Characterization of the new strain of influenza A (H1N1) virus - The elderly aged 90 or older carry high levels of antibodies -

As part of the JST Targeted Basic Research Program, Prof. Yoshihiro Kawaoka of the Institute of Medical Science, University of Tokyo, and others used cultured cells and animal models to characterize the newly emerged strain of influenza A (H1N1) virus originating from swine (Note 1) (hereinafter referred to as swine-origin influenza virus), which has reached pandemic proportion.
Initial speculation regarding this new influenza virus suggested that it was mildly virulent, in accordance with reports that a substantial number of hospitalizations resulting from infections by swine-origin influenza viruses were of patients with no underlying illnesses. Additionally, while it was presumed that persons 60 years old or older had neutralizing antibodies (Note 2) against the swine-origin influenza virus, the existence and levels of neutralizing activity in persons in this age group had not been confirmed. Finally, it was unclear whether licensed and experimental antiviral drugs were effective against this new strain of influenza virus.
To address these gaps in knowledge, the research group inoculated the swine-origin influenza viruses into various animal models. The results showed that compared with seasonal influenza viruses, the swine-origin influenza viruses replicated to higher titers in the lungs of infected animals and caused more severe lung lesions. Additionally, the data demonstrated that currently licensed and experimental antiviral drugs inhibited replication of the new influenza viruses.
Further, analysis of human sera demonstrated that neutralizing antibodies were present in people age 90 or older, but those who were younger possessed little, if any, neutralizing antibodies.
This study yielded valuable information on the characteristics of the swine-origin influenza virus that will be important in the design of countermeasures, including the development of vaccines and new antiviral drugs against the new influenza virus.
This research was conducted jointly by domestic and foreign universities, research institutes, hospitals, and corporations.
The research results were published in the online breaking news version of the British scientific journal "Nature" on July 13, 2009 (U.K. time).

This research was undertaken with funding from the following program:
Exploratory Research for Advanced Technology (ERATO) Type Research in Basic Research Program

Project Name	:	"Kawaoka Network Project on Host Response to Infections"
Research Director	:	Yoshihiro Kawaoka (Professor, Institute of Medical Science, The University of Tokyo)
Research Term	:	October, 2008 through March, 2014

This project aims to create improved strategies for prevention and treatment of viral diseases, as well as clarify the host response to viral infection, using influenza virus as a model.
NOTE: In response to the spreading epidemic/infection by swine-origin influenza viruses, JST expanded this project in June of this year to research related to influenza.

<Background to the Research and its Progress>

The global spread of swine-origin influenza viruses in humans since March, 2009, prompted the World Health Organization (WHO) to declare a pandemic (Note 3) on June 11. Although some became seriously ill, most patients affected by the swine-origin influenza viruses showed mild symptoms. Yet, more than half of the hospitalizations related to infection by this new influenza virus were of patients with no underlying illnesses, calling into question the pathogenicity of the swine-origin influenza virus. Also, as most of the confirmed cases were in young adults, it was suggested that people 60 years old or older had neutralizing antibodies against the swine-origin influenza virus, but this remained unconfirmed. Lastly, it was unclear whether the current registry of antiviral drugs was effective against this new influenza virus.

<Content of the Research>

Mice, ferrets (small mammals in the weasel family), and cynomolgus macaques (Note 4) were infected with swine-origin influenza viruses and examined for replication, tissue tropism, and pathogenicity of the virus. We found that these viruses replicated more efficiently (Figure 1) and caused more severe pathological lesions in the lungs (Figure 2), thus triggering more prominent symptoms, than infection by seasonal influenza viruses.
Also, analysis of sera collected from persons in different age groups showed that people born before 1918, that is to say, those who were 90 years old or older at the time of serum donation have neutralizing antibodies against the new influenza viruses (Figure 3).
We also investigated the sensitivity of the swine-origin influenza viruses to antiviral drugs that are currently licensed or in clinical trials and we found that existing neuraminidase inhibitors (Note 5) and antiviral drugs under development inhibit the replication of the swine-origin influenza viruses (Figure 4).

<Future Developments>

Our findings indicate that the newly emerged H1N1 swine-origin influenza viruses are more pathogenic than seasonal influenza viruses. Moreover, neutralizing antibodies were detected in persons 90 years old or older, indicating the vast majority of the population is susceptible to infection by these viruses. While we did find that currently licensed and experimental anti-viral drugs are effective against the swine-origin influenza viruses, the rapid, unchecked spread of this virus could result in the accumulation of mutations that enhance the pathogenicity of the virus (similar to the Spanish influenza virus, Note 6) or generate drug-resistant strains. Therefore, the characteristics of the swine-origin influenza virus determined in this study provide critical information for the development of countermeasures, including the development of effective vaccines and new antiviral drugs.

<Reference Drawings>

Figure 1: Virus titres in organs of cynomolgus macaques on day 3 after virus infection

Seasonal influenza viruses were found in several organs, including the nasopharynx, trachea, lungs, but high virus titres of the swine-origin influenza viruses were found in various organs of the upper and lower respiratory system such as lungs/bronchus/nasopharynx, indicating the replicative ability of the swine-origin influenza viruses in the lower bronchial pathways, including the lungs.

Figure 2: Pathological lesions in the lungs of infected cynomolgus macaques on day 3 after virus infection

(a) Alveolar spaces filled with air were observed in the lungs of normal macaques.

(b, c) Although the lungs of macaques infected by seasonal viruses showed some thickening of the alveolar walls, there were many regions remaining that contained air.

(d, e) The lungs of macaques infected by the swine-origin influenza viruses were consolidated with inflammatory cells/red blood corpuscles/protein, etc., with little air-filled spaces in the alveoli.

Figure 3: Neutralization antibody titres in human sera from a broad range of age groups against swine-origin or the seasonal influenza viruses

(a) As expected, no antibodies were detected against seasonal influenza viruses isolated in 2009 using human sera collected in 1999, (b) but antibody activity against these seasonal viruses was present in various age groups in sera collected in 2009.

(a, b) In both panels of sera, with few exceptions, neutralizing antibody activity against the swine-origin influenza viruses was detected in those born in or prior to 1918.

Figure 4: Effectiveness of anti-influenza drugs against swine-origin or seasonal influenza viruses

Mice were infected with viruses, administered anti-influenza drugs after one hour, and virus titres in mice lungs were measured on day 3 and 6. With the exception of viruses known to be resistant to oseltamivir, virus replication in the lungs of drug-treated mice was reduced in comparison to that in untreated mice, indicating the effectiveness of anti-viral therapy.

<Explanation of Terminology>

Note 1: Swine-origin influenza A (H1N1) virus
Influenza viruses are classified into 3 genera of A, B and C types; only type A viruses have caused pandemics in humans. Type A influenza viruses are further categorized based on the antigenicity (specificity of antibody bound) their surface proteins hemagglutinin (HA) and neuraminidase (NA); there are currently 16 HA (H1 through H16) and 9 NA subtypes (N1 through N9). Thus, H1N1 refers to a strain of type A influenza virus with H1 HA and N1 NA proteins. H1N1 type viruses have caused two human pandemics—in 1918 and 1957—in addition to the current pandemic of swine-origin influenza virus.
Typically, influenza viruses are recognized as newly emerged when they are of non-human animal-origin and contain genes to which humans lack previous exposure. However, press releases refer to the swine-origin H1N1 influenza virus, which was confirmed to have broken out in Mexico in March 2009 and has rapidly spread around the world, as a new virus strain despite the pre-existence of the H1N1 subtype in humans.

Note 2: Neutralizing antibody
Here, it refers to anti-influenza virus antibodies that inhibit the replication of influenza viruses.

Note 3: Pandemic
A worldwide epidemic of infectious diseases; here, it refers to the global spread of the swine-origin influenza viruses. Human pandemics of influenza in the 20th century were the Spanish flu in 1918, the Asian flu in 1957, and the Hong Kong flu in 1968.

Note 4: Cynomolgus macaque
Cynomolgus macaque is a monkey of the Macaque family, in the parvorder Catarrhini, as are humans. Because of their genetic relatedness, monkeys are considered the definitive standard, when compared to other laboratory animals such as mice or rats, for modeling human infection and are commonly used in research in physiology, behavioral science, pharmacology and medical science. The genome of the cynomolgus macaque is well-characterized and an abundance of research tools exist to facilitate studies with this animal; thus, they are used in both basic and applied research.

Note 5: Neuraminidase inhibitor
A neuraminidase inhibitor is an antiviral drug that blocks the function of the glycoprotein neuraminidase (NA) found on the surface of influenza virus particles, thereby inhibiting the replication of influenza viruses. Neuraminidase inhibitors include oseltamivir, which is most frequently used today, zanamivir, and CS-8958, which is now in clinical trial.

Note 6: Spanish flu
The Spanish flu refers to the 1918-1919 influenza pandemic caused by a new strain of influenza A virus of subtype H1N1. To date, the Spanish flu pandemic has been the most lethal influenza pandemic, claiming the lives of 20-40 million people globally.

<Title of the Journal Article>

"In vitro and in vivo characterization of new swine-origin H1N1 influenza viruses"
doi: 10.1038/nature08260

<For inquiries regarding this press release>

<About research>
Dr. Yoshihiro Kawaoka
Division of Virology, Department of Microbiology and Immunology, Institute of Medical Science, University of Tokyo
4-6-1 Shiroganedai, Minato-ku, Tokyo 108-8639, Japan
Tel: +81-3-5449-5310 Fax: +81-3-5449-5408
E-mail:

<About Japan Science and Technology Agency (JST) projects>
Tadashi Kobayashi
Department of Research Project, Collaborative Development of Innovative Seeds, Office of Basic Research, JST
Sanbancho Building, 3-5, Sanbancho, Chiyoda-ku, Tokyo 102-0075, Japan
Tel: +81-3-3512-3528 Fax: +81-3-3222-2068
E-mail:

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