Molecular change in 1918 pandemic influenza virus stops transmission

Jeremy · Jan 31, 2007

http://www.news-medical.net/?id=21634

Molecular change in 1918 pandemic influenza virus stops transmission

Experts at the Centers for Disease Control and Prevention have shown that a molecular change in the 1918 pandemic influenza virus stops its transmission in ferrets that were in close proximity, shedding light on the properties that allowed the 1918 pandemic virus to spread so quickly and potentially providing important clues that could help scientists assess emerging influenza viruses, such as H5N1.

The study, which is published in the Feb. 5 issue of Science, showed that a modest change of two amino acids in the main protein found on the surface of the 1918 virus did not change the virus's ability to cause disease, but stopped respiratory droplet transmission of the virus between ferrets placed in close proximity. The experiments were conducted with ferrets because their reaction to influenza viruses closely mimics how the disease affects humans.

"With this vital research, we are learning more about what may have contributed to the spread and deadliness of the 1918 pandemic," said CDC Director Dr. Julie Gerberding. "By better understanding how this virus spreads, we can be better positioned to slow down or stop the spread of the pandemic virus and hence be better prepared for the next pandemic."

To spread and cause illness, the influenza virus must first bind to host cells found in humans and animals. The Science study suggests that the hemagglutinin (HA), a type of protein found on the surface of influenza viruses, plays an important role in the 1918 virus's ability to transmit from one host to another efficiently. This research suggests that, for an influenza virus to spread efficiently, the virus's HA must prefer attaching to cells that are found predominately in the human upper airway instead of cells found predominately in the gastrointestinal tracts of birds. Other changes may be necessary as well. Current H5N1 viruses prefer attaching to avian cells, suggesting the virus would need to make genetic changes before it could pass easily between humans.

"Work on the 1918 virus is providing clues that are helping us evaluate other influenza viruses with pandemic potential, such as H5N1, that may emerge," said Dr. Terrence Tumpey, lead author of the paper and a CDC senior microbiologist. "Though we still don't know what changes might be necessary for H5N1 to transmit easily among people, it's likely that changes in more than one virus protein would be required for the H5N1 virus to be transmitted among humans."

Influenza pandemics occur when a new strain emerges to which people have little or no immunity. Most experts believe another pandemic will occur, but it is impossible to predict which strain will emerge as the next pandemic strain, when it will occur or how severe it will be.

The 1918 pandemic caused an estimated 675,000 deaths in the United States and up to 50 million worldwide, in the worst pandemic of the past century.

The research was done in collaboration with Mount Sinai School of Medicine and the Southeast Poultry Research Laboratory. All laboratory work with 1918 virus was conducted at CDC in a high containment Biosafety Level 3 laboratory with enhancements, using stringent biosecurity precautions to protect both laboratory workers and the public from exposure to the virus.

Currently available antiviral drugs have been shown to be effective against the 1918 influenza virus and similar viruses.

AlaskaDenise · Feb 1, 2007

Re: Molecular change in 1918 pandemic influenza virus stops transmission

Where can we find out exactly what those 2 amino acid changes are???

It seems the details would be an important detail to share.

.

Similia · Feb 1, 2007

Re: Molecular change in 1918 pandemic influenza virus stops transmission

Dear all,

a little bit more additional information from today's press:

http://infectious-diseases.jwatch.org/cgi/content/full/2007/131/1?q=etoc

Aberrant Immune Response Key to 1918 Flu Virulence?
Infection of macaques with the 1918 virus led to dysregulation of the animals' immune systems, resulting in enhanced lung injury and a blunted antiviral response.

The influenza strain responsible for the 1918 pandemic was unusually virulent. In previous studies, investigators gained new insight into this virulence by studying events in mice inoculated with a genetically engineered copy of the strain. Now, a multinational research team has extended this approach to nonhuman primates.

Seven macaques were infected with a re-created 1918 influenza virus and three with a modern H1N1 strain (K173). They were then euthanized 3, 6, or 8 days after infection. High titers of the 1918 virus were detected throughout the respiratory tract; in contrast, K173 was largely limited to the upper airway. Inflammation and lung injury were more pronounced with the 1918 virus. Animals infected with the 1918 virus exhibited greater levels of interleukin (IL)-6 in serum. Host genes modulated by the 1918 virus remained relatively stable throughout infection, whereas the response to K173 changed between early and later stages of the infection. Lung IL-6 mRNA remained elevated at day 8 with the 1918 virus but not with K173. Several key cytokine genes including IL-8 and CXCL11 showed delayed activation with 1918-virus infection. K173-infected animals showed high levels of mRNA for "antiviral" type I interferons. These levels were much lower in 1918-virus–infected animals, despite high viral loads. Two additional genes important in antiviral host response, DDX58 and IFIH1, were increased by K173 but not by the 1918 virus.

Comment: These results extend earlier findings in mice to an animal model that should more closely reflect human disease. One or more gene products of the 1918 strain cause unregulated stimulation of tissue-damaging immune responses while preventing responses important in clearing the infection. Additional work is needed to define the mechanisms responsible and to explore the possibility of therapeutically disrupting them.

— Bradley E. Britigan, MD

Published in Journal Watch Infectious Diseases January 31, 2007

Citation(s):
Kobasa D et al. Aberrant innate immune response in lethal infection of macaques with the 1918 influenza virus. Nature 2007 Jan 18; 445:319-23.
[Medline abstract]
_____________________________________
Best,

Gaby

AlaskaDenise · Feb 1, 2007

Re: Molecular change in 1918 pandemic influenza virus stops transmission

Two additional genes important in antiviral host response, DDX58 and IFIH1...

So these are the 2 amino acids referenced in the first article?

Write up on DDX58 at
http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=gene&cmd=Retrieve&dopt=full_report&list_uids=23586

write up on IFIH1 at
http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=gene&cmd=Retrieve&dopt=Graphics&list_uids=64135

.

AlaskaDenise · Feb 1, 2007

Re: Molecular change in 1918 pandemic influenza virus stops transmission

Thanks Gaby. :applause:

I believe this letter is a more detailed version of what you posted....

http://www.nature.com/nature/journal/v445/n7125/full/nature05495.html

Nature 445, 319-323 (18 January 2007) | <ABBR title="Digital Object Identifier" minmax_bound="true">doi</ABBR minmax_bound="true">:10.1038/nature05495; Received 11 September 2006; Accepted 29 November 2006
Aberrant innate immune response in lethal infection of macaques with the 1918 influenza virus

Darwyn Kobasa1, Steven M. Jones2,3, Kyoko Shinya5, John C. Kash6, John Copps8, Hideki Ebihara2,9,10,11, Yasuko Hatta12, Jin Hyun Kim12, Peter Halfmann12, Masato Hatta12, Friederike Feldmann2, Judie B. Alimonti2, Lisa Fernando2, Yan Li1, Michael G. Katze6,7, Heinz Feldmann2,4 and Yoshihiro Kawaoka9,10,11,12

<LI id=a1 minmax_bound="true">Respiratory Viruses, and, <LI id=a2 minmax_bound="true">Special Pathogens Program National Microbiology Laboratory, Public Health Agency of Canada, Winnipeg, Manitoba R3E 3R2, Canada <LI id=a3 minmax_bound="true">Department of Immunology and, <LI id=a4 minmax_bound="true">Department of Medical Microbiology, University of Manitoba, Winnipeg, Manitoba R3E 3R2, Canada <LI id=a5 minmax_bound="true">The Avian Zoonosis Research Centre, Tottori University, Tottori 680-8550, Japan <LI id=a6 minmax_bound="true">Department of Microbiology, School of Medicine, and <LI id=a7 minmax_bound="true">Washington National Primate Research Center, University of Washington, Seattle, Washington 98195, USA <LI id=a8 minmax_bound="true">National Centre for Foreign Animal Diseases, Canadian Food Inspection Agency, Canadian Science Centre for Human and Animal Health, Winnipeg, Manitoba R3E 3M4, Canada <LI id=a9 minmax_bound="true">Division of Virology, Department of Microbiology and Immunology and <LI id=a10 minmax_bound="true">International Research Center for Infectious Diseases, Institute of Medical Science, University of Tokyo, Tokyo 108-8639, Japan <LI id=a11 minmax_bound="true">CREST, Japan Science and Technology Agency, Saitama 322-0012, Japan
Department of Pathobiological Sciences, University of Wisconsin-Madison, Madison, Wisconsin 53706, USA

Correspondence to: Yoshihiro Kawaoka9,10,11,12 Correspondence and requests for materials should be addressed to Y.K. (Email: kawaokay@svm.vetmed.wisc.edu).

Top of page Abstract

The 1918 influenza pandemic was unusually severe, resulting in about 50 million deaths worldwide1. The 1918 virus is also highly pathogenic in mice, and studies have identified a multigenic origin of this virulent phenotype in mice2, 3, 4. However, these initial characterizations of the 1918 virus did not address the question of its pathogenic potential in primates. Here we demonstrate that the 1918 virus caused a highly pathogenic respiratory infection in a cynomolgus macaque model that culminated in acute respiratory distress and a fatal outcome. Furthermore, infected animals mounted an immune response, characterized by dysregulation of the antiviral response, that was insufficient for protection, indicating that atypical host innate immune responses may contribute to lethality. The ability of influenza viruses to modulate host immune responses, such as that demonstrated for the avian H5N1 influenza viruses5, may be a feature shared by the virulent influenza viruses.

Genes of the 1918 virus were constructed from published sequences6, 7, 8, 9, 10, 11, and the virus was generated using plasmid-based reverse genetics, as previously described12. The virus was highly virulent in intranasally inoculated mice with an LD50 (dose required to kill 50% of animals) of 103.5 plaque-forming units (PFU), comparable to that previously reported4. Cynomolgus macaques (Macaca fascicularis), previously used to study H5N1 influenza virus pathogenesis13, 14, were selected as a nonhuman primate model for further analysis of virulence. Macaques (n = 7) were infected with the 1918 virus) or a conventional human virus, A/Kawasaki/173/01 (K173; H1N1) (n = 3), via multiple routes, as in the H5N1 virus experiment13. All 1918-virus-infected animals became symptomatic within 24 h post-infection. They appeared depressed, were hesitant to eat or drink normal food items, and showed respiratory complications such as nasal discharge and non-productive cough. They became progressively more debilitated and eventually developed an acute respiratory distress syndrome. Two macaques infected with the 1918 virus and one with K173 were euthanized on each of days 3 and 6 for analysis.

Of these, one 1918-virus-infected animal had reached the predetermined score for euthanasia on day 6. The remaining animals, originally scheduled for euthanasia on day 21 post-infection, were euthanized on day 8 owing to severity of symptoms in 1918-virus-infected animals.

In 1918-virus-infected animals respiratory signs were the most pronounced indication of illness with an increase in respiration rate, from a post-infection average of 26.4, to a range of 72?84 on day 8. Decreases in lung function shown by a decrease in blood oxygen saturation of as much as 36% compared with pre-infection levels were detected by pulse oximetry.

Consistent changes in heart rate and blood pressure were not observed. In contrast, animals infected with the K173 virus showed few, very mild, clinical signs.

The 1918 virus was present at high titres in both the upper and lower respiratory tissues on days 3, 6 and 8, whereas the K173 virus was isolated mainly from the upper respiratory tissues on days 3 and 6 at appreciably lower titres and on day 8 in only one tonsil (Fig. 1). The 1918 virus was recovered from the heart and spleen of some animals, but neither virus was isolated from the brain, kidneys, liver or colon of any animal. Virus was detected more efficiently with nasal or throat swabs from 1918-virus-infected animals than for K173-virus-infected animals (Supplementary Table 1), but neither virus was isolated in rectal or genital swabs, or in the blood, on any day.
Figure 1: Viral replication in nonhuman primate tissues.

a?c, Virus titres were determined in plaque assays of tissue homogenates on days 3 (a), 6 (b) and 8 (c) post-infection in animals infected with the K173 (control) or 1918 virus (1918-1 to 1918-7). Individual titres for each tissue in each animal are reported as log10 (PFU per g tissue). Virus was not isolated from the colon, liver, kidneys or brain of any animal.
High resolution image and legend (259K)

Lungs of the 1918-virus-infected macaques were the only tissue to exhibit macroscopic pathologic changes as severe lesions, with 60% to 90% of the lung tissue affected by days 6?8 (Supplementary Fig. 1). Profuse watery and bloody liquid filled infected areas, greatly reducing lung function. On day 3 with either virus, there was evidence of some degree of alveolar damage with concomitant detection of viral antigen (Supplementary Fig. 2a?i); however, K173 was not isolated from the lungs, indicating low levels of replication. In contrast to lesions of the K173-infected animal, lungs of 1918-virus-infected animals showed infection of multifarious cells of the alveolar wall on day 3 (Supplementary Fig. 2f, g). Common to infection with either the K173 or 1918 virus were flattened linear alveolar cells that were positive for viral antigen without showing desquamation into the alveolar space (Supplementary Fig. 2d, f). A prominent characteristic of 1918-virus-infected lungs was antigen in plump alveolar cells and desquamation of these cells into the alveolar space (Supplementary Fig. 2g, h). These differences in the early phase of infection between the 1918 and K173 viruses might account for later differences in inflammation and the eventual pathologic state of the lungs.

By days 6 and 8, the lungs of K173-virus-infected animals showed signs of healing, evidenced by thickening of the alveolar wall and no viral antigen expression (Fig. 2a?c, day 8; day 6 not shown). In contrast, the lungs of all 1918-virus-infected animals showed worsening alveolar damage and substantial viral antigen (Fig. 2d?h). Extensive oedema and haemorrhagic exudates were prominent (Fig. 2d?f), as reported for patients who succumbed to the 'Spanish' influenza15. By day 8, expression of viral antigen was diminished or undetectable in consolidated alveolar areas, but still appreciable around consolidated areas (Fig. 2g). Bronchiolitis and bronchitis with expression of viral antigen were prominent at this time (Fig. 2h).
Figure 2: Pathologic examination of lungs infected with the 1918 or K173 virus on day 8 post-infection.

a, In lungs of a K173-virus-infected animal, peribronchiolitis with lymph follicle formation was detected throughout the lung (arrows). b, c, Mild thickening of the alveolar wall was observed in the middle lobes, but antigen was not detected. d?f, Most areas of the lungs of a 1918-virus-infected animal contained consolidated lesions (d) consisting of bronchiolitis and alveolitis (e) with fibrinous and inflammatory exudates (*) and alveolar oedema (f) with proteinaceous fluid and haemorrhage (*). g, h, Viral antigen (brown) was detected in the large regenerative alveolar cells (g) and the bronchiolar epithelial cells in lesions (h). Magnifications are

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25 in a;

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240 in b, c, g and h; and

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120 in d?f.
High resolution image and legend (391K)

Among the chemokines and cytokines tested (see Methods), substantial increases of interleukin-6 (IL-6), IL-8, CCL2 (monocyte chemotactic protein-1) and CCL5 (RANTES) were detected in the sera of infected animals compared with pre-infection levels. No changes in IL-2, IL-4, IFN

or TNF-

were detected. Both IL-8 (day 6) and CCL5 (days 6 and 8) were elevated to similar levels in animals infected with either virus (Fig. 3). CCL2 was elevated 1.4- to 1.8-fold in K173-infected animals on days 3 and 6, respectively, whereas in the 1918-virus-infected animals, increases were higher (2- to 4-fold), but did not achieve statistical significance. The most striking changes were seen in IL-6. On day 0, IL-6 in all macaques ranged from 2.85 to 13.04 pg ml-1, except for one animal with a baseline measurement of 251.32 pg ml-1. In 1918-virus-infected animals, IL-6 increased 3- to 9-fold by day 3 post-infection (P = 0.10), 6- to 19-fold by day 6 (P = 0.07), and 5- to 25-fold by day 8, while remaining throughout the study between 2.85 and 4.59 pg ml-1 in the K173-infected animals. In humans experimentally infected with influenza virus, IL-6 expression at both the site of replication and in sera directly correlated with the extent of viral shedding and fever and is thought to have a role in mediating the clinical manifestations of infection16, 17. A similar relationship may also explain the progression to severe clinical signs in macaques infected with the 1918 virus.
Figure 3: Chemokine and cytokine levels in serum.

The levels of chemokines (CCL2 and CCL5) and cytokines (IL-6 and IL-8) in serum were analysed by a cytometric bead array assay. The levels on days 3, 6 and 8 were compared with the day 0 baseline to determine the relative changes within each animal. The bars represent the average for each group.

High resolution image and legend (73K)

To investigate the regulation of the host response to the 1918 virus, we considered the global gene expression profiles in bronchi, a lower respiratory tissue with substantial replication of both viruses (see Fig. 1a?c), using microarray analysis (Fig. 4a, b). In general, overall gene expression patterns of individual 1918-virus-infected animals were similar, particularly on days 6?8 (Supplementary Fig. 3). One striking observation was the relative constancy in gene expression profiles in the 1918-virus-infected animals at days 3 through 8 post-infection, in marked contrast to the K173 virus-infected animals, which showed an appreciably more dynamic response during the course of infection. The sustained host response in 1918-virus-infected macaques is similar to that recently reported in 1918-virus-infected mice18, indicating that critical decisions influencing the outcome of the infection may occur very early, as recently suggested for H5N1 virus infection in humans19. The functional response associated with the observed gene expression changes was analysed by gene ontology (see Methods). This analysis demonstrated that activation of immune-response-related genes in K173-infected animals was highest at day 3 post-infection, whereas the expression of these genes was relatively constant throughout the course of 1918 virus infection (Supplementary Fig. 4). Interestingly, we observed a dramatic activation of genes involved in cellular metabolism in the K173-infected animals on days 6 to 8 post-infection, probably due to cell proliferation and tissue remodelling after clearance of the virus.
Figure 4: Microarray analysis of gene expression in the bronchi of macaques on days 3, 6 and 8 post-infection.

a, Overall gene expression profiles in K173- and 1918-virus-infected animals. b, Expression of chemokines and cytokines as determined by expression oligonucleotide microarray analysis of bronchial tissue from macaques infected with the K173 or 1918 influenza virus. c, Expression of type I interferon (IFN)-stimulated genes in K173- and 1918-virus-infected macaque bronchi. For each K173 virus infection point, the data presented are gene expression changes calculated from technical replicate arrays (n = 4). The data shown for the day 3 and day 6 1918 virus infections are error-weighted averages for two animals (n = 8 arrays), whereas the day 8 1918 virus infection data are the error-weighted averages for three animals (n = 12 arrays). Genes included in this figure showed

2-fold (P < 0.01) differences in expression in at least one experiment. Genes shown in red were upregulated and those shown in green were downregulated in infected relative to mock-infected animals. See Supplementary Table 2 for full gene names.

High resolution image and legend (468K)

Detailed analysis of immune-response-related gene expression changes (Fig. 4b) demonstrated that bronchi from both groups of animals showed activation of several inflammatory chemokine and cytokine genes, including CCL11 and IL-6. IL-6 messenger RNA was expressed at a high level until day 8 post-infection in the 1918-virus-infected animals, but not the K173-infected animals, consistent with levels detected in serum (Fig. 3). Several key cytokine genes, including IL-8 and CXCL11, showed a delay in activation in 1918-virus-infected animals, although several chemokines important for the activation and recruitment of neutrophils, including CXCL6 and CXCL1, were preferentially upregulated. Strikingly, K173-infected animals showed a marked increase in expression of mRNAs for many type I interferons (IFNs) and a corresponding increase in mRNA expression of type-I-IFN-stimulated genes early in infection (Fig. 4c), coinciding with the greatest load of the virus (Fig. 1). This response was downregulated on days 6 and 8 post-infection, when the K173 virus was not detected. 1918-virus-infection, in contrast, induced much fewer IFN-

genes, suggesting that it caused an altered antiviral response in the bronchus. Accordingly, the 1918-virus-infected animals also showed differential activation of type-I-IFN-stimulated gene expression on days 3 to 8, despite viral titres in bronchi that were 10?5,000-fold higher than in K173-virus-infected animals.

Although the mechanisms underlying the differences in regulation of type I IFN responses remain elusive, our data suggest that the 1918 virus induces an antiviral response different from that of K173, possibly including reduced sensitivity to type I IFN responses. Finally, IFN-

1 mRNA was either not induced or was downregulated in all samples except for a modest induction of IFN-

1 in the K173-infected animal at day 3 post-infection (not shown). Only limited IFN-

1 expression might be expected given that IFN-

1 is a first-wave interferon and would probably be induced earlier than our first sample collection (that is, day 3).

An important pathway in activation of the antiviral response to influenza virus infection occurs through the activities of DDX58 (or retinoic-acid-inducible protein I) and IFIH1 (or melanoma differentiation-associated gene 5)20, 21. Both genes were induced in the K173-virus-infected, but not the 1918-virus-infected, animals (Fig. 4d). The nonstructural protein 1 (NS1) protein of virulent influenza viruses can modulate the IFN-mediated antiviral response22, 23, 24 and RIG-I is a target of NS1 immunosuppressive activity20, 21, 25. Although an immunomodulatory role for 1918 virus NS1 was not shown in mice8, possibly owing to its species specificity, NS1 does influence the virulence of the H5N1 virus in a mammalian model5. This supports the idea that NS1 may be a subject for further investigation as an effector of the aberrations in the macaque immune response to 1918 virus infection and emphasizes the value of development of the macaque model for studying influenza pathogenesis26.

Results of the present study, the first in nonhuman primates, indicate that atypical expression of the innate immune response may be a critical determinant of the severity and outcome of infection by the 1918 virus.

Better understanding of the virus?host interaction will aid development of interventions that can interfere with the virus' ability to modulate host innate immune responses and thus alter outcome of severe infections due to viruses such as the H5N1 viruses circulating at present.
Top of page Methods

Viruses

Genes of the 1918 (GenBank DQ208309, DQ208310, DQ208311, AF117241, AY744935, AF250356, AY130766, AF233238) and K173 virus were constructed with the 5' and 3' noncoding sequences of A/WSN/33 (H1N1) and cloned into plasmid vector pPolI, as previously described3. The 1918 and K173 viruses were generated by reverse genetics12, and titred stocks prepared3, as previously described. All procedures with the 1918 influenza virus were performed in the biosafety level 4 facility of the National Microbiology Laboratory of the Public Health Agency of Canada.

Determination of the lethal dose for the 1918 virus in mice

Isoflurane-anaesthetized 6-week-old female BALB/c mice were intranasally inoculated with 10-fold serial dilutions (five mice per dilution) of virus in 100

l of phosphate-buffered saline and monitored daily for disease symptoms and survival. The LD50 was calculated using the method of Reed and Muench27.

Viral pathogenicity in nonhuman primates

Ten cynomolgus macaques (Macaca fascicularis), 9?19 years old, weighing 3.7?12 kg, were confirmed seronegative against current H1N1 and H3N2 influenza reference viruses by haemagglutination inhibition assay. Animals were infected with K173 (n = 3), as a conventional human virus control, and 1918 (n = 7) viruses through a combination of intratracheal (4 ml), intranasal (0.5 ml per nostril), intraocular (0.5 ml per eye) and oral (1 ml) routes with suspension containing 106 PFU ml-1 (infectious dose by all routes is 7

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106 PFU). Before infection and on days 3, 6 and 8 post-infection, blood and oral, nasal, throat, genital and rectal swabs were collected from anaesthetized animals and suspended in 1 ml of MEM containing 0.3% bovine serum albumin and antibiotics (MEM/BSA). Animals were monitored daily for clinical signs, using an approved scoring sheet, and on days 3, 6 and 8 post-infection vital signs including pulse rate, blood pressure, temperature, respiration rate and blood O2 saturation, as measured by pulse oximetry, were recorded.

One K173- and two 1918-virus-infected animals were euthanized on day 3 and on day 6 post-infection; all remaining animals were euthanized on day 8 post-infection for complete necropsy. Tissue samples were placed in RNAlater (Ambion) for subsequent RNA extraction (Qiagen RNAlater kit). The remaining tissue was fixed in 10% phosphate-buffered formalin. Fixed tissues were dehydrated, embedded in paraffin, cut into 5-

m-thick sections and stained with standard haematoxylin and eosin. For viral antigen detection, sections were processed for immunostaining by the two-step dextran polymer method (DAKO), with a rabbit polyclonal antibody to WSN.

All animal experiments were performed under an approved animal-use document and according to the guidelines of the Canadian Council on Animal Care.

Expression microarrays

Equal masses of total RNA isolated from bronchi collected from infected macaques were amplified with a Low RNA Input Linear Amplification Kit (5188-5339; Agilent Technology) according to the manufacturer's instructions and as previously described28. Global gene expression in infected bronchi was compared to pooled RNA prepared from equal masses of total RNA from whole lung tissue of three uninfected macaques. Mock-infected intact lung was used as a reference for the microarrays because of a lack of mock-infected bronchus tissue. Probe labelling and microarray slide hybridization were performed as previously described28 with custom rhesus macaque (Macaca mulatta) oligonucleotide microarrays containing 22,000 rhesus probes corresponding to

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18,000 unique rhesus genes (designed in collaboration with Agilent Technologies). Raw microarray image files were processed using Feature Extraction 8.1 software (Agilent Technologies) and entered into a custom-designed relational database (Expression Array Manager) and analysed with Rosetta Resolver System 5.1 (Rosetta Biosoftware, Seattle, Washington) and Spotfire Decision Site for Functional Genomics 8.1 (Spotfire, Somerville, Massachusetts). Primary data are available at <http://expression.microslu.washington.edu> in accordance with proposed MIAME standards29. Gene ontology analysis was performed using FatiGO (http://fatigo.bioinfo.cipf.es/) as described in reference 30.

Serum cytokine analysis

Serum was obtained from blood collected in K2EDTA vacutainer tubes (BD Biosciences) that were centrifuged at 1,540g for 10 min and

-irradiated (5 Mrad) for removal from biosafety level 4 containment. Commercial kits for detection of human cytokines and chemokines (IL-2, IL-4, IL-5, IL-6, IFN-

, TNF-

, IL-8, IL-10, IL-1

, IL12p70, CCL2, CCL5, M1G and IP-10) were screened for cross-reactive detection of homologous macaque proteins. Of these, IL-2, IL-4, IL-6, IL-8, CCL2, TNF-

, IFN-

and CCL5 exhibited significant cross-reactivity and were detected in supernatants of lipopolysaccharide or phorbol 12-myristate 13-acetate/ionomycin-stimulated macaque peripheral blood mononuclear cells. Levels of each were assayed using the Human Chemokine CBA Kit I and the human IL-6 Flex set (BD Biosciences) according to the manufacturer's instructions. The samples were acquired with the LSRII (BD Biosciences) flow cytometer.

Chemokine and IL-6 samples were analysed with BD Biosciences' CBA and FCAP software, respectively.

Virus titration

Tissue homogenates (10% w/v) were prepared in MEM/BSA. Debris was pelleted by centrifugation (2,000g, 5 min) and virus titres determined in 10-fold serial dilutions of supernatant by standard plaque assay on MDCK cells, in duplicate for each dilution. Virus was similarly determined in the blood and swab suspensions.

Top of page References

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Top of page Supplementary Information

Supplementary information accompanies this paper.

Top of page Acknowledgements

We thank D. Dick, J. Gren, A. Grolla and P. Melito for help with animal care, and V. Carter, M. Thomas and S. Proll for microarray technical assistance. We also thank J. Gilbert for editing the manuscript. This work was supported by the Public Health Agency of Canada (D.K., S.M.J. and H.F.), by grants-in-aid for scientific research on priority areas from the Ministries of Education, Culture, Sports, Science, and Technology, Japan (Y.K. and K.S.), by CREST (Japan Science and Technology Agency; Y.K.), and by private grants to Y.K.
Microarray data were deposited at Arrayexpress with accession number E-TABM-181.

gsgs · Feb 1, 2007

Re: Molecular change in 1918 pandemic influenza virus stops transmission

more detailled, yes.
But it takes too much time to get any meaning out of it,
so I gave up. Lots of other stuff to read here instead.

Anne · Feb 1, 2007

Re: Molecular change in 1918 pandemic influenza virus stops transmission

thanks a lot..
gsgs, at the end, you will have a diplom...( perhaps

)

scottmcpherson · Feb 1, 2007

Re: Molecular change in 1918 pandemic influenza virus stops transmission

Okee dokee, so it stands to reason that two amino acid changes can also make a virus efficiently deadly to humans. Got it.

Shannon Bennett · Feb 1, 2007

Re: Molecular change in 1918 pandemic influenza virus stops transmission

I'd like to know which strain K173 is from? Vietnam, Turkey, Indonesia all with different surfaces and all with different fatality rates in infected humans. Apparently the reason for deat remains the same though, greatly incrteased cytokines and chemokines. At least that is how I understand the papers.

FluBossie · Feb 1, 2007

Re: Molecular change in 1918 pandemic influenza virus stops transmission

Shannon said:
I'd like to know which strain K173 is from? Vietnam, Turkey, Indonesia all with different surfaces and all with different fatality rates in infected humans. Apparently the reason for deat remains the same though, greatly incrteased cytokines and chemokines. At least that is how I understand the papers.

Shannon,

K173 is: a conventional human virus, A/Kawasaki/173/01 (K173; H1N1) (n = 3) or is that not wat you meant?
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My first thoughts about: Molecular change in 1918 pandemic influenza virus stops transmission

In this news message the researchers at the CDC changed (or delete) two amino acids in the main protein from the 1918 virus, that made the transmission to stop. That is a reverse method with a pandemic virus that already (in the lab) exists. To find out wich change or more changes in amino acids in main protein for H5N1 or other flu's is more complex to find out and yet not to predict, because nature does what it prefers. I don't think that when you put the same amino acids in a main protein in an other virus that you get an increased transmission.

Next research may give us the answer...and hopefully in a very biosafety lab level 4 or 3+.
Nevertheless it a major finding! :tiphat:
But first have to read the full paper and try to understand!

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FluBossie · Feb 1, 2007

Re: Molecular change in 1918 pandemic influenza virus stops transmission

I did not read the other thread about this issue. Yes, maybe we are just one 2.6 amino acid away from the next pandemic!

I have to go shopping and get preps...

Molecular change in 1918 pandemic influenza virus stops transmission

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