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      Evolution of infectious bronchitis virus in Taiwan: Positively selected sites in the nucleocapsid protein and their effects on RNA-binding activity


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          RNA recombination has been shown to underlie the sporadic emergence of new variants of coronavirus, including the infectious bronchitis virus (IBV), a highly contagious avian pathogen. We have demonstrated that RNA recombination can give rise to a new viral population, supported by the finding that most isolated Taiwanese (TW) IBVs, similar to Chinese (CH) IBVs, exhibit a genetic rearrangement with the American (US) IBV at the 5’ end of the nucleocapsid ( N) gene. Here, we further show that positive selection has occurred at two sites within the putative crossover region of the N-terminal domain (NTD) of the TW IBV N protein. Based on the crystal structure of the NTD, the stereographic positions of both predicted selected sites do not fall close to the RNA-binding groove. Surprisingly, converting either of the two residues to the amino acid present in most CH IBVs resulted in significantly reduced affinity of the N protein for the synthetic RNA repeats of the viral transcriptional regulatory sequence. These results suggest that modulating the amino acid residue at either selected site may alter the conformation of the N protein and affect the viral RNA–N interaction. This study illustrates that the N protein of the current TW IBV variant has been shaped by both RNA recombination and positive selection and that the latter may promote viral survival and fitness, potentially by increasing the RNA-binding capacity of the N protein.

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          Codon-substitution models for heterogeneous selection pressure at amino acid sites.

          Comparison of relative fixation rates of synonymous (silent) and nonsynonymous (amino acid-altering) mutations provides a means for understanding the mechanisms of molecular sequence evolution. The nonsynonymous/synonymous rate ratio (omega = d(N)d(S)) is an important indicator of selective pressure at the protein level, with omega = 1 meaning neutral mutations, omega 1 diversifying positive selection. Amino acid sites in a protein are expected to be under different selective pressures and have different underlying omega ratios. We develop models that account for heterogeneous omega ratios among amino acid sites and apply them to phylogenetic analyses of protein-coding DNA sequences. These models are useful for testing for adaptive molecular evolution and identifying amino acid sites under diversifying selection. Ten data sets of genes from nuclear, mitochondrial, and viral genomes are analyzed to estimate the distributions of omega among sites. In all data sets analyzed, the selective pressure indicated by the omega ratio is found to be highly heterogeneous among sites. Previously unsuspected Darwinian selection is detected in several genes in which the average omega ratio across sites is 1. Genes undergoing positive selection include the beta-globin gene from vertebrates, mitochondrial protein-coding genes from hominoids, the hemagglutinin (HA) gene from human influenza virus A, and HIV-1 env, vif, and pol genes. Tests for the presence of positively selected sites and their subsequent identification appear quite robust to the specific distributional form assumed for omega and can be achieved using any of several models we implement. However, we encountered difficulties in estimating the precise distribution of omega among sites from real data sets.
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            Coronavirus avian infectious bronchitis virus.

            Infectious bronchitis virus (IBV), the coronavirus of the chicken (Gallus gallus), is one of the foremost causes of economic loss within the poultry industry, affecting the performance of both meat-type and egg-laying birds. The virus replicates not only in the epithelium of upper and lower respiratory tract tissues, but also in many tissues along the alimentary tract and elsewhere e.g. kidney, oviduct and testes. It can be detected in both respiratory and faecal material. There is increasing evidence that IBV can infect species of bird other than the chicken. Interestingly breeds of chicken vary with respect to the severity of infection with IBV, which may be related to the immune response. Probably the major reason for the high profile of IBV is the existence of a very large number of serotypes. Both live and inactivated IB vaccines are used extensively, the latter requiring priming by the former. Their effectiveness is diminished by poor cross-protection. The nature of the protective immune response to IBV is poorly understood. What is known is that the surface spike protein, indeed the amino-terminal S1 half, is sufficient to induce good protective immunity. There is increasing evidence that only a few amino acid differences amongst S proteins are sufficient to have a detrimental impact on cross-protection. Experimental vector IB vaccines and genetically manipulated IBVs--with heterologous spike protein genes--have produced promising results, including in the context of in ovo vaccination.
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              Severe acute respiratory syndrome vaccine development: experiences of vaccination against avian infectious bronchitis coronavirus

              Vaccines against infectious bronchitis of chickens (Gallus gallus domesticus) have arguably been the most successful, and certainly the most widely used, of vaccines for diseases caused by coronaviruses, the others being against bovine, canine, feline and porcine coronaviruses. Infectious bronchitis virus (IBV), together with the genetically related coronaviruses of turkey (Meleagris gallopavo) and ring-necked pheasant (Phasianus colchicus), is a group 3 coronavirus, Severe acute respiratory syndrome (SARS) coronavirus being tentatively in group 4, the other known mammalian coronaviruses being in groups 1 and 2. IBV replicates not only in respiratory tissues (including the nose, trachea, lungs and airsacs, causing respiratory disease), but also in the kidney (associated with minor or major nephritis), oviduct, and in many parts of the alimentary tract—the oesophagus, proventriculus, duodenum, jejunum, bursa of Fabricius, caecal tonsils, rectum and cloaca, usually without clinical effects. The virus can persist, being re-excreted at the onset of egg laying (4 to 5 months of age), believed to be a consequence of the stress of coming into lay. Genetic lines of chickens differ in the extent to which IBV causes mortality in chicks, and in respect of clearance of the virus after the acute phase. Live attenuated (by passage in chicken embryonated eggs) IBV strains were introduced as vaccines in the 1950s, followed a couple of decades later by inactivated vaccines for boosting protection in egg-laying birds. Live vaccines are usually applied to meat-type chickens at 1 day of age. In experimental situations this can result in sterile immunity when challenged by virulent homologous virus. Although 100% of chickens may be protected (against clinical signs and loss of ciliary activity in trachea), sometimes 10% of vaccinated chicks may not respond with a protective immune response. Protection is short lived, the start of the decline being apparent 9 weeks after vaccination with vaccines based on highly attenuated strains. IBV exists as scores of serotypes (defined by the neutralization test), cross-protection often being poor. Consequently, chickens may be re-vaccinated, with the same or another serotype, two or three weeks later. Single applications of inactivated virus has generally led to protection of <50% of chickens. Two applications have led to 90 to 100% protection in some reports, but remaining below 50% in others. In practice in the field, inactivated vaccines are used in laying birds that have previously been primed with two or three live attenuated virus vaccinations. This increases protection of the laying birds against egg production losses and induces a sustained level of serum antibody, which is passed to progeny. The large spike glycoprotein (S) comprises a carboxy-terminal S2 subunit (approximately 625 amino acid residues), which anchors S in the virus envelope, and an amino-terminal S1 subunit (approximately 520 residues), believed to largely form the distal bulbous part of S. The S1 subunit (purified from IBV virus, expressed using baculovirus or expressed in birds from a fowlpoxvirus vector) induced virus neutralizing antibody. Although protective immune responses were induced, multiple inoculations were required and the percentage of protected chickens was too low (<50%) for commercial application. Remarkably, expression of S1 in birds using a non-pathogenic fowl adenovirus vector induced protection in 90% and 100% of chickens in two experiments. Differences of as little as 5% between the S1 sequences can result in poor cross-protection. Differences in S1 of 2 to 3% (10 to 15 amino acids) can change serotype, suggesting that a small number of epitopes are immunodominant with respect to neutralizing antibody. Initial studies of the role of the IBV nucleocapsid protein (N) in immunity suggested that immunization with bacterially expressed N, while not inducing protection directly, improved the induction of protection by a subsequent inoculation with inactivated IBV. In another study, two intramuscular immunizations of a plasmid expressing N induced protective immunity. The basis of immunity to IBV is not well understood. Serum antibody levels do not correlate with protection, although local antibody is believed to play a role. Adoptive transfer of IBV-infection-induced αβ T cells bearing CD8 antigen protected chicks from challenge infection. In conclusion, live attenuated IBV vaccines induce good, although short-lived, protection against homologous challenge, although a minority of individuals may respond poorly. Inactivated IBV vaccines are insufficiently efficacious when applied only once and in the absence of priming by live vaccine. Two applications of inactivated IBV are much more efficacious, although this is not a commercially viable proposition in the poultry industry. However, the cost and logistics of multiple application of a SARS inactivated vaccine would be more acceptable for the protection of human populations, especially if limited to targeted groups (e.g. health care workers and high-risk contacts). Application of a SARS vaccine is perhaps best limited to a minimal number of targeted individuals who can be monitored, as some vaccinated persons might, if infected by SARS coronavirus, become asymptomatic excretors of virus, thereby posing a risk to non-vaccinated people. Looking further into the future, the high efficacy of the fowl adenovirus vector expressing the IBV S1 subunit provides optimism for a live SARS vaccine, if that were deemed to be necessary, with the possibility of including the N protein gene.

                Author and article information

                Vet Microbiol
                Vet. Microbiol
                Veterinary Microbiology
                Elsevier B.V.
                29 October 2012
                23 March 2013
                29 October 2012
                : 162
                : 2
                : 408-418
                [a ]Department of Life Sciences, Agricultural Biotechnology Center, National Chung Hsing University, Taichung 40227, Taiwan
                [b ]Institute of Genomics and Bioinformatics, Agricultural Biotechnology Center, National Chung Hsing University, Taichung 40227, Taiwan
                [c ]School of Veterinary Medicine, National Taiwan University, Taipei 10617, Taiwan
                [d ]Department of Physical Therapy, Graduate Institute of Rehabilitation Science, China Medical University, Taichung, Taiwan
                Author notes
                [* ]Corresponding author at: 250 Kuo Kuang Road, Taichung 40227, Taiwan. Tel.: +886 4 22840416; fax: +886 4 22854391. suhonglin@ 123456nchu.edu.tw

                These authors contributed equally.

                Copyright © 2012 Elsevier B.V. All rights reserved.

                Since January 2020 Elsevier has created a COVID-19 resource centre with free information in English and Mandarin on the novel coronavirus COVID-19. The COVID-19 resource centre is hosted on Elsevier Connect, the company's public news and information website. Elsevier hereby grants permission to make all its COVID-19-related research that is available on the COVID-19 resource centre - including this research content - immediately available in PubMed Central and other publicly funded repositories, such as the WHO COVID database with rights for unrestricted research re-use and analyses in any form or by any means with acknowledgement of the original source. These permissions are granted for free by Elsevier for as long as the COVID-19 resource centre remains active.

                : 27 April 2012
                : 15 October 2012
                : 17 October 2012

                Veterinary medicine
                infectious bronchitis virus,rna recombination,coronavirus,nucleocapsid,positive selection


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