Introduction to the CAE Virus

Viruses are very small, simple organisms that have only one goal in life—to make more of themselves. They are so small they can carry only a small number of genes plus a few important molecules all wrapped up in a protein coat. For this reason, viruses need to invade a cell and exploit the machinery of that cell to replicate. Viruses are completely dependent on the genetics and function of the cell that plays host to them.

The virus that causes inflammation of the brain (encephalitis) in young goats and degenerative arthritis and mastitis in adult goats, is a member of the retrovirus family of viruses. These viruses carry their genes in the form of RNA instead of DNA. While there are other types of RNA viruses, retroviruses are unique in that the RNA is converted into double-strand DNA. This DNA is then inserted into the genome of the living cell to become a permanent gene within the chromosome of the host cell. The CAE virus is classified as belonging to subgroups of retroviruses called lentiviridae. Lenti is the Latin word for slow, describing the long disease process which can sometimes takes years to develop. Another virus that is very similar to the CAE virus is the Maedi-visna virus (MVV). MVV, the first lentivirus to be discovered in 1954 in Icelandic sheep, causes inflammation of the lungs and wasting of the spinal cord and brain. In the U.S. the disease is called ovine progressive pneumonia (OPP). The CAE and Maedi-visna viruses are so similar, and since recent evidence shows that both viruses can cross-infect both sheep and goats, CAEV and MVV are now more often referred to as Small Ruminant Lentiviruses (SRLVs).

The CAE virus is most commonly passed through milk and especially colostrum from an infected dam to her kids. This is called vertical transmission. The virus can also be passed, but only rarely, from one goat to another which, is called horizontal transmission. In sheep, horizontal transmission of MVV appears to be a more important source of infection.

Structure of the CAE Virus

The CAE virus has a similar structure to all other lentiviruses. On the surface it is quite simple. The outer membrane is called the "envelope" and is studded with a protein called the envelope (Env) protein. (Figure 1.) The virus uses this protein to gain access to the inside of its target cell. (Figure 2.)

If we peel open the envelope we find inside a cone-shaped structure. This is called the capsid. (Figure 3.) Its outer surface is made up of a protein with the strange name "gag," which comes from the fact that this protein is specific to a group of virus so it is called Group specific AntiGen. You can also see that the envelope is double. It came originally from the cell that gave birth to the virus.

Inside the capsid we find the RNA genome. (Figure 4.) Lentiviruses are unique in the world of RNA viruses in that they carry two copies of RNA. It isn't known exactly why they do this but since converting RNA back into DNA is a very complex process, which is prone to lots of errors, it is probable that having an extra copy is for backup in case something goes wrong.

There are also several other important proteins packaged inside the capsid. One, reverse transcriptase, converts RNA into DNA once the capsid enters the cell. The other, integrase is involved with integrating the DNA into the cellular chromosome.

In order to replicate all viruses need to get their genome inside of a host cell. To do this viruses make use of an assortment of proteins that exist on the surface of cells which the cell normally uses for its own functions.

The envelope protein of the CAE virus, shown here, binds only with specific proteins on the surface of the cell that it infects. The envelope protein has two components, one called the surface component (SU) shown in green and the other is the transmembrane component (TM) shown in blue which projects through the membrane. The entire protein exists in groups of three (a trimer) on the surface of the virus. The envelope protein is similar but not exactly the same in all other lentiviruses.

Life Cycle of the CAE Virus

The CAE virus infects monocytes and macrophages. Monocytes are a type of white blood cell which circulate in the blood. The CAE virus cannot replicate inside a monocyte but its genome (called a provirus) is carried to all tissues of the body without being detected by the immune system. When a monocyte migrates to body tissues it matures into a macrophage. When this happens, factors are expressed by the cell which the provirus requires for replication. The macrophage then becomes a factory for production of infectious viruses which attack more monocytes and mature macrophages. (Figure 5.)

There are a few studies which indicate that the CAE virus may also be able to infect other types of cells. However, since the expression of virus proteins in these cases is restricted and the number of cells infected is very low this is probably not a common source of CAE infection.

The First Step of Infection: Gaining Access to the Cell

The virus first binds to a surface molecule of a monocyte/macrophage using the envelope protein. Cells have many kinds of proteins on their surface which they use for their normal activities. Viruses exploit these receptors to gain entrance to the cell. The CAE virus will know that it is near a monocyte or macrophage because the envelope protein on the surface of the virus will fit only with a cell surface protein that exists only on goat (and sheep) monocyte/ macrophage cells. The envelope protein on the virus is a key that must fit exactly to a cellular lock to gain entrance to the cell. The reason that the CAE virus cannot infect humans is because human cells do not have the correct receptor on the cell surface.

Second Step of Infection: Converting RNA Into DNA

Fusion of the membranes of the virus and host cell allow the the capsid to enter the cell. The capsid undergoes a structural change which partially opens it up. (Figure 6.) This allows access to required cell components. The capsid is now called a preintegration complex or PIC.

Inside the PIC reverse transcriptase converts the single strand of RNA into double-strand complementary DNA (cDNA). This is a complex, error prone process which leaves mutations in the newly created DNA. This is the reason that there are many variant forms of the CAE retrovirus.

Third Step of Infection: Integration of DNA Into Cell Chromosome

The PIC migrates to the nucleus of the cell carrying the cDNA and another of the proteins the virus brought along called integrase. CDNA and integrase is inserted into the nucleus through a nuclear pore. (Figure 7.)

Integrase clips off a base pair at the end of the viral cDNA, cuts open the strands of host DNA, then binds the ends of the viral DNA into the host DNA. Repair proteins supplied by the cell come along and clean up the loose ends.

The genome of the CAE virus is now a permanent part of the goat's monocyte/macrophage DNA. At this stage it's called a provirus.

Fourth Step of Infection: Replication of New Viruses

The proviral DNA is transcribed into RNA which is transferred from the nucleus to the cytoplasm.

Viral RNA is either used as a template to make viral proteins with the help of cell machinery or is packaged into the newly made viral capsid. Viral mRNA is transcribed into envelope protein, which is transferred to the cell membrane. Capsid proteins, reverse transcriptase, integrase, protease and a few other proteins are also created from transcription of RNA.

Finally, two copies of RNA plus proteins are packaged into the newly created capsid and the capsid buds out from the cell through the modified membrane. The membrane of the virus came from the cell so this is why the CAE retrovirus is called an enveloped virus.

The new, free virus must go through a maturation period before it can become infective which among other things prevents it from immediately reinfecting the cell that produced it.

The Genome of the CAE Virus

This is a graphic representation of the genome of the CAE virus. The genome shown in Figure 8 has sequences on each end called LTR for Long Terminal Repeats. LTRs, present in all retroviruses, are important in establishing the provirus in the host cell DNA and contain signals that are involved in expressing the viral genes. The colored bars show the location of the genes of the CAE virus. Gag encodes for the capsid protein. Pol encodes a polyprotein precursor which become protease, reverse transcriptase, and integrase proteins. Env encodes for both the surface and membrane components of the envelope protein. The linear order of gag, pol and env is the same in all retroviruses. Vif, tat and rev code for regulatory and accessory proteins necessary for efficient viral replication including transport of transcribed proviral RNA to the cytoplasm (rev), influencing the cell cycle (tat) and protecting the viral genome from attack by cellular defenses (vif). With only a few other exceptions, these are the only genes the virus has to work with. It requires necessary proteins and enzymes found in the cell in order to replicate.

The CAE Virus and the Immune System

The surface and transmembrane portions of the CAE envelope protein antigen is shown here as simple, smooth shapes. In reality antigens are large proteins composed of strings of amino acids that are folded tightly into specific, complex conformations.

For instance an imaginary protein. It consists of a long string of 64 amino acids each represented by a ball.

The envelope protein of the CAE virus has over 900 amino acids, which are folded into an extremely complex shape.

The immune system can make many different antibodies to various regions within the antigen protein. These regions are called epitopes. Researchers have so far identified at least 11 different antibody binding epitopes on the envelope protein of the CAE virus. In the image below, the envelope protein of the CAE virus is shown untangled and stretched out with the approximate antibody binding regions. At this time six different antibodies have been identified that bind to regions of the transmembrane portion of the env protein (called TM1 through TM6), and five different antibodies that bind to the surface component (called SU1 through SU5).

Seroconversion

When the goat is first infected with CAE virus the immune system will slowly begin to produce antibodies to various regions of the envelope protein and to the capsid protein. Called seroconversion, this process may take only a few weeks or as long as several months. Goats develop a strong and early antibody response to the surface epitopes SU3 and SU5 with the levels remaining constant over a long time. Antibody production toward the capsid protein gag also occurs early but the level of anti-gag antibody decreases slowly over time. Goats develop antibodies toward the transmembrane portion of the envelope protein much later in the course of infection but an early strong response to several transmembrane (TM) epitopes can predict the appearance of severe symptoms.

Antibodies to different antigens of the CAE virus can rise or fall (sometimes to undetectable levels) over the course of time. It isn't certain why this happens. It could be due to the high variability of field strains of the virus or to the genetic make up of individual goats. Antigens developed from experimental strains of the virus may be different from antigen of the more variable field strains. In addition, antibodies can disappear in the case of severe clinical symptoms, yet animals with no symptoms have detectable antibody levels.

Symptoms of CAE and antibody production

There is a strong correlation between the development of severe arthritis and high levels of the TM3 and TM4 antibodies, especially TM3. It is not very well understood why high levels of TM3 antibodies cause the severe symptoms of CAE but it is suggested that the antibodies somehow play an important role in the disease process possibly enhancing the infectivity of the virus. It has been shown in other animal models that vaccination with similar retroviral antigens result in a high viral load with more severe symptoms rather than preventing viral infection. Vaccination attempts in goats have not been promising in that the vaccines do not control or prevent virus replication and may even enhance or cause severe symptoms of arthritis.

CAEV Antibodies and Immunity

Goats that have been infected with the CAE virus for a long time but do not have symptoms have lower anti-CAEV antibody titers. Antibodies against the CAE virus are not able to neutralize the virus for reasons that are not understood. Instead, the presence of CAE virus is very persistant in spite of the fact that the immune system mounts a strong immune response. Even in a small minority of goats that are able to produce antibodies that seem capable of neutralizing the CAE virus, these neutralizing antibodies are produced very slowly and in very small amounts and free CAE virus can be found in synovial fluid and blood.

References

Demonstration of Coinfection with and Recombination by Caprine Arthritis-Encephalitis Virus and Maedi-Visna Virus in Naturally Infected Goats. Giuliano Pisoni, Giuseppe Bertoni, Maria Puricelli, Marina Maccalli, and Paolo Moroni, Journal of Virology, May 2007, p. 4948-4955, Vol. 81, No. 10

Direct Evidence for Natural Transmission of Small-Ruminant Lentiviruses of Subtype A4 from Goats to Sheep and Vice Versa.Cyril Shah, et. al.Journal of Virology, July 2004, p. 7518-7522, Vol. 78, No. 14

Evidence for interference, coinfections, and intertypic virus enhancement of infection by ovine-caprine lentiviruses.P. E. Jolly and O. Narayan J. Virol. 1989 November; 63(11): 4682-4688

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Relative contribution of colostrum from Maedi-Visna virus (MVV) infected ewes to MVV-seroprevalence in lambs. Alvarez V., Arranz J., Daltabuit-Test M., Leginagoikoa I., Juste R.A., Amorena B., de Andrés D., Luján L.L., Badiola J.J., Berriatua E. Instituto Vasco de Investigación y Desarrollo Agrario (NEIKER), Sanidad Animal, 48160-Derio, Vizcaya, Spain. Res Vet Sci. 2005 Jun;78(3):237-43

Lack of Functional Receptors is the Only Barrier That Prevents Caprine Arthritis-Encephalitis Virus from Infecting Human Cells. Laila Mselli-Lakhal, et. al. Journal of Virology, September 2000, p. 8343-8348, Vol. 74, No. 18

Variability and Immunogenicity of Caprine Arthritis-Encephalitis Virus Surface. Glycoprotein S. Valas, C. Benoit, C. Baudry, G. Perrin, and R.Z. Mamoun. Journal of Virology, July 2000, p 6178-6185

Antibody Reactivity to the Immunodominant Epitopes of the Caprine Arthritis-Encephalitis Virus gp38 Transmembrane Protein Associates with the Development of Arthritis. G. Bertoni, et. al. Journal of Virology, Nov. 1994, p. 7139-7147

B-cell epitopes of the envelope glycoprotein of caprine arthritis-encephalitis virus and antibody response in infected goats. G. Bertoni, et. al. Journal of General Virology (2000), 81, 2929-2940

The illustration of the genome of CAEV was adapted from: Caprine Arthritis Encephalitis Complex. G. Bertoni In: Recent Advances in Goat Diseases, Tempesta M. (editor) International Veterinary Information Service, Ithaca, NY Jul 42, 2007 www.ivis.org

Basic Virology, Third Edition, 2008 Edward K. Wagner, Martinez J. Hewlett, David C. Bloom, and David Camerini, Karin Christensen, illustrator Blackwell Publishing

Immunobiology, Third Edition, 1997. Charles A. Janeway, Jr. and Paul Travers. Garland Publishing, Inc.