Purging the PERVs

pigThere aren’t enough human organs to meet the needs for transplantation, so we have turned to pigs. Unfortunately pig cells contain porcine endogenous retroviruses, PERVS, which could infect the transplant recipient, leading to tumor formation. But why worry? Just use CRISPR to purge the PERVs.

The genomes of many species on Earth are littered with endogenous retroviruses. These are DNA copies of retroviral genomes from previous infections that are integrated into germ line DNA and passed from parent to offspring. About 8% of the human genome consists of ERVs. The pig genome is no different – it contains PERVs (an acronym made to play with). The genome of an immortalized pig cell line called PK15 contains 62 PERVs. Human cells become infected with porcine retroviruses when they are co-cultured with PK15 cells.

The presence of PERVS is an obvious problem for using pig organs for transplantation into humans – a process called xenotransplantation. The retroviruses produced by pig cells might infect human cells, leading to problems such as immunosuppression and tumor formation. No PERV has ever been shown to be transmitted to a human, but the possibility remains, especially with   the transplantation of increasing numbers of pig organs into humans.

The development of CRISPR/Cas9 gene editing technology made it possible to remove PERVs from pigs, potentially easing the fears of xenotransplantation. This technology was first used to remove all 62 copies of PERVS from the PK15 cell line. But having PERV-free pig cells doesn’t help humans in need of pig organs – for that you need pigs.

To make pigs without PERVs, CRISPR/Cas9 was used to remove the PERVs from primary (that is, not immortal) pig cells in culture. Next, the nuclei of these PERV-less cells was used to replace the nucleus of a pig egg cell. After implantation into a female, these cells gave rise to piglets lacking PERVs.

In theory such PERV-less piglets can be used to supply organs for human transplantation, eliminating the worrying about infecting humans with pig retroviruses. But first we have to make sure that the PERV-free pigs, and their organs, are healthy. The more we study ERVs, the more we learn that they supply important functions for the host. For example, the protein syncytin, needed to form the placenta, is a retroviral gene, and the regulatory sequences of interferon genes come from retroviruses. There are likely to be many more examples of essential functions provided by ERVs. It would not be a good idea to have transplanted pig organs fail because they lack an essential PERV!

Transgenic pigs resistant to foot-and-mouth disease

Foot-and-mouth disease virus (FMDV) infects cloven-hoofed animals such as cattle, pigs, sheep, goats, and many wild species. The disease caused by this virus is a substantial problem for farmers because infected animals cannot be sold. Transgenic pigs have now been produced which express a short interfering RNA (siRNA) and consequently have reduced susceptibility to infection with FMDV.

FMDV is classified in the picornavirus family which also contains poliovirus and rhinoviruses. The virus is highly contagious and readily spreads long distances via wind currents, and among animals by aerosols and contact with farm equipment. Infection causes a high fever and blisters in the mouth and on the feet – hence the name of the disease. When outbreaks occur, they are economically devastating. The 2001 FMDV outbreak in the United Kingdom was stopped by mass slaughter of all animals surrounding the affected areas – an estimated 6,131,440 – in less than a year.

Vaccines against the virus can be protective but they are not an optimal solution. One problem is that antigenic variation of the virus may thwart protection. In addition, countries free of FMDV generally do not vaccinate because this practice would make the animals seropositive and prevent their export (it is not possible to differentiate between antibodies produced by natural infection versus immunization). Furthermore, if there were an outbreak of foot-and-mouth disease in such countries, the rapid replication and spread of the virus would make vaccination ineffective – hence culling of animals as described above is required. Clearly other means of protecting animals against FMDV are needed.

Synthetic short interfering RNAs (siRNA) have been shown to block viral replication in cell culture and in animals. To achieve such inhibition, short synthetic RNAs complementary to viral sequences are produced in cells. Upon infection, these siRNAs combine with the cellular RNA-induced silencing complex (RISC) which then targets the viral RNA for degradation.

To determine if siRNA could be used to protect pigs from foot-and-mouth disease, a complementary viral sequence was first identified that blocks FMDV replication in cell culture by ~97%. A vector containing this siRNA sequence was then used to produce transgenic pigs. Such animals not only express the antiviral siRNA, but as the encoding vector is present in germ cells, it is passed on to progeny pigs.

Expression of the siRNA was confirmed in a variety of transgenic pig tissues, including heart, lung, spleen, liver, kidney, and muscle. In fibroblasts produced from transgenic pigs, virus replication was reduced 30 fold. When transgenic pigs were inoculated intramuscularly with FMDV, none of the animals developed signs of disease such as fever or blisters of the feet and nose. In contrast, control non-transgenic pigs developed high fever and lesions. Viral RNA levels in the blood of transgenic pigs were 100-fold lower than in control animals. At 10 days post-infection no viral RNA was detected in heart, lung, spleen, liver, kidney, and muscle, while high levels were observed in these organs from non-transgenic controls.

These results show that siRNAs can protect transgenic pigs from FMDV induced disease. An important question that must be answered is whether transgenic pigs still contain enough virus to transmit infection to other animals. In addition, siRNAs are short – 21 nucleotides – and a mutation in the viral genome can block their inhibitory activity. Therefore it would be important to determine if mutations arise in the FMDV genome that lead to resistance to siRNAs.

Even if transgenic siRNA pigs do not transmit infection, and viral resistance does not arise, I am not sure that consumers are ready to accept such genetically modified animals.

Influenza A/Mexico/2009 (H1N1) update

reassortment-swine-300x157Here is an update on the global swine flu situation as of 4 May 2009, and comments on interesting unresolved questions.

There have been laboratory confirmed cases of infection in over half of the United States (30), with a total of 226 cases and the one death in Texas last week. Globally, 20 countries have reported 985 cases of infection. The highest numbers are in Mexico, with 590 cases and 25 deaths.

According to CDC, very few of the American cases are in individuals who are over 50 years of age. This observation suggests that exposure to previous H1N1 strains might confer some protection against infection, as suggested by Dr. Peter Palese. Individuals who are over 50 today were born in 1959 or earlier. Recall that from 1918 to 1957, H1N1 influenza viruses circulated globally. Those who are older than 50 were likely to have been infected with H1N1 influenza virus strains during those years. Whether or not there is any protection from such previous exposure will likely be determined by laboratory tests in the near future.

Also very interesting is the report from Alberta, Canada that pigs have been infected with the human H1N1 virus. It is believed that the animals were exposed to the virus by a Canadian who had returned from Mexico with flu-like symptoms. This report raises more questions than it answers. But if the new H1N1 virus can readily infect pigs, this could serve as a new source of the virus for future outbreaks. Promedmail poses the tough but important questions:

First, how was directionality of infection established? Was the worker sick when he came in contact with pigs? If so, what lapse in biosecurity allowed a sick human worker to even be on a swine farm as standard biosecurity practices on progressive or up to date swine farms would screen such an individual out and prevent him or her from coming into contact with pigs? Has the worker tested positive for the novel influenza A H1N1 virus? What is the prevalence of the new virus in the swine herd and finally, but most importantly, what quarantine and traceback procedures are in place to make sure that the swine herd does not infect other swine farms? Finally, although we know animal diagnostic laboratories have never seen this virus before in pigs, what surveillance efforts are being made to look at previous swine serum banks or test apparently healthy swine herds on a population basis to actively ensure swine populations are free of this novel influenza A H1N1 virus?

Another important question concerns influenza vaccines. According to Health and Human Services Secretary Kathleen Sebelius, vaccines for the new strains, as well as last season’s, will be ready this fall. Clearly we need a vaccine containing the new H1N1 strain and the relevant influenza B virus strain. But what about last season’s strains – there were two, an H1N1 and an H3N2. If these were also included, that would make for a tetravalent influenza virus vaccine.

Here is one bit of information which would argue against including last year’s H3N2 and H1N1 strains in this fall’s vaccine. In each previous pandemic, the newly emerging viruses replaced the previous circulating strain. For example, in 1968, the H3N2 virus replaced H2N2 viruses that had been causing influenza since 1957. Whether or not the new H1N1 viruses replace the two previous strains will be an important question to address in the coming months. Such information will be used to make decision about the components of the influenza virus vaccine used this fall.

Update: terrific map from WHO on laboratory confirmed cases.


Swine influenza A/Mexico/2009 (H1N1) update

phases5-6Here is an update on the global swine flu situation as of 29 April 2009.

Not surprisingly, laboratory confirmed case counts continue to rise globally. There are 91 cases in the US in 10 states (Arizona, California, Indiana, Kansas, Massachusetts, Michigan, Nevada, New York, Ohio, Texas). There has been a laboratory confirmed fatal case in Texas, in a Mexican toddler visiting the US with his family. Eight other countries report a total of 57 laboratory confirmed cases, with no changes in the numbers in Mexico. Other countries reporting cases are Austria (1), Canada (13), Germany (3), Israel (2), New Zealand (3), Spain (4) and the United Kingdom (5). Globally nine countries have confirmed 148 cases with 2 deaths.

These numbers might not appear to justify the enormous reactions of health agencies such as CDC and WHO. Remember that there are probably many more infections with this virus, not only in the countries that report isolation of the virus but in other areas as well. It will likely take several weeks before we have a good appreciation for how extensively the virus has spread.

On Tuesday WHO raised the level of influenza pandemic alert from phase 4 to phase 5. According to WHO, “Phase 5 is characterized by human-to-human spread of the virus into at least two countries in one WHO region. While most countries will not be affected at this stage, the declaration of Phase 5 is a strong signal that a pandemic is imminent and that the time to finalize the organization, communication, and implementation of the planned mitigation measures is short.” WHO regions include African, European, Eastern Mediterranean, Americas, Southeast Asia, and Western Pacific. The cases in 4 European countries fulfilled the requirement for moving to phase 5.

Incredibly, Egypt began slaughtering 300,000 pigs as a ‘precautionary measure’ against the spread of swine flu. Farmers will not be compensated because the meat will be sold for consumption. This is a somewhat controversial move, because it is highly unlikely that pigs anywhere will play a role in further spread of the virus, which is now adapted to humans.

Sequences of viral RNAs from 20 swine flu isolates have now been posted on the NCBI website. Included are isolates from California, Texas, New York, Ohio, Kansas, and Germany (taken from a tourist who returned from Mexico). It is difficult to understand why RNA sequences of none of the Mexican isolates have  been posted, which would enable us to determine if the viruses in that country are different from the others. However, examination of the sequences of the New York and German isolates, which presumably originated in Mexico, reveal no significant differences with sequences from other isolates. From this information I conclude that the apparent higher virulence of swine flu in Mexico is not a consequence of a genetically diverged virus.

Other interesting information that can be gleaned from sequence information is contained in a statement from CDC: “…the HA, PB2, PB1, PA, NP, NS genes
contain gene segments from influenza viruses isolated from swine in North America [such as, A/swine/Indiana/P12439/00], while the NA and M genes are most closely related to corresponding genes from influenza viruses isolated in swine population in Eurasia.
However, the NA and M genes from 2 swine virus isolates from America are also closely related to the novel H1N1 virus (A/swine/Virginia/670/1987, A/swine/Virginia/67a/1987), if a reasonable nucleotide substitution rate is accepted. Thus, H1N1 from Mexico may be a swine flu virus strain of entirely American origin, possibly even of relatively ancient origin.” In the coming days I will attempt to construct a history of the evolution of swine influenza. In the meantime it may well be that this new human strain emerged from the US, as did the 1918-19 pandemic virus.

It is curious that CDC originally asserted that the new swine influenza virus inherited genes from human, pig, and bird viruses. Dr Anne Schuchat made this statement during a press conference on 23 Apr 2009, noting that  “Preliminary testing of viruses from the 1st 2 patients shows that they are very similar. We know so far that the viruses contain genetic pieces from 4 different virus sources. This is unusual. The 1st is our North American swine influenza viruses. North American avian influenza viruses, human influenza viruses, and swine influenza viruses found in Asia and Europe. That particular genetic combination of swine influenza virus segments has not been recognized before in the US or elsewhere.”

I am not sure why the sequence information now available indicates a very different origin for these viruses.

Although many still describe this virus as swine flu, it is technically no longer a pig virus – having acquired the ability to be transmitted among humans and cause disease, it is now a human virus. I realize that the official strain names are cumbersome (A/Mexico/4482/2009 [H1N1]), and therefore it is likely that we will be using ‘swine flu H1N1’ at least until the next pandemic.

New swine influenza viruses in humans

swineA new strain of swine influenza virus has been recently isolated from seven persons in the US. Is it time to break out the swine flu vaccine of 1976?

Last week the CDC reported that swine influenza virus had been isolated from two children with respiratory illness in California. The cases were not linked and the children recovered from the illness. The virus was identified as a swine influenza H1N1 strain, similar to viruses that have circulated in American pigs for the past ten years. However some of the viral genes are derived from Eurasian swine influenza viruses. The isolates are new because this particular combination of swine influenza virus RNAs has not been observed before among swine or human viruses.

A similar virus was subsequently identified in five additional individuals in Texas. It’s curious that one of the California children had traveled to Texas before becoming ill, but whether or not the cases are related has not been revealed.

What is the origin of these new swine viruses? None of the people who were infected had known contact with pigs. Others must have acquired the virus from pigs, who then passed it on – demonstrating that the virus can be transmitted among humans.

At the moment these infections don’t seem to be cause for alarm. Because influenza virus surveillance is more intense than ever before, it is likely that new viruses will always be detected. Furthermore, respiratory disease caused by these new viruses has not been very severe. Another mitigating factor is that the influenza season is nearly over – viral transmission wanes when the weather becomes warmer and more humid.

It is believed that swine influenza originated in 1918-19, when pigs became infected with the pandemic influenza virus strain. Since that time, the H1N1 swine virus has been transmitted back to humans. The hypothesis for the origin of swine influenza is supported by the finding that pigs can be experimentally infected with the human 1918 pandemic influenza virus strain. Furthermore, other human influenza virus strains are known to infect pigs. For example, in the early 1970s, a human H3N2 subtype entered the European swine population.

Pigs can be infected with both human and avian influenza virus strains because the cells of their respiratory tract bear receptors for both kinds of viruses. Based on this observation, it has been suggested that influenza viruses pass from birds through pigs on their way to infecting people. For example, if a pig is infected with avian and human influenza A viruses, reassortment of the viral RNAs occurs, leading to new virus strains to which humans are not immune. The 1957 and 1968 human pandemic viruses were reassortants of human and bird strains, although there is no evidence that these viruses arose in pigs. The role of pigs as a ‘mixing vessel’ for influenza virus has been questioned in view of the recent transmission of avian influenza viruses directly to humans.

Swine influenza viruses probably routinely pass among humans and swine; in this case they were detected as a consequence of heightened surveillance. Gerald Ford won’t be rolling over in his grave over this incident.

Weingartl, H., Albrecht, R., Lager, K., Babiuk, S., Marszal, P., Neufeld, J., Embury-Hyatt, C., Lekcharoensuk, P., Tumpey, T., Garcia-Sastre, A., & Richt, J. (2009). Experimental Infection of Pigs with the Human 1918 Pandemic Influenza Virus Journal of Virology, 83 (9), 4287-4296 DOI: 10.1128/JVI.02399-08

de Jong, J., Smith, D., Lapedes, A., Donatelli, I., Campitelli, L., Barigazzi, G., Van Reeth, K., Jones, T., Rimmelzwaan, G., Osterhaus, A., & Fouchier, R. (2007). Antigenic and Genetic Evolution of Swine Influenza A (H3N2) Viruses in Europe Journal of Virology, 81 (8), 4315-4322 DOI: 10.1128/JVI.02458-06

Van Reeth, K. (2007). Avian and swine influenza viruses: our current understanding of the zoonotic risk Veterinary Research, 38 (2), 243-260 DOI: 10.1051/vetres:2006062

Reverse zoonoses: Human viruses that infect other animals

bonoboThe many human viral diseases that have crossed from other animal species  – such as AIDS, Ebola, SARS, encephalitis and respiratory diesease caused by henipaviruses – demonstrate the pathogenic potential of the zoonotic pool. Are there also reverse zoonoses – diseases of humans that are transferred to other animal species?

An example of a reverse zoonosis occurred just last week at Lincoln Park Zoo in Chicago, where a 9 year old chimpanzee died of respiratory disease caused by human metapneumovirus. This member of the paramyxovirus family is responsible for approximately 10% of all respiratory tract infections. All seven members of a group of chimpanzees were infected with the virus, but only one became ill. The virus was likely transmitted to the chimps by humans, but precisely how and when is not known. Outbreaks of fatal respiratory disease in wild chimpanzees have been reported previously, and human metapneumovirus has been one of several human viruses isolated from these primates. Such infections are expected to occur more frequently in zoological parks, and ando also in game preserves as human encroachment of these facilities increases.

Influenza virus may also be frequently transmitted from humans to other animal species. An outbreak of influenza recently lead to the death of 6 bonobos (a species of chimpanzee) in a Congo wildlife sanctuary. Although the influenza subtype responsible for the outbreak has not been identified, it has been suggested that the source was one or more visitors during the February epidemic of influenza in Kinshasha.

Human influenza viruses are frequently isolated from pigs. Since its emergence in 1968, the H3N2 subtype has infected pigs many times throughout the world, and has often caused serious outbreaks. More recently the influenza H1N1 subtype has been shown to infect pigs. Influenza viruses remain in the pig population for long periods of time, and may serve as reservoirs for the recycling of older influenza virus strains back into the human population.

Just as increasing human population, travel, and the global food business increase the likelhood that viruses will jump from animals into humans, the same factors ensure the reverse spread as well – often with dire consequences for zoological parks and wild and domestic animals. And the viruses we pass on may come back to haunt us another day.

Kaur, T., Singh, J., Tong, S., Humphrey, C., Clevenger, D., Tan, W., Szekely, B., Wang, Y., Li, Y., Alex Muse, E., Kiyono, M., Hanamura, S., Inoue, E., Nakamura, M., Huffman, M., Jiang, B., & Nishida, T. (2008). Descriptive epidemiology of fatal respiratory outbreaks and detection of a human‐related metapneumovirus in wild chimpanzees at Mahale Mountains National Park, Western Tanzania. American Journal of Primatology, 70 (8), 755-765 DOI: 10.1002/ajp.20565

Yu, H., Zhou, Y., Li, G., Zhang, G., Liu, H., Yan, L., Liao, M., & Tong, G. (2009). Further evidence for infection of pigs with human-like H1N1 influenza viruses in China Virus Research, 140 (1-2), 85-90 DOI: 10.1016/j.virusres.2008.11.008

de Jong, J., Smith, D., Lapedes, A., Donatelli, I., Campitelli, L., Barigazzi, G., Van Reeth, K., Jones, T., Rimmelzwaan, G., Osterhaus, A., & Fouchier, R. (2007). Antigenic and Genetic Evolution of Swine Influenza A (H3N2) Viruses in Europe Journal of Virology, 81 (8), 4315-4322 DOI: 10.1128/JVI.02458-06

Ebola in pigs – Nipah redux?

10816_loresEbola virus infection was reported in Phillipine pigs in 2008. This past week it was revealed that the virus was apparently transmitted from Phillipine pigs to a pig farmer. Why do these events bring Nipah virus to mind?

The Ebola virus that was found in 4 of 6,000 pigs in the Phillipines last year is the Reston strain. This isolate was first identified in Reston, VA, in 1989, in monkeys in a primate facility that had been shipped from the Phillipines. It infected at least 20 lab workers, but did not cause severe disease. In contrast, African strains of Ebola, such as Zaire and Sudan, are associated with high fatality rates.

It is not known how Ebola-Reston infected the Phillipine pigs. There is evidence that the reservoir for African Ebola strains is the fruit bat (or flying fox). It is possible that the virus is transmitted to primates in Africa through contaminated fruit (upon which the bats urinate or defecate while feeding) or bat meat. A recent article in Microbe Magazine explores bats as reservoirs of viruses.

Yes, there are similar fruit bats in the Phillipines. And they have been implicated previously in the transmission of other virus diseases. For example, Nipah virus, a member of the Paramyxoviridae, was first identified as the agent of respiratory illness and encephalitis in Malaysian pig farmers in 1999. This outbreak lead to 105 human deaths; one million pigs were slaughtered to stop the spread of infection. It is believed that fruit bats infected the pigs by contaminating them with urine, feces, or fruit. The pigs, in turn, infected their handlers, who are in close contact with them each day.

Given the Nipah virus scenario, it is not difficult to imagine the spread of Ebola-Reston from fruit bats, to pigs, and then to pig farmers. The pig farms are here to stay, and they only grow larger – increasing the contact between pigs and mobile wild animals. The conclusion is inescapable – more frequent contact between pigs and wild animals, more  future infection of pigs with Ebola.

Dr. Pierre Rollin of the Centers for Disease Control and Prevention has said that “This virus is very stable, not like flu or HIV”. He indicated that there is no change of the virus when it travels from primates to humans. Limited genetic change is typical of a dead-end zoonotic infection: one example is the identical genome sequence of the NYC 1999 West Nile virus isolate with that of a virus from a goose in Israel. Since then, West Nile virus has undergone enormous variation as it has spread across the United States. This is typical of RNA viruses, whose genomes undergo extensive mutation. I seriously doubt that Ebola is genetically ‘very stable’ as implied by Dr. Rollin’s statement above. Rather, we have not yet seen extensive circulation in humans which would lead to a more diverse sequence pool.

I also find curious the statement by Dr. Thomas G. Ksiazek, from the University of Texas Medical School in Galveston, that “It’s probably a rare event that pigs get infected”. Perhaps so far it has been rare. But the rapid growth of farming is leading to more overlap between bat habitats and pig farms. It seems likely that farm animals will be taking more frequent dips in the zoonotic pool, with dire consequences.