Our laboratory serves as the national avian influenza reference laboratory in China. That gives us a chance to collect all the viruses that cause outbreaks in China and to analyse the evolution of the viruses, and so I am able to give you the whole picture about how the H5N1 avian influenza viruses are changing in China. I am also going to tell you something about vaccine development in China – what kinds of vaccines have been developed and used, and why.
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China has a long history of H5N1 virus research. The first virus was isolated from sick geese in Guangdong Province in 1996. The virus is highly pathogenic for chickens, and since the detection of this virus we have started active surveillance of poultry in the five provinces of southern China, as indicated on the map. That surveillance was conducted by our laboratory. From 1999 to 2002 we were able to isolate 21 H5N1 viruses from apparently healthy ducks.
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But the viruses are lethal for chickens, based on the intravenous injection index value, which is the World Organisation for Animal Health (OIE) standard.
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When we ran the full genome sequence analyses of these viruses, we found that these duck viruses could be divided into different genotypes, based on the diversity of the internal genes. But, as you can see here, most of those viruses still contain genes from the goose/Guangdong virus.
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In 2004 we had 50 outbreaks in China.
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And when we sequenced those viruses we found that the majority of them were descended from the duck virus that we detected in 2001. But we did isolate two viruses that showed some kind of HA variations.
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As you may know, in China we started to use a killing plus vaccination strategy to control those outbreaks in 2004. But, in 2005 we still had 30 outbreaks. And I should point out that those outbreaks all occurred in the non-vaccinated poultry. All of the 10 outbreaks in Xinjiang Province occurred in small-scale farms or in backyard chickens. And we also had some outbreaks in southeastern China from the waterfowl.
We had huge outbreaks in northern China in 2005. That situation is quite different from others because those birds were kind of vaccinated. People used an unqualified vaccine on their farm, and when their birds were sick they thought that perhaps it was something else, until they sent samples to my laboratory two weeks later and we found it was the H5N1 virus. By that time the virus had already spread, and we slaughtered a lot of chickens to control this outbreak. And the virus that caused this outbreak is very similar to the virus that caused the outbreaks in Qinghai Lake.
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When we ran sequence analyses on the viruses that caused outbreaks in 2005 in China, we found that those viruses could be divided into three clearly different clades. One is the wild bird clade containing the virus that caused the wild bird outbreaks and the huge outbreak in North China as I mentioned; the second one is the waterfowl clade that contains the viruses from the outbreaks in southern China; and the other one is the Xinjiang clade, the viruses were all from chickens in Xinjiang.
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I would like to give you a little bit more information about the wild bird outbreaks in Qinghai. This is Qinghai Lake, and the place shown in the slide is called Egg Island. Every year, during April to June, a lot of migratory birds stay here to hatch new birds.
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This slide shows the disease course over the two months. The disease was first observed in birds called bar-headed geese. These birds came to this lake from other places (most likely from other countries) about one week before they showed the disease.
Within less than 10 days the disease was transmitted to two other species, the great black-headed gulls and the brown-headed gulls. In the lake those three species live very close together, but it took eight days for the disease to be transmitted from the bar-headed geese to those two birds.
And then it took about another 10 days before the disease was transmitted to another two species. In total, over 6000 wild birds were found dead in this outbreak.
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When we ran the sequence analyses we detected four genotypes of viruses from this outbreak. And at least three of them were isolated from bar-headed geese. This epidemiology and virology information suggests that the viruses may have been carried by the bar-headed goose from other places to the lake and spread to other species of the wild birds in the lake.
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The viruses in one genotype – I call it genotype C – was spread and carried by the wild birds to Europe and Africa.
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The wild bird outbreak occurred in May. Then in October and November we had an outbreak in northern China, as I mentioned before. That outbreak was caused by the genotype C wild bird virus also.
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In 2006 the similar genotype C virus caused wild bird outbreaks in Qinghai and Tibet.
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And I have to mention that we also detected some outbreaks in 2006 in northern China, mainly in chickens in the Shanxi and Ningxia provinces. Those viruses show some antigenic drift, and are genetically different from the viruses detected in wild birds, waterfowls and the viruses from chickens in Xingjiang province.
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From these data, I would like to summarise that the H5N1 avian influenza viruses that circulated in China during 2005-2007 are classified into different clades based on the HA gene, and some viruses isolated in northern China display antigenic drift, suggesting that attention is needed for vaccine development and application.
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Currently we have developed different inactivated vaccines and live virus vectored vaccines. Of the inactivated vaccines, one is an H5N2 subtype vaccine, which was mentioned in Peter Daniels’s talk. Another one is an artificially generated high-growth reassortant H5N1 subtype vaccine.
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The H5N2 vaccine is immunogenic for chickens, but I should say that it is not a perfect vaccine.
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For a perfect inactivated vaccine, the seed virus should be a low pathogenic strain and be safe for the environment, it should be able to grow well in eggs to ensure enough antigen in the vaccine product, and it should be antigenically well matched with the prevalence viruses.
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We have analysed the H5N1 viruses that we isolated from 1996-2002 and found that although those viruses showed different genotypes, the HA genes are kind of conserved. So we used the goose/Guangdong virus as the HA gene donor for vaccine seed virus generation.
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We cloned the HA genes and deleted the four basic amino acids at the cleavage site, which is a virulence factor for the H5N1 viruses.
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We used reverse genetics to generate a high-growth virus. The six internal genes of the goose/Guangdong virus was replaced with the high-growth virus PR8.
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This virus can grow to very high titres in chicken eggs, and it cannot kill eggs, but the wildtype goose/Guangdong virus can kill eggs within thirty hours.
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This vaccine can induce very good HI antibody titres and the duration is also much longer than that induced by the H5N2 vaccine.
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When we challenged the vaccinated chickens at different time points, we found that even after 43 weeks of one dose of the vaccine inoculation the chickens could be completely protected from highly pathogenic H5N1 virus challenge.
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China has a lot of waterfowl, and the waterfowl are really important intermediate hosts to introduce the H5N1 viruses from wild birds to other domestic poultry. Therefore, we also tested the vaccines in waterfowl. We performed this study in a small-scale farm – the titres shown here are in a goose farm – and we found that multiple doses are needed to induce high level and long duration titres. We challenged at the end of the observation, and found that the goose could be completely protected.
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This shows the titres we got from the ducks. In ducks we gave two doses of the vaccines. The second dose was given three months after the first dose, and then we followed the antibody titres for one year. At the end of the observation we shipped the ducks to the laboratory for the challenge study, and we found that the vaccinated ducks could be completely protected – no virus shedding, no disease, no deaths.
We also developed two kinds of live virus vectored vaccines. One is a recombinant fowlpox vaccine, the other is a recombinant Newcastle disease virus (NDV) vaccine.
The recombinant fowlpox vaccine is a very conventional technique and the vaccine works very well, and it has been used in China since 2005.
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As we know, the Newcastle disease virus holds great promise as a vector for human emerging infectious disease vaccine development. But we thought it would be very suitable to be a vector for H5 avian influenza virus vaccine development, because it can prevent two List A poultry diseases, Newcastle and H5N1 influenza. Newcastle is also a very lethal disease for chickens.
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So for one of our studies we chose the wild bird virus as the HA gene donor, because we thought this virus is a geographically widely distributed virus.
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We first established the reverse genetics for the Newcastle disease virus.
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The virus we chose was the live attenuated vaccine strain LaSota, and we inserted the HA genes to this site.
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We found that the recombinant virus was very attenuated in chickens and chicken eggs.
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Then we tested whether this recombinant virus could work as a vaccine for the Newcastle disease virus. We challenged the vaccinated birds and we found that all the birds could be completely protected.
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And then we challenged the vaccinated birds with two different H5N1 viruses, the index strain GS/GD/96 and the homologous strain BHG/QH/05. We found that although antigenetically they show some difference, for the challenge study all the birds survived and there was no virus shedding.
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This shows the virus shedding – the vaccinated birds did not shed any virus.
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This vaccine is really a very cost-effective vaccine. Currently, in avian influenza vaccines and Newcastle disease vaccines production, the main substrates are egg. One egg can only produce a maximum of 50 doses of inactivated vaccine for NDV or avian influenza. But one egg can produce 4000 doses of the recombinant Newcastle and avian influenza vaccines.
As I mentioned, the H5N1 viruses isolated in China show changes both antigenetically and genetically. So we have chosen different representative strains from different clades for vaccine seed virus generation.
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Currently we have different seed viruses available, but only the GS/GD/96 vaccine has been widely used in China, Vietnam and Mongolia, and also in Egypt, I think. The CK/SX/06 vaccine has been used in a limited area in northern China where the genetic drift of virus in birds was detected.
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This slide shows the dosages and the kinds of vaccines that have been used in China during 2004-2006, and I have not updated this slide with the vaccines used in 2007.
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I would like to say that vaccines are playing a very important role for the control of highly pathogenic H5N1 avian influenza in China, and in other countries as well.
I think that effective vaccination could protect the birds from virus infection, reduce the disease and death, and reduce the virus loading in the environment. Therefore, it will reduce or eliminate human infection by H5N1 influenza.
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The international scientists listed on the left of the slide have provided plasmids and suggestions for our research. And the people listed on the right are the people in my laboratory who did the large amount of work I presented.
Discussion
Question: I have a comment about the screening system during the development of the vaccine. Commonly virologists use the increase of the antibody titre in the host, but in the case of the tumour system of clinical trials for tumour immunotherapy, the increase of the antibody titre actually correlates with the increase of the tumour-specific CTL over the helper T cell generation. But it is very difficult to induce the final effector cells of the specific CD8 T cell. That is essential for the concrete elimination of the virus-infected host cells. So have you ever tried, using your vaccine, monitoring the increase of the cellular system, CD8? I think it is very important for development of the vaccination for the virus.
Hualan Chen: Maybe, but we didn’t try it. Thank you.
Question: What is the process for registering an avian vaccine in China? Do you have to demonstrate protection?
Hualan Chen: Yes. We have a very complicated process to do that. We need to show the protective efficacy and how to produce the vaccine.
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