Reverse-transcriptase polymerase chain reactions (RT-PCRs) were used to examine RNA extracted from mouth/nasal swabs from pheasants exhibiting signs of respiratory disease. The oligonucleotides used were based on sequences of infectious bronchitis virus (IBV), the coronavirus of domestic fowl. A RT-PCR for the highly conserved region II of the 3' untranslated region of the IBV genome detected a coronavirus in swabs from 18/21 estates. Sequence identity with the corresponding region of IBVs and coronaviruses from turkeys was > 95%. A RT-PCR for part of the S1 region of the spike protein gene was positive with 13/21 of the samples. Sequence analysis of the RT-PCR products derived from nine of the pheasant viruses revealed that some of the viruses differed from each other by approximately 24%, similar to the degree of difference exhibited by different serotypes of IBV. Further analysis of the genome of one of the viruses revealed that it contained genes 3 and 5 that are typical of IBV but absent in both the transmissible gastroenteritis virus and murine hepatitis virus groups of mammalian coronaviruses. The nucleotide sequences of genes 3 and 5 of the pheasant virus had a similar degree of identity (approximately 90%) with those of coronaviruses from turkeys and chickens, as is observed when different serotypes of IBV are compared. This work: (a) confirms that coronaviruses are present in pheasants (indeed, commonly present in pheasants with respiratory disease); (b) demonstrates that their genomes are IBV-like in their organization; and (c) shows that there is sequence heterogeneity within the group of pheasant coronaviruses, especially within the spike protein gene. Furthermore, the gene sequences of the pheasant viruses differed from those of IBV to similar extents as the sequence of one serotype of IBV differs from another. On the genetic evidence to date, there is a remarkably high degree of genetic similarity between the coronaviruses of chickens, turkeys and pheasants.
Avipoxvirus infections have been observed in an extensive range of wild, captive and domesticated avian hosts, yet little is known about the genome diversity and host-range specificity of the causative agent(s). Genome-sequence data are largely restricted to Fowlpox virus (FWPV) and Canarypox virus (CNPV), which have been sequenced completely, showing considerable divergence between them. It is therefore proving difficult, by empirical approaches, to identify pan-genus, avipoxvirus-specific oligonucleotide probes for PCR and sequencing to support phylogenetic studies. A previous preliminary study used the fpv167 locus, which encodes orthologues of vaccinia virus core protein P4b (A3). PCR per se did not discriminate between viruses, but restriction-enzyme or sequence analysis indicated that the avipoxviruses clustered either with FWPV or with CNPV. Here, further study of the P4b locus demonstrated a third cluster, from psittacine birds. A newly identified locus, flanking fpv140 (orthologue of vaccinia virus H3L), confirms the taxonomic structure. This locus is particularly useful in that viruses from the fowlpox-like and canarypox-like clusters can be discriminated by PCR on the basis of fragment size, whilst sequence comparison allows discrimination for the first time between Pigeonpox virus and Turkeypox virus. Except within the psittacines, virus and avian host taxonomies do not show tight correlation, with viruses from the same species located in very different clades. Nor are all the existing recognized avipoxvirus species, defined primarily by avian host species (such as CNPV and Sparrowpox virus), resolved within the present structure.
In 1992 we began an investigation into incidents of unusual and mass mortalities of the common frog (Rana temporaria) in Britain which were being reported unsolicited to us in increasing numbers by members of the public. Investigations conducted at ten sites of unusual mortality resulted in two main disease syndromes being found: one characterized by skin ulceration and one characterized by systemic haemorrhages. However, frogs also were found with lesions common to both of these syndromes and microscopic skin lesions common to both syndromes were seen. The bacterium Aeromonas hydrophila, which has been described previously as causing similar lesions, was isolated significantly more frequently from haemorrhagic frogs than from those with skin ulceration only. However, as many of the latter were euthanased, this may have been due to differences in post mortem bacterial invasion. An iridovirus-like particle has been identified on electron microscopical examination of skin lesions from frogs with each syndrome and iridovirus-like inclusions have been detected in the livers of frogs with systemic haemorrhages. Also, an adenovirus-like particle has been cultured from one haemorrhagic frog. A poxvirus-like particle described previously from diseased frogs has now been found also in control animals and has been identified as a melanosome. Both the prevalence of the iridovirus-like particle and its association with lesions indicate that it may be implicated in the aetiology of the disease syndromes observed. Specifically, we hypothesize that primary iridovirus infection, with or without secondary infection with opportunistic pathogens such as A. hydrophila, may cause natural outbreaks of 'red-leg', a disease considered previously to be due to bacterial infection only.
Eighteen isolates of infectious bronchitis virus (IBV) from
Intestinal contents of 13-day-old turkey poults in Great Britain were analysed as the birds showed stunting, unevenness and lameness, with 4% mortality. At post mortem examination, the main gross features were fluid caecal and intestinal contents. Histological examination of tissues was largely unremarkable, apart from some sections that showed crypt dilation and flattened epithelia. Negative contrast electron microscopy of caecal contents revealed virus particles, which in size and morphology had the appearance of a coronavirus. RNA was extracted (turkey/UK/412/00) and used in a number of reverse transcription-polymerase chain reactions (RT-PCRs) with the oligonucleotides based on sequences derived from avian infectious bronchitis virus (IBV), a coronavirus of domestic fowl. The RT-PCRs confirmed that turkey/UK/412/00 was a coronavirus and, moreover, showed that it had the same partial gene order (S-E-M-5-N-3' untranslated region) as IBV. This gene order is unlike that of any known mammalian coronavirus, which does not have a gene analogous to the gene 5 of IBV.The gene 5 of the turkey virus had two open reading frames, 5a and 5b, as in IBV and the coronaviruses isolated from turkeys in North America. The turkey/UK/412/00 also resembled IBV, but not mammalian coronaviruses, in having three open reading frames in the gene encoding E protein (gene 3). The percentage differences between the nucleotide sequences of genes 3 and 5 and the 3' untranslated region of turkey/UK/412/00 when compared with those of IBVs were similar to the differences observed when different strains of IBV were compared with each other. No sequences unique to the turkey isolates were identified. These results demonstrate, for the first time, that a coronavirus was associated with disease in turkeys outside of North America and that it is a Group 3 coronavirus, like IBV.
The degree of variation exhibited within the 793/B serotype (also known as 4/91 and CR88 serotypes) was investigated with nine French and 10 British isolates, collected between 1985 and 1994. The S1 part (1644 nucleotides) of the spike protein gene of the first known isolate of this serotype, FR/CR85131/85, had 95.9% to 97% nucleotide identity with the other isolates. Partial sequencing of isolates from Iran and Saudi Arabia, isolated in 2000, revealed approximately 95% nucleotide identity with European isolates, including the two live 793/B vaccinal strains, showing that they were not re-isolations of vaccinal virus. The data indicates that strains within the 793/B serotype have > or =96% nucleotide identity within the whole S1 gene and > or =93% nucleotide identity within the first 560 nucleotides, and > or =92% and > or =86% amino acid identities in the corresponding protein regions. This is similar to the identities exhibited within the Massachusetts serotype. Sequence analysis of a 793/B field isolate after passage in embryonated eggs, then in chickens and then again in eggs revealed selection for a serine and alanine at S1 amino acid position 95 in chicken-passaged and egg-passaged virus, respectively. There was no change in pathogenicity. This is the first demonstration at gene sequence level of host-driven selection for infectious bronchitis virus.
SUMMARYSince the winter of 1990/91 respiratory disease of poultry in Great Britain has commonly been associated with the 793/B (or 4/91) serotype of infectious bronchitis virus (IBV). We have sequenced a variable part of the S1 region of the spike protein (5) gene. Comparison of up to 270 nucleotides of 12 British 793/B isolates, obtained in 1991 and 1993, revealed 94 to 100% nucleotide identity with each other. Eleven of them fell into one of two subgroups, A and B, one isolate forming subgroup C. Identity within subgroups A and B was > 98%. The whole S1 gene sequence (1617 nucleotides) was determined for five 793/B isolates, two from each of subgroups A and B and one from subgroup C; nucleotide identity between any two isolates was > 97%. A large proportion of the nucleotide differences corresponded to amino acid changes. The whole S1 amino acid sequence differed by 21 to 25% or more from that of all other published IBV sequences. This extensive difference has probably contributed to the persistence of the 793/B serotype in Britain even though heterologous vaccines have been used.The finding that the 793/B isolates could be placed into three subgroups suggests that either (a) they had diverged from a common progenitor present, but undetected, in Britain prior to 1990/91 or (b) at least three different strains of the 793/B serotype had entered Britain in or prior to 1990/91.
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