A new influenza virus discovered in Missouri pigs has a combination of genes from avian and swine flu viruses, supporting the theory that pigs can serve as a mixing vessel for flu viruses and a potential source for a human pandemic strain, according to a report published yesterday. 

Researchers found that the virus, an H2N3 subtype, caused illness in experimentally infected mice and was transmissible in swine and ferrets, suggesting it has adapted to mammals, according to the report, published in the Proceedings of the National Academy of Sciences (PNAS). In addition, genetic analysis showed the virus has a mutation linked with an increased ability to infect mammals. 

The discovery marks the first identification of an H2 virus in swine, according to the authors of the report. The flu pandemic of 1957-58 was caused by an H2 virus, namely H2N2. That virus was replaced by an H3N2 strain in the pandemic of 1968-69, and H2 viruses have not circulated in humans since then. Hence, people younger than 40 have little immunity to H2 viruses, scientists say. 

"Our results provide further evidence for the potential of swine to promote reassortment between different influenza viruses, and the genetic and biologic properties of the H2N3 viruses described suggest that it would be prudent to establish vigilant surveillance in pigs and in workers who have occupational exposure," states the PNAS report. 

It was written by scientists from Iowa State University and the US Department of Agriculture's (USDA's) Agricultural Research Service (ARS), both in Ames, Iowa; the University of Minnesota in St. Paul; and St. Jude Children's Research Hospital and the University of Tennessee, both in Memphis. The lead author is Wenjun Ma of Iowa State and the USDA. 

Two incidents 5 months apart
The discovery of the new virus began with an illness outbreak in pigs at a Missouri swine nursery in September 2006, according to the report. The pigs' lungs showed obvious signs of pneumonia, and tests showed the presence of an influenza gene, but the subtype could not be determined. Samples were submitted to the ARS in Ames, where genetic sequencing and a search of a flu sequence database showed the virus subtype to be H2N3. 

After the virus was subtyped, a record search revealed that another unidentified virus had been submitted in April 2006, from a 12-week-old pig at another Missouri swine farm, according to the report. Analysis by the ARS showed that this isolate too was an H2N3, and the two viruses were nearly identical. 

The two outbreak sites are about 8 miles apart, according to Marie Gramer of the University of Minnesota College of Veterinary Medicine in St. Paul, a coauthor of the report. She said there was no connection between the two operations. 

Phylogenetic analysis showed that the virus's hemagglutinin (HA) gene most closely matched the genes of H2 viruses isolated from North American mallard ducks, while the neuraminidase (NA) gene was closely related to that of an H4N3 virus found in blue-winged teal. Five of the other six genes were derived from swine flu viruses currently circulating in the United States, the scientists determined. 

The source of the virus is unknown, but the likeliest possibility is pond water, which was used to clean barns and water the animals on both farms, according to the report. That transmission pathway has been described before. 

Looking for genetic evidence of the new viruses' adaptation to mammals, the authors compared their HA genes with the HA genes from avian H2N3 viruses. They found an amino-acid substitution at position 226 that, in other H2 and H3 viruses, has been associated with adaptation of avian viruses to humans, according to the report. 

To assess the new isolate's pathogenicity in swine, the scientists inoculated 20 4-week-old pigs with one of the viruses, and 3 days later housed 10 other pigs with the inoculated pigs. On necropsy, the inoculated pigs were found to have mild to moderate pneumonia, while the contact pigs had antibodies to the virus but slight or no signs of pneumonia. 

The researchers also exposed mice to one of the H2N3 viruses at three different doses. Mice that received the middle or highest doses showed signs of illness, and 75% of those with the highest dose died, but none exposed to the lower doses died. 

To test the virus's effect in ferrets, the scientists inoculated three 18-week-old, separately caged ferrets with it and then placed an uninfected ferret in each cage a day later. Subsequent tests showed the virus was present in all the animals, but none showed obvious signs of illness. 

Reason for public health concern
The authors write that their findings are "of considerable concern to public health." In particular, the H2N3 viruses' mutation associated with an affinity for human-type cell-surface receptors and their ability to replicate and spread in swine and ferrets "suggests that the swine H2N3 viruses have undergone adaptation to the mammalian host and as such have the ability for sustained transmission." 

However, the report also says that "receptor-binding changes" are not sufficient by themselves to permit avian flu viruses to successfully adapt to humans. For example, swine H1N1 flu viruses prefer human-type receptors and have occasionally infected humans but have not become established in human populations. 

Gramer told CIDRAP News that no illnesses were reported among farm workers in connection with the swine cases, and no workers were tested at the time. However, serologic testing of the workers is currently being conducted under a grant from the Centers for Disease Control and Prevention, she reported. 

When asked whether the newfound virus currently represents a threat to humans, Gramer replied, "No more than any other influenza virus in the world. Transmission of flu from pigs to humans is likely rare. This flu itself is rare and we don't know if it is currently circulating in this farm or any other farm in the USA. We haven't found it again." 

Although pigs have long been regarded as a viral mixing vessel that could potentially produce human pandemic strains, whether this has ever happened is unclear, the PNAS report states. The 1957 and 1968 pandemic viruses both resulted from genetic reassortment of human and avian strains, but the mixing might have occurred in humans, pigs, quail, or some other host, it says. 

Gregory C. Gray, MD, MPH, an Iowa infectious disease expert who was not involved in the study, says the new findings should cause public health officials "much concern" and point up the need for careful and coordinated flu surveillance in swine, poulty, and humans who work with them. 

The report suggests that state and federal public health officials should work "aggressively" with the swine and poultry industries to study the prevalence of the new virus and find out whether it is infecting humans and to set up coordinated surveillance programs, said Gray, who is director of the Center for Emerging Infectious Diseases at the University of Iowa in Iowa City. 

Much effort is currently devoted to the hunt for highly pathogenic flu viruses in migratory and aquatic birds, Gray continued. "Influenza surveillance in agriculture is largely managed by the industries. Our agriculture industry and human influenza surveillance are not well coordinated. We might wake up one day to find a virus like the one in this report has become endemic in our agriculture industries and a major threat to the industries as well as to man." 

He added that numerous reports show that common swine flu viruses can cause severe disease in humans. "A novel virus like the one in the PNAS report, if highly transmissible between people, has potential to cause markedly more morbidity, even a pandemic," he said. 

Gray has previously written about the need to include agricultural workers in seasonal flu vaccination programs, flu surveillance, and pandemic planning. "The PNAS report greatly increases our concern," he commented. 

Another expert, Michael T. Osterholm, PhD, MPH, generally concurred with Gray. "I think it’s a potentially serious threat," said Osterholm, director of the University of Minnesota Center for Infectious Disease Research and Policy, publisher of CIDRAP News. "I think one of the problems we have is that we tend to think of influenza today, because of H5N1, as an Asian disease, with the roots of any future pandemic planted deep in Asia. But we have to be very careful because we don't know that." 

Gramer commented that flu is very common in pigs, particularly in the spring, fall, and early winter, and is typically mild and self-limiting. "Flu surveillance in pigs is entirely voluntary," she said. Some surveillance for flu in farm workers is carried on under research grants from the CDC and the National Institutes of Health, she added. 

Ma W, Vincent AL, Gramer MR, et al. Identification of H2N3 influenza A viruses from swine in the United States. Proc Natl Acad Sci 2007 Dec 26;104(52):20949-54