Virus Structure and Antigens

Virus particles, called virions, consist of a nucleic acid core surrounded by a layer of proteins (see Figures 9-2 and 38-2). This protein layer, called the capsid, is made up of subcomponents called capsomeres. An envelope containing lipoprotein may also surround virions. The complexity of viruses varies. Some, such as poxviruses, are complex, whereas others, such as foot-and-mouth disease virus, are relatively simple. Antibodies can be produced against epitopes on all the proteins situated inside and on the surface of the virion. Antibodies against the nucleoprotein components are not usually significant from a protective point of view, but they may be useful for serologic diagnosis.

Pathogenesis of Virus Infections

Adsorption, the first step in the invasion of a cell by a virus, occurs when a virus binds to receptors on the cell surface. These receptors have not evolved for the convenience of viruses but have some other physiological function. The rabies virus binds to the receptor for acetylcholine, a neurotransmitter. The Epstein-Barr virus (the cause of infectious mononucleosis) binds to a receptor for C3. Rhinoviruses that cause the common cold bind to cell-surface integrins. The chemokine receptor CCR5 has been identified as the receptor used by West Nile virus. The nature, number, and distribution of host cell receptors determine the host range and tissue tropism of a virus. The bound virion is taken into the cell through endocytosis or by fusion with the plasma membrane. Once inside a cell, the capsid is dismantled so that its nucleic acid is released into the cell cytoplasm, a process called uncoating. Once the virus genome is uncoated, replication begins (Figure 26-1). The host cell DNA, RNA, and protein synthesis are usually inhibited so that only viral genetic information is processed. If the virus, for example, a herpesvirus, contains DNA, this viral DNA is replicated. The new viral DNA is then transcribed into viral messenger RNA (mRNA), and this RNA is translated into new capsid proteins. These new proteins are then assembled into virions. The host cell also replicates the viral nucleic acid so that large quantities of viral DNA are produced. The viral DNA is packaged inside the new capsids so that complete virions are formed. If the virus is unenveloped, the infected cells rupture, and the virions are released into the environment. If the virions are enveloped, they leave the cell by budding through the cell surface. The cell membrane that encloses them serves as the new envelope. The released virions may then spread to nearby cells and invade them in turn.

If a virus contains RNA rather than DNA, its replication takes a slightly different course. For most RNA viruses, such as Newcastle disease or foot-and-mouth disease virus (FMDV), viral DNA is not used. Thus in FMDV infection, the viral single-stranded RNA (the “plus strand”) is used as a template to synthesize a complementary “minus strand” of RNA. These minus strands are then used to generate new plus strands that can be translated into viral proteins. Some viruses contain double-stranded RNA (dsRNA) and use only one of the strands generated during replication. In other RNA viruses, the infecting virus RNA may be complementary to the newly synthesized viral RNA that will translate into viral proteins.

A different replication mechanism is employed in the case of some RNA tumor viruses and immunodeficiency viruses (Figure 26-2). These are called retroviruses since their RNA is first reversely transcribed into DNA by an enzyme called a reverse transcriptase. The new viral DNA is then integrated into the host cell genome as a provirus. This proviral DNA can then be transcribed into RNA, as well as being able to copy itself. The proteins and RNA can then be packaged into a complete new virion.

Changes in virus-infected cells may be minimal, perhaps detectable only by the expression of new proteins on the cell surface. Sometimes, however, the changes may be extensive and result in either cell death or malignant transformation and the development of tumors.

Innate Immunity

Rapid, powerful innate immune responses limit many viral infections. Interferons are especially important in this process. Lysozyme can destroy several viruses, as can many intestinal enzymes and bile. Collectins bind to viral glycoproteins and block virus interaction with host cells. For example, conglutinin, MBL, SP-A, and SP-D can all inactivate influenza viruses. Defensins from leukocytes and mucosal epithelial cells play a dual role in antiviral defenses since they can act both on the virus and on the host cell. Thus defensins can inactivate enveloped virions by disrupting their envelopes or by interacting with their glycoproteins. Some defensins can act on virus-infected cells by blocking intracellular signaling pathways and interfering with transcription of viral RNA. Finally, cells invaded by viruses may undergo premature apoptosis, preventing successful viral invasion and replication.

Interferons

The interferons are cytokines that protect other cells against viral, bacterial, and protozoan invasion. They are all glycoproteins of 20 to 34 kDa. They are classified into three major types: I, II, and III. There are many type I interferons, each denoted by a greek letter. These include IFN-α, produced in large quantities by plasmacytoid dendritic cells and in much smaller amounts by lymphocytes, monocytes, and macrophages. Most mammals produce multiple isoforms of IFN-α. (There are 18 different isoforms in humans, 12 in pigs and cattle, 4 in horses, 2 in dogs). IFN-β is derived from virus-infected fibroblasts. (There are 5 isoforms in cattle and pigs and 1 in dogs and humans.) IFN-ω is produced by lymphocytes, monocytes, and human, horse, pig, rabbit, and dog trophoblast cells (6 to 7 in pigs, 5 in humans, 2 in horses, and none in dogs). A distinct form of type I interferon, IFN-τ, has been isolated from the ruminant trophoblast, and IFN-δ has been isolated from the pig trophoblast. IFN-δ is only distantly related to the other type I interferons. IFN-ε is a member of the type I family whose expression is limited to reproductive and brain tissues. It appears to play a role in mucosal and nervous system immunity. In most cases these molecules act on virus-infected cells to inhibit viral growth. The trophoblast interferons also regulate the maternal immune response to a fetus (see Figure 32-9).

There is only one type II interferon, IFN-γ, a produced by antigen-stimulated T cells. It is also produced in pig trophoblast cells (Box 26-2).

Box 26-2   Measuring Interferons

Interferons may be assayed by measuring their antiviral effects. For example, serum samples to be tested for interferon activity are added to fibroblast cultures at various dilutions and incubated for 18 to 24 hours. The fibroblast monolayers are then washed, and a standard quantity of bovine vesicular stomatitis virus is added to each culture. After 48 hours of incubation, the monolayers are stained, and virus plaques are seen as cleared areas in the monolayer. The presence of interferons in the test serum will reduce the number of plaques formed. This is called a plaque reduction assay.

Three type III interferons have been identified, called IFN- λ1, IFN-λ2, and IFN-λ3 (also known as interleukin-29 [IL-29], IL-28A, and IL-28B). They are expressed in response to viral infections and TLR ligands. They signal through a unique receptor complex consisting of IL-10Rβ and IL-28Rα. While structurally unrelated to the type I interferons, they induce similar intracellular signals and a similar gene expression profile and can be produced by most appropriately stimulated cell types. Despite this, the type III interferons are probably more important as immune regulators. For example, IFN-λ1 inhibits Th2 responses, whereas IFN-λ2 enhances Th1 responses. Pigs lack IFN-λ2.

Antiviral Activities

The two major type I interferons (IFN-α and IFN-β) are produced by virus-infected cells within a few hours after viral invasion, and high concentrations may be achieved in vivo within a few days, long before adaptive immunity develops. For example, in cattle infected intravenously with bovine herpesvirus-1 (BHV-1), peak interferon levels in serum are reached 2 days later and then decline, but they are still detectable by 7 days (Figure 26-3). In contrast, antibodies are not usually detectable in serum until 5 to 6 days after the onset of a virus infection.

Type I IFN-α and IFN-β are secreted by virus-infected cells, and both bind to a heterodimeric receptor (IFNAR) on nearby cells activating a JAK/STAT signaling pathway (see Figure 8-8) (Figure 26-4). Receptor binding results in the development of an “antiviral state” within a few minutes that peaks 5 to 8 hours later. This virus resistance is mediated through four major pathways.

• The 2′5′ A pathway: Type I interferons upregulate transcription of the genes coding for 2′5′-oligoadenylate synthetases (2′5′-OAS). Expressed OAS enzymes are then activated by exposure to dsRNA. The activated enzymes act on adenosine triphosphate (ATP) to form 2′5′ adenylate oligomers. These oligomers in turn activate a latent ribonuclease called RNAase L (Figure 26-5). RNAase L degrades viral RNA and inhibits viral growth.

• The Mx guanosine triphosphatase (GTPase) pathway: Mx proteins are interferon-induced GTPases that accumulate as oligomers on intracellular membranes such as the smooth endoplasmic reticulum. Following viral infection, Mx monomers are released. These bind and trap viral nucleocapsids and other essential viral components and so block the assembly of new viruses. Mx proteins are expressed in many different cell types such as hepatocytes, endothelial cells, and immune cells. They inhibit a wide range of RNA viruses, including the influenza viruses.

• The protein kinase R (PKR) pathway: PKR is induced by type I interferons. The inactive kinase accumulates in the cell nucleus and cytoplasm, where it is activated directly by viral RNAs. Activated PKR regulates several cell signaling pathways and phosphorylates an initiation factor called eIF2α, which then prevents translation initiation of viral mRNA.

• The ISG15 pathway: One of the most prominent of the interferon-stimulated genes is ISG15. This codes for a 17-kDa, ubiquitin-like protein that binds to many different proteins and enhances their destruction. It is not known how this results in increased antiviral resistance and reduced viral replication.

The ability of cells to produce interferons varies. Virus-infected leukocytes, especially plasmacytoid dendritic cells, produce large amounts of IFN-α; virus-infected fibroblasts produce IFN-β; and antigen-stimulated T cells are the major source of IFN-γ (Chapter 14). Kidney cells are poor interferon producers, and neutrophils produce no interferon.

Lymphocytes from normal, unsensitized donors can kill virus-infected cells. This innate cytotoxicity is due to natural killer (NK) cells (Chapter 19). NK cell cytotoxicity is stimulated by type I interferons and, as a result, is important early in a virus infection. Indeed NK cells provide a first line of defense against many viruses. NK cells also produce IFN-γ, and this too has a direct antiviral effect. NK cells may therefore reduce the severity of viral infections long before the development of adaptive immunity and the appearance of specific cytotoxic T cells.

IFN-α not only activates NK cells but it also stimulates the differentiation of monocytes into dendritic cells, as well as the maturation and activity of dendritic cells. IFN-α participates in the transition from innate to adaptive immunity and drives some γ/δ T cell responses. It stimulates memory T cell proliferation, activates naïve T cells, and enhances antigen-specific T cell priming.

Virus capsid and envelope proteins are antigenic, and it is against these that antiviral antibody responses are largely mounted (Figure 26-6). Antibodies can prevent cell invasion by blocking the adsorption of virions to target cells, by stimulating phagocytosis of viruses, by triggering complement-mediated virolysis, or by causing viral clumping and thus reducing the number of infectious units available for cell invasion. Binding of antibodies alone does not destroy viruses since the splitting of virus-antibody complexes may release infectious virions.

Antibodies are not only directed against proteins on free virions but are also directed against viral proteins expressed on infected cells. As a result, these infected cells may also be destroyed. Virus infections in which antibody-mediated destruction of infected cells occurs include Newcastle disease, rabies, bovine virus diarrhea, infectious bronchitis of birds, and feline leukemia. Antibodies may kill infected cells by complement-mediated cytolysis or by antibody-dependent cell-mediated cytotoxicity (ADCC). The cytotoxic cells include lymphocytes, macrophages, and neutrophils with Fc receptors through which they can bind to antibody-coated target cells.

Virus-neutralizing antibodies include immunoglobulin G (IgG) and IgM in serum and IgA in secretions. IgE may also be protective since IgE-deficient humans suffer from severe respiratory infections. As in antibacterial immunity, IgG is quantitatively the most significant immunoglobulin, whereas IgM is qualitatively superior.

Although most viruses infect cells by binding directly to receptors on target cells, some use an intermediate molecule. For instance, some antibody-coated viruses bind to cells through Fc receptors. This, of course, facilitates endocytosis of the virus and may thus enhance virus infection. Complement may enhance some virus infections in a similar fashion. Examples of viruses whose infections are enhanced by antibodies include feline infectious peritonitis, Aleutian disease of mink, African swine fever, and human immunodeficiency virus (HIV).