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Abstract

Human-to-human transmission raises demand for DNA data.

A strain of avian flu that spread through a family in Indonesia, killing seven of the eight people infected, was accumulating mutations as it spread from person to person, according to confidential sequence data seen by Nature. The functional significance of the mutations isn't clear — most of them seem unimportant. But influenza researchers say the finding reiterates the need for sequence data to be made more widely available, if the virus is to be better understood.

The cluster of cases of the deadly H5N1 strain, which occurred earlier this year, is the first in which the World Health Organization (WHO) has admitted that human-to-human transmission was the most likely cause of spread (see Nature 441, 554–555; 2006). Eight members of an extended family in Kubu Sembelang, in north Sumatra, were affected. The first patient, a 37-year-old woman who became ill on 24 April and died on 4 May, is thought to have caught the disease from poultry, then transmitted it to six relatives. One of these, her 10-year-old nephew, who died on 13 May, is thought to have passed the disease to his 32-year-old father (see 'Flu cluster in Indonesia').

Virus isolates from six of the eight family members have been sequenced, but the WHO has not released the data, saying that they belong to Indonesia. Instead, the agency released a statement on 23 May stating that there was "no evidence of genetic reassortment with human or pig influenza viruses and no evidence of significant mutations".

Nature has now obtained more detail on the genetic changes, which suggest that although the WHO statement was not incorrect, plenty more could have been said. Viruses from five of the cases had between one and four mutations each compared with the sequence shared by most of the strains. In the case of the father who is thought to have caught the virus from his son — a second-generation spread — there were twenty-one mutations across seven of the eight flu genes. This suggests that the virus was evolving rapidly as it spread from person to person.

One of the mutations confers resistance to the antiviral drug amantadine, a fact not mentioned in the WHO statement. The data were presented by Malik Pereis, a virologist at the University of Hong Kong, at a closed meeting of around a dozen international experts in animal and human health, held in Jakarta, Indonesia, in late June.

The virus did not evolve into a pandemic strain — the combination of mutations was not even enough to allow it to spread beyond close family. Many of the genetic changes did not result in the use of different amino acids by the virus. And there were no amino-acid changes in key receptor binding sites known to affect pathogenicity and transmissibility.

But experts say they cannot conclude that the changes aren't significant. "It is interesting that we saw all these mutations in viruses that had gone human-to-human," says one scientist who was present at the Jakarta meeting but did not wish to be named because he was commenting on confidential data. "But I don't think anyone knows enough about the H5N1 genome to say how significant that is."

Elodie Ghedin, a genome scientist at the University of Pittsburgh School of Medicine in Pennsylvania, says she's surprised that the virus from the father had so many mutations compared with others in the cluster, apparently arising in just a few days. "I have a hard time believing that the father acquired the virus from his son," she says, adding that the nine mutations in one gene in the father's virus are almost identical to those in viruses isolated from human cases in Thailand and Vietnam in 2004.

Flu researchers don't all look at data from the same angle.

One possibility is that the father simply caught a different strain of virus from birds, although other mutations in his virus are similar to those in the strain isolated from his son. Or perhaps the virus from the son reassorted with another flu strain circulating in his father at the time, Ghedin says.

Part of the reason the picture is so unclear, say virologists contacted by Nature, is that the continued withholding of genetic data is hampering study of the virus. None of the sequence data from the Indonesian cluster has been deposited in public databases — access is restricted to a small network of researchers linked to the WHO and the US Centers for Disease Control and Prevention in Atlanta, Georgia.

Ghedin, for example, works on how mutations in one area of a genome can predispose other areas to further changes. She is part of a project started in 2004 to sequence thousands of human and bird-flu strains, but she has little access to H5N1 virus from humans. "Flu researchers don't all look at the data from the same angle," she says. "The more diverse analyses that are performed, the better we will understand the evolution of this virus."

"If all of the H5N1 isolates were available, there'd be quite a few people focused on understanding these data," agrees David Lipman, director of the US National Center for Biotechnology Information in Bethesda, Maryland.

But Paul Gully, who joined the WHO two months ago as senior adviser to Margaret Chan, head of the agency's pandemic-flu efforts, defends the agency's position. He points out that the WHO's priority is investigating outbreaks, not academic research. And he adds that although calls for more complete genome data and wider sharing of samples are "a valid point", labs are stretched during outbreaks, and don't have the time or resources to do high-quality sequencing.

He agrees that sharing samples with other researchers would allow such work to be done. But he says the WHO must work within the constraints set by its member states — they own the data, and decide whether to share it. "As more countries share data, hopefully that research will get done," he says.

The WHO has not formally asked Indonesia to share the sequences, Gully adds. "We would rather wait and see what Indonesia decides."

 

 

 

niman – at 08:08


The distribition of the changes in the Karo cluster were far from random. Even though each round of replication requires the copying of over 12,000 letters into the eight gene segments, this was done quite faithfully in the H5N1 isolated from the first series of transmissions.

These first cases all were infected between May 2–4 and all died about a week a after infection. The fatalities included the index case’s two sons, a sister, her daughter, and the 10 year old son of another brother ($#2). The sequence of the H5N1 of the surviving brother #1 was not presented and brother #2 was not initially infected.

Thus, initially there were 5 sequences (then initial post that started this thread mistakenly said six different individuals) and all were vitually identical. The sister and nephew sequences were the same. The two sons had one difference and the niece had another. Thus, the five sequences from the five relatives were 0/12,000, 0/12,000, 1/12,000. 1/12,000, 1/12,000 for the five individuals. A secoind H5N1 isolate was obtained from the nephew and it had an additinal change at position 29 of HA.

After the nephew died, his father developed symptoms and died. H5N1 from the father (brother #2 of the index case) had the same change as his son at position 29 of HA. However, that H5N1 also had 8 more changes in HA, three changes in NA, and 2 changes in M. The other 5 genes were exactly the same as his son and the consensus for the family.

Thus, while all 6 versions of H5N1 from the initial group of 5 people differed by at most 1 letter, and the brother #2 had 14 changes, 14 random mutations suddenly appearing in brother #2 is a HIGHLY unlikely explanation.

These 14 changes however, are EASILY explained by a second H5N1 infection in brother #2.
And:

niman – at 08:44


Back to the DATA. The second line of evidence against random mutation is the distribution among the family members. Even though the H5N1 has the same mutation and all eight individuals were blood relatives and died from the H5N1, one has 14 changes and all others had zero or one change.

The simplest explanation for the sudden appearance of 14 (or 13 new) changes is a second H5N1 infection. The evidence for dual infections in flu is pretty overwhelming. One piece of evidence the number of flu serotypes. There are 16 H’s and 9 N’s so there are 144 theoretical combinations. A look at the list of flu serotpes at GenBank shows that most combinations have been found and the combinations are formed by reassortment in dual infections, which creates new combinations, but not new H’s or N’s.
And:
Here is more on the analysis. The 14 changes probably represent both recombination and reassortment. The only hard evidence for recombination is in HA. The father had the C29T polymorphism found in the second H5N1 collection from his son. The other 8 changes could have come from the second H5N1. That distribution would only require a single crossover somewhere between position 29 and 150, the location of the next novel polymorphism. The remainder of HA probably matched the second H5N1. Similarly, all of NA and M could have come from the second parent. PB2, PB1, PA, NP, and NS matched the son.

Thus, the data can be easily explained by a dual infection by the son's H5N1 and a separate H5N1. The new info in the H5N1 from the father would come from the parent via recombination in HA and reassortment of NA and M.

therefore, only one new gene needed to be created. HA was the only gene with a marker fro the son and 8 new markers. The other changes could have been acquired via acquisition of the entire gene.

Thus, the gene pool may not have dramatically changed, but the genes in the cluster changed via a second infection.

Infection of the father by two separate sources would be a major worry, indicating H5N1 was widespread and other family members may have been infected with a milder version. There was a ninth family member hospitalized, but released after testing negative and thee were media reports of others hospitalized and under observation, but those reports were not confirmed.

Since a reporter noticed the symptoms in the father and the family did not trust the WHO investigators, it is not all that clear on who was infected and who was not. The dual infection in the father may have been a signal of infections in more family members or neighbors.

 

If there is a recombination with H5 with another flu strain like H1 would this change the H and or the N? Is this what he's talking about here?