Contact infection of mink with influenza A viruses of avian and mammalian origin

Summary Avian influenza A virus Hav 7 N 2 was transmitted to mink by contact. Other avian influenza A viruses, Hav 4 Nav 1 and Hav 6 Nav 5, were not transmitted, and human, swine and equine influenza A viruses were transmitted to mink by a similar contact.     

Replication of avian influenza A viruses in mammals.

The recent appearance of an avian influenza A virus in seals suggests that viruses are transmitted from birds to mammals in nature. To examine this possibility, avian viruses of different antigenic subtypes were evaluated for their ability to replicate in three mammals-pigs, ferrets, and cats. In each of these mammals, avian strains replicated to high titers in the respiratory tract (10(5) to 10(7) 50% egg infective doses per ml of nasal wash), with peak titers at 2 to 4 days post-inoculation, similar to the pattern of human and other mammalian viruses in these animals. Most avian strains were recovered for 5 to 9 days post-inoculation. One avian H1N1 virus initially replicated poorly in pigs, but was adapted to this host and even transmitted to other pigs. Replication of the avian viruses occurred in the respiratory tracts of mammals, whereas, in birds, they replicate in the intestinal tract as well. The infected mammals had no significant disease signs and produced low levels of humoral antibodies; however, challenge experiments in ferrets indicated that they were immune. These studies suggest that influenza A viruses currently circulating in avian species represent a source of viruses capable of infecting mammals, thereby contributing to the influenza A antigenic pool from which new pandemic strains may originate.     

Pandemic threat posed by avian influenza A viruses

Influenza pandemics, defined as global outbreaks of the disease due to viruses with new antigenic subtypes, have exacted high death tolls from human populations. The last two pandemics were caused by hybrid viruses, or reassortants, that harbored a combination of avian and human viral genes. Avian influenza viruses are therefore key contributors to the emergence of human influenza pandemics. In 1997, an H5N1 influenza virus was directly transmitted from birds in live poultry markets in Hong Kong to humans. Eighteen people were infected in this outbreak, six of whom died. This avian virus exhibited high virulence in both avian and mammalian species, causing systemic infection in both chickens and mice. Subsequently, another avian virus with the H9N2 subtype was directly transmitted from birds to humans in Hong Kong. Interestingly, the genes encoding the internal proteins of the H9N2 virus are genetically highly related to those of the H5N1 virus, suggesting a unique property of these gene products. The identification of avian viruses in humans underscores the potential of these and similar strains to produce devastating influenza outbreaks in major population centers. Although highly pathogenic avian influenza viruses had been identified before the 1997 outbreak in Hong Kong, their devastating effects had been confined to poultry. With the Hong Kong outbreak, it became clear that the virulence potential of these viruses extended to humans.     

Temperature sensitivity in maturation of mammalian influenza A viruses

In contrast to avian influenza viruses, mammalian influenza viruses are restricted in growth at 41°C compared to 37°C. We have shown that the block in the replication cycle at the nonpermissive temperature is late. Although all virus-specific components (vRNA, cRNA, proteins) are produced, there is no maturation to infectious particles.In the case of the virus strains A/FM/1/47 (H1N1) and A/Eq/Miami/63 (H3N8) no budding is observed. With A/PR/8/34 (H1N1) hemagglutinating, non-infectious, filamentous particles are produced at 41 °C which have a length of up to 6 μm and a markedly reduced content of NP- and M-proteins.     

Replication of avian influenza viruses in humans

Summary Volunteers inoculated with avian influenza viruses belonging to subtypes currently circulating in humans (H1N1 and H3N2) were largely refractory to infection. However 11 out of 40 volunteers inoculated with the avian subtypes, H4N8, H6N1, and H10N7, shed virus and had mild clinical symptoms: they did not produce a detectable antibody response. This was presumably because virus multiplication was limited and insufficient to stimulate a detectable primary immune response. Avian influenza viruses comprise hemagglutinin (HA) subtypes 1–14 and it is possible that HA genes not so far seen in humans could enter the human influenza virus gene pool through reassortment between avian and circulating human viruses.     

Genetic diversity among avian influenza viruses

The RNAs of a series of avian influenza viruses of the subtype Hav7Neg2 were examined to determine if their antigenic similarity reflected an overall conservation of their RNA sequences. Genetic analysis by gel electrophoresis showed a marked variability in all of the RNA segments of the isolates. Analysis by competitive hybridization indicated that with some genome segments this variability represented major genetic differences. These differences were present even among concurrent isolates from one geographical area. In contrast, similar analysis of human H3N2 influenza virus isolates showed that viruses isolated 9 years apart were much more similar than the cocirculating avian viruses. The genetic diversity in avian influenza viruses may result from the cocirculation of many different influenza A viruses in ducks and their ability to recombine in nature.     

Development and application of monoclonal antibodies against avian influenza virus nucleoprotein

Rapid and accurate diagnosis of avian influenza (AI) infection is important for an understanding epidemiology. In order to develop rapid tests for AI antigen and antibody detection, two monoclonal antibodies (mAbs) against influenza nucleoprotein (NP) were produced. These mAbs are designated as F26-9 and F28-73 and able to recognize whole AI virus particles as well as the recombinant NP. Both of the mAbs were tested in a slot blot for their reactivity against 15 subtypes of influenza virus; F28-73 reacted with all tested 15 subtypes, while F26-9 failed to react with H13N6 and H15N8. The mAb binding epitopes were identified using truncated NP recombinant proteins and peptide array techniques. The mAb F26-9 reacted with NP-full, NP-1 (638bp), NP-2 (315bp), NP-4 (488bp), and NP-5 (400bp) in the Western blot. The peptide array results demonstrated that the mAb F26-9 reacted with NP peptides 15-17 corresponding to amino acids 71-96. The mAb F28-73 recognized the NP-full, -1 and -4 fragments, but failed bind to NP-2, -3, -5, and any peptides. This antibody-binding site is expected to be contained within 1-162 amino acids of AI NP, although the exact binding epitope could not be determined. The two mAbs showed reactivity with AI antigen in immunofluorescence, immunohistochemistry and immune plaque assays. Immune response of AI infected animals was determined using the mAb F28-73 in a cELISA. All tested chickens were positive at 11 days post-infection and remained positive until the end of the experiment on day 28 (>50% inhibition). The two mAbs with different specificities are appropriate for developing various tests for diagnosis of AI infection.     

流感/禽流感病毒及其致病力鉴别的基因芯片技术研究

目的 建立流感/禽流感病毒及其致病力鉴别的基因芯片检测技术.方法 以血凝素(HA)、神经氨酸酶(NA)、核蛋白(NP)基冈作为靶片段,设计病毒检测和致病力特异性鉴别探针,建立基因芯片鉴别检测技术,采用单引物扩增法(SPA)处理样本核酸,分别对此芯片进行特异性、敏感性和符合率评价.结果 此芯片能够特异性的检测H1N1、H3N2、B型流感病毒及H5N1、H9N2禽流感病毒,敏感性分别为8HAU、16HAU、32HAU及8HAU、8HAU.致病力鉴别探针敏感性为32HAU.同RT-PCR方法比较,检测灵敏度为83.9%.结论 建立的常见流感病毒检测基因芯片特异性高、敏感性高、灵敏度高,更能够对致病力进行有效甄别,可作为临床诊断、传染病防控等方面的有益补充.     

Inhibition of influenza A virus replication by influenza B virus nucleoprotein: an insight into interference between influenza A and B viruses

Given that co-infection of cells with equivalent titers of influenza A and B viruses (FluA and FluB) has been shown to result in suppression of FluA growth, it is possible that FluB-specific proteins might hinder FluA polymerase activity and replication. We addressed this possibility by individually determining the effect of each gene of FluB on the FluA polymerase assay and found that the nucleoprotein of FluB (NP_FluB) inhibits polymerase activity of FluA in a dose-dependent manner. Mutational analyses of NP_FluB suggest that functional NP_FluB is necessary for this inhibition. Slower growth of FluA was also observed in MDCK cells stably expressing NP_FluB. Further analysis of NP_FluB indicated that it does not affect nuclear import of NP_FluA. Taken together, these findings suggest a novel role of NP_FluB in inhibiting replication of FluA, providing more insights into the mechanism of interference between FluA and FluB and the lack of reassortants between them.     

Attachment of infectious influenza A viruses of various subtypes to live mammalian and avian cells as measured by flow cytometry

At present there is much interest in the cell tropism and host range of influenza viruses, especially those of the H5N1 subtype. We wished to develop a method that would enable investigation of attachment of infectious virus through the interaction of the hemagglutinin molecule and live mammalian and avian cells and the subsequent infection of these cells. To this end, influenza viruses of various HA subtypes were constructed that either carry the green fluorescent protein (GFP) instead of the neuraminidase protein, or that express GFP in the cytoplasm of infected cells. The HA genes were derived from influenza viruses A/PR/8/34 (H1N1), A/Netherlands/178/95 (H3N2) and A/Vietnam/1194/04 (H5N1). Using these pairs of viruses, attachment and post-attachment events in the virus replication cycle can be distinguished. In general, the expression of NeuAc(alpha2-3)Gal or NeuAc(alpha2-6)Gal receptors on the cells tested corresponded with the attachment of the viruses that were studied with respect to predicted receptor specificity. Virus attachment was not always predictive for efficient infection of the cells.     

Mutants and revertants of an avian influenza A virus with temperature-sensitive defects in the nucleoprotein and PB2

ts19 is a temperature-sensitive (ts) mutant of the influenza A fowl plague virus with a defect in the nucleoprotein (NP). In ts19-infected chicken embryo cells all viral components are synthesized in normal yields at the nonpermissive temperature, but infectious virus is not formed. Under these conditions the migration of the NP and M of ts19 from the cell nucleus to the cytoplasm is affected. This ts defect is due to a single amino acid replacement (R162K) in a completely conserved region of the NP. Another mutant with a different defect in the NP is ts81. After infection with ts81 at 40 degrees no vRNA is being synthesized. By backcross of a revertant derived from ts81 many isolates with a ts defect in the PB2 protein were obtained. This ts defect seems to extragenically suppress the ts defect in the NP gene and to be dominant in a wild-type background.     

High-throughput neuraminidase substrate specificity study of human and avian influenza A viruses

Despite the importance of neuraminidase (NA) activity in effective infection by influenza A viruses, limited information exists about the differences of substrate preferences of viral neuraminidases from different hosts or from different strains. Using a high-throughput screening format and a library of twenty α2-3- or α2-6-linked para-nitrophenol-tagged sialylgalactosides, substrate specificity of NAs on thirty-seven strains of human and avian influenza A viruses was studied using intact viral particles. Neuraminidases of all viruses tested cleaved both α2-3- and α2-6-linked sialosides but preferred α2-3-linked ones and the activity was dependent on the terminal sialic acid structure. In contrast to NAs of other subtypes of influenza A viruses which did not cleave 2‐keto‐3‐deoxy‐d-glycero-d-galacto-nonulosonic acid (Kdn) or 5-deoxy Kdn (5d-Kdn), NAs of all N7 subtype viruses tested had noticeable hydrolytic activities on α2-3-linked sialosides containing Kdn or 5d-Kdn. Additionally, group 1 NAs showed efficient activity in cleaving N-azidoacetylneuraminic acid from α2-3-linked sialoside.     

Genetic divergence of the NS genes of avian influenza viruses

The nucleotide sequences of the NS genes of avian influenza A viruses, A/Chicken/Japan/24, A/Duck/England/56, A/Tern/South Africa/61, A/Duck/Ukraine/1/63, and A/Mynah/Haneda-Thai/76, were determined and compared among themselves and with two reported NS sequences of the avian viruses, A/FPV/Rostock/34 and A/Duck/Alberta/60/76. Thirty-six to two hundred forty base differences in the NS genes were found in pairwise comparisons among the viruses. The numbers of base differences in the NS genes increased with time, except A/Duck/Alberta/60/76 virus. However, the NS genes of the avian viruses did not change sequentially with time and were arranged in separate evolutionary lineages. When the NS genes of avian viruses employed in the present study were compared with those of human viruses, sequence similarity was confirmed (M. Baez, R. Taussig, J. J. Zarza, J. F. Young, P. Palese, A. Reisfield, and A. M. Skalka, 1980, Nucleic Acids Res. 8, 5845-5858). The numbers of base differences in the NS genes between avian viruses and the A/PR/8/34 virus were 61 to 83, and the NS gene of the oldest avian isolate, A/Chicken/Japan/24, was most closely related to that of the A/PR/8/34 virus. It was hypothesized that NS genes of human influenza viruses and those of some avian influenza viruses had been derived from a common ancestor gene.     

An avian influenza A virus killing a mammalian species — the mink

Summary During October of 1984 an influenza epidemic occurred on mink farms in the coastal region of South Sweden. Six strains of an influenza A virus were isolated. All six isolates were of the H10 subtype in combination with N4. The H10 subtype in combination with various N subtypes was hitherto only known to occur in avian strains, the prototype being the A/chicken/Germany/N/49 (H10N7) virus.With 1 Figure