I've been very excited to tell you about a new paper that has just been published by PLoS Biology. This paper nicely unites the two fields of evolutionary biology that I am most passionate about, the evolution of viruses and evolution of birds.

According to this newly published paper, fragments of ancient viruses have been discovered hiding in the chromosomes of songbirds, including popular pets like zebra finches, Taeniopygia guttata (top). DNA analysis indicates these viral remnants are ancient hepadnaviruses, a family of DNA viruses that includes the hepatitis B virus (HBV), which infects roughly one-third of the world's human population, causing a variety of acute and chronic liver diseases.

These ancient viruses are endogenous viruses: they insert themselves or pieces of themselves into the genome of an organism, and are then passed from one generation to the next. Up until now, nearly all endogenous viruses found have been retroviruses. This is not surprising since insertion of the retrovirus into the host's genome is an essential part of its life cycle. But when deactivated fragments of these viral freeloaders reside in a host's genome for millions of years, they are referred to as "fossil viruses" and the scientists who study them are palaeovirologists.

As genome sequencing becomes easier and cheaper, palaeovirologists are now uncovering remnants of other types of virus such as hepadnaviruses in a variety of organisms ranging from fungi to plants - and even including humans [DOI: 10.1038/nature08695].

Even more interesting, this discovery shows that hepadnaviruses,

originally thought to be 6000 years old, are much older than scientists realised.

"They've been sitting there for at least 19 million years, far longer than anyone previously thought this family of viruses had been in existence," said Cédric Feschotte and Clément Gilbert, the paper's two co-authors. Dr Feschotte, an associate professor and Dr Gilbert, a post-doctoral research associate, are members of the Genome Biology Group at the University of Texas at Arlington, where they study the evolution of the genome and how ancient viruses have shaped their hosts' immune systems over millions of years.

But Dr Gilbert and Dr Feschotte didn't know to look at avian DNA to find this particular fossil virus. Instead, they started by searching for HBV in GenBank, a public database that contains more than 100 million DNA sequences.

"We screened all complete genomes that are available in public web databases using the human hepatitis B sequence as a query," Dr Gilbert explained to me in an email. "The zebra finch was the only species where we found integrated HBV-like fragments."

The team found 15 of these HBV-like fragments sprinkled throughout 10 of the zebra finch's 33 chromosomes, most with one or more mutations that rendered them inactive. Thus, these fossil viruses are functionally dead, but their imprint remains, ghost-like, in their host's genome, marking the passage of time by accumulating random errors when copied, as does the surrounding genomic DNA.

Analysis of differences between these 15 HBV-like fragments and hepadnaviruses show that the zebra finch HBV-like fragments fell into one of two groups (collectively known as endogenous zebra finch HBVs [eZHBVs]), suggesting that the virus inserted itself twice into the bird's DNA before being shuffled around the genome (red branches in Figure 2, below). While similar, the endogenous fossil viruses that are embedded in the songbird genomes were distinct from free-roaming extant avian hepadnaviruses (blue branches) while mammalian hepadnaviruses segregated into their own distinct groups (black branches):

But how long had these two eZHBVs been present in the finch genome? To answer this question, Dr Gilbert and Dr Feschotte first had to compare the eZHBVs from closely related songbird species (black throated finch, Poephila cincta; scaly breasted munia, Lonchura punctulata; and gouldian finch, Chloebia gouldiae) and from two more distantly related birds (dark-eyed junco, Junco hyemalis and the olive sunbird, Cyanomitra olivaceus).

Comparing differences between the eZHBVs (red branches) to those in

free-roaming extant avian hepadnaviruses (black branches), the team used their data to construct a family tree for the fossil virus. They then compared the fossil virus tree to their avian hosts' family tree, using them as an evolutionary clock, and found the two trees looked the same (see right side in Figure 3, below):

Comparing the family trees for the fossil virus and the bird species,

the team found that the most distantly related of the birds, the olive sunbird, lacked the virus, whereas it was present in all the other birds' genomes. Based on other scientists' work documenting the timing for when these species arose, they concluded that the first viral insertion occurred between 35m and 25m years ago, when the dark-eyed junco, the next oldest relative, split off.

Dr Gilbert and Dr Feschotte then used the molecular clock method, which verified their findings and indicated that the first fossil virus had inserted itself into the birds' genome between 19m and 40m years ago.

Together, these data strongly suggest that the fossil eZHBVs integrated into the genome of the songbird's common ancestor that lived more than 19m years ago, prior to the evolution of these different bird species.

According to Dr Gilbert, the team was also surprised to find "a strikingly slow, long-term mutation rate that is 1,000 times slower than the viral [mutation] rates that had previously been estimated based on comparisons of currently circulating viral sequences only."

When the team compared the fossil virus to extant hepadnaviruses, they were surprised to learn that, despite their antiquity, the fossil hepadnaviruses are remarkably similar to the modern viruses. Dr Feschotte thinks that the slow evolution of the hepadnaviruses observed in birds indicates that these viruses are better adapted to their avian hosts than what is suggested by research into the disease-causing HBVs.

"Genomic fossils like the remarkable hepadnaviral fossils found by Gilbert and Feschotte have the prospect of completely revising our preconceived notions about the age and evolution of such viruses," said Harmit Singh Malik, who was not part of the study. Dr Malik is an associate member of the Fred Hutchinson Cancer Research Center in Seattle and is one of the leaders in the emerging field of palaeovirology.

"They provide an unexpectedly clear lens on an ancient time when these viruses were prevalent and abundant."

Eddie Holmes, a distinguished professor of biology at Pennsylvania State University's Eberly College of Science and an expert in the field of viral evolution, said the team's work "provides a glimpse into an ancient viral world that we never knew existed".

"The results they obtained were remarkable; whereas we previously thought of hepadnavirus evolution on timescales of only a few thousand years, this paper shows that the true timescale is in fact many million years," said Dr Holmes, who is currently traveling, in a press release. "Therefore, hepadnavirues, and likely many other viruses as well, are far older than we previously thought."

These fossil viruses are not capable of causing disease in birds, nor infecting humans.

"The viruses that we found are very old, are integrated in the bird genome, and do not have the potential to encode any functional protein product," said Dr Gilbert. "So they do not have any effect in songbirds."

"Finding these viruses fossilised in songbird genomes tells us that they were once infecting these birds, like hepatitis B is infecting humans today," said Dr Gilbert.

Although it is not possible to know whether these ancient viruses caused the same disease in birds as HBV does in humans today, "it is possible that some of these birds may still be infected by HBV-like viruses today."

This study will catalyse new lines of inquiry that may help scientists predict and prevent future human viral pandemics that originate in birds.

"We hope our study will encourage people to screen for the presence of such circulating viruses in this group of birds," said Dr Gilbert. "We can therefore use this discovery as a guide to screen targeted groups of bird species for the presence of new circulating hepatitis B-like viruses."

Sources:

Gilbert C & Feschotte C (2010). Genomic Fossils Calibrate the Long-Term Evolution of Hepadnaviruses. PLoS Biology, 8 (9): e1000495. DOI: 10.1371/journal.pbio.1000495

Dr Clement Gilbert [personal email, 28 September 2010]

Traci Peterson, UTA publicity agent.