Infectious viruses

Initially, it was assumed that the HlV-1 population is infinite, evolution is deterministic, and antiretroviral resistance development is definite (Coffin 1995). However, our research amongst others has demonstrated that the effective population size, defined as the average number of HIV variants that produces infectious progeny is relatively small (Leigh Brown 1997 Leigh Brown and Richman 1997 Nijhnis et al. 1998). This can be explained because the majority of virus particles that are produced harbor deleterious mutations resulting in noninfectious virus. Also limited target cell availability and inactivation of potentially infectious viruses by the host... 

Complex viruses Some virions are even more complex, being composed of several separate parts, with separate shapes and symmetries. The most complicated viruses in terms of structure are some of the bacterial viruses, which possess not only icosahedral heads but helical tails. In some bacterial viruses, such as the T4 virus of Escherichia coli, the tail itself is a complex structure. For instance, T4 has almost 20 separate proteins in the tail, and the T4 head has several more proteins. In such complex viruses, assembly is also complex. For instance, in T4 the complete tail is formed as a subassembly, and then the tail is added to the DNA-containing head. Finally, tail fibers formed from another protein are added to make the mature, infectious virus particle. 

The eclipse is the period during which the stages of virus multiplication occur. This is called the latent period, because no infectious virus particles are evident. Finally, maturation begins as the newly synthesized nucleic acid molecules become assembled inside protein coats. During the maturation phase, the titer of active virus particles inside the cell rises dramatically. At the end of maturation, release of mature virus particles occurs, either as a result of cell lysis or because of some budding or excretion process. The number of virus particles released, called the burst size, will vary with the particular virus and the particular host cell, and can range from a few to a few thousand. The timing of this overall virus replication cycle varies from 20-30 minutes in many bacterial viruses to 8-40 hours in most animal viruses. We now consider each of the steps of the virus multiplication cycle in more detail. 

Researches have developed methods to test for HIV and estimate the amounts of infectious virus present in various body fluids and secretions. HIV can be isolated relatively easily from blood, semen, and vaginal/cervical secretions (including menstrual fluid). When blood and semen are examined closely, the great majority of HIV is associated with infected cells (mostly macrophages) present in these fluids. In blood, if the cells are removed, low levels of HIV are present in the cell-free serum. It has also been isolated from breast milk. With much greater difficulty, the virus has on occasion been isolated from saliva, tears, urine, perspiration, and feces. 

Viruses, like all pathogens, show host specificity, usually infecting only one or a restricted range of host species. The initial basis of specificity is the ability of the virus particle to attach to the host cell. If the amount of infectious virus is measured over a period of time in the host, it is seen to fall, after an initial lag period, remain low for a period of time, and then rise to even higher levels. The period during which the amount of virus is low is referred to as the eclipse period. The virus infection cycle can be divided into several events. 

Penetration. After fusion of viral and host membranes, or uptake into a phagosome, the virus particle is carried into the cytoplasm across the plasma membrane. This penetration process is an active one that requires expenditure of energy by the cell. At this stage the envelope and the capsid are shed, and the viral nucleic acids are released. The uncoating of virus accounts for the drop in infectious virus assayed, because the uncoated virus cannot withstand the assay conditions. 

Expression of the Viral Genetic Material. This occurs during the eclipse period, when the amount of infectious virus appears low. Several events occur during the eclipse phase ... 

One of the paradoxes about HIV infection is that most infected individuals contain HIV antibodies, but the disease eventually occurs in most cases, even in the presence of these antibodies. This means that HIV antibodies are unable to prevent the onset of AIDS. This may be due to several factors. First, the levels of antibodies raised might be insufficient to block the spread of infectious virus. In addition, antibodies can be produced against different parts of virus. Only some of these antibodies (neutralizing antibodies) can inactivate virus and prevent infection. Finally, several unique features of HIV infection provide the virus with ways to evade the immune system. 

The HIV antibody test measures whether an individual has circulating antibodies to HIV. However, strictly speaking, the test does not indicate if an antibody positive individual still harbors infectious virus. Some individuals who are exposed to HIV might have raised a successful immune response and completely eliminated the infection. However, by and large, most HIV-antibody positive individuals turn out to be still infected. 

Currently, there is only one antisense drug on the market— Vitravene (active ingredient fomivirsen) for the treatment of cytomegalovirus (CMV)-induced retinitis (inflammation of the retina) in AIDS patients. Fomivirsen has 21 nucleotides complementary to a CMV mRNA sequence, which is necessary for the production of infectious virus. Two examples of experimental antisense drugs are provided in Exhibit 3.15, while Table 3.1 lists other antisense drugs in clinical phase. 

The amount of infectious virus in lungs was defined by titration of 10% homogenates in tissue cultures of chorioallantoic membranes (CAM) of 11-13-days-old chicken embryos. TID50 was calculated by the modified Kerber s method using the formula mentioned above. 

These results show that the maximum amount of virus was found in the lungs of the animals of the control group on Day 3, after which it gradually decreased by Day 14, tbe virus was not detected even in the experimental group. The animals receiving Grinization had much lower levels of infectious virus in lungs since Day 1 to Day 10 of infection... 

Fig 2, The effect of Grinization application on dynamics of infectious virus accumulation in lungs of the infected mice. 

Pharmacology Saquinavir is an inhibitor of HIV protease which cleaves viral polyprotein precursors to generate functional proteins in HIV-infected cells. The cleavage of viral polyprotein precursors is essential for maturation of infectious virus. Saquinavir is a synthetic peptide-like substrate analog that inhibits the activity of HIV... 

These drugs inhibit the activity of HIV protease. This enzyme, which is required for the production of a mature infectious virus, cleaves the gag-pol polyprotein into structural proteins and active enzymes. The pharmacokinetic parameters of the protease inhibitors are listed in Table 51.5. 

The viral protease enzyme, human immunodeficiency virus type 1 (HIV-1), is a causative agent of AIDS. HIV-1 mediates the processing of the viral precursor polyproteins. This process is essential for the production and maturation of infectious viruses [1,2]. CR1XIVAN is a specific and potent inhibitor of the HIV-1 protease, and is used in the treatment of AIDS. 

Cello, J., Paul, A. V., and Wimmer, E. (2002). Chemical synthesis of poliovirus cDNA generation of infectious virus in the absence of natural template. Science, 297, 1016-18. 

Poliomyelitis is caused by a highly infectious virus known to affect only... 

A similar strategy has been used to optimize a number of peptide-based compounds with therapeutic potential, including tachykinins, enkephalins, and protease inhibitors. HIV protease is essential for producing mature, infectious virus, and two protease molecules are carried in each mature virion (Figure 4.7). With inhibitors developed specifically for HIV protease, but not human protease, one hopes to halt virus replication. Peptides with HIV protease inhibitor activity have been further refined by means of computer- and structure-based design strategies, leading to the development of new molecular entities that are stable to proteases and compact so that they can be administered orally (Table 4.6). Some protease inhibitors (e.g., ritonavir and... 

Figure 4.7. a schematic presentation of the role of protease in processing of HIV gag (structural protein) and polymerase pol essential for producing infectious virus. With inactive HIV protease, the virus generated is immature and hence not infectious. The i indicates where HIV protease cleavage occurs in producing the pol gene product essential for viral replication. 

The process of actually getting the recombinant DNA into a cell will depend on the type of vector used. If the vector is a plasmid, the DNA is now ready for transfection into the host cells. If the vector is a virus, it may be necessary to package the DNA into infectious virus particles before it can be introduced into the host cells. Infection is usually a fairly efficient means of introducing the recombinant DNA into the host, while transfection tends to be quite inefficient. Transfection involves special chemical and enzymatic treatment of the host cell to make its cell membrane permeable to the DNA, and such treatment tends to reduce the viability of the host. 

To study HIV-related pathogenesis, transgenic mice harbouring the complete HIV proviral genome have been generated (Leonard et al., 1988). These mice developed a fatal disease reminiscent of human AIDS, and infectious virus could be isolated from skin, spleen and lymph nodes. HIV is... 

---