fig 473. Viral hemagglutination. Virus-containing fluid js iluted in doubling dilutions and 0.5% suspension of chick red cells is added. Where no virus is present the calls settle down to a button-like aggregate with sharp Where the virus is present, there isa difuse wide spread shield-like pattern on the bottom of the wells in the plastic plate VIRAL MULTIPLICATION ‘The genetic information necessary for viral replication is contained in the viral nucleic acid, but lacking bio- synthetic enzymes, the virus depends on the synthetic machinery of the host cell for replication, Early studies on viral replication employed the bacteriophage as the model, While there are general similarities in the pat- tern of multiplication of bacterial and animal viruses, there are also important differences, The viral multipli- cation cycle can be divided into six sequential phases (Fig. 47.4), though the phases may sometimes overlap. 1. Adsorption: Virions may come into contact with cells by random collision but adsorption takes place only if there is affinity between the two, The cell surface Table 47.2 Characteristics of hemagglutination by viruses should contain specific receptor sites to which the virus can ain attachment, §n influenza viruses, the hemagglutinin on the virus surface gets attached to glycoprotein receptor sites on the surface of the respiratory epithelium, Destruction of the receptor sites by RDE prevents viral adsorption. With HIV, attachment is between the CD4 receptor on host cells and the viral surface glycoprotein gp 120. In the ease of polioviruses, the receptor is the lipoprotein Present on the surface of primate but not rodent cells. ‘The poliovirus can, therefore, attach itself to primate cells but not to rodent cells, Differences in suscept bility to viral infection are to a large extent based on the presence or absence of receptors on cells. If the phase of adsorption can be bypassed, cells normally insusceptible to viruses may be rendered susceptible to them, Thus, infectious nucleic acid extracted from picornaviruses can infect rodent cells, which are resist- ant to infection by the whole virus. 2. Penetration: Bacteria possess rigid cell walls Bacterial viruses cannot, therefore, penetrate into bacte- rial cell, and only the nucleic acd is introduced intra- celluarly by a complex mechanism. Animal cells do not ‘have rigid cell walls and the whole virus can enter into ‘them. Virus particles may be engulfed by a mechanism resembling phagocytosis, a process known as ‘viropexis’. Alternatively, in enveloped viruses, the viral envelope ‘may fuse wit the plasma membrane of the host cell and ‘id into the cytoplasm. . Uncoating: This is the process of stripping the outer layers and capsid so that the nucleic acid is released into the cell. With most viruses, uncoat- ing is effected by the action of lysosomal enzymes of the hostcell In poxviruses, uncoating isa two-step process. In the first step, the outer coat is removed by lysosomal enzymes in the phagocytic vacuole. The inner core of the virus, containing the internal protein and nucleic Influenza virus Fowl, human, guinea pig, others elution at 37°C Parainfluenza, mumps; NOV Fowl, human, guinea pig, others; elution at 37°C; hemolysin present Measles Monkey, 37°C “Togavirus—several groups of Arbovirus Goose, pigeon, one-day-old chick: pH and temperature critical ; Rubella Goose, pigeon, one-day-old chick; 4°C Enterovirus, some Coxsackle and ECHO Rhinovirus, some serotypes Rabies Reovirus Human; 4°C and 37°C © scanned with OKEN Scannertesting sin Attachmant \ Ss Receptor - ci ct ee @ xting ration Reeare OOO & ef 4 Uncoat Assemty ting Capnes tom anun nuec ACKE t ‘Synthesis of viral messanger RNA J ) Synthesis of val prota or (Girect or via host machi Rew capsids ‘Synthesis of viral nucleic acid Fig. 47.4 Stages in the infection of a host's cell and replication of a virus acid, is released into the cytoplasm where the second sep of uncoating is effected by a viral uncoating enzyme and the DNA is liberated. 4. Biosynthesis: This phase includes the synthesis not merely of the viral nucleic acid and capsid protein but also of enzymes necessary in the various stages of viral synthesis, assembly and release. In addition, certain ‘regulator protcins' are also synthesised which serve to shut down normal cellular metabolism and direct the sequential production of viral components, The site of viral synthesis depends on the type of virus. In general, most DNA viruses synthesise their nucleic acid in the host cell nucleus. The exceptions are the poxviruses, which synthesise all their components in the host cell cytoplasm. Most RNA viruses synthesise all their com- ponents in the cytoplasm. Exceptions are orthomyxo- Viruses, some paramyxoviruses and retroviruses which are synthesised partly in the nucleus. Viral prot synthesised only in the cytoplasm. Steps in biosynthesis: ‘Transcription of messenger RNA (mRNA) from the Viral nucleic acid, ‘Capsid shed * Transation ofthe mRNA into ‘early protein’. They ‘early or non-structural proteins’ ae eneymes wig initiate and maintain the synthesis of virus nents. They may also induce shutdown of host rota and nucleic acid synthesis © Replication of viral nucleic acid, © Synthesis of ‘late’ or structural proteins, which a the components of daughter virion capsids, ‘The critical step in viral biosynthesis is the transcrip tion of mRNA from the viral nucleic acid. Once tis is achieved, the host cell resources can be utilised fr translating mRNA into viral components. Depening on the structure of their genome, viruses use diferat strategies for the transcription of mRNA. Replication mechanisms: Viruses have been catego- rised into six classes by Baltimore (1970) based oa + In the case of fully double-stranded DNA iruses (such as adeno, herpes, papovavruse) DNA enters the host cell nucleus and uses the hot | cell enzymes for transcription, The extracted ON from these viruses is infectious. With hepadns\" | © scanned with OKEN Scannera rel which have a partially double-stranded DNA, tre aple is compete by a viral DNA polymerase, ipa the host eytophasm, The mature DNA then imp in the lew, to be transcribed. by host seriptases. Extracted hepadnaviras: DNA is not Ietious. Powtuses which replicate in the eytox plasm form mRNA using polymerases contained in jon ise Poxvirus DNA is not infectious. «e class 2: With single-stranded DNA. viruses. (for example, ) the DNA molecule moves into ‘heh cellnweleus and is converted into the duplex form. Transcription i achieved by host enzymes, «s Class 5: In wovirases, the double-stranded RNA is rranscribed 10 MRNA by viral polymerases. Class 4: Depending on the method of mRNA tran- scription single-stranded RNA viruses ae classified into evo categories. In the positive strand (plus ‘trend, positive sense) RNA viruses, the viral RNA rif act as the MRNA. Viral RNA is infectious by eal and is translated directly into viral proteins jn the host cell cytoplasm (for example, picorna, togaviruses)- Class 5: In the negative strand (minus sense) RNA sinuses (lor example, thabdo, orthomyxo, para- yxoviridae), the RNA is ‘antisense’, with polarity opposite to mRNA. They possess their own RNA polymerases for mRNA transcription, Extracted nucleic acids from these viruses are not infectious. + Class 6: Retroviridae exhibit a unique replicati strategy. Their single-stranded RNA genome is con- verted into an RNA:DNA hybrid by the viral reverse transcriptase (RNA directed DNA polymerase) ‘enzyme. Double-stranded DNA is then synthesised from the RNA:DNA hybrid. The double-stranded DNA form of the virus (provirus) is integrated into the host cell chromosome. This integration may lead to transformation of the cell and development of neoplasia 5, Maturation: Assembly of daughter virions follows the synthesis of viral nucleic acid and proteins. Virion assembly may take place in the host cell nucleus or cyto plasm, Herpes and adenoviruses are assembled in the nucleus, while picorna and poxviruses are assembled in the cytoplasm. At this stage, the non-enveloped viruses are present intracellularly as fully developed virions, but in the case of enveloped viruses, only the nucleocapsid 4s complete, Envelopes are derived from the host cell membrane during the process of budding. ‘The host call membrane which becomes the envelope is modi- tran the vii parvouitu General Properties of Viruses fied by incorporation of virus-specific antigens. Herpes Viruses assembled in the nucleus acquire their envelope from the nuclear membrane as they are released into the cytoplasm enclosed in a vesicle. Myxoviruses bud from the cell surface and their envelope is formed by the modified cytoplasmic membrane of the host cell. ‘The incorporation of viral antigen (hemagglutinin) on the coll membrane endows the cell with the property of hemadsorption, 6, Release: Inbacterialviruses, therelease of progeny virions takes place by lysis of the infected bacterium. However, in animal viruses, release usvally occurs with ‘out cell Isis. Myxoviruses are released by a process of ‘budding from the cell membrane over a period of time. ‘The host cell is unaffected and may even divide, the daughter cells, continuing to release virions. Progeny virions are released into the surrounding medium and may infect other cells. In some viruses (for example, varicella), transmission occurs direetly from cell to cell, very little free virus being demonstrable extracellularly in the medium, Not all animal viruses spare the host cell, The poliovirus causes profound damage to the ‘host cell and may be released by cell lysis. Eclipse phase: From the stage of penetration till the appearance of mature daughter virions, the virus cannot be demonstrated inside the host cell. This period during which the virus seems to disappear or go ‘underground? is known as the ‘eclipse phase’. The time taken for a single cycle of replication is about 15-30 minutes for bacteriophages and about 15-30 hours for animal viruses. A single infected cell may release a large number of progeny virions. While this can be determined readily in bacteriophages (burst size), itis difficult to assess in animal viruses that are released over a prolonged period. Abnormal replicative eycles: ‘e A proportion of daughter virions produced may not be infective. This is due to defective assembly. Such ‘incomplete viruses’ are seen in large proportions ‘when cells are infected with a high dose of the influ- enza virus. The virus yield will have a high hemag- glutinin ttre but low infectivity. This is known as the von Magnus phenomenon. Virus infection in some cells does not lead to production of infectious progeny. In such cells (non-permissive cells), the viral components may be synthesised but maturation or asserubly is defec- tive, and either no release occurs or the progeny is © scanned with OKEN Scannerenemies eee non-infectious. This is known as abortive infection. Here, the defect is in the type of cel] and not in the parental viruses, e Some viruses are genetically defective in that when they infect cells, they are unable to give rise to fully formed progeny. Yield of progeny virions occurs only if the cells are simultaneously infected with a helper virus, which can supplement the genetic deficiency. For example, some strains of the Rous sarcoma virus (RSV) cannot code for the synthesis of the viral envelope. When RSV infects a cell that harbours a helper virus (for example, avian leukosis virus), infectious progeny result, the helper virus contributing to the synthesis of the envelope. The envelope antigen of progeny RSV will therefore be determined by the type of helper virus. Other exam- ples of defective viruses are the hepatitis D virus and adeno-associated satellite viruses which replicate only in the presence of their helper viruses—hepa- titis B and adenoviruses, respectively. Viruses which are genetically deficient and therefore incapable of producing infectious daughter virions without the helper activity of another virus are known as ‘defec- tive viruses’, CULTIVATION OF VIRUSES As viruses are obligate intracellular not be grown on any inanimate cult oe wnathnAn anna. Parasites, they can- ure medium. Three © scanned with OKEN Scanner