Ribosome profiling of porcine reproductive and respiratory syndrome virus reveals novel features of viral gene expression.

The arterivirus porcine reproductive and respiratory syndrome virus (PRRSV) causes significant economic losses to the swine industry worldwide. Here we apply ribosome profiling (RiboSeq) and parallel RNA sequencing (RNASeq) to characterise the transcriptome and translatome of both species of PRRSV and to analyse the host response to infection. We calculated programmed ribosomal frameshift (PRF) efficiency at both sites on the viral genome. This revealed the nsp2 PRF site as the second known example where temporally regulated frameshifting occurs, with increasing -2 PRF efficiency likely facilitated by accumulation of the PRF-stimulatory viral protein, nsp1ß. Surprisingly, we find that PRF efficiency at the canonical ORF1ab frameshift site also increases over time, in contradiction of the common assumption that RNA structure-directed frameshift sites operate at a fixed efficiency. This has potential implications for the numerous other viruses with canonical PRF sites. Furthermore, we discovered several highly translated additional viral ORFs, the translation of which may be facilitated by multiple novel viral transcripts. For example, we found a highly expressed 125-codon ORF overlapping nsp12, which is likely translated from novel subgenomic RNA transcripts that overlap the 3' end of ORF1b. Similar transcripts were discovered for both PRRSV-1 and PRRSV-2, suggesting a potential conserved mechanism for temporally regulating expression of the 3'-proximal region of ORF1b. We also identified a highly translated, short upstream ORF in the 5' UTR, the presence of which is highly conserved amongst PRRSV-2 isolates. These findings reveal hidden complexity in the gene expression programmes of these important nidoviruses.

Viruses have tiny genomes. Rather than carry all the genetic information they need, they rely on the cells they infect. This makes the few genes they do have all the more important. Many viruses store their genes not in DNA, but in a related molecule called RNA. When the virus infects cells, it uses the cells' ribosomes ­ the machines in the cells that make proteins ­ to build its own proteins. One of the central ideas in biology is that one molecule of RNA carries the instructions for just one type of protein. But many viruses break this rule. The ribosomes in cells read RNA instructions in blocks of three: three RNA letters correspond to one protein building block. But certain sequences in the RNA of viruses act as hidden signals that affect how ribosomes read these molecules. These signals make the ribosomes skip backward by one or two letters on the viral RNA, restarting part way through a three-letter block. Scientists call this a 'frameshift', and it is a bit like changing the positions of the spaces in a sentence. The virus causes these frameshifts using proteins or by folding its RNA into a knot-like structure. The frameshifts result in the production of different viral proteins over time. The porcine reproductive and respiratory syndrome virus (PRRSV) uses frameshifts to cause devastating disease in pigs. Besides the sequences in its RNA that allow the ribosomes to skip backwards, the viral enzyme that copies the RNA can also skip forward. This results in shortened copies of its genes, which also changes the proteins they produce. To find out exactly how PRRSV uses these frameshifting techniques, Cook et al. examined infected cells in the laboratory. They monitored the RNA made by the virus and looked closely at the way the cells read it using a technique called ribosome profiling. This revealed that frameshifting increases over the course of an infection. This is partly because the viral protein that causes frameshifts builds up as infection progresses, but it also happened with frameshifts caused by RNA knots. The reason for this is less clear. Cook et al. also discovered several new RNAs made later in infection, which could also change the proteins the virus makes. RNA viruses cause disease in humans as well as pigs. Examples include coronaviruses and HIV. Many of these also have frameshift sites in their genomes. A better understanding of how frameshifts change during infection may aid drug development. Future work could help researchers to understand which proteins viruses make at which stage of infection. This could lead to new treatments for viruses like PRRSV.

Mycoplasma gallisepticum in pheasants and the efficacy of tylvalosin to treat the disease.

Infectious sinusitis, a common condition seen in adult pheasants, is primarily caused by Mycoplasma gallisepticum. The aims of the present study were to investigate the pathogenicity of M. gallisepticum in 14-day-old pheasants and evaluate the macrolide antibiotic tylvalosin (TVN) as a treatment for infectious sinusitis. The minimum inhibitory concentration of TVN for five isolates of M. gallisepticum taken from pheasants confirmed their susceptibility to TVN (range: 0.002 to 0.008 µg/ml). One of the isolates (G87/02) was inoculated intranasally into 72 pheasants (two groups of 36) at 14 days of age. Eight days later, when 18/72 (25%) of the pheasants showed clinical signs, one group was treated with 25 mg TVN/kg bodyweight daily in drinking water for three consecutive days. An uninfected, unmedicated control group (n=12) was also included. In contrast to the uninfected control group, a range of clinical signs typical of infectious sinusitis with varying severity was observed in challenged birds and M. gallisepticum was re-isolated from the infraorbital sinus and the eye/conjunctiva at necropsy, 22 days post challenge. In comparison with untreated birds, medication with TVN significantly reduced clinical signs and the re-isolation/detection of M. gallisepticum (P≤0.0021). The daily liveweight gain of treated birds was significantly increased in comparison with untreated birds (P=0.0002), and similar to daily liveweight gains observed in the uninfected control group. In conclusion, TVN at 25 mg/kg bodyweight daily for three consecutive days in drinking water was efficacious in the treatment of M. gallisepticum infection induced by challenging 14-day-old pheasants.

Avian encephalomyelitis virus is a picornavirus and is most closely related to hepatitis A virus.

The complete RNA genome of avian encephalomyelitis virus (AEV) has been molecularly cloned and sequenced. This revealed AEV to be a member of the Picornaviridae and consequently it is the first avian picornavirus for which the genome has been sequenced. Excluding the poly(A) tail the genome comprises 7032 nucleotides, which is shorter than that of any mammalian picornavirus sequenced to date. An open reading frame commencing at nucleotide 495 and terminating at position 6896 (6402 nucleotides) potentially encodes a polyprotein of 2134 amino acids. The polyprotein sequence has 39% overall amino acid identity with hepatitis A virus (HAV; genus Hepatovirus), compared to 19 to 21% for viruses from the other five picornavirus genera. Eleven cleavage products were predicted. The highest identity (49%) with HAV was in the P1 region, encoding the capsid proteins. The 5' and 3' untranslated regions (UTRs) comprise 494 and 136 nucleotides, respectively. The 5' UTR is the shortest of any picornavirus sequenced to date and, unlike HAV, it does not contain a long polypyrimidine tract.

A comparison of three serological methods to detect chicken and turkey antibodies to avian nephritis virus and the use of virus-specific monoclonal antibodies.

Avian nephritis virus (ANV) strain G-4260 was inoculated orally in to 1-day-old and 3-week-old chickens and the sequential antibody response to the virus was monitored by serum neutralization, ELISA and immunofluorescence tests. The order of sensitivity of the serological tests was in the sequence given above, with the neutralization test being by far the most sensitive. There was no obvious difference in the antibody titres produced by either age group. Virus was recovered from kidney tissue, the highest titres being obtained 3 to 5 days post-inoculation. Histological examination revealed mainly lymphocytic infiltration of the interstitium and degenerative changes in the tubular epithelium of the kidney. The G-4260 strain of ANV was given orally to 1-day-old turkey poults, but no serological response was induced. Virus was not recovered from the kidneys and no histological lesions were produced in this organ. Monoclonal antibodies were produced which neutralized the infectivity of ANV. An antigen trap ELISA was developed using a monoclonal antibody and infected chicken kidney tissue cultures. However, this assay did not detect ANV in kidney samples taken directly from infected chickens, with the exception of one sample which was shown to contain the highest concentration of infectious virus.

Avian herpesvirus as a live viral vector for the expression of heterologous antigens.

Control of Marek's disease in the poultry industry has been successfully achieved for several decades by large-scale vaccination of day-old chickens with live herpesvirus of turkeys (HVT) strains. Several features of this virus including lack of pathogenicity and long-term immune protection due to a persistent viraemic infection made us decide to use HVT as a live viral vector for the expression of foreign antigens. Potential sites for the integration of foreign DNA in the unique short region of the HVT genome were identified by the insertion of a beta-galactosidase expression cassette. Vaccination trials with recombinant virus strains indicated that the marker gene was expressed and stably maintained during animal passage. Based on an insertion site mapping in one of the open reading frames of the unique short region, a general recombination vector was designed for the integration of foreign genes into HVT. Recombinant virus-directed expression of individual antigens from Newcastle disease virus was driven by a strong promoter element derived from the lung terminal repeat sequence of Rous sarcoma virus.

The immunoglobulin M response in chicken serum to infectious bursal disease virus.

Despite the severe bursal damage observed in 5-week-old chicks as a result of the replication of a virulent strain of infectious bursal disease virus, the virus-specific antibody response in serum followed a typical pattern. Three methods were used to monitor the IgM response, an indirect ELISA using serum and ELISAs, carried out on gel chromatography fractions and eluants from affinity chromatography of serum. The profile of IgM production was found to be similar by each method. Possible future uses for the indirect ELISA are discussed.

Local and systemic antibody class responses to an infectious laryngotracheitis virus vaccine strain.

Chickens infected with infectious laryngotracheitis virus (ILTV) responded by producing virus-specific IgG in their sera, which increased steadily in concentration, but with slight fluctuations, until peak titres were reached 40 days post-inoculation (pi), immediately prior to the second challenge. Thereafter, following an initial lag, concentrations continued to increase for 21 days before falling slightly at the end of the experiment. In contrast, peak concentrations of ILTV-specific IgM were reached 6 days pi falling to their lowest levels by day 16, before increasing to a second peak and trough on days 26 and 32, respectively. This cyclical production of ILTV-specific IgM was confirmed in a second experiment. The pattern of production of ILTV-specific IgG, IgM and IgA, detected in tracheal washings, occurred in the same cyclical manner. IgM was produced first, peak concentrations being detected 5 days pi, whereas IgG and IgA did not peak until 10 days pi, with second peaks of each class being detected 25-30 days pi. The possibility that the cyclical antibody class response to ILTV infection is related to the previously reported intermittent pattern of re-excretion of the virus is discussed.

Infection and reinfection of chickens with Salmonella typhimurium: bacteriology and immune responses.

Four-day-old chickens infected orally with a spectinomycin-resistant (Spcr) mutant of a highly invasive avian Salmonella typhimurium strain excreted salmonellae in the feces for at least 10 weeks. When these chickens were reinfected at this time with a nalidixic acid-resistant (Nalr) mutant of the same strain, they excreted this mutant in significantly smaller numbers (P less than 0.01) than did a previously uninfected control group. The Nalr mutant had a shorter survival rate in the tissues of the immunized chickens than in tissues of the control birds. The Spcr mutant stimulated strong IgG, IgA, and IgM responses in serum, small-intestinal contents, and bile. These were detected by enzyme-linked immunosorbent assay (ELISA) against antigens of crude whole bacterial cell protein sonicate, lipopolysaccharide, flagella, and outer-membrane proteins. There was some evidence of an anamnestic response with IgA in bile following reinfection with the Salmonella. The peak response of antibody-producing cells from the spleens of infected chickens, assayed by solid-phase ELISA, occurred at 3 weeks postinoculation. A strong delayed hypersensitivity reaction, detected by foot-pad swelling after inoculation with either whole-cell or outer-membrane proteins, was observed between 2 and 5 weeks after infection with the Spcr mutant. The data indicate that outer-membrane proteins are major immunogens for both humoral and cell-mediated arms of the immune system.

Molecular cloning and sequence analysis of the genome of chicken anaemia agent.

The replicative form (RF) DNA of chicken anaemia agent (CAA) was isolated and cloned into bacterial plasmids. After religation of the cloned CAA DNA and transfection into MDCC-MSB1 cells, the DNA could induce c.p.e. characteristic of that caused by CAA, and an antigen was produced which gave positive immunofluorescence when detected with an anti-CAA serum. Sanger sequencing of the 2298 bp genome revealed several open reading frames (ORFs); the major ORF encoded a polypeptide of 51.8K. In SDS-PAGE of CAA viral particles a 50K protein has been reported as the only detectable viral protein. The genomic region downstream of the major ORF had several predicted GC-rich inverted repeats, a poly(A) signal and four copies of an 18 bp repeat element. Database searches did not reveal any sequence with homology to the viral genomic DNA, nor to the amino acid sequence of any of the ORFs, apart from the N-terminal 40 amino acids of the major ORF which showed a limited similarity to the structure of protamines.

IgM responses in chicken serum to live and inactivated infectious bronchitis virus vaccines.

Intramuscular (i.m.) administration of infectious bronchitis virus (IBV) oil-emulsion vaccine (OEV) to IBV-primed or unprimed chickens resulted in the production of zero or minimal concentrations of IBV-specific IgM in the serum, as measured by enzyme-linked immunosorbent assay of gel chromatography fractions. Live-attenuated infectious bronchitis (IB) vaccine given i.m. or by eyedrop stimulated the production of IBV-specific IgM in similar amounts following inoculation by both routes. These levels were comparable to those found in earlier studies following intranasal inoculation with a virulent strain of IBV and confirm that the detection of IBV-specific IgM is a valuable aid to the diagnosis of recent infection. As expected, administration of live-attenuated IB vaccines i.m. or by eyedrop protected the respiratory tract against challenge with virulent virus 24 days later; however, OEV given i.m. did not.

Indirect antigen-trap ELISAs using polyclonal antisera for detection of group B and D Salmonellas in chickens.

Indirect antigen-trap ELISAs able to detect Salmonella typhimurium and S. enteritidis are described. Their limits of sensitivity were about 10(7) organisms in nutrient broth culture. With the ELISA developed for S. typhimurium, 76 of 77 strains gave optical density (OD) readings of 1.06 to 1.40. Three other serotypes from group B in the Kaufmann-White scheme gave high values: S. saintpaul produced an OD of 1.40, S. schwarzengrund 0.93 and S. derby 0.88. Twenty other serotypes all produced OD values of 0.39 to 0.66. Four Citrobacter strains and an E. coli produced lower values. With the S. enteritidis ELISA, seven strains of this serotype produced OD values of 1.13 to 1.23 and 13 other serotypes gave OD values of 0.14 to 0.87. There was good correlation between the ELISA and bacteriological culture after examining selenite broth cultures of cloacal swabs taken from experimentally infected chickens. A few samples were positive by ELISA and negative by culture and vice versa. Similar results were obtained from cloacal swabs taken from a commercial flock known to be infected with S. typhimurium. Much closer correlation existed between the two methods for identifying artificially contaminated eggs or for spleens taken from experimentally infected birds.

Fowlpox virus: pathogenicity and vaccination of day-old chickens via the aerosol route.

Day-old chickens were given a single fowlpox virus vaccination (strain HP201) either via the aerosol or wing-web route. Both methods induced protective immunity against a wing-web or intravenous challenge with virulent fowlpox virus at 47 days old, although high titred virus preparations were required for successful aerosol vaccination. However, no clinical signs of infection were observed as a result of aerosol vaccination even if invasive strains of Escherichia coli were administered simultaneously. The use of aerosol fowlpox virus vaccination of day-old chicks has been shown to be a possible means of mass vaccination and could be applied to the use of fowlpox virus in recombinant vaccines.

Fowlpox vaccination: routes of inoculation and pathological effects.

Chickens were vaccinated against fowlpox via the wing web, oral route, drinking water or by aerosol. Using two inoculations of virus, at 5 and 26 days of age, protective immunity was induced in chickens which resisted challenge with a pathogenic fowlpox virus given either via the wing web or intravenously at 46 days of age. Aerosol and wing web vaccination induced slightly better protective immunity than drinking water or oral vaccination. Virus was detected in lung cells of chicks vaccinated by either oral, drinking water or aerosol routes. Virus was also recovered from the lungs of these chicks, maximum titre being obtained 6 days post vaccination. Although a marked histological reaction was present in the lung no clinical signs were observed in the chicks.

A method for the rapid purification of serum IgM for the diagnosis of recent viral infections of chickens.

The rapid purification of chicken IgM from serum was achieved by affinity chromatography. IgM immunoadsorbent gels were prepared using monoclonal antibodies specific to chicken IgM. Five different eluting agents were compared for the dissociation of the adsorbed IgM; the most convenient for routine purposes was 2 M NaCl, Tris-HCl, EDTA (NTE), as this enabled direct assay of eluents by ELISA without requiring the intermediate step of dialysis, which the other eluting agents did. Eluents prepared from sera obtained from infectious bronchitis virus (IBV) and infectious laryngotracheitis (ILTV)-infected chickens, together with samples of the same serum fractionated by gel chromatography, were tested by ELISA for virus-specific antibodies and to confirm the identity of the antibody class. In the case of both IBV and ILTV, similar results were obtained using immunoaffinity and gel chromatography. IBV-specific IgM, as determined by both methods in the ELISA, reached its highest concentration at the 8th day after inoculation and was virtually absent by the 24th day, whilst the highest concentration of ILT-specific IgM was detected at 6 days and no or little IgM was present at 16 days after inoculation. Purification of serum IgM by affinity chromatography followed by ELISA was considered suitable for routine serological diagnosis of IB and ILT, since the time required to complete the assay (3 hours) was considerably less than that for gel chromatography and many samples could be assayed simultaneously.

Antibody response to experimental Salmonella typhimurium infection in chickens measured by ELISA.

An indirect ELISA has been developed to detect Salmonella typhimurium antibodies in chicken sera, using whole bacterial cell protein, flagellar protein or lipopolysaccharide as antigens. In experimental infections high concentrations of S typhimurium-specific IgG persisted after the faecal excretion of S typhimurium had ceased, whereas the specific IgM response was transitory. Some uninfected chickens placed in contact with experimentally infected birds developed high IgG titres in the absence of detectable faecal excretion. Other S typhimurium strains, which varied in their invasive abilities, also induced high titres of IgG. The ELISA allowed chickens infected experimentally with S typhimurium to be differentiated from chickens infected with 10 other serotypes, including S enteritidis. The use of whole blood in place of serum in the ELISA reduced the titres slightly. The storage of serum dried on to filter paper strips for four weeks produced little change in ELISA antibody titre, and the treatment of such strips with phenol or chloroform vapour had little or no effect on the antibody titre.

Development of immunoglobulin class-specific enzyme-linked immunosorbent assays for measuring antibodies against avian rotavirus.

Immunoglobulin class-specific enzyme-linked immunosorbent assays were developed for detecting antibodies against avian rotavirus in serum, intestinal contents, and bile from experimentally infected specific-pathogen-free (SPF) chickens. Both indirect and antibody-capture (AbC) assays were developed based on monoclonal antibodies specific for chicken IgG, IgM, and IgA. Treatment of purified rotavirus with sodium thiocyanate before coating the plate improved the rotavirus-specific reading in the indirect assay. Use of Immunolon 2 plates facilitated attachment of monoclonal antibodies to the plate in the AbC assay. Addition of 5% powdered skim milk to the diluent buffer reduced nonspecific background readings. The indirect assay was superior for detecting rotavirus-specific IgG, whereas the AbC assay was better for detecting rotavirus-specific IgM and IgA. The presence of intestinal contents in the assay wells did not reduce the measurable titers of IgG, IgM, or IgA. These assays showed that SPF chickens produced systemic and mucosal antibodies against avian rotavirus.

Amino acids within hypervariable region 1 of avian coronavirus IBV (Massachusetts serotype) spike glycoprotein are associated with neutralization epitopes.

The spike glycoprotein (S) gene of IBV codes for a precursor protein which is cleaved into the N-terminal S1 and C-terminal S2 glycopolypeptides. The S1 glycopolypeptide, which induces neutralizing antibody, comprises approximately 520 amino acid residues. We have determined the nucleotide sequence of S1 of seven strains of the Massachusetts (Mass) serotype and the first 337 bases of two additional Mass strains. Despite the fact that the strains had been isolated over three decades in Europe and the U.S.A. there was only 4% base and 6% amino acid variation within the group. Nearly one third of the 32 amino acid differences in S1 were in two hypervariable regions (HVRs 1 and 2) comprising residues 38-51 and 99-115, identified by Niesters et al. (1986), showing that HVRs 1 and 2 are a feature of the Mass serotype. Amino acid variation within HVRs 1 and 2 was 29% and 40% respectively. Five vaccine strains could be distinguished from each other by sequencing of the first 337 nucleotides. Variants of M41 which resisted neutralization by two monoclonal antibodies (A13 and A38) had the same, single base change at position 134, resulting in substitution of proline residue 45 by histidine. This indicates that residues within HVR 1 are associated with epitopes which induce neutralizing antibody.

Demonstration of antibodies to turkey rhinotracheitis virus in serum from commercially reared flocks of chickens.

Using both an ELISA and a serum neutralisation test, antibodies to turkey rhinotracheitis (TRT) virus were found in sera collected from commercial flocks of chickens after the initial appearance of TRT in turkeys in Britain in mid-1985 but not in chicken sera collected before that time. Good correlation was found between the results of the two assays and antibodies were found in chickens of all commercial types and of all ages, ranging from 31 days to 56 weeks. Apparently healthy flocks appeared to have been infected with the virus, as did flocks which had recovered from a variety of disease outbreaks including ones attributed to infectious bronchitis, and severe tracheitis as well as swollen head syndrome. The results presented here do not provide conclusive proof of an association between the presence of TRT antibodies in chicken sera and any particular disease condition and it appears that TRT virus can infect chickens without necessarily being responsible for clinical disease.