Infectious bronchitis virus (IBV) is a gammacoronavirus in the subfamily Coronavirinae, family Coronaviridae and order Nidovirales (http://www.ictvonline.org/virusTaxonomy.asp?version=2009). IBV is endemic throughout the world and is the causative agent of an economically important poultry disease, infectious bronchitis (IB). IB is a highly contagious respiratory disease resulting in watery eyes, snicking, wheezing and rales. In addition, virus replication causes ciliostasis in the trachea. As a consequence, IB is associated with bacterial secondary infections that can lead to increased morbidity and mortality 1. IB results in poor egg quality, reduced egg production, poor meat quality and reduced weight gain. This, along with necessary disease control by vaccination, puts a large economic burden on the global poultry industry. In the UK alone, IB was estimated to cost the poultry industry £24 million per year in disease losses during the period 2000-2002 2.
Genome organisation and viral proteins
IBV has a large, single-stranded, positive sense RNA genome of approximately 27 Kb. The genome has a 5ʹ cap and a 3ʹ poly-A tail. The first two-thirds of the genome contains 2 large overlapping open reading frames, ORF 1a and ORF1b, that encode the replicase polyproteins, pp1a and pp1ab. Pp1ab is generated via a ribosomal frameshift towards the end of pp1a 3. These 2 large polyproteins are cleaved by viral proteases encoded within the polyprotein into constituent polypeptides, including the viral enzymes and other machinery required for synthesising viral RNA. Coronaviruses encode among other enzymes an RNA-dependent RNA polymerase (non-structural protein, nsp, 12), an RNA helicase (nsp13), capping machinery (nsp10, nsp14 and nsp16) and, unusually for an RNA virus, a proof-reading enzyme (nsp14) 4-6. The remaining one-third of the genome encodes four structural proteins and at least four accessory proteins 7-9. The structural and accessory proteins are translated from sub-genomic mRNAs transcribed by the viral machinery. Currently, two mRNAs are known to be polycistronic with additional open reading frames translated via leaky ribosomal scanning or via an internal ribosome entry site (IRES) 10-12.
The IBV particle is approximately 120 nm in diameter and has a lipid envelope. The four structural proteins assemble to generate the virus particle and three of these are glycoproteins that are inserted into the envelope. The spike (S) glycoprotein is the viral attachment and fusion protein 13. It exists as a trimer with a large head domain on the outside of the virus particle. This forms a “corona” or crown around the particle that gives coronaviruses their name. Another major component of the envelope is the membrane (M) protein, which plays a role in particle assembly 14,15. The final viral protein inserted into the envelope is the envelope (E) protein. This is a minor constituent of the envelope and plays a role during viral budding 16-18. Within the particle, the viral genome is bound by the nucleocapsid (N) protein 19,20. Other viral and cellular proteins may also be incorporated into the particle 21.
IBV attaches, via the S protein, to a receptor on the cell surface. Sugars are known to play a role in viral attachment to cells, although other unidentified receptor proteins may also be important 13. Following attachment, the virus particle is taken into the cell and the viral envelope fuses with the cell membrane thereby releasing the genome into the cytoplasm. As the genome has a 5′ cap and a 3′ poly-A tail, it resembles cellular mRNA and is directly translated by the ribosome. This results in expression of the viral nsps and assembly of the viral transcription machinery, the replication-transcription complex (RTC), which synthesises viral RNAs. Like the vast majority of positive sense RNA viruses, during replication IBV induces the rearrangement of cellular membranes 22. The viral RTC is thought to localise to these specialised membrane structures within the infected cell, which are termed replication organelles. This is thought to protect nascent RNA from detection by the host and provide a platform for the assembly of the large multi-component RTC. The membrane rearrangements induced by IBV include double membrane vesicles, regions of ER that become zippered together and small open-neck vesicles bound by two membranes that remain tethered to the zippered ER, called spherules 23,24.
Viral RNA is synthesised via a negative sense intermediate in a discontinuous manner (reviewed in 25). The transcription machinery proceeds along the template RNA until it reaches the genomic transcription regulatory sequence (TRS) located at the 5′ end of each ORF. Here, the transcription machinery either reads through the TRS and continues transcribing along the genome until the next TRS or detaches and reattaches at a complementary sequence called the leader TRS near the 5′ end of the genome. Once reattached, the transcription machinery continues until the end of the template. This generates the 3′ co-terminal nested set of RNAs. Positive sense viral mRNAs are then synthesised by the transcription machinery from these sub-genomic negative sense copies.
Following RNA synthesis, viral structural and accessory proteins are translated and new viral particles assemble. The envelope glycoproteins are inserted into the ER and trafficked to the ER-Golgi intermediate compartment and this is where new particles form. Viral budding occurs in a process that requires interactions between S, M, E and N 15,17,18,26-28. Finally, enveloped particles are released from the cell via exocytosis. Progeny virions begin to be released from cells in culture by 6 hours post infection and progeny continues to be released until the cell dies 23.
The virus initially infects the respiratory tract. Following infection here, some strains of IBV are able to spread to secondary sites of infection including the oviducts and the kidneys. Virus replication in the oviducts affects egg production with fewer eggs being laid and an increase in poor quality or shell-less eggs. In addition, some more recent highly pathogenic strains of IBV result in severe damage to the oviducts and generation of “false-layers” or birds with large numbers of abdominal cysts creating a distended abdomen and a “penguin-like stance” 29.
Several vaccines are available for prevention of IBV. There are both inactivated vaccines and live-attenuated vaccines. Live-attenuated vaccines are produced by serial passage in embryonated eggs to reduce pathogenicity in adult birds. However, there is a delicate balance between reducing pathogenicity and maintaining immunogenicity. The molecular basis of attenuation in these vaccine strains is not understood and will likely be different in each new vaccine produced. Despite the number of available vaccines, IBV continues to cause new outbreaks. This is due to the very large number of different strains and poor-cross protection between different strains (http://www.infectious-bronchitis.com/ib-virus-classification.asp). In addition, new viral strains emerge on a regular basis due to the relatively high mutation rate of the genome and recombination between co-circulating IBV strains 30-33. Development of novel ways to produce vaccines is a major area of research in the field.
Contributed by: Helena Maier (The Pirbright Institute)
Copyright © 2016 Helena Maier
First posted: 16-Sept-2016
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