Panel Session I: Cross-Species Transmission – Species Specificity and Tropism

Dr. Preston Marx spoke on SIV Infection of Macaques: A Model of Cross-Species Transmission and Pathogenesis. The infection of Asian rhesus macaques by simian immunodeficiency viruses (SIVs) derived from sooty mangabeys is a model for cross-species viral infection. SIV sooty mangabey (SIV/SM) has been conclusively shown to be the origin of SIV/MAC. While no illness due to SIV/SM infection has been detected in sooty mangabeys, its transmission into the cross-species host (macaque) induces AIDS fairly rapidly. SIV/MAC uses "analogous cell surface receptors in either host." SIV/PBJ is another pathogenic SIV derived from sooty mangabey. PBJ contains a mutation in the nef gene that allows the virus to induce an acutely lethal gastrointestinal (GI) disease. Animals that survive the initial GI attack later develop AIDS.

SIV/MAC has been developed as a model of mucosal transmission in order to understand early transmission pathogenesis. An aim is to use this model to develop possible viral vaccines. Characteristics of SIV/MAC include that it is secreted in body fluids and crosses genital and rectal epithelium. In addition, cell-free transmission of SIV is much easier than cell-associated SIV transmission. Moreover, the intact vaginal mucosa is a partial, not complete, barrier to transmission and some naturally occurring attenuated SIVs transmit without causing disease.

Vaginally introduced virulent SIV/MAC strain 239 exhibits a clear distinction between rapid and slow progressors in the macaque. Rapid progressors have plasma RNA levels that achieve 10⁶ to 10⁷ copies, have no detectable antibodies, and die in 1 to 6 months, whereas slow progressors generate a strong antibody response and generally live 2 to 3 years, with some animals surviving for 6 to 7 years.

Target regions of the female reproductive tract include the vaginal mucosa, vaginal epithelium, the ectocervix, and the transition region where the tissue goes from stratified squamous epithelium to cervical single-cell epithelium. Dendritic cells, antigen-presenting cells, of the cervix have been shown to be an SIV/MAC target bothin vitro in humans and in vivo in macaques. Dendritic cells drain to lymph nodes, providing direct shuttles for virus to enter and replicate in the immune system. SIV-positive dendritic cells are seen in the vagina at day 2 post-infection by in situ PCR.

"Receptors and coreceptors are interchangeable in vitro between macaque and human cells and between SIV and HIV." CCR5 and CXCR4 are coreceptors generally used by HIV-1 strains that are macrophage (M) tropic or T-cell (T) tropic. However, CCR5 is the coreceptor for SIV strains that are either M tropic or T tropic: SIV does not use CRCX4. CCR5 is highly conserved across primate species and will act as a coreceptor for SIV, HIV-1, and HIV-2 according to tropism. Yet, human cells with a deficient CCR5 support SIV. Hence, there is clearly an unknown coreceptor for SIV.

Dr. Marx has also found a new red capped mangabey virus, an SIV with the HIV-1 pol gene. It grows in human and rhesus peripheral blood mononuclear cells (PBMCs) and it does not use CCR5! This is the first known monkey virus in the HIV-1 lineage.

Propagation and Replication Across Species Barriers

Drs. Robin Weiss, Carolyn Wilson, David Onions, and Jonathan Stoye each spoke on this topic.

Dr. Weiss spoke on endogenous Retroviruses That Jump Host Species. His early research revealed that an endogenous viral envelope gene could complement an envelope-deficient sarcoma virus and thus augment infection. The subsequent discovery of reverse transcriptase clarified how this could come about.

Endogenous retroviruses generally do not cause disease in their host population, because "the host, which has acquired the virus genetically, evolves resistance to it." Murine leukemia virus demonstrates this. However, host species can change over time. One feline endogenous virus entered the cat population after exogenous infection from a baboon endogenous C type retrovirus. Moreover, the baboon endogenous retrovirus appears to have resulted from a recombination between an initial C type virus and a portion of a D type simian retrovirus. Furthermore, this virus was first identified incorrectly as a human retrovirus (Nature, 1971) when human tumor cells were grown as a xenograft in fetal cat brains. The virus, which is present in all cats, came out in human cells, since the "human cells did not restrict the replication of this virus."

More current work growing human tumor cells in nude and other immunodeficient mice demonstrates host into graft retrovirus colonization. Approximately one in three serially transplanted tumors are productively infected with these xenotropic viruses. The question to address in the context of xenotransplantation is whether graft into host virus transfer is likely.

Researchers are beginning to assess pig viruses for pig into human viral transmission. A porcine kidney cell line PK15 releases C type viral particles that can infect pig testes, ST-IOWA cells, mink cells, and human 293 cells. However, most other human cells are not infected. Yet, cocultivation with irradiated PK15 cells led to infection of a greater number of human cell types. On the other hand, virus from swine kidney cells (MPK) infects pig cells but not human cells. Pigs appear to have several copies of this endogenous C type virus, but there is some polymorphism that might make it possible to breed out this virus. It is possible that genetically modified pigs would be more capable of cross-species viral transmission. Abrogation of hyperacute rejection will make pig enveloped viruses more resistant to inactivation by human complement, and some viruses may be preadapted for transfer to humans. In fact, retroviral vectors produced in mouse or dog packaging cell lines are complement-inactivated when introduced in vivo. This inactivation occurs by the exact same mechanism that causes hyperacute rejection in xenotransplantation. Humans are genetic knockouts for the a1,3-galactosyl-transferase gene and make antibodies against this enzyme. It is under investigation whether viruses released from cells of transgenic pigs with human cell membrane proteins are more resistant to inactivation by human complement. This could affect the selection of pigs as xenotransplant donors.

Dr. Wilson spoke on Induction and Isolation of a Retrovirus with a Human Host. NIH mini-pig peripheral blood mononuclear cells (PBMC) were used to develop an in vitro model that would mirror a xenotransplant setting. PBMCs mitogenically activated with phytohemagglutinin (PHA) and phorbol myristate acetate (PMA) had a reverse transcriptase (RT) activity peak at 5 days post-activation. These PBMCs were cocultivated with human 239 cells and ST-IOWA cells after the 5-day period. Both cell types showed increasing RT activity after a lag period between 20 and 40 days as well as a productive infection that spread efficiently. Thus, it has been shown that infectious retrovirus can be isolated from at least two separate strains of the NIH mini-pig by mere mitogenic stimulation of PBMCs. Virus released from these activated cells directly infected both pig and human embryonic kidney cells.

The RTs isolated from three pig-infected cell lines showed a high degree of homology. ST cells infected with NIH mini-pig virus, ST cells infected with Yucatan PBMCs, and 293 cells infected with NIH mini-pig virus have identical RTs at the amino acid level and there is only one nucleotide difference in ST Yucatan RT compared to the other RTs.

Dr. Onions spoke on Cross-Species Transmission of Viruses: Implications for Xenotransplantation with Porcine Tissue. Zoonotic viruses from pigs that can replicate in human cells are of particular concern in the context of xenotransplantation. In a xenograft, viruses (such as herpes and paramyxoviruses) may also spread by cell-to-cell contact and syncytia formation. The H1N1 swine flu virus caused one of the greatest pandemics of this century in 1918 and 1919. With regard to influenza, in fact, pigs appear to act "as a mixing vessel for both avian and human viruses; avian viruses do not seem to go directly to man." Viral groups associated with cross-species transmission include parvoviruses, coronaviruses, rotaviruses, influenza viruses, retroviruses, adenoviruses, morbillivirus, herpesviruses, and papillomaviruses.

Reassortment and emergence of novel porcine viruses continues today. Porcine reproductive respiratory syndrome virus, which probably arose from a rodent arteritis virus in the past decade, causes a new emerging disease in pigs. Since influenza virus H1N1, which is a constantly changing virus, is still endemic in the pig population, it is possible that these two viruses could further combine.

The alpha herpesvirus, otherwise called pseudorabies, seems to have limited cross-species transmission. The GE glycoprotein of this virus is important for it to exit the cell. While this virus replicates in human cells, "there is no evidence that it is zoonotic."

It has been observed that many large complex viruses have evolved genetic mechanisms for modulating immune responses that would otherwise attack them. Some of them carry genes that block tumor necrosis factor induction of infected cell apoptosis. And the gamma herpesviruses carry an IL-10 homologue, which switches the anti-viral, cytotoxic T helper 1 response to an antibody producing T helper 2 response.

Canine adenovirus 1 exhibits another type of transmission block. While the degree of homology between some regions of this virus and human adenovirus 5 is high enough to indicate that the canine virus could infect humans, the two viruses' E3 regions contain immune-modulating genes that have hardly any identity. Hence, even if the canine virus infected humans, it probably could not establish a persistent infection. In sharp contrast, a significant cross-species jump occurred with adenovirus 76. Adenovirus 76 is normally found in ducks, but contaminated a manmade chicken vaccine and killed hundreds of millions of chickens. This virus is now established as a chicken-to-chicken infection.

One gamma herpesvirus, bovine herpesvirus II, is a lymphoma-inducing fatal virus for cows. It originated in sheep where it appears to do no harm, but it jumped host species to cows when sheep and cows were grazed together in the same pasture. This would be a dead-end transmission. Some porcine viruses could be unproductive but also be oncogenic. Viruses that are important in veterinary medicine should be tested for in donor animals. But, "What do you do about the unknown?" The pig could carry a gamma herpesvirus that is unknown due to its benign state in pigs, but it could "come out" in xenotransplantation.

The condition of decreased immune surveillance could lead to graft damage. Porcine cytomegalovirus may act this way, and human viruses like flu, adenoviruses, rhinoviruses, or hepatitis C could infect pig cells.

Minor changes in some viruses can result in a change in tissue or species tropism. Coronaviruses are particularly susceptible to these events. In addition, one feline parvovirus changes into a canine parvovirus with relatively few nucleotide changes. FeLV-A, feline leukemia virus A, does not infect human cells, but it can undergo a few mutations and deletions to become FeLV-C and gain the ability to infect human cells. And in a pig parvovirus, a five amino acid mutation changes a nonpathogenic virus into one that is highly pathogenic.

Hysterectomy or hysterotomy followed by early or segregated weaning for consecutive generations is proposed as one method to create optimal xenotransplant donor pigs. This would require close monitoring to prevent the reintroduction of unwanted viral strains through various vectors, including the staff and the housing. Viruses that cross the placenta require special attention. But endogenous retroviruses stand out as the current major barrier.

"The dooms day scenario is . . . that you take a porcine organ, you transplant it into a person, a porcine cell expresses a retrovirus, it infects a human cell . . . (and) produces a virus capable of transmission to the general public. Complex recombinations do occur, but they are not common."

Porcine kidney PK15 cells do express a retrovirus capable of infecting human cells. The viral polymerase (pol) region has the highest identity, and the envelope (env) region has significant identity to human retroviral equivalents. The porcine endogenous virus envelope does confer the ability to infect human cells, but it is not very efficient. Scientists have developed an ELISA assay to detect antibodies to this virus and have the RT-PCR tools to detect viral expression; therefore, it is likely that this virus would be detected prior to use of porcine organs.

In monitoring possible infection of xenotransplant recipients, the infectious state needs to be considered. For example, FeLV-A can infect a cat in such a way that the cat has both antigen and antibody, but the virus may be sequestered and not appear in PBMCs. Moreover, a cat may appear to be in recovery but have provirus-positive cells within the bone marrow. Therefore, it will be important to have a range of tests to determine infection states.

To determine whether xenotransplantation is safe, it needs to be established whether porcine infectious virions are produced in vivo in pig organs and whether they infect primates, and it will be necessary to look for antibody as well as PCR positivity. The safest solution is to breed pigs lacking expressible provirus or use gene knockouts to get rid of unwanted sequences. Potentially, xenograft recipients could also be vaccinated against unwanted viruses.

Dr. Stoye spoke on Distribution and Host Range Properties of Two Classes of Pig Endogenous Retroviruses. Endogenous retroviruses became established in the germ line after germ cell infection; thereafter, they are inherited as Mendelian genes. Host species evolved mechanisms, such as Fv1 and Fv4 genes, to control replication of these endogenous sequences; so it is probably important to restrict their replication. While most of these viruses are defective, some of them can give rise to infectious virus or "contain partial gene products that can recombine with infecting exogenous retroviruses." Endogenous retroviruses produced by the pig kidney cell line, PK15, were examined. Studies were focused on the viral envelope gene, because this is the region that determines the retroviral host range.

Two PK15 porcine endogenous retrovirus (PERV) strains, PERV-A and PERV-B, were isolated. They belong to the mammalian C type retrovirus family. PERV-A and PERV-B showed 92 percent amino-acid identity in the transmembrane region of the envelope protein. Analysis of the cDNA clones isolated from pig cells showed that 29 out of 32 clones were PERV-A, and only 3 clones were PERV-B. However, cDNA clones from human 293 cells were exclusively PERV-B. So, initially, PERV-A was thought to be ecotropic, and PERV-B was believed to be xenotropic. Though PERV-A and PERV-B are virtually identical in the transmembrane (TM) region, they have significant differences in the SU region (cell attachment region) of the env protein. The proviruses have major differences in the VRA, VRB, and polypurine-rich regions, suggesting that they will bind two different receptors.

Neither PERV-A nor PERV-B was present in uninfected human 293 cells. RT-PCR studies revealed that 293 cells infected with PK15 expressed both viruses, which showed that PERV-A was transferred with PERV-B in these cells. PERV-A could be a pseudotype of PERV-B. Alternatively, both viruses can infect human cells. Recent experiments to generate cell lines infected with only one of these proviruses are consistent with the latter possibility. It has now been shown that both PERV-A and PERV-B can infect pig and human cells, i.e., are polytropic.

It would be desirable to selectively breed pigs lacking viruses capable of infecting human cells. This will be difficult, because all pig varieties tested contain 20 to 40 PERVs, and at least eight PERV proviruses are shared among all varieties of pigs tested. The Meishan pig does seem to have slightly fewer, so it may be a good breeding candidate. Yet, it is not clear what portion of these endogenous proviruses can give rise to infectious virus. This needs to be determined. The overall picture indicates that retroviruses present in donor pig herds will be expressed in transplant tissue. It also seems likely that the recipient will become infected, particularly with immunosuppression to prevent organ rejection; the immunosuppression will allow the virus to evade human immune responses. Currently, the key issues appear to be whether infection leads to high levels of viral replication in the transplant recipient and, if so, whether any pathology results. Infection will most probably occur without associated pathology. However, infections could result in pathology that manifests many years after the transplant; some may give rise to cancers. The greatest risk would be the generation of "a highly transmissible pathogenic virus, which could affect all of us." While this may be possible, it seems very, very unlikely.

Panel Discussion I:

An open panel discussion with comments and questions from attendees ensued. (1) The concern about generating knockout pigs was allayed. (2) The relationship between viremia and virus transmissibility was addressed. Dr. Onions took the position that viremia is a "danger point" from the standpoint that a "reasonably hot" virus that is constantly bombarding cells will probably eventually hit and activate an oncogene, not because viremia will "itself lead to transmissibility." (3) It was also asked why the infectivity of PK15 virus has not been explored in human PBLs. This work, as well as determining the virus' specific cellular tropism, is planned.