Filament-Producing Mutants of Influenza A/Puerto Rico/8/1934 (H1N1) Virus Have Higher Neuraminidase Activities than the Spherical Wild-Type

Influenza virus exhibits two morphologies – spherical and filamentous. Strains that have been grown extensively in laboratory substrates are comprised predominantly of spherical virions while clinical or low passage isolates produce a mixture of spheres and filamentous virions of varying lengths. The filamentous morphology can be lost upon continued passage in embryonated chicken eggs, a common laboratory substrate for influenza viruses. The fact that the filamentous morphology is maintained in nature but lost in favor of a spherical morphology in ovo suggests that filaments confer a selective advantage within the infected host that is not necessary for growth in laboratory substrates. Indeed, we have recently shown that filament-producing variant viruses are selected upon passage of the spherical laboratory strain A/Puerto Rico/8/1934 (H1N1) [PR8] in guinea pigs. Toward determining the nature of the selective advantage conferred by filaments, we sought to identify functional differences between spherical and filamentous particles. We compared the wild-type PR8 virus to two previously characterized recombinant PR8 viruses in which single point mutations within M1 confer a filamentous morphology. Our results indicate that these filamentous PR8 mutants have higher neuraminidase activities than the spherical PR8 virus. Conversely, no differences were observed in HAU:PFU or HAU:RNA ratios, binding avidity, sensitivity to immune serum in hemagglutination inhibition assays, or virion stability at elevated temperatures. Based on these results, we propose that the pleomorphic nature of influenza virus particles is important for the optimization of neuraminidase functions in vivo.     

新甲型H1N1流感病毒小鼠致死模型的建立

建立新甲型H1N1流感病毒小鼠致死模型,为研究致病性、宿主适应性以及疫苗保护性提供动物模型,并寻找病毒在适应宿主过程中影响毒力和适应性的关键位点。将新甲型H1N1流感病毒A/四川/SWL1/2009 H1N1在小鼠中连续传15代,各代次毒株均在MDCK细胞上增殖后进行测序,根据序列分析结果选择6个传代毒株感染小鼠,连续监测14 d体重和死亡情况;并对第14代和15代病毒在噬斑实验纯化后克隆和测序分析。原代病毒不致死BABL/C小鼠,经动物体内连续传代适应宿主动物后,其毒力增强,具体表现为所选的6个传代毒株中第7、11、15代毒株可以100%致死试验小鼠;分析这6个传代毒株的全基因组表明这些毒株的部分氨基酸位点发生突变。新甲型H1N1流感病毒经小鼠体内连续传代后,建立了小鼠致死模型,病毒毒力增强可能与某些氨基酸位点的改变有关。     

Enhanced pathogenicity and transmissibility of H9N2 avian influenza virus in mammals by hemagglutinin mutations combined with PB2-627K

H9N2 avian influenza viruses (AIVs) circulate globally in poultry and have become the dominant AIV subtype in China in recent years. Previously, we demonstrated that the H9N2 virus (A/chicken/Eastern China/SDKD1/2015) naturally harbors a mammalian-adaptive molecular factor (627K) in the PB2 protein and is weakly pathogenic in mice. Here, we focused on new markers for virulence in mammals. A mouse-adapted H9N2 virus was serially passaged in mice by infecting their lungs. As expected, infected mice showed clinical symptoms and died at passage six. A comparison between the wild-type and mouse-adapted virus sequences identified amino acid substitutions in the hemagglutinin (HA) protein. H9N2 viruses with the T187P ?+ ?M227L double mutation exhibited an increased affinity to human-type (SAα2,6Gal) receptors and significantly enhanced viral attachment to mouse lung tissues, which contributed to enhancing viral replication and virulence in mice. Additionally, HA with the T187P ?+ ?M227L mutation enabled H9N2 viral transmission in guinea pigs via direct contact. AIV pathogenicity in mice is a polygenic trait. Our results demonstrated that these HA mutations might be combined with PB2-627K to significantly increase H9N2 virulence in mice, and this enhanced virulence was achieved in other H9N2 AIVs by generating the same combination of mutations. In summary, our study identified novel key elements in the HA protein that are required for H9N2 pathogenicity in mice and provided valuable insights into pandemic preparedness against emerging H9N2 strains.     

Serial passage of a Helicoverpa armigera nucleopolyhedrovirus in Helicoverpa zea cell cultures

The serial passaging of baculoviruses in cell lines numerous times can result in a variety of mutations or defective viral populations becoming predominant in the cultures. The generation of these mutants during cell culture passage, also known as "the passage effect," can seriously hinder the use of in vitro methods for large-scale production of baculoviruses for use as biopesticides. In an effort to develop a large-scale in vitro method of producing Helicoverpa armigera singly enveloped nucleopolyhedrovirus (HaSNPV), it was essential to determine whether or not the passage effect was evident when this virus is serially passaged in cell cultures. An isolate of HaSNPV was serially passaged in Helicoverpa zea cell cultures up to 10 times. The production of occlusion bodies decreased with increasing passage number and there was evidence of defective viruses becoming predominant in cultures after 5 passages. The number of virions present within cross sections of passage 3 occlusion bodies was 1.5 times higher than those from passage 10 occlusion bodies when quantified using electron microscopy. A laboratory bioassay showed that potencies of passage 3 isolates against H. armigera larvae were 8 times higher than potencies of passage 10 isolates. This study indicated that changes typical of the passage effect were evident when HaSNPV was serially passaged in H. zea cell cultures up to 10 times.     

Intraepidemic heterogeneity of influenza A (H3N2) viruses in 1985: antigenic analysis and sensitivity to non-specific inhibitors

During the influenza outbreak of 1984-85 22 strains of H3N2 viruses were isolated in Finland. An intra-epidemic heterogeneity was demonstrated in an antigenic analysis by haemagglutination inhibition test with antisera produced in rats. The strains could be classified into three groups which corresponded to the following reference strains: group I: A/Hong Kong/1/84, A/Hong Kong/3/84; group II: A/Philippines/2/82; group III: A/Caen/1/84. Seven of the isolates were entirely insensitive to gamma-inhibitors of guinea-pig sera, which is in contrast to the small number of these viruses found among H3N2 strains isolated in the 1970s. The insensitive strains could not be isolated until the second or third passage through the eggs, whereas about half of the sensitive and intermediate strains were already isolated during the first passage. Conversions in reactivity with gamma-inhibitors could be detected only from an intermediate or an insensitive virus to a sensitive virus when several strains were passed serially in ovo and in MDCK cultures. The findings suggest that the gamma-inhibitor-insensitive strains corresponded well to the viruses of the human host or arose from dimorphic virus populations under an arbitrary selection of terminal dilution conditions prevailing during isolation in eggs. The insensitive strains did not differ substantially from the sensitive viruses in their ability to agglutinate erythrocytes of different laboratory animals or in their disagglutination patterns. On the other hand, propagation of viruses in MDCK cultures had an effect on these properties. The results are discussed with respect to Q phase variants and receptor binding properties.     

Egg‐ or cell culture‐derived hemagglutinin mutations impair virus stability and antigen content of inactivated influenza vaccines

Egg‐derived viruses are the only available seed material for influenza vaccine production. Vaccine manufacturing is done in embryonated chicken eggs, MDCK or Vero cells. In order to contribute to efficient production of influenza vaccines, we investigate whether the quality of inactivated vaccines is influenced by the propagation substrate. We demonstrate that H3N2 egg‐derived seed viruses (A/Brisbane/10/07, IVR147, and A/Uruguay/716/07) triggered the hemagglutinin (HA) conformational change under less acidic conditions (0.2–0.6 pH units) than antigenically similar primary isolates. This phenotype was associated with HA₁ (A138S, L194P) and HA₂ (D160N) substitutions, and strongly related to decreased virus stability towards acidic pH and elevated temperature. The subsequent propagation of H3N2 and H1N1 egg‐derived seed viruses in MDCK and Vero cells induced HA₂ N50K (H1N1) and D160E (H3N2) mutations, improving virus growth in cell culture but further impairing virus stability. The prevention of the loss or recovery of stability was possible by cultivation at acidified conditions. Viruses carrying less stable HAs are more sensitive for HA conformational change during concentration, purification and storage. This results in decreased detectable HA antigen content – the main potency marker for inactivated influenza vaccines. Thus, virus stability can be a useful marker for predicting the manufacturing scope of seed viruses.     

Selection of a single amino acid substitution in the hemagglutinin molecule by chicken eggs can render influenza A virus (H3) candidate vaccine ineffective.

This study investigated whether a single amino acid change in the hemagglutinin (HA) molecule influenced the efficacy of formalin-inactivated influenza A (H3N1) vaccine candidates derived from high-growth reassortants between the standard donor of high-yield genes (A/PR/8/34 [H1N1]) and host cell variants generated from the same clinical isolate (A/Memphis/7/90 [H3N2]) by passage in embryonated chicken eggs. Two clones of the isolate generated by growth in eggs differed from the parent virus (represented by an MDCK cell-grown counterpart) solely by the presence of Lys (instead of Glu) at position 156 or Ile (instead of Ser) at position 186 in the HA1 subunit. The protective efficacy of egg-grown HA Lys-156 and HA Ile-186 reassortant variants was compared with that of the MDCK cell-grown reassortant vaccine. Classically, antibody titers in serum have been used to demonstrate vaccine efficacy. Here, parameters of B-cell responsiveness were monitored, including the kinetics, character, and localization of the primary antibody-forming cell (AFC) response and the development of B-cell memory in lymphoid tissues associated with the priming site (spleen) and responsive to pulmonary challenge with infectious virus (upper and lower respiratory tract lymph nodes). We show that the egg-grown HA Lys-156 variant induced an AFC profile vastly different from that elicited by the other two reassortant vaccines. The vaccine was poorly immunogenic; it induced antibodies that were cross-reactive prior to challenge but which, postchallenge with a lethal dose of the MDCK cell-grown reassortant virus, were targeted primarily to the HA Lys-156 variant, were of the immunoglobulin M isotype, were nonprotective, and were derived from the spleen. In contrast, the egg-grown HA Ile-186 variant was remarkably like the MDCK cell-grown virus in that protective immunoglobulin G antibodies were unaffected by the Ile-186 substitution but poorly recognized HA with Lys-156. Furthermore, memory AFC responsiveness was localized to regional lymphoid tissue in the upper respiratory tract, where challenge HA was found. Thus, it is recommended that in the selection of vaccine candidates, virus populations with the egg-adapted HA Lys-156 substitution be eliminated and that, instead, egg-grown isolates which minimally contain Ile-186 be used as logical alternatives to MDCK cell-grown viruses.     

Host Adaptation and the Alteration of Viral Properties of the First Influenza A/H1N1pdm09 Virus Isolated in Japan

A/Narita/1/2009 (A/N) was the first H1N1 virus from the 2009 pandemic (H1pdm) to be isolated in Japan. To better understand and predict the possible development of this virus strain, the effect of passaging A/N was investigated in Madin-Darby canine kidney cells, chicken eggs and mice. A/N that had been continuously passaged in cells, eggs, or mice obtained the ability to grow efficiently in each host. Moreover, A/N grown in mice had both a high level of pathogenicity in mice and an increased growth rate in cells and eggs. Changes in growth and pathogenicity were accompanied by amino acid substitutions in viral hemagglutinin (HA) and PB2. In addition, the adapted viruses exhibited a reduced ability to react with ferret antisera against A/N. In conclusion, prolonged passaging allowed influenza A/N to adapt to different hosts, as indicated by a high increase in proliferative capacity that was accompanied by an antigenic alteration leading to amino acid substitutions.     

Oseltamivir-resistant influenza A viruses are transmitted efficiently among guinea pigs by direct contact but not by aerosol

Influenza viruses resistant to the neuraminidase (NA) inhibitor oseltamivir arise under drug selection pressure both in vitro and in vivo. Several mutations in the active site of the viral NA are known to confer relative resistance to oseltamivir, and influenza viruses with certain oseltamivir resistance mutations have been shown to transmit efficiently among cocaged ferrets. However, it is not known whether NA mutations alter aerosol transmission of drug-resistant influenza virus. Here, we demonstrate that recombinant human influenza A/H3N2 viruses without and with oseltamivir resistance mutations (in which NA carries the mutation E119V or the double mutations E119V I222V) have similar in ovo growth kinetics and infectivity in guinea pigs. These viruses also transmit efficiently by the contact route among cocaged guinea pigs, as in the ferret model. However, in an aerosol transmission model, in which guinea pigs are caged separately, the oseltamivir-resistant viruses transmit poorly or not at all; in contrast, the oseltamivir-sensitive virus transmits efficiently even in the absence of direct contact. The present results suggest that oseltamivir resistance mutations reduce aerosol transmission of influenza virus, which could have implications for public health measures taken in the event of an influenza pandemic.     

Efficacy of a Parainfluenza Virus 5 (PIV5)-Based H7N9 Vaccine in Mice and Guinea Pigs: Antibody Titer towards HA Was Not a Good Indicator for Protection

H7N9 has caused fatal infections in humans. A safe and effective vaccine is the best way to prevent large-scale outbreaks in the human population. Parainfluenza virus 5 (PIV5), an avirulent paramyxovirus, is a promising vaccine vector. In this work, we generated a recombinant PIV5 expressing the HA gene of H7N9 (PIV5-H7) and tested its efficacy against infection with influenza virus A/Anhui/1/2013 (H7N9) in mice and guinea pigs. PIV5-H7 protected the mice against lethal H7N9 challenge. Interestingly, the protection did not require antibody since PIV5-H7 protected JhD mice that do not produce antibody against lethal H7N9 challenge. Furthermore, transfer of anti-H7 serum did not protect mice against H7N9 challenge. PIV5-H7 generated high HAI titers in guinea pigs, however it did not protect against H7N9 infection or transmission. Intriguingly, immunization of guinea pigs with PIV5-H7 and PIV5 expressing NP of influenza A virus H5N1 (PIV5-NP) conferred protection against H7N9 infection and transmission. Thus, we have obtained a H7N9 vaccine that protected both mice and guinea pigs against lethal H7N9 challenge and infection respectively.     

Specific Residues in the 2009 H1N1 Swine-Origin Influenza Matrix Protein Influence Virion Morphology and Efficiency of Viral Spread In Vitro

In April 2009, a novel influenza virus emerged as a result of genetic reassortment between two pre-existing swine strains. This highly contagious H1N1 recombinant (pH1N1) contains the same genomic background as North American triple reassortant (TR) viruses except for the NA and M segments which were acquired from the Eurasian swine lineage. Yet, despite their high degree of genetic similarity, we found the morphology of virions produced by the pH1N1 isolate, A/California/04/09 (ACal-04/09), to be predominantly spherical by immunufluorescence and electron microscopy analysis in human lung and swine kidney epithelial cells, whereas TR strains were observed to be mostly filamentous. In addition, nine clinical pH1N1 samples collected from nasal swab specimens showed similar spherical morphology as the ACal-04/09 strain. Sequence analysis between TR and pH1N1 viruses revealed four amino acid differences in the viral matrix protein (M1), a known determinant of influenza morphology, at positions 30, 142, 207, and 209. To test the role of these amino acids in virus morphology, we rescued mutant pH1N1 viruses in which each of the four M1 residues were replaced with the corresponding TR residue. pH1N1 containing substitutions at positions 30, 207 and 209 exhibited a switch to filamentous morphology, indicating a role for these residues in virion morphology. Substitutions at these residues resulted in lower viral titers, reduced growth kinetics, and small plaque phenotypes compared to wild-type, suggesting a correlation between influenza morphology and efficient cell-to-cell spread in vitro. Furthermore, we observed efficient virus-like particle production from cells expressing wild-type pH1N1 M1, but not M1 containing substitutions at positions 30, 207, and 209, or M1 from other strains. These data suggest a direct role for pH1N1 specific M1 residues in the production and release of spherical progeny, which may contribute to the rapid spread of the pandemic virus.     

Scanning-Beam Electron Microscopy of Mycoplasma pneumoniae

The morphology and the existence of a growth cycle of Mycoplasma pneumoniae have not been clearly established. There is disagreement as to whether this organism exists as a spherical or filamentous form, and whether it progresses from filamentous to spherical forms as the organism ages. A scanning-beam electron microscope (SEM) was utilized to provide detailed observations of the cycle of morphological changes during growth phases of M. pneumoniae. Cultures of cells grown and fixed in liquid suspension displayed morphological changes from spherical to filamentous and then to larger round forms. After 8 hr to 2 days of growth (phase I), spherical forms and aggregates were revealed. Two- to 6-day-old growth (phase II) was composed of both straight and branching filaments with bulbous elements situated at intervals along their lengths, and microcolonies composed predominantly of intertwined filaments. Six- to 10-day-old growth (phase III) was characterized by flattened, spherical organisms larger than those observed in phase I, occasional membranes or ghosts, and a paucity of aggregates or microcolonies. Thus, stereo-scan electron microscopic studies suggest that M. pneumoniae undergoes an orderly and sequential metamorphosis during its life cycle.     

Novel Avian-Origin Influenza A (H7N9) Virus Attachment to the Respiratory Tract of Five Animal Models

We determined the pattern of attachment of the avian-origin H7N9 influenza viruses A/Anhui/1/2013 and A/Shanghai/1/2013 to the respiratory tract in ferrets, macaques, mice, pigs, and guinea pigs and compared it to that in humans. The H7N9 attachment pattern in macaques, mice, and to a lesser extent pigs and guinea pigs resembled that in humans more closely than the attachment pattern in ferrets. This information contributes to our knowledge of the different animal models for influenza.     

Adaptation of high-growth influenza H5N1 vaccine virus in Vero cells: implications for pandemic preparedness

Current egg-based influenza vaccine production technology can't promptly meet the global demand during an influenza pandemic as shown in the 2009 H1N1 pandemic. Moreover, its manufacturing capacity would be vulnerable during pandemics caused by highly pathogenic avian influenza viruses. Therefore, vaccine production using mammalian cell technology is becoming attractive. Current influenza H5N1 vaccine strain (NIBRG-14), a reassortant virus between A/Vietnam/1194/2004 (H5N1) virus and egg-adapted high-growth A/PR/8/1934 virus, could grow efficiently in eggs and MDCK cells but not Vero cells which is the most popular cell line for manufacturing human vaccines. After serial passages and plaque purifications of the NIBRG-14 vaccine virus in Vero cells, one high-growth virus strain (Vero-15) was generated and can grow over 10(8) TCID(50)/ml. In conclusion, one high-growth H5N1 vaccine virus was generated in Vero cells, which can be used to manufacture influenza H5N1 vaccines and prepare reassortant vaccine viruses for other influenza A subtypes.     

Chimeric influenza A viruses with a functional influenza B virus neuraminidase or hemagglutinin

Reassortment of influenza A and B viruses has never been observed in vivo or in vitro. Using reverse genetics techniques, we generated recombinant influenza A/WSN/33 (WSN) viruses carrying the neuraminidase (NA) of influenza B virus. Chimeric viruses expressing the full-length influenza B/Yamagata/16/88 virus NA grew to titers similar to that of wild-type influenza WSN virus. Recombinant viruses in which the cytoplasmic tail or the cytoplasmic tail and the transmembrane domain of the type B NA were replaced with those of the type A NA were impaired in tissue culture. This finding correlates with reduced NA content in virions. We also generated a recombinant influenza A virus expressing a chimeric hemagglutinin (HA) protein in which the ectodomain is derived from type B/Yamagata/16/88 virus HA, whereas both the cytoplasmic and the transmembrane domains are derived from type A/WSN virus HA. This A/B chimeric HA virus did not grow efficiently in MDCK cells. However, after serial passage we obtained a virus population that grew to titers as high as wild-type influenza A virus in MDCK cells. One amino acid change in position 545 (H545Y) was found to be responsible for the enhanced growth characteristics of the passaged virus. Taken together, we show here that the absence of reassortment between influenza viruses belonging to different A and B types is not due to spike glycoprotein incompatibility at the level of the full-length NA or of the HA ectodomain.     

Novel H7N9 Influenza Virus Shows Low Infectious Dose,High Growth Rate,and Efficient Contact Transmission in the Guinea Pig Model

The zoonotic outbreak of H7N9 subtype avian influenza virus that occurred in eastern China in the spring of 2013 resulted in 135 confirmed human cases, 44 of which were lethal. Sequencing of the viral genome revealed a number of molecular signatures associated with virulence or transmission in mammals. We report here that, in the guinea pig model, a human isolate of novel H7N9 influenza virus, A/Anhui/1/2013 (An/13), is highly dissimilar to an H7N1 avian isolate and instead behaves similarly to a human seasonal strain in several respects. An/13 was found to have a low 50% infectious dose, grow to high titers in the upper respiratory tract, and transmit efficiently among cocaged guinea pigs. The pH of fusion of the hemagglutinin (HA) and the binding of virus to fixed guinea pig tissues were also examined. The An/13 HA displayed a relatively elevated pH of fusion characteristic of many avian strains, and An/13 resembled avian viruses in terms of attachment to tissues. One important difference was seen between An/13 and both the H3N2 human and the H7N1 avian viruses: when inoculated intranasally at a high dose, only the An/13 virus led to productive infection of the lower respiratory tract of guinea pigs. In sum, An/13 was found to retain fusion and attachment properties of an avian influenza virus but displayed robust growth and contact transmission in the guinea pig model atypical of avian strains and indicative of mammalian adaptation.     

Guinea pig model for evaluating the potential public health risk of swine and avian influenza viruses

Background

The influenza viruses circulating in animals sporadically transmit to humans and pose pandemic threats. Animal models to evaluate the potential public health risk potential of these viruses are needed.

Methodology/Principal Findings

We investigated the guinea pig as a mammalian model for the study of the replication and transmission characteristics of selected swine H1N1, H1N2, H3N2 and avian H9N2 influenza viruses, compared to those of pandemic (H1N1) 2009 and seasonal human H1N1, H3N2 influenza viruses. The swine and avian influenza viruses investigated were restricted to the respiratory system of guinea pigs and shed at high titers in nasal tracts without prior adaptation, similar to human strains. None of the swine and avian influenza viruses showed transmissibility among guinea pigs; in contrast, pandemic (H1N1) 2009 virus transmitted from infected guinea pigs to all animals and seasonal human influenza viruses could also horizontally transmit in guinea pigs. The analysis of the receptor distribution in the guinea pig respiratory tissues by lectin histochemistry indicated that both SAα2,3-Gal and SAα2,6-Gal receptors widely presented in the nasal tract and the trachea, while SAα2,3-Gal receptor was the main receptor in the lung.

Conclusions/Significance

We propose that the guinea pig could serve as a useful mammalian model to evaluate the potential public health threat of swine and avian influenza viruses.     

Synonymous codon usage of the VP2 gene of a very virulent infectious bursal disease virus isolate serial passaged in chicken embryos

Three very virulent infectious bursal disease virus (vvIBDV) strains were isolated from a single farm and shown to be phylogenetically related to the vvIBDV isolate UK661. In this study, a comparative analysis of the synonymous codon usage in the hypervariable region of theVP2 (vVP2) gene of the vvIBDV strains was done on viruses serially passaged in chicken embryos. Sequencing demonstrated that codons change during the serial passage in the vVP2 gene of the viruses. Nine codon mutations resulted in amino acids changes. The amino acid changes were I256V, I296L 6in isolate XA1989, A222P, I242V, Q253H, I256V in isolate XA1998, and Q253H, I256V, I296L in isolate XA2004. Three of the nine amino acid changes occurred at residue 256. The codons of the amino acids A232, N233, I234, T269, T283 and H338 changed to the synonymous codons in XA1989 after the 16th passage, in XA1998 after the 24th passage and in XA2004 22nd passage viruses. These mutations change the key amino acid residues Q253H and I256V in the domains which are essential for its virulence, and the synonymous codons were observed compared to classical virulent IBDV. The results indicated that the codon changes during the serial passage comprised of synonymous codon usage in the vVP2 gene of IBDV, and this synonymous codon bias was correlated with pathotypes. The extent of synonymous codon usage bias in the IBDV-vVP2 gene maybe influence the gene expression level and secondary structure of protein as well as hydrophobicity, therefore the results provide useful perspectives for evolution and understanding of the pathogenesis of IBDV.     

用8质粒病毒拯救系统产生H9N2/WSN重组A型流行性感冒病毒

把禽流行性感冒(流感)病毒A／Chicken／Shanghai／F／98(H9N2)的血凝素(HA)和神经氨酸酶(NA)基因cDNA克隆至polⅠ-pol Ⅱ双向转录和表达载体pHW2000,用这两种质粒与8质粒病毒拯救系统中流感病毒A／WSN／33(H1N1)6个内部基因cDNA的质粒组合(6 2重排),共转染COS-1细胞,产生了能在鸡胚中高滴度增殖的H9N2／、WSN重组病毒。用A／WSN／33的8个基因cDNA质粒作对照,也产生了转染子病毒。经过EID50测定和MDCK感染实验,新基因型H9N2／WSN病毒感染鸡胚的能力强(EID50为10^-11／0．2m1),而且对鸡胚的毒力弱,在不加胰酶的情况下不使MDCK细胞产牛病变。经电镜观察,两个转染子病毒的形态与野生型流感病毒相似。反向遗传操作技术的建立,为对禽流感病毒基因功能和疫苗构建等方面的研究提供了新的手段。