Commentary
H5N1
Transport and Transmission By Wild Birds
Recombinomics Commentary
13:53
December 2, 2008
India might be due to migratory birds, there is as yet very little
evidence for them playing a meaningful part in the epidemiology of
this disease, in fact, rather to the contrary, as commercial
movements of poultry and such have been found to be the consistent
cause.
The above remarks on the current outbreak of H5N1 in Assam, India ignore the temporal and spatial data, as well as sequence data, which support a wild bird origin for the spread of H5N1 west of China, including south Asia, since the outbreak at the Qinghai Lake Nature Reserve in the spring of 2005. Prior to that outbreak H5N1 had been reported in wild birds, but the direction of transmission was unclear, due in part to the frequent poultry outbreaks of H5N1 in China and countries to the east of China.
In May, 2005 China reported large scale deaths of long range migratory birds at Qinghai Lake. One hypothesis, which was published in detail on ProMED held that the deaths at Qinghai Lake represented “wild birds as victims” and the spread from Qinghai Lake was unlikely because “dead birds don’t fly”. However, the nature reserve at Qinghai Lake is large, with well over 100 species of birds representing 100’s of thousands of birds.
The initial OIE report described five H5N1 infected species of long range migratory birds, including bar-headed geese, which can migrate 1000 miles in 24 hours, allowing for significant spread by birds that were lethally infected. Moreover, low path H5 is present in wild migratory birds offering cross-reactive immunity to H5N1. Therefore, many H5N1 produced mild symptoms or were asymptomatic in waterfowl. The outbreak at Qinghai Lake represented a new sub-clade (2.2 or Qinghai strain), which allowed the spread to be monitored by phylogenetic analysis of the sequences from the isolates. The sequences had many of the hallmarks of highly pathogenic avian influenza from Asia, including a polybasic cleavage site in HA, and 20 aa deletion in NA, a 5 aa deletion in NS. Moreover, clade had a number of distinguishing markers, including a distinctive HA polybasic cleavage site of GERRRKKR, as well as E627K in PB2, which had not been previously reported in H5N1 in birds (prior H5N1 isolates with E627K were mammalian, which was also true for seasonal flu).
Since clade 2.2 was associated with waterfowl deaths, following its spread was relatively straightforward, even in the absence of confirming sequence data. After the May / June outbreak at Qinghai Lake, farms in northwest China reported H5N1 outbreaks in waterfowl in June, suggesting the clade 2.2 at Qinghai Lake did not burn itself out. This was confirmed by a large outbreak of H5N1 in wild bird and poultry near Chany Lake in Russia in July. The H5N1 was sequenced and was clade 2.2. Moreover the isolates included a healthy crested grebe, providing direct evidence for asymptomatic infection of migratory birds. Previously Russia had not reported HPAI H5N1, which was also true of Kazakhstan, which also had H5N1 in adjacent regions in August, 2005.
In addition to the outbreaks in Russia and Kazakhstan in August, 2005, Mongolia reported wild bird deaths at the remote Erhel Lake. Bird conservation groups aided in the investigation and the comments on results linked to Erhel Lake were telling. Initial comments expressed doubts that the dead birds would be H5N1 positive, because the number of dead birds was markedly lower than Qinghai Lake. Even after initial data showed that the dead waterfowl was H5 positives, doubts were expressed that the H5 birds would be H5N1 infected. After the birds were H5N1 confirmed, doubts were expressed that H5N1 would spread, because cloacal swabs from healthy birds were H5N1 negative.
However, H5N1 had clearly migrated into Erhel lake, and the negative data on healthy birds was not compelling because the H5N1 testing was initially designed to detect H5 in dead or dying poultry, which would have a higher viral load and individual tissues with signs of pathology could be assayed. Moreover, although detection of H5N1 in healthy birds was a challenge, H5N1 had been isolate from a asymptomatic crested grebe at Chany Lake.
The ability of the testing procedures to detect H5N1 in infected birds was experimentally tested. Although the birds had been experimentally infected, most H5N1 detection was limited to nasopharyngeal swabs. Cloacal swabs were negative for H5N1 isolates, and generally negative in PCR tests. Virus isolation from nasopharyngeal swabs was limited to a 24 hour period several days post-infection. PCR tests detected H5 for a slightly longer time period, but most daily collections post infection were negative.
Thus, detection of H5N1 in healthy wild birds is rare, especially when cloacal swabs or fecal samples are collected. However, Russia published a Mission report in the 2005 outbreak and found H5N1 in hunter killed birds including over two dozen wild bird species. These data once again demonstrated that H5N1 was widespread in wild birds, but detected under a limit set of circumstances.
Russia reported multiple outbreaks in southern Siberia in the summer of 2005, raising concerns that H5N1 would spread to Europe, the Middle East, and Africa as birds migrated south from Russia and Mongolia via well characterized flyways.
The concerns were realized in the following months. H5N1 was first reported in the Volga Delta in late August, followed by outbreaks in Romania, Turkey, and Ukraine in late 2005.
However, the most compelling data was the detection of H5N1 in a healthy teal in the Nile Delta in December, 2005. Although the level of H5N1 was too low for isolation of the virus, repeated extraction and amplification of the RNA yielded HA and NA sequences which were Qinghai H5N1 and most closely related to H5N1 in Austria. In December, 2005 all countries in western Europe, including Austria, denied H5N1 infection, as did countries in the Middle east and Africa.
In late 2005, family members in eastern Turkey began developing bird flu symptoms. Four siblings were transferred to Van and three were unconscious. Initial throat swabs were negative, but as siblings died, fluid from their lungs were H5N1 positive and sequence analysis showed that these were clade 2.2. Human cases were subsequently confirmed in Iraq, Azerbaijan, Egypt, and Djibouti. All were clade 2.2.
In January, 2005 reports of confirmed H5N1 in wild birds and poultry were submitted by countries in Europe and the Middle East and by the spring of 2005 approximately 50 countries reported confirmed H5N1. All isolates were clade 2.2 and many countries, especially in Europe, reported H5N1 exclusively in wild birds.
This sequence of events was repeated a year later. In the summer of 2006 there was a massive H5N1 outbreak at Uvs Lake in Mongolia and adjacent areas in southern Russia. H5N1 is not a reportable disease in wild birds and neither country filed an OIE report, but media reports indicted the bird deaths were on a par with Qinghai Lake and sequences from Mongolia and Russia (Tyva) were published. The sequences represented one of the clade 2.2 sub-clades circulating in eastern Europe and south Asia in 2006 (clade 2.2.3), but additional changes were found in these isolates, which phylogenetically defined a Uvs Lake strain. These sequences were subsequently reported in South Korea and Japan at the end of 2006, followed by Kuwait in early 2007.
Although the Uvs Lake strain was not reported in Europe at the end of 2006 or beginning of 2007, it appeared in wild birds in the summer of 2007. Following a poultry outbreak in the Czech Republic, H5N1 was reported in wild birds in Germany at multiple location in four states, as well as wild birds in the Czech Republic and France. Although all isolates were the Uvs Lake strain, each location had unique sequences indicating that a common poultry source was not responsible for the multiple outbreaks. Earlier, in 2007 the outbreaks in Bernard Mathew turkeys in England was virtually identical (99.96% identity) with sequences from Hungary, where the same country had operations, demonstrating the level of identity expected for isolates from a common source. This level of identity was not present in the multiple wild bird isolates. Thus although all were the Uvs Lake strain, all were due to independent introductions, which was also reported for multiple outbreaks in Nigeria in 2006, which were initially speculated to be due to trade.
In late 2007 / early 2008 the Uvs Lake strain spread throughout Europe. Recently released sequences from a German wild bird outbreak in Bavaria in the summer of 2007, as well as free range turkeys in England at the end of 2007, and wild swans in England at the beginning of 2008 were all the Uvs Lake strain, but each outbreak represented an independent introduction.
Thus, sequence analysis can easily distinguish between a common source or independent introductions, but this type of analysis is rare for countries like India or Bangladesh. A recent report on the 2007 outbreak in Bangladesh had only one full sequence. Although all HA cleavage sites were clade 2.2, the lack of full sequences precluded determination of whether the multiple outbreaks were due to independent introductions. For the outbreaks that began at the end of 2007 in Bangladesh, no sequences have been made public, but are clade 2.2 and closely related. However, it is unlikely that the level of identity is above 99.9% as was seen in the England / Hungary isolates which were linked by the same company with facilities in both countries.
Thus, which sequence data from the current outbreak in India is unlikely to be forthcoming, sequence analysis of prior outbreaks in Europe, the Middle East, Africa, and south Asia support independent introductions of clade 2.2 by resident or migratory birds.
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