Eurosurveillance, Volume 14, Issue 21, 28 May 2009 CLUSTER ANALYSIS OF THE ORIGINS OF THE NEW INFLUENZA A(H1N1) VIRUS A Solovyov1

Physics Department, Princeton University, Princeton, United States

Center for Infection and Immunity, Mailman School of Public Health, Columbia University, New York, United States

Department of Biomedical Informatics, Center for Computational Biology and Bioinformatics, Columbia University College of Physicians and Surgeons, New York, United States

Date of submission: 27 May 2009

In March and April 2009, a new strain of influenza A(H1N1) virus has been isolated in Mexico and the United States. Since the initial reports more than 10,000 cases have been reported to the World Health Organization, all around the world. Several hundred isolates have already been sequenced and deposited in public databases. We have studied the genetics of the new strain and identified its closest relatives through a cluster analysis approach. We show that the new virus combines genetic information related to different swine influenza viruses. Segments PB2, PB1, PA, HA, NP and NS are related to swine H1N2 and H3N2 influenza viruses isolated in North America. Segments NA and M are related to swine influenza viruses isolated in Eurasia.

Introduction

Influenza A virus is a single stranded RNA virus with a segmented genome. When different influenza viruses co-infect the same cell, progeny viruses can be released that contain a novel mix of segments from both parental viruses. Since the first reported pandemic in 1918, there have been two other pandemics in the 20th century. In both cases, the pandemic strains presented a novel reassortment of genome segments derived from human and avian viruses [1-3]. The origins of the 1918 strain are so not clear, although different analyses suggest that this virus had an avian origin [4,5].

When and where pandemic reassortments happen remains a mystery. Avian viruses often undergo reassortment events among different subtypes. Several reports suggest that reassortments are also frequent between human viruses [6,7]. Swine have been found frequently with co-infections and reassortment of swine, human, and avian viruses has been reported [8-10,3]. In addition, cell surface oligosaccharide receptors of the swine trachea present both, a N-acetylneuraminic acid-alpha2,3-galactose (NeuAcalpha2,3Gal) linkage, preferred by most avian influenza viruses, and a NeuAcalpha2,6Gal linkage, preferred by human viruses [11]. Co-infection combined with co-habitation of swine and poultry on small family farms all over Asia, and the presence of avian as well as human receptor types in pigs have led to the “mixing vessel” conjecture [12,13] that suggests that most of the inter-host reassortments are produced in pigs.

Recently, a new A(H1N1) subtype strain has been identified initially in Mexico, then rapidly reported in all continents. As of 27 May, 12,954 cases of the new influenza A(H1N1) virus infection, including 92 deaths have been reported to the World Health Organization [14,15]. Several approaches have been used to understand the origins of this strain. Searches in public databases containing influenza A genomes using sequence alignment tools indicated that the closest relatives for each of the eight genomic segments are from viruses circulating in swine for the past decade [16-19]. These include genome segments derived from “triple reassortant” swine viruses that combined in the late 1990s genome segments from viruses previously identified in humans, birds, and swine [20]. Similar conclusions were drawn by the application of phylogenetic techniques [16,21].

Here we present a cluster analysis using Principal Component Analysis and unsupervised clustering. Clustering methods are particularly robust under changes in the underlying evolutionary models. Our results substantiate previous reports [16,21], and demonstrate that for each of the genome segments of the new influenza A(H1N1) virus the closest relative was most recently identified in a swine, compatible with a reassortment of Eurasian and North American swine viruses (Figure 1).

Figure 1. Origins of the new influenza A(H1N1) virus

Materials and methods

Influenza sequences were obtained from the National Center for Biotechnology Information (NCBI) [22] in the United States. We performed a search using Basic Local Alignment Search Tool (BLAST) for each of the eight A/California/04/2009(H1N1) segments separately, recording the 50 best matches. Then we constructed the union of all these matches, taking the sequences for all their segments available in the database. We aligned these sequences using the stretcher algorithm as implemented in the EMBOSS package.

After the alignment we translate the sequences into the binary data, comparing them to the reference sequence site by site. A mutation maps to 1, while a nucleotide identical to that in a reference sequence maps to 0. Whenever there are masks, they map to the corresponding fractional numbers. Gaps are not counted as polymorphisms. Therefore, if there are the S sequences restricted to the P polymorphic sites, these data translate to the SxP matrix. Each row of this matrix can be thought of as a vector in a P-dimensional space, and it represents one of the sequences.

We perform the Principal Component Analysis (PCA) in order to determine the most significant coordinates in this P-dimensional space. After this we leave the principal components which capture 85% of the total variance, discard the remaining ones and project the data onto this relevant coordinate subset.

This procedure is followed by the consensus K-means clustering. Namely, if one targets for K clusters, one repeats the K-means clustering procedure N times, and forms the matrix n whose elements nij (i,j=1,…,S) represent the number of times out of the N trials when the i-th and j-th sequences were clustered together. In our analysis we set N=100. The matrix of the distances between the samples is:

One then performs the standard hierarchical clustering with this matrix, targeting for the K clusters. This procedure does not depend on any assumptions made by the phylogenetic models. Note that these techniques can be used for inferring phylogenies as well [23], though this is beyond the scope of the present note.

Results

Sequence comparison of available sequences of the new A(H1N1) virus (as of 27 May 2009) did not identify significant sequence variation, except for a few point mutations. Hence A/California/04/2009(H1N1) was chosen as the representative for further analyses. There are many different phylogenetic techniques, each of them with their own assumptions about evolutionary models that vary in the way of computing genetic distances, probabilities, etc. As opposed to phylogenetic techniques, cluster methods do not have a need for evaluation of a tree, which is a more complicated structure than a set of clusters. Clustering techniques do not provide a detailed phylogenetic structure because they analyse group features of the sequence data. That is why the clustering analysis is more robust to the assumptions we make, for instance, the choice of genetic distance. Unsupervised methods provide a way of identifying clusters without relying on previous information about the origins, host and time isolation.

Figures 2a-2h show the data projected onto the first two principal components with the corresponding percentage of variation. The figures clearly show that in all cases the new virus sequences clustered with those of swine viruses. The closest matches for each of the segments are summarised in the Table.

Our analyses support the hypotheses whereby the 2009 pandemic influenza A(H1N1) virus derives from one or multiple reassortment(s) between influenza A viruses circulating in swine in Eurasia and in North America. It is schematically illustrated in the Figure 1.

Supplementary Tables 1 to 8 show the results of the clustering for each of the eight segments (PB2, PB1, PA, HA, NP, NA, M NS):

http://www.eurosurveillance.org/public/public_pdf/Table_1_Cluster_analysis_HA.pdf

http://www.eurosurveillance.org/public/public_pdf/Table_2_Cluster_analysis_NA.pdf

http://www.eurosurveillance.org/public/public_pdf/Table_3_Cluster_analysis_PB2.pdf

http://www.eurosurveillance.org/public/public_pdf/Table_4_Cluster_analysis_PB1.pdf

http://www.eurosurveillance.org/public/public_pdf/Table_5_Cluster_analysis_PA.pdf

http://www.eurosurveillance.org/public/public_pdf/Table_6_Cluster_analysis_NP.pdf

http://www.eurosurveillance.org/public/public_pdf/Table_7_Cluster_analysis_MP.pdf

http://www.eurosurveillance.org/public/public_pdf/Table_8_Cluster_analysis_NS.pdf

Acknowledgements

The work of T. Briese, G. Palacios and W. I. Lipkin was supported by National Institutes of Health awards HL083850 and AI57158 (Northeast Biodefense Center - Lipkin). The work of A. Solovyov has been supported by grant NSF PHY-0756966.

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Swine flu: students at university investigated

Fears have been raised that swine flu is circulating in Edinburgh after two students tested positive for the disease but have not been in contact with any known cases or travelled to the United States or Mexico.

By Rebecca Smith, Medical Editor

Last Updated: 6:48PM BST 29 May 2009

The two students, a 20-year-old man and a 21-year-old man from Greece, who both study in Edinburgh, developed symptoms of the disease last Sunday while in Scotland.

They were tested in Greece and found to be positive for H1N1 swine flu but they have not travelled to countries where the disease is widespread and to their knowledge have not been in contact with anyone confirmed or suspected of having the disease.

The cases could mark a significant shift in the swine flu outbreak in Scotland as doctors in Greece have questioned whether the virus is spreading more widely but current testing methods are not detecting it.

A spokesman for Heriot Watt University, where the two students are studying, said: "There were seven potential contacts who have all been cleared. We are continuing to operate as normal and we are in close contact with the relevant health and governmental bodies who are monitoring the situation."

Health Protection Scotland is still investigating the source of the infections, a spokesman for the agency said.

There are also three cases in England where the source of the infection has not yet been established. One in the North West and two in the East of England region.

Clusters of cases have occurred in schools in London and Birmingham and earlier this week Eton College confirmed it will remain closed for an extra week after half term after a pupil tested positive.

Ministers and health officials have said that if swine flu continues to spread it is to be expected that so-called 'de novo' cases will appear.

However a spokesman said the Health Protection Agency said: "There is currently no widespread community transmission."

Dr Takis Panagiotopoulos, of the Hellenic Centre for Disease Control and Prevention (KEELPNO), Athens, Greece, has written a research paper published online in Eurosurveillance, the scientific journal on the epidemiology of communicable diseases in Europe.

He said the 21-year-old patient had not travelled outside of Scotland and had not been in contact with any known H1N1 cases to his knowledge. "However he met a large number of people, mainly students and attended three student parties. Furthermore he spent a lot of time with his two room-mates and at least two other close friends, one of whom is case two."

The research paper adds that the source of the infection could be an as yet unidentified traveller returning from Mexico or the United States with the infection and has passed it on unwittingly to the two students, or: "There is a chance that institutionwide transmission has been taking place in the university the cases attend or widespread transmission exists in the community in the specific geographical area in Scotland that has led to the exposure of the two cases," the paper said.

Dr Panagiotopoulos wrote: "Cases of the new influenza A (H1N1) infection are for the first time confirmed in travellers from one European country to another, with no specific history of exposure to a traveller from Mexico or the United States and no traceable link to the source of infection.

"It is probably necessary to modify the present EU definition of "cases under investigation" to also include clusters of patients with influenza-like illness, irrespective of travel history."

He pointed out that under the official EU-wide criteria for a suspected case, these students would not have been tested because of the lack of travel history to an affected area and no contact with a confirmed or probable case and this means 'we are by definition going to miss cases infected locally in the event of established community transmission without known and identified chain(s) of transmission'.

A spokesman for the Health Protection Agency said a selection of GPs across the country are taking random swabs from any patient presenting with flu-like illness to check if the disease is more widespread and so far there is no evidence of this.

New swine flu cases point to invisible pandemic

13:17 29 May 2009 by Debora MacKenzie

Hospitals in Greece have identified H1N1 swine flu in two students who had no contact with known cases of the virus and had not been in countries with widespread infection. The infections were discovered even though the students should not have been tested for swine flu under European rules. The Greek authorities say this shows the rules must change.

Indeed, an investigation by New Scientist earlier this month showed that the EU rules would exclude exactly such cases and could make H1N1 appear much less widespread in Europe than it is.

Takis Panagiotopoulos of the Hellenic Centre for Disease Control and Prevention in Athens and colleagues reported on 28 May in Eurosurveillance, a weekly bulletin published by the European Centre for Disease Prevention and Control (ECDC) in Stockholm, Sweden, that two Greek men returning home from Scotland had tested positive this week for H1N1 swine flu.

Chance test

The two go to university in Edinburgh and had attended term-end parties at the end of last week. Both developed coughs and fevers at the weekend before flying back to Greece, where one went to hospital in Athens on Tuesday.

"The examining physician decided to take a pharyngeal swab, which was tested at the National Influenza Reference Laboratory for Southern Greece, although the patient did not meet the European Union and national criteria for the new influenza A (H1N1) testing," the team reports.

The swab was tested with a kit for H1N1 distributed by the US Centers for Disease Control and Prevention (CDC), and was positive for swine flu. The student in Athens warned the second student, who was now in Thesaloniki. He also tested positive. Both cases were mild.

Contacts of the two in Greece and Scotland and on the flights are being traced.

'Missing cases'

The Greek cases are "community acquired", meaning they have no contacts with known cases or countries with swine flu. The ECDC guidelines adopted by most EU countries, including Greece, recommend testing for H1N1 only when people have such contacts, excluding community acquired cases.

"It is of concern that with the present EU [testing criteria] we are by definition going to miss cases infected locally in the event of established community transmission," the Greek team warns. "It is probably necessary to modify the present EU definition … to also include clusters of patients with influenza-like illness, irrespective of travel history," they say, especially as the tourist season is getting under way.

Officially, swine flu has increased very slowly in Britain, even though the virus appears to be as contagious as ordinary flu. John Oxford of the University of London says the UK may have tens of thousands of mild, untested cases. The US CDC says there could be 100,000 cases in the US, even though only a few thousand, mostly severe, cases have been tested.

Finding community acquired cases outside the Americas is a requirement for declaring H1N1 swine flu an official pandemic, which the WHO has not yet done.

Eton College shut as pupil contracts swine flu

Eton College has been forced to shut for an extra week after the half-term break as a pupil tested positive for swine flu.

by Rebecca Smith, Medical Editor
Last Updated: 5:51PM BST 28 May 2009

Eton College has been forced to shut for an extra week after the half-term break as a pupil tested positive for swine flu.

The student, who is mildly unwell, tested positive on Wednesday and has been recovering at home.

The source of the infection is being investigated by health officials and after taking advice from the Health Protection Agency the school will remain closed until June 7th.

Following discussion with the HPA the school plans to remain closed until 7 June, and boys who are due to sit public examinations will be allowed to return under controlled conditions.
"The HPA is following up pupils who have been in contact with the boy that tested positive. The objective is to minimise the risk of spread of infection, while allowing boys to take their public examinations."

The Department of Health has secured contracts with vaccine manufacturers for 90m doses, enough for half the population as it is thought each person will need two jabs, to be delivered by December.

School swine flu hits 50 people

Donna Pendley's nine-year-old son attends Welford School Fifty children and adults connected with a Birmingham primary school are being treated for swine flu. Forty-four new cases of the disease, linked to Welford School in Handsworth, have been confirmed by the Health Protection Agency. The agency said that of the total, 45 were children and five were adults, including at least one teacher. The number of cases is expected to rise. It is the single largest outbreak of the virus in the UK. The total number of cases in the West Midlands region is now 57 with 184 confirmed cases in the UK.  You wonder how it got to the school - I really am nervous  Donna Pendley, parent The 420-pupil school is closed for the half-term holiday. It closed on Thursday, a day earlier than scheduled, after a higher than normal number of illnesses was noted. A statement issued by the school at the time said the school would have a "deep-clean" during the holiday. The school was informed on Thursday evening that one of those taken ill was a confirmed case of swine flu. All parents and staff were contacted and asked to collect anti-viral drugs. The HPA said all the confirmed cased were being treated at home with anti-virals and were responding well to treatment. Professor Anthony Kessel, of the Health Protection Agency, said case numbers at the school were not a surprise It added there were a number of laboratory tests outstanding and the number of confirmed cases at the school were expected to rise. HPA regional director Sue Ibbotson said: "While the illness can be unpleasant for those suffering none of the cases have been hospitalised. "Viruses spread easily in schools and the HPA is working hard with the school and parents to limit its further spread." She advised that unless people have flu-like symptoms, or are being tested for swine flu, there was no need to stop their normal activities. Anyone displaying flu-like symptoms should phone rather than visit their surgery. Donna Pendley, whose nine-year-old son Kenyjah attends the school, said Tamiflu had been given to him, but not to her or her two-year-old daughter. She said: "We got the leaflet about swine flu some time ago, but when it appears in your area it makes you nervous. "You wonder how it got to the school, how did it get to Birmingham and Handsworth? "I am nervous, I really am nervous."

Europe may be blind to swine flu cases

A doctor performs a swab test, to test the patient for H1N1. At present, only high-risk patients are being tested in Europe (Image: Burger / Phanie / Rex Features)

Editorial: Ignoring flu won't make it go away

EUROPE might have more H1N1 swine flu than it knows. The virus could be circulating widely but not being spotted simply because people are not being tested.

• 20 May 2009 by Debora MacKenzie
• Magazine issue 2709. Subscribe and get 4 free issues.
• For similar stories, visit the Epidemics and Pandemics Topic Guide

As New Scientist went to press, the World Health Organization was still undecided about declaring a full-blown pandemic, despite a surge in swine flu cases in Japan. To do this it needs evidence of "sustained transmission" outside the Americas, where the virus originated. This means finding cases in the general population that have not had known contact with places or people confirmed to have the virus. Japan found H1N1 this week in over 100 people, many without known contact.

But European countries are using a case definition from the European Centre for Disease Prevention and Control (ECDC) in Stockholm, Sweden, that virtually precludes discovering such cases. It recommends testing people with symptoms only if they have been to affected countries or had contact with a known or suspected case in the past seven days.

"We can't test every mild case of flu symptoms," says Johan Giesecke, chief scientist at ECDC. "But it's true, we might not be seeing community spread because we aren't looking." On 18 May, the UK had 101 confirmed cases of H1N1, of which only three fell outside the case definition.

The UK Health Protection Agency's criteria are similar (see diagram). Doctors "are encouraged to use this algorithm", an HPA spokesperson told New Scientist, but they can "use their clinical discretion" to test anyone.

An anonymous UK New Scientist reader, and two family members, had flu symptoms after one returned ill from New York on 10 April. They were not tested for H1N1. "My general practitioner is horrified that I am not even eligible for a test because I have not returned from Mexico in the last seven days, nor been in contact with someone who has been diagnosed."

A New Scientist reader had flu symptoms on returning from New York but was not tested for H1N1

Tests may simply be unavailable. "I was given only two swabs [for H1N1] initially," says Laurence Buckman, head of the GP committee of the British Medical Association. More are available now, "but if you can't do many tests you save them for people who meet the case definition".

Any others, says Buckman, will be picked up by "sentinel" clinics that compile weekly statistics. The ECDC claims this system "would detect circulation of the new H1N1 virus before any major outbreaks occur".

However, such sentinel systems are designed to track ordinary flu, not to detect a new infection that is initially highly localised. "It may take weeks before the numbers indicate an epidemic," warns Dick Wenzel of Virginia Commonwealth University in Richmond, past president of the International Society for Infectious Diseases. He advises testing clusters of flu and all severe cases.

Hong Kong is testing all hospitalised cases of flu and pneumonia. Belgium, departing from ECDC advice, is testing flu-like clusters and deaths. But without more tests, Europe may be missing an epidemic.

Editorial: Ignoring flu won't make it go away

Ignoring flu won't make it go away

• 20 May 2009
• Magazine issue 2709. Subscribe and get 4 free issues.
• For similar stories, visit the Epidemics and Pandemics Topic Guide

H1N1 flu is still spreading. In North America, the number of cases may have passed the 100,000 mark; and cases in Japan may tip us into a pandemic. Yet Europe claims it doesn't have evidence of "sustained transmission" of the virus.

That's hardly surprising, as Europe isn't doing the relevant tests (see "Europe is failing to test for circulating swine flu"). Do governments fear that if they discover the virus is spreading, people with sniffles will swallow antivirals unnecessarily and spawn a drug-resistant strain? Whatever the reason, mad cows taught the UK that refusing to see - and tell - the truth about disease is unwise. If H1N1 is spreading elsewhere, it is unlikely to peter out in Europe. The authorities have had years to draw up pandemic plans. Yet they appear as ill-prepared to track the spread of this virus as they are to make a vaccine for it.

Beware the return of swine flu

• 19 May 2009
• Magazine issue 2708. Subscribe and get 4 free issues.

THE backlash was predictable. Around the world, pundits and even some politicians are calling for an end to "crying wolf" over swine flu. It's no worse than ordinary flu, they say, brandishing news reports of mild cases. Why are we wasting money on this nonsense?

Bluster of this kind will be with us as long as it attracts newspaper readers and TV viewers. But let's be clear: nobody who knows the first thing about flu is crying wolf. It is an inherently unpredictable disease, and the fact that the H1N1 virus has not yet wreaked obvious havoc doesn't mean there is no cause for serious concern. Yes, it is possible that the virus will fizzle out completely. Yes, it is possible that if or when it comes back for another go, it will - unlike the last H1N1 pandemic in 1918 - be no more lethal than it is now.

The fact that the H1N1 virus has not yet wreaked obvious havoc doesn't mean there is no cause for concern

If this virus fails to persist, so much the better. But there is a real danger that it will launch another wave - perhaps in as little as a few months - and it makes sense to do everything to prepare for the worst. The appearance of the Mexican virus has exposed our inability to make timely quantities of vaccine (see "'Pandemic' flu vaccine will be too late for most") despite the best efforts of the industry and five years of angst over bird flu - a threat which, incidentally, remains real.

It is also wrong to assert that Mexican H1N1 is little more serious than ordinary flu. Yes, the raw mortality figures make it seem that way. Ordinary flu kills about half a million people a year - about 0.2 to 0.5 per cent of those who catch it. For the Mexican virus the current, early estimate looks only slightly worse: something between 0.3 to 1.5 per cent of people who caught it have died.

But behind these figures there is a crucial difference. Ever since we got antibiotics to defeat the bacterial pneumonia that can follow flu, nearly all deaths from regular flu have been in the elderly, and they are now almost exclusively among the over-75s. It may sound callous, but the truth is that most of those who die of flu have a good chance of dying soon anyway; it just happens to be flu that strikes the final blow.

But in Mexico it has been those aged 15 to 54 who have got really sick with this virus. These are people who would expect to have many productive years ahead of them: the teenage students, the twenty-something parents, the farmers and doctors and truckers who make the world work. Even if this virus gets no worse, the burden of illness and death rates shifted onto this age group would have a very different impact from ordinary flu.

We need to take the new H1N1 flu seriously. And then hope the wolf really does slink away. Either way, we need to make sure we're better prepared next time.