The Publisher, 5th Estate was the English editor for this report
Report of Academic Workshop on Influenza
“Preparing for the Next Influenza Outbreak”
1) Date: 26 March 2010
2) Venue: Lecture Room, School of Medicine, Kobe University
3) Language: English
4) Objectives: We are now entering the new millennium where both weaponized and “novel” infectious diseases are being experimented with by many world governments and universities. As these diseases now threaten the lives and health of people on a scale never before imagined, it is essential to create and implement protocols to respond promptly and appropriately to these threats. Therefore, in order to realize a relatively safe and anxiety-free society it is also necessary to build a protective system from a global perspective, to counter infectious diseases which spread with lightning speed as modes of transportation become ever faster and travel by citizens of the world increases exponentially every
year.
This workshop, “Preparing for the Next Influenza Outbreak,” is organized to present a venue allowing researchers from the EU Member States and Japan to exchange their experiences and useful information. This information may then be shared with colleagues and health care professionals from a broader range as we prepare for the next pandemic.
5) Program:
13:10 -13:40 “Experience of pandemic (H1N1)2009 outbreak in Kobe”.
Dr. Michio HAYASHI, Director, Division of Infectious Diseases and
Division of Respiratory Medicine, Division of Infection control and
Prevention, Kobe City Hospital Organization Kobe City Medical Center
General Hospital
Chair: Dr. Kentaro IWATA, Professor, Division of Infectious Disease
Therapeutics, Center for Infectious Disease (CID), Kobe University
13:40 - 14:25 “2009 Influenza Pandemic”
Prof. Yoshihiro KAWAOKA, Director, International Research Center
for Infectious Diseases and Division of Virology, Department of
Microbiology and Immunology, Institute of Medical Science,
University of Tokyo
Chair: Dr. Yoshitake HAYASHI, Professor, Division of Infectious
Disease Pathology, Center for Infectious Diseases (CID), Kobe
University
14:25 - 15:10 “Determinants of Virulence of Zoonotic and Pandemic Influenza Viruses"
Prof. Dr. Ron FOUCHIER, Professor in Molecular Virology at the
Department of Virology, Erasmus Medical Center Rotterdam
Chair: Dr. Hak HOTTA, Professor, Division of Microbiology, Centre
for Infectious Diseases (CID), Kobe University
15:30 - 16:15 “Clinical Aspects of Pandemic Influenza H1N1 2009”
Dr. Norio SUGAYA, Director, Department of Pediatrics, Keiyu Hospital
Chair: Dr. Soichi Arakawa, ICD, Infection Control Team, Kobe
University Hospital
16:15 - 17:00 “Adaptation Mechanisms of Influenza A Viruses”
Dr. Kyoko SHINYA, Associate Professor, Division of Zoonosis,
Graduate School of Medicine, Kobe University
Chair: Dr. Takaaki NAKAYA, Associate Professor, International
Research Center for Infectious Diseases, Research Institute for
Microbial Diseases, Osaka University
17:00 - 17:45 “Novel Influenza Virus-Specific Polymerase Inhibitors”
Dr. Martin Schwemmle, Professor of Virology at the Department of
Virology, University of Freiburg
Chair: Dr. Yasuko MORI, Professor, Division of Clinical Virology,
Center for Infectious Diseases (CID), Kobe University
Coordinator: Prof. Akiko MAKINO, Research Associate, Division of
Zoonotic Virology, Center for Infectious Diseases (CID), Kobe
University
5) Results:
I.
Dr. Michio Hayashi, during the session of “Experience pandemic (2009) in Kobe” disclosed the strategy taken in controlling influenza pandemic of 2009 in Kobe City Hospital, Kobe, Japan. The first outbreak of pandemic 2009 flu in Kobe was confirmed on May 15 when seven people were confirmed to have been infected. Since then the rate of infection rose steadily and reached its peak on May 21 with 446 confirmed cases for the entire city.
Responding to the outbreak of pandemic flu, immediate action and countermeasures were taken. The major efforts by Kobe city were the establishment of isolation units for outpatient with fever or “Hatsu Netsu Gairai” in several hospitals such as Kobe City Hospital; the set up a 24-hour hotline center for fever consultation. Outpatient and fever isolation units were built as emergency external installations outside the hospital, along with a considerable bio-security level facility. Samples from patients with fever were examined with real time PCR, and any positive result would be subjected to a second test for confirmation.
Kobe city hotline center for fever consultation provided information for citizens about the things related to pandemic 2009 flu infection. Most of the questions were “what should I do when I catch fever?” The peak of these calls was reached on May 19 with a number of 2,678 callers. Approximately 20% of the callers were calling from their homes.
These prevention and control actions during 2009 influenza pandemic in Kobe demonstrated the preparedness of the Kobe city government in facing the possibly incoming pandemic.
II.
Dr. Yoshihiro Kawaoka, at the second session titled “2009 Influenza Pandemic” spoke about important knowledge of the novel H1N1 2009 influenza virus. The virus was first identified to have infected people in Mexico at the end of March 2009, and has since been rapidly circulating worldwide. This condition had urged the World Health Organization (WHO) to raise the world pandemic alert to level four. This had been the first world pandemic for over 40 years.
The cause of the disease pandemic flu 2009 is a new influenza A virus subtype H1N1. The virus was rapidly circulating among the human population all over the world and caused relatively mild illness. Of all cases, 1 to 10% of the patients were hospitalized. The susceptible age novel H1N1 2009 infection was different compared to the 2007-2008 flu season infection. The most susceptible ages of novel H1N1 2009 ranged from 24 to 40 years old, meanwhile the 2007-2008 flu tended to infect babies (around 0-4 years old) and elders (over 80 years old). Mortality was mostly caused by secondary bacterial infection, and also in people with risk factors such as pregnancy and young age. Among children, mortality was mostly identified around the ages 5 to 9 years for novel H1N1 2009 infection, and 0 to 4 years old for 2007-2008 seasonal flu infection.
Phylogenetic study of novel H1N1 2009 revealed that the virus consisted of the genes from a re-assortant swine flu virus (combination of classic swine, North America avian and H3N2 human flu type) and Eurasia avian-like swine flu virus. Receptor analysis showed that the virus had preference to both human-type and avian-type receptors even though the human-type receptor was most determined.
A pathogenicity testing of novel H1N1 2009 flu compared to seasonal flu virus had showed that novel H1N1 2009 flu was able to cause severe inflammation in most lobes of the deep lung, whereas seasonal flu infection mostly showed only partial mild changes in the deep lung.
Currently, the control and treatment of pandemic 2009 flu infection relies on a neuraminidase inhibitor known as oseltamivir or Tamiflu, since the virus found having resistance to M2 blockers such as adamantenes. However, to anticipate the development of oseltamivir resistant pandemic 2009 flu strains, further research is needed to find alternative drugs while preparing for the next pandemic. Other compounds such as T705 and CS-8959 had been evaluated and seemed to be promising as the next anti influenza drugs.
II.
Dr. Ron Fouchier at the third session, “Determinants of Virulence of Zoonotic and Pandemic Influenza Viruses" describes the virulence markers of influenza virus and circumstances of 2009 pandemic in the Netherlands. Based on its pathogenicity, avian influenza (AI) is divided into highly pathogenics (HPAI), for example H5 and H7 subtypes, and low pathogenic (LPAI) strains. HPAI H5N1 had decimated poultry industries and caused human casualties in many countries, while the H7N7 subtypes had also proved to have been transmitted to humans.
The most distinctive virulence marker is found in the cleavage site of the HA gene, where the site will be recognized and cleaved by certain protease to promote replication. Cleavage of HPAI has a pattern of multiple basic amino acids, RERKKR, which is cleavable by multi proteases, while LPAI has a pattern of PQIETR that is cleavable only by protease trypsin-like protease limited in human upper respiratory tract. However, studies showed that a LPAI strain might have increased its pathogenicity when multiple basic amino acids were experimentally introduced into its HA cleavage site.
In 2003, an outbreak of AI H7N7 subtype had been reported in the Netherlands. The virus infected 255 farms that caused approximately 31 million birds to be culled. A total of 89 H7N7 human infections were reported. Human cases showed conjunctivitis, and one was reported to have died. Amino acid sequence from the outbreak showed amino acid changes from glutamic acid (E) to lysine (K) in PB2 segment, or also called E627K. Further study showed this amino acid substitution related to high pathogenicity of the virus in infected mice. This substitution had also been previously known as one of the virulence markers in the H5N1 virus.
In the case of novel H1N1 2009 flu, the virus had characteristics of being able to reach lower airways faster, replicate faster and reach higher titer. These findings suggest that the virus has reached complete adaptation. Sequence analysis of novel H1N1 2009 flu fatal cases from the Netherlands showed amino acid mutation of the HA segment at position 222 from aspartic acid (D) to glycine (G), which has been studied as well as one of the HA receptor binding sites. Furthermore, this amino acid mutation was also found in Ukraine and Norway fatal cases.
Further study in mice and ferrets showed that virus having HA D222G did not show any remarkable virulence change when compared to wild virus. However, a receptor binding experiment showed that HA D22G showed binding preference to 2-3 alpha sialic acid which is commonly found in the colons of birds and the human deep lung.
IV.
Dr. Norio SUGAYA at the session titled “Clinical Aspects of Pandemic Influenza H1N1 2009” spoke about the success of Japan in limiting the mortality of pandemic flu in 2009.
The influenza pandemic has caused millions of deaths in most countries, including Japan. Records mention that during the “Spanish flu” pandemic in 1918, Japan suffered a 0.87% death rate, about 0.45% during 1957-58 pandemic and about 0.076% during 1968-1970 pandemic. In the 2009 pandemic, about 1-2% of patients were hospitalized and 0.1-0.2% patients died globally. Of the total world death, 0.001% was reported from Japan, or in other words, equal to one death in Japan per 10,000 deaths globally. Compared to influenza season 2008 which caused 272 deaths, the pandemic influenza H1N1 2009 caused relatively fewer deaths, with 186 deaths. Based on these facts, Japan is ranked as the country with the lowest mortality rate caused by H1N1 2009 in the world. The contributing factors of the mortality rate of H1N1 2009 pandemic flu infection are pregnancy, secondary infection, accompanying disease and young age.
WHO recommendation for H1N1 2009 virus drugs are Tamiflu (per oral) and Peramivir (intra-vein). During the 2009 pandemic in Japan, doctors prescribed Tamiflu for patients according to the WHO guidelines even though WHO guidelines for the antiviral drug mentions that only patient-at-risk groups should be treated with antiviral drugs. Tamiflu is a commercial name of oseltamivir phosphate, a neuraminidase inhibitor. It works by blocking the site of enzymatic activity of neuraminidase, so that the virus loses the ability to replicate further. In Japan, Tamiflu has been the main prescription for seasonal flu long before and it was used in pandemic H1N1 2009 influenza patients. Dosage recommendation for treatment is 75 mg twice a day for 5 days, while dosage for prophylaxis is 75 mg per day for a week after possible exposure. The use of Tamiflu during the 2009 pandemic in Japan seemed to not only have suppressed the number of deaths among patients, but also saved the life of patients-at-risk, as a pregnant woman had survived after being treated with Tamiflu.
V.
Dr. Kyoko Shinya at the session titled “Adaptation Mechanism of Influenza Virus” presented the results of her studies of influenza A receptors in human airways and ostrich involvement in the selection H5N1 avian influenza virus.
Avian influenza or bird flu virus H5N1 subtype had infected over 100 people worldwide. Most of the cases had a contact history with birds, and human to human transmission was relatively rare. Sialic acid with sugar linkage has been known as the influenza virus host receptor. Human type receptors consist of sialic acid with 2-6 alpha sugar linkage, meanwhile the bird type receptor consists of sialic acid with 2-3 alpha sugar linkage. Dr Shinya studied the type of sialic acid in human airways epithelial cells and found that human upper and lower airways had different types of sialic acid. Human upper airways were found to have sialic acid 2-3 sugar linkage or human type receptors, but lower airways from the branch of bronchus to alveolus sialic acid were dominated with 2-3 sugar linkage or avian type receptors. This finding explained why avian influenza virus airborne transmission is relatively limited among humans. However, when an eventual transmission from bird to human occurred, it might reach human deeper lung and efficient viral replication would occur.
Amino acid at position 627 in the influenza A virus PB2 segment has been known to affect the temperature sensitivity of the virus. Virus with lysine at PB2 627 (PB2-627Lys) has been known to have efficiently replicated in the upper respiratory organs in mice. From the Influenza Virus Resource Database, it was found that PB2-627Lys appeared in isolates from Ratitae birds (e.g. ostrich, emu, rhea) in high prevalence. When placed in ostrich and chicken embryo fibroblast, the H5N1 virus developed PB2-627Lys mutation in ostrich lines after six passages, unlike when it was passed in the chicken line. Infection experiments with the ostrich chicks with the H5N1 virus in the absence of PB2-627Lys, showed that PB2-627Lys developed in the virus, isolated from several organs of the ostrich chicks.
VI.
Dr Martin Schwemmle at the session of “Novel Influenza Virus-Specific Polymerase Inhibitor” spoke about polymerase inhibitors as future anti-influenza drugs.
Knowledge of influenza replication cycle has become the basis for anti-influenza drugs. Known anti-influenza drugs have a neuraminidase inhibitor mode of action along with M2 channel blockers and polymerase inhibitors. Consequently, the future target for new drug development such as cap-binding inhibitors, endonuclease activity etc. may be promising.
The correct assembly of three viral polymerase subunits PB1, PB2 and PA is required for viral RNA synthesis and infectivity. Influenza A PA had been studied to bind with N-terminus PB1 at a small binding location with 1-25 amino acid residues. Polymerases are mostly conserved among influenza viruses, however some different amino acids are found at the PB1 binding sites of different viral sub-types. The initial study was done by using small fluorescence peptide (Green Fluorescence Protein/GFP) to mimic a peptide which is intended to bind to the PB1 binding location that interrupted the formation of polymerase complex. This experiment was proven to inhibit the polymerase activity of the influenza A and B viruses.
Because several amino acids are present in the PB1 binding site, further experimentation is needed to find out which amino acid, and which location will produce the maximum virus inhibition effect. After locating the suitable peptide, the screening of compounds specifically blocking PA-PB1 interaction and inhibiting growth of influenza A and B virus may be promising for the discovery new anti influenza drugs.
6) Conclusions:
The characteristics of influenza viruses are different among subtypes. Adaptation of influenza viruses in certain host cells reflect mutations that change the virus characteristics and finally resulted as a species-barrier leap. The influenza pandemic of 2009 was caused by the influenza A virus subtype H1N1 which is a re-assortment of human, swine and bird flu segments. The causes relatively mild disease but spreads very quickly among human populations.
Currently available antiviral drugs such as oseltamivir and peramivir are still effective for the current 2009 pandemic, however consecutive research in antiviral drugs is still ongoing while anticipating the next pandemic. Polymerase inhibitors are one of the effective anti influenza drugs. The PB1-binding domain is small and unique peptide as a site where PA binds and activates the polymerase complex. A single amino acid change in the PB1-binding domain alters the pathogenicity of the virus. Therefore, compounds which target PB1 binding sites can be isolated for the next anti-influenza drugs.
Japan has experienced influenza pandemic since the 1918 “Spanish flu,” until the 2009 H1N1 flu. For the current 2009 influenza pandemic, Japan follows WHO guidelines for influenza control and treatment, and one of those is the use of oseltamivir (Tamiflu) for treatment and prevention. Tamiflu recommended usage is 75 mg Tamiflu twice a day for six days for treatment, or 75 mg per day for seven days after exposure for prevention. Using this guideline, Japan has suppressed the number of pandemic flu deaths in the country, and also recorded the lowest death rate globally.
Local and regional governments in Japan attempted several control measures during the pandemic flu 2009. Kobe city, for example has succeeded in constructing an outpatient ward with the fever isolation unit/”Hatsu Netsu Gairai” to triage, give first aid action for incoming patients with fever, and a hotline center for fever consultation. Current experience should be applied as one of the strategies to combat the next pandemic.
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