designed in this study can be used to detect and quantify the order-level (family-level in the case of Methanosarcinales) methanogenic groups in anaerobic biological processes and various environments.     

新世纪流感大流行的思考

2009年从墨西哥开始暴发了一场席卷全世界的流感疫情．此次大流行的毒株,甲型H1N1病毒,包含了猪源、禽源和人源流感病毒的基因片段．研究该毒株的基因重配、进化历程及其生物学特性,将对防控此次流行具有重要意义．目前,该毒株的遗传进化关系已明确,通过遗传性状分析可获知该毒株可能的生物学性状,但流感大流行动向、毒株遗传变化、毒力及致病性变化仍在密切监控中．流感病毒生态系统具有复杂性,其基因组易突变、易重配、易在自然宿主保存,使得流感大流行存在一定的必然性．正视流感大流行的威胁,积极提高流感病毒在生态系统中的监控,加强流行病学调查,发展疫苗与药物,建立有效公共卫生保障体系,才能降低流感大流行的破坏性．     

A quantitative polymerase chain reaction assay for detecting and identifying fungal contamination in human allograft tissue

To complement donor selection and tissue processing, rapid and reliable detection, discrimination, and quantification of fungal pathogens are extremely important for tissues destined to be implanted into humans. The current detection method for fungal pathogens, in particular, is difficult and time-consuming. Quantitative polymerase chain reaction (qPCR) technology is considered one of the most sensitive methods to detect low levels of DNA. Here a qPCR method is described that can detect clinically relevant, pathogenic fungal organisms. The assay allowed the quantification of fungal organisms within a tissue implant and provides a means to identify the contaminating species. The primers for the qPCR assay were designed to amplify a conserved region of the L2 region of the large ribosomal subunit (LSU) gene. This set of primers was able to detect fewer than 10 colony forming units from Aspergillus and Candida species in spiked samples. Clinical samples were also evaluated using this method and the data compared positively to the existing accepted 28-day fungal culture method for fungal detection. The qPCR method described herein significantly reduced the time required to identify fungal contamination in tissue implants.     

The use of a quantitative real-time polymerase chain reaction assay for identification and enumeration of Lactobacillus buchneri in silage

Aims:  To detect and quantify Lactobacillus buchneri in plant samples with the aid of polymerase chain reaction (PCR) methods.
Methods and Results:  DNA from silage samples spiked with different amounts of L. buchneri cells was isolated using a lysozyme/sodium dodecyl sulfate lysis and phenol/chloroform extraction method. The DNA served as a template for PCR amplification with primers specific for the bacterium. The primers were developed by comparison of 16S rDNA sequences from different lactic acid bacteria (LAB) and testing for specificity with 11 different strains of LAB. As few as 100 L. buchneri colony-forming units per gram of silage could be detected. Additionally, the technique was successfully applied to quantify the population of L. buchneri in two cultivars of corn with or without inoculation.
Conclusions:  The PCR assay provided a specific and rapid tool for identifying and enumerating L. buchneri in silage samples.
Significance and Impact of the Study:  The use of microbial inoculants for silage production is a safe and environment friendly practice, but the full potential of such additives can only be achieved with a better understanding of the fate and activity of the microbes involved. The current study describes a methodology to detect and enumerate L. buchneri , a micro-organism used as an inoculant.     

Measurement of Pseudomonas aeruginosa multidrug efflux pumps by quantitative real-time polymerase chain reaction

Multidrug efflux pumps contribute to multiple antibiotic resistance in Pseudomonas aeruginosa. Pump expression usually has been quantified by Western blotting. Quantitative real-time polymerase chain reaction has been developed to measure mRNA expression for genes of interest. Whether this method correlates with pump protein quantities is unclear. We devised a real-time PCR for mRNA expression of MexAB-OprM and MexXY-OprM multidrug efflux pumps. In laboratory strains differing in MexB and MexY expression and in several clinical isolates, protein and mRNA expression correlated well. Quantitative real-time PCR should be a useful alternative in quantitating expression of multidrug efflux pumps by P. aeruginosa isolates in clinical laboratories.     

Detection of Norwalk virus in the UK by the polymerase chain reaction

Abstract We have developed a polymerase chain reaction for the detection of Norwalk virus using the published sequence of the virus RNA dependent RNA polymerase gene and have used this to clone and sequence this region of a virus from a UK outbreak. We have applied this method to a panel of UK Norwalk-like viruses using both Tet-z and Taq DNA polymerases and found that amplification produces a multiplicity of bands from stool samples. However, combination with Southern blotting. Taq polymerase amplification detected virus in 13 of a panel of 30 clinical samples known to contain these viruses and also detected astroviruses in a mixed infection. Amplification using Tet-z DNA polymerase was less efficient (6/30) and detected predominantly viruses typed as UK type 2 by solid phase immune electron microscopy.     

Measuring growth of a phenanthrene-degrading bacterial inoculum in soil with a quantitative competitive polymerase chain reaction method

We measured growth of a phenanthrene-degrading bacterium, Arthrobacter, strain RP17, in Forbes soil, amended with 500 μg g(-1) phenanthrene using a quantitative competitive polymerase chain reaction method. The inoculum, which was not indigenous to Forbes soil, grew from 5.55x10(5) colony forming units (cfu) g(-1) to 1.97x10(7) cfu g(-1) within 100 h after the cells were added to the soil. Maximum population density was reached before the highest degradation rate was observed 150 h after the cells were added to soil. Population density remained stable even after 56% of the phenanthrene had mineralized. This study is one of the few documented examples of growth by a non-indigenous bacterium in a non-sterile soil amended with a pollutant.     

Genomic signature analysis of the recently emerged highly pathogenic A(H5N8) avian influenza virus: implying an evolutionary trend for bird-to-human transmission

In early 2014, a novel subclade (2.3.4.4) of the highly pathogenic avian influenza (HPAI) A(H5N8) virus caused the first outbreak in domestic ducks and migratory birds in South Korea. Since then, it has spread to 44 countries and regions. To date, no human infections with A(H5N8) virus have been reported, but the possibility cannot be excluded. By analyzing the genomic signatures of A(H5N8) strains, we found that among the 47 species-associated signature positions, three positions exhibited human-like signatures (HLS), including PA-404S, PB2-613I and PB2-702R and that mutation trend of host signatures of avian A(H5N8) is different before and after 2014. About 82% of A(H5N8) isolates collected after January of 2014 carried the 3 HLS (PA-404S/PB2-613I/PB2-702R) in combination, while none of isolates collected before 2014 had this combination. Furthermore, the HA protein had S137A and S227R substitutions in the receptor-binding site and A160T in the glycosylation site, potentially increasing viral ability to bind human-type receptors. Based on these findings, the newly emerged HPAI A(H5N8) isolates show an evolutionary trend toward gaining more HLS and, along with it, the potential for bird-to-human transmissibility. Therefore, more extensive surveillance of this rapidly spreading HPAI A(H5N8) and preparedness against its potential pandemic are urgently needed.     

Triplex real‐time polymerase chain reaction assay for detection and quantification of norovirus (GI and GII) and sapovirus

To improve detection of norovirus (NoVGI, NoVGII) and sapovirus (SaV), a simultaneous quantitative RT‐PCR method was established. This triplex real‐time PCR method was evaluated using a combination of optimized specific primers and probes. The performance of the developed PCR assay was equivalent to that of monoplex real‐time PCR across a broad dynamic range of 10²–10⁷ copies/assay using plasmid DNA standards. The limit of detection was 10² copies/assay. The quantitative value was comparable with that of monoplex real‐time PCR of stool samples. Our triplex real‐time PCR is useful for detection of NoV and SaV infections.     

H5N1亚型禽流感病毒进化的研究进展

由H5N1流感病毒引起的高致病性禽流感,在禽类之间广泛传播。当人类接触这些禽类时,可能会被感染并产生严重的呼吸道症状,且死亡率高达60%。血凝素(hemagglutinin,HA)是H5N1病毒中和抗体的主要抗原,为了便于对病毒的HA突变进行研究,根据HA遗传基因的差异远近,所有的H5病毒株都被划分在20个分支内。对于H5N1病毒进化的研究在禽流感疫苗的研制、禽流感大流行的预防等方面均具有重要意义。现对禽流感、H5N1病毒特征、血凝素的结构功能、H5N1病毒的分支以及病毒进化的研究进行概述。     

21世纪的首次大流行：2009甲型（H1N1）流感

2009年4月初,在墨西哥和美国出现一种新型甲型（H1N1）流感病毒。该病毒通过人-人传播迅速在全球范围蔓延。该病毒拥有来自人流感病毒、禽流感病毒和猪流感病毒的基因片段,其HA基因与引发1918年大流行的流感病毒株的HA基因同源性很高。该病毒倾向于感染儿童、青少年、孕妇,以及具有心肺疾病的人。据观察,它在人群中的传播能力高于季节性流感。部分感染患者具有在季节性流感中罕见的呕吐和腹泻症状。先前的流感病毒大流行和2009年爆发的甲型H1N1流感病毒大流行表明,由于流感病毒变异速度快、容易发生基因重排,新产生的变异毒株很可能造成新的大流行,威胁人类健康。由于禽流感病毒和人流感病毒都能感染猪,猪被认为是通过基因重排生成新的大流行病毒的＂混合容器＂。     

Human monoclonal antibodies derived from a patient infected with 2009 pandemic influenza A virus broadly cross-neutralize group 1 influenza viruses

Influenza viruses are a continuous threat to human public health because of their ability to evolve rapidly through genetic drift and reassortment. Three human monoclonal antibodies (HuMAbs) were generated in this study, 1H11, 2H5 and 5G2, and they cross-neutralize a diverse range of group 1 influenza A viruses, including seasonal H1N1, 2009 pandemic H1N1 (H1N1pdm) and avian H5N1 and H9N2. The three HuMAbs were prepared by fusing peripheral blood lymphocytes from an H1N1pdm-infected patient with a newly developed fusion partner cell line, SPYMEG. All the HuMAbs had little hemagglutination inhibition activity but had strong membrane-fusion inhibition activity against influenza viruses. A protease digestion assay showed the HuMAbs targeted commonly a short α-helix region in the stalk of the hemagglutinin. Furthermore, Ile45Phe and Glu47Gly double substitutions in the α-helix region made the HA unrecognizable by the HuMAbs. These two amino acid residues are highly conserved in the HAs of H1N1, H5N1 and H9N2 viruses. The HuMAbs reported here may be potential candidates for the development of therapeutic antibodies against group 1 influenza viruses.     

Detection of bovine kappa-casein genomic variants by the polymerase chain reaction method

Summary A method is described for identifying variants at the bovine κ-casein locus by combining polymerase chain reaction (PCR) amplification and restriction enzyme digests.