Identification and characterization of a novel alpaca respiratory coronavirus most closely related to the human coronavirus 229E.

In 2007, a novel coronavirus associated with an acute respiratory disease in alpacas (Alpaca Coronavirus, ACoV) was isolated. Full-length genomic sequencing of the ACoV demonstrated the genome to be consistent with other Alphacoronaviruses. A putative additional open-reading frame was identified between the nucleocapsid gene and 3'UTR. The ACoV was genetically most similar to the common human coronavirus (HCoV) 229E with 92.2% nucleotide identity over the entire genome. A comparison of spike gene sequences from ACoV and from HCoV-229E isolates recovered over a span of five decades showed the ACoV to be most similar to viruses isolated in the 1960's to early 1980's. The true origin of the ACoV is unknown, however a common ancestor between the ACoV and HCoV-229E appears to have existed prior to the 1960's, suggesting virus transmission, either as a zoonosis or anthroponosis, has occurred between alpacas and humans.

Reverse restriction fragment length polymorphism (RRFLP): A novel technique for genotyping infectious laryngotracheitis virus (ILTV) live attenuated vaccines.

A novel technique, the reverse restriction fragment length polymorphism (RRFLP) assay, was developed as a means of detecting specific informative polymorphic sites in the infectious laryngotracheitis virus (ILTV) genome. During the RRFLP procedure, DNA is digested with restriction enzymes targeting an informative polymorphic site and then used as template in a real-time polymerase chain reaction (PCR) with primers flanking the informative region. The analysis of the DeltaC(t) values obtained from digested and undigested template DNA provides the genotype of the DNA. In this study, the RRFLP assay was applied as a method to differentiate between the two types of infectious laryngotracheitis virus attenuated live vaccines. Sequence analysis of ILTV vaccines revealed an informative polymorphic site in the 5'-non-coding region of the infected cell protein (ICP4) gene. Unique AvaI and AlwI restriction enzyme sites were identified in the tissue culture origin and chicken embryo origin attenuated vaccines, respectively. These two informative polymorphic sites were used in a RRFLP assay to genotype rapidly and reproducibly ILTV attenuated live vaccines.

Strain differentiating real-time PCR for Mycoplasma gallisepticum live vaccine evaluation studies.

Mycoplasma gallisepticum causes respiratory disease and production losses in poultry. Vaccination of poultry with M. gallisepticum live vaccines is an approach to reduce susceptibility to infection and to prevent the economic losses. The development and evaluation of live vaccines usually requires the involvement of several vaccine and challenge strains in the same experimental setup. Our goal was to develop a tool to allow the differentiation between a set of known M. gallisepticum strains in a quantitative manner. We developed 5 real-time PCR assays that absolutely differentiated between one of the five commercial and laboratory vaccine strains: F, ts-11, 6/85, K5831, K5054, and the challenge strain R low when tested on in vitro cultures. The assay K5831 vs. R low was also tested on specimens from live birds that were vaccinated with K5831 and challenged with R low, and successfully differentiated between the vaccine and the challenge strains in a quantitative manner. This preliminary in vivo application of the method also shed light on possible protection mechanisms for the M. gallisepticum K5831 vaccine strain.

Development and evaluation of a real-time Taqman RT-PCR assay for the detection of infectious bronchitis virus from infected chickens.

It is important to rapidly differentiate infectious bronchitis virus (IBV) from disease agents like highly pathogenic avian influenza virus and exotic Newcastle disease virus, which can be extremely similar in the early stages of their pathogenesis. In this study, we report the development and testing of a real-time RT-PCR assay using a Taqman-labeled probe for early and rapid detection of IBV. The assay amplifies a 143-bp product in the 5'-UTR of the IBV genome and has a limit of detection and quantification of 100 template copies per reaction. All 15 strains of IBV tested as well as two Turkey coronavirus strains were amplified, whereas none of the other pathogens examined, tested positive. Evaluation of the assay was completed with 1329 tracheal swab samples. A total of 680 samples collected from IBV antibody negative birds were negative for IBV by the real-time RT-PCR assay. We tested 229 tracheal swabs submitted to two different diagnostic laboratories and found 79.04% of the tracheal swabs positive for IBV by real-time RT-PCR, whereas only 27.51% of the samples were positive by virus isolation, which is the reference standard test. We also collected a total of 120 tracheal swabs at six different time points from birds experimentally infected with different dosages of IBV and found that, independent of the dose given, the viral load in the trachea plateau at 5 days post-inoculation. In addition, an inverse relationship between the dose of virus given and the viral load at 14 days post-inoculation was observed. Finally, we tested 300 total tracheal swab samples, from a flock of commercial broilers spray vaccinated for IBV in the field. The percentage of birds infected with the IBV vaccine at 3, 7, and 14 days post-vaccination was 58%, 65%, and 83%, respectively, indicating that only slightly more than half the birds were initially infected then the vaccine was subsequently transmitted to other birds in the flock. This observation is significant because coronaviruses, which have a high mutation rate, can revert to pathogenicity when bird-to-bird transmission occurs. The real-time RT-PCR test described herein can be used to rapidly distinguish IBV from other respiratory pathogens, which is important for control of this highly infectious virus. The test was extremely sensitive and specific, and can be used to quantitate viral genomic RNA in clinical samples.

In vitro analysis of a hammerhead ribozyme targeted to infectious bronchitis virus nucleocapsid mRNA.

Hammerhead ribozymes are catalytic RNA molecules that specifically cleave a target RNA molecule. Herein, we report the design, synthesis, and in vitro analysis of a hammerhead ribozyme targeted to the infectious bronchitis virus (IBV) nucleocapsid mRNA. At a concentration of 0.5 or 10 microM, the ribozyme, designated IBV-N-Rz, effectively cleaved target RNAs in trans (37 C, 10 mM MgCl2, 50 mM Tris). Cleavage products were visualized by agarose gel analysis. The time course of the ribozyme reaction was monitored by agarose gel analysis and relative quantitative reverse transcription-polymerase chain reaction. The amount of target RNA continually declined over a 5-hr period, indicating that the ribozyme was truly catalytic. Although stability and delivery problems must be overcome, a hammerhead ribozyme targeted to the IBV nucleocapsid mRNA most likely has antiviral activity and may be an effective therapeutic/prophylactic reagent in the future.

Data from 11 years of molecular typing infectious bronchitis virus field isolates.

In 1993, a new molecular typing method for infectious bronchitis virus (IBV) was introduced. This method uses reverse transcriptase-polymerase chain reaction (RT-PCR) and restriction fragment length polymorphism (RFLP) analysis of the spike gene to obtain RFLP patterns that correlate with serotype. Using that test at the Poultry Diagnostic and Research Center (PDRC, University of Georgia, Athens, GA), we have identified a total of 1523 IBV isolates in the past 11 yr. The data were obtained from clinical samples submitted to our laboratory from birds with clinical signs characteristic of IBV infection. The samples are primarily from the southeastern United States but are also from many other states as well as from outside the United States. Most of the isolations occurred during July, followed by May, April, November, October, and January. The fewest number of isolates identified on an annual basis was 20 in 2003. An unusually high number of isolations occurred in 1997 (318 isolations) and 1999 (246 isolations), which coincided with the GAV variant virus and GA98 variant virus outbreaks respectively. By far, the Ark-DPI strain was the most frequently identified type of IBV and ranged from 23% to 65% of total isolations per year. Ark-like isolates, defined as having a similar but unique RFLP pattern from the Ark-DPI vaccine strain were identified every year of the study except in 1996. In addition, new Ark-like isolates continued to emerge each year (except in the year 2000) beginning in 1997, reflecting the ability of that IBV type to undergo genetic drift. Eighty-two different variant viruses were identified although only two (GAV and GA98) became persistent and caused widespread disease. Some viruses tended to be geographically restricted to a given area (CAV in California and MX97-8147 in Mexico), whereas others were widespread (Ark-DPI, Conn, DE072, and Mass). The Florida, Gray, Holte, Iowa, and JMK types were not detected during the 11-yr period, and no foreign virus types were detected in the United States. These data show that IBV variant viruses are consistently circulating in commercial poultry and are capable of causing disease outbreaks. Our observations highlight the importance of constantly monitoring IBV as well as other coronaviruses like severe acute respiratory syndrome-coronavirus that have the ability to change and emerge to cause disease in a susceptible host.