Chapter 60

The name orthomyxovirus is derived from two Greek words, orthos meaning correct or proper and myxa meaning mucus. The family includes the viruses responsible for influenza. Influenza is the Italian form of the Latin word influentia meaning influence because epidemics of infectious disease were believed to result from astrological or occult influences. Orthomyxoviruses are spherical or pleomorhic, enveloped viruses, 80–120 nm in diameter (Fig. 60.1). Extremely long filamentous forms also occur. The envelope is derived from the host cell membrane into which viral glycoproteins and non-glycosylated proteins have been incorporated. The glycoproteins form surface projections, termed ‘spikes’ or peplomers, and are of two types in influenza A and B viruses: a haemagglutinin (H) which is involved in virus attachment and envelope fusion and a neuraminidase (N) which enzymatically destroys viral receptors and promotes both entry and release of virus from infected cells. Influenza viruses haemagglutinate erythrocytes from a wide variety of species. Antibody to the H glycoprotein neutralizes infectivity. The nucleocapsid is helical in symmetry and the genome of linear, negative-sense, single-stranded RNA is segmented into six to eight pieces depending on the genus. Replication occurs in the nucleus of the cell with release by budding from the plasma membrane. Virions are very labile under ordinary environmental conditions, being sensitive to heat, lipid solvents, detergents, irradiation and oxidizing agents.

The family consists of five genera: Influenzavirus A, Influenzavirus B, Influenzavirus C, Thogotovirus and Isavirus (Fig. 60.2). Influenza A virus is the most important member of the family and a significant pathogen of animals and man. Influenza B and C viruses are pathogens of man while Thogoto virus and Dhori virus are tick-borne arboviruses isolated from camels, cattle and humans in parts of Africa, Europe and Asia. The genus Isavirus has one member, infectious salmon anaemia virus, which is a cause of disease in Atlantic salmon.

Influenza A virus isolates are divided into subtypes on the basis of the H and N antigens. Seventeen H and nine N subtypes have been described to date (Fouchier et al. 2005). New variant influenza A virus isolates emerge at frequent intervals. Two mechanisms are involved in this process, point mutations (antigenic drift) which effect variation within a subtype and genetic reassortment (antigenic shift) which gives rise to novel subtypes. In order to assess the risk posed by the emergence of new variant viruses a precise system of classification of isolates, promoted by the World Health Organization, has been adopted. This system is based on the type, host, geographic origin, strain number, year of isolation and subtype. For example, the prototype equine influenza A virus subtype 1 isolate from 1956 is designated: influenza virus A/equine/Prague/1/56 (H7N7). Isolates with a wide range of H and N combinations have been detected in birds whereas only a few combinations circulate in mammalian species (Table 60.1). Aquatic birds, particularly ducks, are the reservoirs of influenza A virus providing a genetic bank for the generation of novel subtypes capable of infecting mammals. The viruses replicate in the intestinal tract of birds resulting in a faecal–oral transmission pattern. Migratory birds facilitate the dissemination of virus across borders and between continents. Influenza A virus isolates are generally species-specific, but there are many well-documented incidences of transfer between species. In particular, incidences of direct transfer of H7 and H5 subtypes from birds to humans have given rise to concern. Interest has also focused on low pathogenic H9N2 isolates (Lupiani and Reddy 2009). A new pandemic strain of H1N1 appeared in people in Mexico in 2009, spreading rapidly to other parts of the world. The virus contained genetic elements from avian, swine and human isolates. Pigs, the likely source of this virus, are susceptible to infection with this strain.

Interest has focused on southeast Asia due to the human pandemics in 1957 and 1967, referred to as ‘Asian’ and ‘Hong Kong’ influenza respectively, which arose in that part of the world. It is thought that the large human population present there and traditional agricultural practices which bring humans, ducks and pigs into close contact, facilitate mixed infections. Avian influenza viruses are capable of infecting humans but generally replicate poorly in humans. However, both human and avian influenza viruses replicate in pigs, which may serve as a ‘mixing vessel’ for genetic reassortment. Due to the segmented nature of the genome, mixed infections of influenza A virus subtypes frequently give rise to genetic reassortment. Novel subtypes generated in this way are responsible for the serious pandemics that occur in the human population at approximately 20-year intervals. There is little or no immunity in the human population to each new subtype which consequently spreads rapidly throughout the world, usually replacing the older subtype. In the period between pandemics more subtle antigenic changes occur probably due to the inherent error rate of the viral RNA polymerase and the accumulation of mutations. Variants produced in this way may become dominant if sufficient amino acid changes have occurred at antigenic sites of the H molecule, permitting escape from the effects of neutralizing antibody. Influenza outbreaks follow in the proportion of the population that is immunologically susceptible. Such outbreaks occur abruptly, typically in the winter months in temperate regions.

Avian influenza

Influenza A virus subtypes have a worldwide distribution and are frequently recovered from clinically normal birds. Outbreaks of severe clinical disease accompanied by high mortality occur from time to time in chickens and turkeys, associated with H5 and H7 subtypes. The acute condition in these species is often referred to as fowl plague or highly pathogenic avian influenza (HPAI) and is designated as a listed disease by the OIE. Work with HPAI isolates should be carried out in facilities meeting OIE requirements for containment group 4 animal pathogens. In addition, appropriate precautions should be taken to protect workers handling infectious material.

Infection is maintained in the avian population by low-level circulation within a large wild bird population. Waterfowl are considered the prime candidates for the spread of virus to domestic species during their migrations. Imported cage birds and live-bird markets are other possible sources of infection.

Pathogenesis

The ability of influenza virus to spread in the body is determined by the proteases present in a given tissue and the structure of the haemagglutinin molecule. The production of infectious virions in a tissue is dependent on post-translational cleavage of the precursor haemagglutinin molecule HA0 by host proteases. In the epithelium of the intestinal and respiratory tracts, trypsin and trypsin-like enzymes are capable of cleaving the haemagglutinin molecule of all influenza subtypes. In contrast, protease enzymes present in other tissues are only capable of cleaving the HA0 molecules of virulent subtypes which possess a multiple basic amino acid sequence at the cleavage site. Therefore only virulent subtypes are capable of producing generalized infection characterized by haemorrhages and multifocal necrosis. It is thought that HPAI isolates arise from low-virulence isolates by mutation and that such mutations only take place following the transfer of virus from the natural wild bird host to poultry (Alexander & Capua 2004).

Diagnosis

Clinical signs may vary from inapparent or mild to almost 100% mortality. The highly virulent subtypes cause sudden onset of high mortality. A wider range of clinical signs are seen in birds that survive for a few days. Respiratory signs, cessation of egg laying, greenish diarrhoea, oedema of the head, cyanosis, sinusitis and excessive lachrymation are frequent signs. The severe form of the disease could be confused with velogenic, viscerotropic Newcastle disease or fowl cholera while milder forms of the disease are indistinguishable clinically from other respiratory conditions. Diagnosis requires laboratory confirmation involving virus isolation and characterization.

• Suitable specimens include tracheal and cloacal swabs, faeces and pooled samples of organs. Suspensions in antibiotic solution are inoculated into nine- to 11-day-old specific pathogen-free or specific antibody-free embryonated hens’ eggs. Allantoic fluid is harvested after four to seven days’ incubation and tested for haemagglutinating activity.

• Confirmation of the presence of influenza A virus can be achieved with an immunodiffusion test using a suspension of chorioallantoic membrane from infected eggs and positive antiserum to the nucleocapsid or matrix antigens common to all influenza A viruses.

• Commercial antigen-detection immunoassays have been used to detect influenza A viruses in poultry (Slemons & Brugh 1998, Cattoli et al. 2004). The tests are rapid and should detect any influenza A virus as they are generally based on a monoclonal antibody against the conserved nucleoprotein. However, such assays are probably best used as a flock test as they may lack sensitivity.

• Isolates may be roughly typed by haemagglutination inhibition or immunodiffusion using broadly reactive antisera. Subtyping is carried out by reference laboratories using monospecific antisera prepared against the 16 haemagglutinin and nine neuraminidase subtypes.

• All of the highly pathogenic avian influenza isolates to date have possessed either H5 or H7. However, numerous isolations of low-virulence H5 and H7 subtypes have been made. In order to assess pathogenicity eight to 10 chicks at four to eight weeks of age are inoculated by the intravenous route. Isolates causing more than 75% mortality within eight days (intravenous pathogenicity index of greater than 1.2) are considered highly pathogenic. In addition, genomic sequencing can be used to determine the amino acid sequences at the cleavage site of the haemagglutinin molecule (Wood et al. 1993, Senne et al. 1996). Both HPAI and LPAI isolates of subtypes H5 and H7 are notifiable to the OIE.

• Reverse transcription-PCR techniques (Senne et al. 1996, Starick et al. 2000, Munch et al. 2001) as well as real time RT-PCR (Spackman et al. 2002) have been developed for the detection and subtype identification of virus in clinical samples. These methodologies are particularly useful for the rapid detection of subsequent outbreaks once the primary infected premises has been identified and the virus isolate fully characterized. Reverse transcription-PCR based on primers for conserved sequences of the matrix gene has proved useful for screening for all subtypes in samples from a range of different species (Fouchier et al. 2000). Rapid assays for the detection of H5 and H7 virus have also been developed (Slomka et al. 2007) including nucleic acid sequence-based amplification (NASBA) with electro-chemiluminescent detection (Collins et al. 2002, 2003).

• Serological testing for antibodies to influenza virus can be done using an agar gel immunodiffusion test, haemagglutination inhibition or competitive ELISA (Shafer et al. 1998). A neuraminidase inhibition test has been developed as part of a strategy to differentiate infected from vaccinated animals (DIVA) (Capua et al. 2003).

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