Prospective evaluation of a new automated nucleic acid extraction system using routine clinical respiratory specimens

The aim of the study was to evaluate the MagNA Pure 96? nucleic acid extraction system using clinical respiratory specimens for identifying viruses by qualitative real‐time PCR assays. Three extraction methods were tested, that is, the MagNA Pure LC?, the COBAS Ampliprep?, and the MagNA Pure 96? with 10‐fold dilutions of an influenza A(H1N1)pdm09 sample. Two hundred thirty‐nine respiratory specimens, 35 throat swabs, 164 nasopharyngeal specimens, and 40 broncho‐alveolar fluids, were extracted with the MagNA Pure 96? and the COBAS Ampliprep? instruments. Forty COBAS Ampliprep? positive samples were also tested. Real‐time PCRs were used to identify influenza A and influenza A(H1N1)pdm09, rhinovirus, enterovirus, adenovirus, varicella zoster virus, cytomegalovirus, and herpes simplex virus. Similar results were obtained on RNA extracted from dilutions of influenza A(H1N1)pdm09 with the three systems: the MagNA Pure LC?, the COBAS Ampliprep?, and the MagNA Pure 96?. Data from clinical respiratory specimens extracted with the MagNA Pure 96? and COBAS Ampliprep? instruments were in 98.5% in agreement (P < 0.0001) for influenza A and influenza A(H1N1)pdm09. Data for rhinovirus were in 97.3% agreement (P < 0.0001) and in 96.8% agreement for enterovirus. They were in 100% agreement for adenovirus. Data for cytomegalovirus and HSV1‐2 were in 95.2% agreement (P < 0.0001). The MagNA Pure 96? instrument is easy‐to‐use, reliable, and has a high throughput for extracting total nucleic acid from respiratory specimens. These extracts are suitable for molecular diagnosis with any type of real‐time PCR assay. J. Med. Virol. 84:906–911, 2012. © 2012 Wiley Periodicals, Inc.     

Comparative analytical sensitivities of six rapid influenza A antigen detection test kits for detection of influenza A subtypes H1N1, H3N2 and H5N1.

BACKGROUND: Rapid and simple methods for diagnosing human influenza A (H5N1) disease urgently needed. The limited data so far suggest that the currently available rapid antigen detection kits have poor clinical sensitivity for diagnosis of human H5N1 disease. OBJECTIVES: To compare the analytical sensitivity of six commercially available rapid antigen detection kits for the detection of "human" (subtypes H1N1, H3N2) and "avian" (subtype H5N1) influenza A viruses. STUDY DESIGN: Six commercially available test kits for the detection of influenza A were investigated. Analytic sensitivity for the detection of two contemporary H1N1, two H3N2 and three H5N1 viruses was determined using virus culture as a reference method. RESULTS AND CONCLUSIONS: Each test kit detected the H5N1 virus subtypes as efficiently as they detected conventional human viruses of subtypes H1N1 or H3N2. However, limits of detection of influenza viruses of all subtypes by antigen detection kits were >1000-fold lower than virus isolation. Thus, the reportedly poor clinical sensitivity of these antigen detection kits for diagnosis of patients with H5N1 disease is not due to a difference of sensitivity for detecting avian influenza H5N1 compared to human influenza viruses.     

Viral elution and concentration method for detection of influenza A viruses in mud by real-time RT-PCR

The role of environmental reservoirs in avian influenza virus (AIV) transmission has been investigated during AIV-associated outbreaks. To date, no method has been defined for detection of AIV from mud samples. A procedure using elution and polyethylene glycol (PEG) concentration steps was designed to detect AIV by RT-PCR from 42 g of raw mud, corresponding to 30 g of the solid fraction of mud. RNA was recovered with MagMAX AI/ND Viral RNA Isolation kit (Ambion, Austin, TX). Three elution buffers were studied and viral recoveries higher than 29% were yielded by elution with a 10% beef extract solution (pH 7). The overall method showed that, under some conditions, virus was not detectable in PEG samples, whereas viruses were detected in the elution fractions. PCR curves were improved significantly by running the amplification reaction with a mixture containing a PCR additive for inhibitor removal, such as T4 gene 32 protein (Gp32), although PCR inhibitors from mud were removed partially from PEG samples. A theoretical detection threshold of 5 × 10⁵ RNA copies of H5N1 virus per 30 g of solid mud could be obtained by elution. The overall method has proved successful for detecting H5N1 virus contamination of mud specimens collected during outbreak investigations of avian influenza in Cambodia.     

Comparison of commercial systems for extraction of nucleic acids from DNA/RNA respiratory pathogens

This study compared six automated nucleic acid extraction systems and one manual kit for their ability to recover nucleic acids from human nasal wash specimens spiked with five respiratory pathogens, representing Gram-positive bacteria (Streptococcus pyogenes), Gram-negative bacteria (Legionella pneumophila), DNA viruses (adenovirus), segmented RNA viruses (human influenza virus A), and non-segmented RNA viruses (respiratory syncytial virus). The robots and kit evaluated represent major commercially available methods that are capable of simultaneous extraction of DNA and RNA from respiratory specimens, and included platforms based on magnetic-bead technology (KingFisher mL, Biorobot EZ1, easyMAG, KingFisher Flex, and MagNA Pure Compact) or glass fiber filter technology (Biorobot MDX and the manual kit Allprep). All methods yielded extracts free of cross-contamination and RT-PCR inhibition. All automated systems recovered L. pneumophila and adenovirus DNA equivalently. However, the MagNA Pure protocol demonstrated more than 4-fold higher DNA recovery from the S. pyogenes than other methods. The KingFisher mL and easyMAG protocols provided 1- to 3-log wider linearity and extracted 3- to 4-fold more RNA from the human influenza virus and respiratory syncytial virus. These findings suggest that systems differed in nucleic acid recovery, reproducibility, and linearity in a pathogen specific manner.     

Automated extraction of avian influenza virus for rapid detection using real-time RT-PCR.

BACKGROUND: Highly pathogenic H5N1 avian influenza (AI) poses a grave risk to human health. An important aspect of influenza control is rapid diagnosis. OBJECTIVES: This study describes the efficiency of AI-RNA extraction utilizing silica-based magnetic beads with robotics and its detection with an influenza A matrix gene real-time RT-PCR from tracheal swabs, and compares it to virus isolation and manual spin column extractions. STUDY DESIGN: Analytical sensitivity was assessed by performing dilution analysis and detection of H2N2 AI viral RNA. Diagnostic sensitivity and specificity was assessed by analyzing tracheal swabs collected from H7N2 infected and uninfected chickens. RESULTS: Both manual and robotic extractions detected AI virus at 1log(10)EID(50)/ml. Diagnostic sensitivity and specificity of matrix gene detection with the automated extraction method for chicken tracheal swab specimens was similar to that of virus isolation and the manual extraction method. There were only three discordant results among 212 tested specimens. CONCLUSION: The main advantages of automated robotic viral nucleic acid extraction are high throughput processing; hands-free operation; and reduction in human and technical error. This study demonstrates successful detection of influenza A virus with magnetic beads utilizing the Qiagen MagAttract cell kit on a BioRobot M48 platform.     

流感及H5N1亚型禽流感病毒液相检测芯片的制备

目的 建立利用液相芯片技术检测甲、乙型流感和H5N1亚型高致病性禽流感病毒的方法,并对该方法进行评价。方法 对GenBank中甲型流感病毒的NP、乙型流感病毒的HA以及高致病性禽流感病毒（H5N1）的H5、N1基因片段序列进行同源性比对,根据保守序列,设计针对各基因的简并引物和寡核苷酸探针,制备探针偶联微球,将样本核酸多重PCR扩增产物与微球进行杂交,以Bio-Plex液相芯片检测系统进行芯片检测。结果 该方法可以对甲型流感病毒的NP基因、乙型流感病毒的HA基因以及高致病性禽流感病毒（H5N1）的H5、N1基因同时进行检测,病毒核酸的最低检出量为1pg,检测特异性高。结论 成功构建了甲、乙型流感病毒和H5N1亚型高致病性禽流感病毒液相芯片检测系统,为流感、禽流感的快速检测、诊断奠定了基础。     

Development of two types of rapid diagnostic test kits to detect the hemagglutinin or nucleoprotein of the swine-origin pandemic influenza A virus H1N1

Since its emergence in April 2009, pandemic influenza A virus H1N1 (H1N1 pdm), a new type of influenza A virus with a triple-reassortant genome, has spread throughout the world. Initial attempts to diagnose the infection in patients using immunochromatography (IC) relied on test kits developed for seasonal influenza A and B viruses, many of which proved significantly less sensitive to H1N1 pdm. Here, we prepared monoclonal antibodies that react with H1N1 pdm but not seasonal influenza A (H1N1 and H3N2) or B viruses. Using two of these antibodies, one recognizing viral hemagglutinin (HA) and the other recognizing nucleoprotein (NP), we developed kits for the specific detection of H1N1 pdm and tested them using clinical specimens of nasal wash fluid or nasopharyngeal fluid from patients with influenza-like illnesses. The specificities of both IC test kits were very high (93% for the HA kit, 100% for the NP kit). The test sensitivities for detection of H1N1 pdm were 85.5% with the anti-NP antibody, 49.4% with the anti-HA antibody, and 79.5% with a commercially available influenza A virus detection assay. Use of the anti-NP antibody could allow the rapid and accurate diagnosis of H1N1 pdm infections.     

Singleplex real-time RT-PCR for detection of influenza A virus and simultaneous differentiation of A/H1N1v and evaluation of the RealStar influenza kit

BackgroundA novel influenza A virus, subtype A/H1N1v emerged in April 2009 and caused the first influenza pandemic of the 21st century. Reliable detection and differentiation from seasonal influenza viruses is mandatory for appropriate case management as well as public health.ObjectivesTo develop and technically validate a novel one-step real-time RT-PCR assay which can be used for influenza A virus screening and subtyping of A/H1N1v in a singleplex fashion. To assess the clinical performance of a novel commercial influenza RT-PCR kit based on the in-house version.Study designA real-time RT-PCR assay targeting the matrix gene of influenza A viruses was developed and validated using in vitro transcribed RNA derived from influenza A/H1N1v, A/H1N1 and A/H3N2 virus as well as plaque-quantified influenza A/H1N1v, A/H1N1 and A/H3N2 virus samples. After validation of the in-house version the commercial RealStar kit was used to assess the clinical performance and specificity on a panel of influenza viruses including A/H1N1v, A/H1N1, swine A/H1N1, A/H3N2, avian A/H5N1 as well as patient specimens.ResultsThe lower limit of detection of the in-house version was 2149, 1376 and 2994 RNA copies/ml for A/H1N1v, A/H1N1 and A/H3N2, respectively. The RealStar kit displayed 100% sensitivity and specificity and could reliably discriminate influenza A viruses from A/H1N1v. No cross reaction with swine A/H1N1 and A/H1N2 was observed with the RealStar A/H1N1v specific probe.ConclusionBoth assays demonstrated high sensitivity and specificity and might assist in the diagnosis of suspected influenza cases.     

Evaluation of live avian-human reassortant influenza A H3N2 and H1N1 virus vaccines in seronegative adult volunteers.

An avian-human reassortant influenza A virus deriving its genes coding for the hemagglutinin and neuraminidase from the human influenza A/Washington/897/80 (H3N2) virus and its six "internal" genes from the avian influenza A/Mallard/NY/6750/78 (H2N2) virus (i.e., a six-gene reassortant) was previously shown to be safe, infectious, nontransmissible, and immunogenic as a live virus vaccine in adult humans. Two additional six-gene avian-human reassortant influenza viruses derived from the mating of wild-type human influenza A/California/10/78 (H1N1) and A/Korea/1/82 (H3N2) viruses with the avian influenza A/Mallard/NY/78 virus were evaluated in seronegative (hemagglutination inhibition titer, less than or equal to 1:8) adult volunteers for safety, infectivity, and immunogenicity to determine whether human influenza A viruses can be reproducibly attenuated by the transfer of the six internal genes of the avian influenza A/Mallard/NY/78 virus. The 50% human infectious dose was 10(4.9) 50% tissue culture infectious doses for the H1N1 reassortant virus and 10(5.4) 50% tissue culture infectious doses for the H3N2 reassortant virus. Both reassortants were satisfactorily attenuated with only 5% (H1N1) and 2% (H3N2) of infected vaccines receiving less than 400 50% human infectious doses developing illness. Consistent with this level of attenuation, the magnitude of viral shedding after inoculation was reduced 100-fold (H1N1) to 10,000-fold (H3N2) compared with that produced by wild-type virus. The duration of virus shedding by vaccines was one-third that of controls receiving wild-type virus. At 40 to 100 50% human infectious doses, virus-specific immune responses were seen in 77 to 93% of volunteers. When vaccinees who has received 10(7.5) 50% tissue culture infectious doses of the H3N2 vaccine were experimentally challenged with a homologous wild-type human virus only 2 of 19 (11%) vaccinees became ill compared with 7 of 14 (50%) unvaccinated seronegative controls ( P < 0.025; protective efficacy, 79%). Thus, three different virulent human influenza A viruses have been satisfactorily attenuated by the acquisition of the six internal genes of the avian influenza A/Mallard/NY/78 virus. The observation that this donor virus can reproducibly attenuate human influenza A viruses indicates that avian-human influenza A reassortants should be further studied as potential live influenza A virus vaccines.     

一种新型甲型H1N1流感核酸双检诊断试剂盒的验证评价

目的验证一种“甲型流感通用型及H1N1型病毒核酸双检试剂盒（PCR-荧光探针法）”。方法连续收集流感样患者咽拭子标本150例,提取RNA后,以北京市CDC配发的“甲型H1N1流感病毒Real—timePCR诊断试剂盒”为对照,同时用待评价的双检试剂盒平行扩增,对结果进行Kappa一致性检验和McNemar x2差异性检验以及计算两种方法的检测一致率。结果待验证的双检试剂盒与北京市CDC配发的Real—timePCR诊断试剂盒的一致率,通用引物M为97．33％,H1N1为98．67％。经Kappa一致性检验和MeNemarX。差异性检验,两种方法诊断试剂盒有较高的一致性（分别为Z=10．6466,P〈0．0001和Z=11．3402,P〈0．0001）,且待验证双检试剂盒相对于CDC配发试剂的“假阴性率”和“假阳性率”很低（分别为P=0．3173〉0．05和P=1．000〉0．05）。结论上海科华生物工程股份有限公司生产的“甲型流感通用型及H1N1型病毒核酸双检试剂盒（PCR．荧光探针法）”对于甲型流感通用型和H1N1型的诊断均有良好的诊断特性。     

Experimental evaluation of the FluChip diagnostic microarray for influenza virus surveillance

Global surveillance of influenza is critical for improvements in disease management and is especially important for early detection, rapid intervention, and a possible reduction of the impact of an influenza pandemic. Enhanced surveillance requires rapid, robust, and inexpensive analytical techniques capable of providing a detailed analysis of influenza virus strains. Low-density oligonucleotide microarrays with highly multiplexed "signatures" for influenza viruses offer many of the desired characteristics. However, the high mutability of the influenza virus represents a design challenge. In order for an influenza virus microarray to be of utility, it must provide information for a wide range of viral strains and lineages. The design and characterization of an influenza microarray, the FluChip-55 microarray, for the relatively rapid identification of influenza A virus subtypes H1N1, H3N2, and H5N1 are described here. In this work, a small set of sequences was carefully selected to exhibit broad coverage for the influenza A and B viruses currently circulating in the human population as well as the avian A/H5N1 virus that has become enzootic in poultry in Southeast Asia and that has recently spread to Europe. A complete assay involving extraction and amplification of the viral RNA was developed and tested. In a blind study of 72 influenza virus isolates, RNA from a wide range of influenza A and B viruses was amplified, hybridized, labeled with a fluorophore, and imaged. The entire analysis time was less than 12 h. The combined results for two assays provided the absolutely correct types and subtypes for an average of 72% of the isolates, the correct type and partially correct subtype information for 13% of the isolates, the correct type only for 10% of the isolates, false-negative signals for 4% of the isolates, and false-positive signals for 1% of the isolates. In the overwhelming majority of cases in which incomplete subtyping was observed, the failure was due to the nucleic acid amplification step rather than limitations in the microarray.     

Human and avian influenza viruses target different cells in the lower respiratory tract of humans and other mammals

Viral attachment to the host cell is critical for tissue and species specificity of virus infections. Recently, pattern of viral attachment (PVA) in human respiratory tract was determined for highly pathogenic avian influenza virus of subtype H5N1. However, PVA of human influenza viruses and other avian influenza viruses in either humans or experimental animals is unknown. Therefore, we compared PVA of two human influenza viruses (H1N1 and H3N2) and two low pathogenic avian influenza viruses (H5N9 and H6N1) with that of H5N1 virus in respiratory tract tissues of humans, mice, ferrets, cynomolgus macaques, cats, and pigs by virus histochemistry. We found that human influenza viruses attached more strongly to human trachea and bronchi than H5N1 virus and attached to different cell types than H5N1 virus. These differences correspond to primary diagnoses of tracheobronchitis for human influenza viruses and diffuse alveolar damage for H5N1 virus. The PVA of low pathogenic avian influenza viruses in human respiratory tract resembled that of H5N1 virus, demonstrating that other properties determine its pathogenicity for humans. The PVA in human respiratory tract most closely mirrored that in ferrets and pigs for human influenza viruses and that in ferrets, pigs, and cats for avian influenza viruses.     

流感病毒A/广州/333/99(H9N2)毒株基因组特性的研究

目的 了解一株再次从流感患儿中分离出禽H9N2流感毒株的基因组特性，并弄清它的来源。方法 病毒在鸡胚中传代，从收获的尿囊液中提取RNA，通过逆转录合成cDNA，cDNA用PCR扩增。PCR产物用纯化试剂盒纯化，接着做核苷酸序列测定，然后用Meg Align(Version 1.03)和Editseg(Version 3.69)软件进行基因进化树分析。结果 A／广州／333／99（H9N2）毒株的基因组属于禽流感病毒，但它明显不同于A／Duck/Hong Kong/Y439/97毒株。同时不含有任何人流感病毒基因节段，其基因组中有4个基因节段（分别编码HA、NA、NP和NS蛋白）来自G9毒株基因系，而其余4个基因节段（分别编码PB2、PB1、PA和M蛋白）来自G1毒株基因系。结论 A／广州／333／99（H9N2）病毒是G9和G1毒株通过基因重配而来的重配株，它最大可能性直接来自禽。进一步证实了禽H9N2毒株能感染人，同时首次证实了H9N2不同基因系毒株间，在自然界中也能发生基因重配。     

Coinfection of avian influenza virus (H9N2 subtype) with infectious bronchitis live vaccine

Avian influenza virus of H9N2 subtype is pathotyped as a non-highly pathogenic virus. However, frequent incidences of avian influenza of high mortality that are caused by H9N2 viruses have been observed in broiler chicken farms in Iran and some other Asian countries. Coinfections or environmental factors may be involved in such cases. Infectious microorganisms have been implicating in taking part in the cases of coinfection. We studied the effect of experimental coinfection of H9N2 avian influenza virus with infectious bronchitis live vaccine, which is used extensively in chicken farms in Iran. Clinical signs, gross lesions, viral shedding and mortality rate of the experimentally infected birds were examined. Coinfection of infectious bronchitis live vaccine and H9N2 avian influenza virus led to an extension of the shedding period of H9N2 virus, increasing the severity of clinical signs and mortality rates, causing macroscopic lesions in the embryos.     

1994～1997年深圳地区流感病毒活动及H3N2亚型毒株HA1基因演变特点

目的　了解近几年流感病毒在深圳地区活动的特点及甲３（Ｈ３Ｎ２） 亚型毒株ＨＡ１ 基因演变概况。方法　病毒分离采用常规的鸡胚双腔接种,毒株检定用常量半加敏ＨＩ测定。新鲜收获含病毒粒的鸡胚尿囊液用来提取ＲＮＡ,经逆转录合成ｃＤＮＡ,经聚合酶链反应（ＰＣＲ） 扩增,产物纯化,采用双脱氧链末端终止法进行核苷酸序列测定。结果　近几年来深圳地区流感活动概况与全国情况相一致：在人群中仍同时流行Ｈ３Ｎ２,Ｈ１Ｎ１ 亚型和乙型毒株,当甲型毒株活动减弱时,乙型毒株活动就增强,反之,甲型毒株增强时,乙型毒株就减弱。随着时间的推移,Ｈ３Ｎ２ 亚型毒株ＨＡ１ 基因不断地发生点突变,这种突变严重受人群免疫压力所影响,１９９６ 年的毒株与１９９５ 的毒株相比,不仅氨基酸替换点中多数是位于抗原决定簇区或受体结合部位上,并增加两个糖基化位点,故导致Ｈ３Ｎ２ 毒株於１９９６ 年活动明显增强。结论　近来在深圳地区人群中仍同时流行着Ｈ３Ｎ２,Ｈ１Ｎ１ 亚型和乙型流感病毒。然而,不同年其优势毒株是不一样的。１９９６ 年Ｈ３Ｎ２ 毒株活动增强是由于其ＨＡ１ 区氨基酸序列发生替换所造成。     

2016—2018年长沙市人群及活禽市场环境H5N6亚型禽流感病毒监测分析

目的:开展2016—2018年长沙市人群感染和活禽市场(live poultry markets, LPMs)环境污染H5N6亚型禽流感病毒(avian influenza virus, AIV)监测,为防控人感染H5N6亚型AIV提供实验室数据。方法:采集2016—2018年长沙市流感样病例和不明原因肺炎病例咽拭子6...     

1994～1997年深圳地区流感病毒活动及H3N2亚型毒 …

目的 了解几年流感病毒在深圳地区活动的特点及甲３（Ｈ３Ｎ２）亚型毒株ＨＡ１基因演变概况。方法 病毒分离采用常规的鸡胚双腔接种,毒株检和常量半加敏ＨＩ测定。新鲜收获含病毒粒的鸡胚尿囊液用来提取ＲＮＡ,经逆转录合成ｃＤＮＡ,经聚合酶链反应（ＰＣＲ）扩增,产物纯化采用双脱氧链末端终止法进行核苷酸序列测定。结果 近几年来深圳地区流感活动概况与全国情况相一致;在人群中仍同时流行Ｈ３Ｎ２,Ｈ１Ｎ１ 型和乙型毒     

Development of a novel rapid immunochromatographic test specific for the H5 influenza virus

Three anti-H5 influenza virus monoclonal antibody (mAb) clones, IFH5-26, IFH5-115 and IFH5-136, were obtained by immunising a BALB/C mouse with inactivated A/duck/Hokkaido/Vac-1/04 (H5N1). These mAbs were found to recognise specifically the haemagglutinin (HA) epitope of the influenza H5 subtypes by western blotting with recombinant HAs; however, these mAbs have no neutralising activity for A/duck/Hokkaido/84/02 (H5N3) or A/Puerto Ric/8/34 (H1N1). Each epitope of these mAbs was a conformational epitope that was formed from the regions located between 46 to 60 amino acids (aa) and 312 to 322 aa for IFH5-115, from 101 to 113 aa and 268 to 273 aa for IFH5-136 and from 61 to 80 aa and 290 to 300 aa for IFH5-26. The epitopes were located in the loop regions between the receptor region and alpha-helix structure in haemagglutinin 1 (HA1). Influenza A virus H5-specific rapid immunochromatographic test kits were tested as solid phase antibody/alkaline phosphate-conjugated mAb in the following three combinations: IFH5-26/IFH5-115, IFH5-136/IFH5-26 and IFH5-136/IFH5-115. In every combination, only influenza A H5 subtypes were detected. For effective clinical application, rapid dual discrimination immunochromatographic test kits in combination with H5 HA-specific mAb, IFA5-26 and IFA5-115 and the influenza A NP NP-specific mAb, FVA2-11, were developed. The dual discrimination immunochromatographic tests kits detected influenza A virus H5 subtypes as H5 line-positive and all influenza A subtypes as A line-positive simultaneously. The dual discrimination immunochromatographic test kits may be useful for discriminating highly pathogenic avian influenza A H5N1 viruses from seasonal influenza A virus, as well as for confirming influenza infection status in human, avian and mammalian hosts.     

Pathology, molecular biology, and pathogenesis of avian influenza A (H5N1) infection in humans

H5N1 avian influenza is a highly fatal infectious disease that could cause a potentially devastating pandemic if the H5N1 virus mutates into a form that spreads efficiently among humans. Recent findings have led to a basic understanding of cell and organ histopathology caused by the H5N1 virus. Here we review the pathology of H5N1 avian influenza reported in postmortem and clinical studies and discuss the key pathogenetic mechanisms. Specifically, the virus infects isolated pulmonary epithelial cells and causes diffuse alveolar damage and hemorrhage in the lungs of infected patients. In addition, the virus may infect other organs, including the trachea, the intestines, and the brain, and it may penetrate the placental barrier and infect the fetus. Dysregulation of cytokines and chemokines is likely to be one of the key mechanisms in the pathogenesis of H5N1 influenza. We also review the various molecular determinants of increased pathogenicity that have been identified in recent years and the role of avian and human influenza virus receptors in relation to the transmissibility of the H5N1 virus. A comprehensive appreciation of H5N1 influenza pathogenetic mechanisms should aid in the design of effective strategies for prevention, diagnosis, and treatment of this emerging disease.