Taxonomic Structure of the Family
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Orthomyxoviridae |
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Virions are spherical or pleomorphic, and 80-120 nm in diameter (Fig. 1). Filamentous forms several micrometers in length also occur. The virion envelope is derived from cell membrane lipids, incorporating variable numbers of virus glycoproteins (1-3) and non-glycosylated proteins (1-2). Virion surface glycoprotein projections are 10-14 nm in length and 4-6 nm in diameter. The viral nucleocapsid is segmented, has helical symmetry and consists of different size classes, 150-130 nm in length, with loops at one end.
Physicochemical and Physical Properties
Virion Mr is 250 106. Virion buoyant density in aqueous sucrose is 1.19 g/cm3. S20w of non-filamentous particles is 700-800 S. Virions are very sensitive to heat, lipid solvents, non-ionic detergents, formaldehyde, irradiation or oxidizing agents.
Depending on the genus, virions contain different numbers of segments of linear, negative sense ssRNA [8 segments: Influenza A virus (FLUAV) and Influenza B virus (FLUBV); 7 segments: Influenza C virus (FLUCV) and Dhori virus (DHOV); 6 segments: Thogoto virus (THOV)]. Segment lengths range from approximately 900 to 2350 nts. Genome size ranges from 10.0-14.6 kb. RNA segments possess conserved and partially complementary 5- and 3-end sequences with promoter activity. Defective (shorter, occasionally chimeric) viral RNAs may occur.
Structural proteins common to all genera include: three polymerase proteins (e.g., PA, PB1, PB2 in FLUAV); a nucleocapsid protein (NP), a group-specific protein that is phosphorylated and is associated with each genome ssRNA segment in the form of ribonucleoprotein; a hemagglutinin (HA, HEF or GP), an integral, type I membrane glycoprotein involved in virus attachment, envelope fusion and neutralization); and a non-glycosylated membrane or matrix protein (M1 or M). The HA of FLUAV is acylated at the membrane-spanning region and has N-linked glycans at a number of sites. In addition to its hemagglutinating and fusion properties the HEF protein of FLUCV has esterase activity that functions as a receptor-destroying enzyme. In contrast, the GP of THOV is unrelated to influenzavirus proteins, but shows sequence homology to a baculovirus surface glycoprotein. Depending on the genus, other virion proteins may include an integral, type II envelope glycoprotein (neuraminidase, NA), and an integral, type III membrane protein (M2, NB, or CM2) that may be glycosylated, and may function as an ion channel. In addition to the structural proteins, and depending on the genus, viruses may code for 2 nonstructural proteins (NS1, NS2). Virion enzymes (variously represented and reported among genera) include a transcriptase (PB1 in influenzaviruses A, B, C and thogotoviruses), an endonuclease (PB2 in influenzaviruses A, B, C), and a receptor-destroying enzyme (neuraminidase for FLUAV and FLUBV or 9-0-acetyl-neuraminyl esterase in the case of the FLUCV HEF protein).
Lipids in the virion envelope constitute about 18-37% of the particle weight. They resemble lipids of the host cell plasma membrane.
Carbohydrates in the form of glycoproteins and glycolipids constitute about 5% of the particle weight. They are present as N-glycosidic side chains of glycoproteins, as glycolipids, and as mucopolysaccharides. Their composition is host- and virus-dependent.
Genome Organization and Replication
The genome codes for up to 10 proteins (Mr 14-96 103). The 5 largest genome segments encode 1 protein each, whereas smaller segments often code for additional proteins from spliced or bicistronic mRNAs. Generally the three largest RNAs encode the P proteins, the 4th and 5th the viral HA (HEF, GP) and NP proteins. Depending on the virus, the smaller RNA species encode the NA protein (FLUAV NA and FLUBV NA, NB: 6th RNA), the membrane proteins (FLUAV M1, M2 and FLUBV M1, BM2: 7th RNA; FLUCV M1, CM2 and DHOV M1: 6th RNA) and NS proteins (FLUAV and FLUBV NS1, NS2: 8th RNA; FLUCV NS1, NS2: 7th RNA; putative DHOV 7th RNA: unknown). Gene reassortment occurs during mixed infections involving viruses of the same genus, but not between viruses of different genera (e.g., FLUAV and FLUBV).
Virus entry involves the HA (HEF, GP) and occurs by receptor-mediated endocytosis. The receptor determinant of influenzaviruses is sialic acid bound to glycoproteins or glycolipids. In endosomes low pH-dependent fusion occurs between viral and cell membranes. For influenzaviruses, fusion (and infectivity) depends on cleaved virion HA (FLUAV and FLUBV: HA1, HA2; FLUCV: HEF1, HEF2). In addition to the activity of signal peptidases, the HA of the influenzaviruses must undergo post-translational cleavage by cellular proteases to acquire infectivity and fusion activity. Cleavability depends, among other factors, on the number of basic amino acids at the cleavage site. It produces a hydrophobic amino terminal HA2 molecule. No requirement for glycoprotein cleavage has been demonstrated for the GP species of Thogotoviruses. Integral membrane proteins migrate through the Golgi apparatus to localized regions of the plasma membrane. New virions form by budding, thereby incorporating matrix protein and the viral nucleocapsids which align below regions of the plasma membrane containing viral envelope proteins. Budding is from the apical surface in polarized cells.
Viral nucleocapsids are transported to the cell nucleus where the virion transcriptase complex synthesizes mRNA species. For influenzaviruses, mRNA synthesis is primed by capped RNA fragments 10-13 nts in length that are generated from host heterogeneous nuclear RNA species by viral endonuclease activity associated with the PB2 protein. Thogotoviruses differ from influenzaviruses in having capped viral mRNA without host-derived sequences at the 5-end. Virus-specific mRNA synthesis is inhibited by actinomycin D or -amanitin due to inhibition of host DNA-dependent RNA transcription and a (presumed) lack of newly synthesized substrates that allow the viral endonuclease to generate the required primers. Virus-specific mRNA species are polyadenylated at the 3-termini, and lack sequences corresponding to the 5-terminal (approx. 16) nucleotides of the viral RNA segment. Certain mRNAs are spliced to provide alternative products (Fig. 2). Protein synthesis occurs in the cytoplasm. However, NP, M1, and NS1 proteins accumulate in the cell nucleus during the first few hours of replication, then migrate to the cytoplasm. Cytoplasmic inclusions of NS1 may be formed.
Complementary RNA molecules which act as templates for new viral RNA synthesis are full-length transcripts and are neither capped nor polyadenylated. These RNAs exist as nucleocapsids in the nucleus of infected cells.
The best studied antigens are the NP, M1, HA and NA proteins of FLUAV and FLUBV. NP and M1 are genus specific for the influenzaviruses. Considerable variation occurs among the FLUAV HA and NA antigens, less for FLUBV or the HEF surface antigens of FLUCV. THOV and DHOV do not cross-react in standard serologic tests, while DHOV and Batken virus do. Antibody to HA, HEF, and GP neutralizes virus infectivity.
Influenzaviruses agglutinate erythrocytes of many species. Serotype-specific antibodies may block agglutination. The NA or HEF of attached influenza virions may destroy sialic acid containing virus receptors of erythrocytes, resulting in elution of virus. Hemolysis of erythrocytes may be produced by HA at acid pH. By comparison to the influenzaviruses, thogotoviruses exhibit limited hemagglutination with certain erythrocyte species.
Certain influenzaviruses A naturally infect humans and cause respiratory disease. Particular influenzaviruses A infect other mammalian species and a variety of avian species. Interspecies transmission, though rare, is well documented. Influenza B virus strains appear to naturally infect only humans and cause epidemics every few years. Influenzaviruses C cause more limited outbreaks in humans and also infect pigs. Influenzaviruses A and B replicate in the amniotic cavity of embryonated hen eggs, and after adaptation they can also be propagated in the allantoic cavity. Influenzaviruses C replicate only in the amniotic cavity. Primary kidney cells from monkeys, humans, calves, pigs, and chickens support replication of many FLUAV and FLUBV strains. The host range of these viruses may be extended by addition of trypsin to the growth medium, so that multiple cycle replication can occur in some continuous cell lines. Clinical specimens from influenza-infected hosts often contain sub-populations of virus with minor sequence differences in HA proteins. These subpopulations may differ in their receptor specificity or their propensity for growth in different host cells.
Natural transmission of the influenzaviruses is by aerosol (human and most non-aquatic hosts) or is water-borne (ducks). Thogoto and Dhori viruses are transmitted by ticks and replicate in both ticks and a variety of tissues and organs in mammalian species as well as in mammalian cell cultures. In some laboratory species (e.g., hamsters for THOV) these infections have a fatal outcome. Unlike influenzaviruses, these viruses do not cause respiratory disease and do not replicate in embryonated hens’ eggs. All known orthomyxoviruses have an MX1-sensitive step in their multiplication cycle.
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