In order to verify a commonly held assumption that only Massachusetts (Mass) serotype of infectious bronchitis virus (IBV) was prevalent in the United States between the 1930s (when IBV was first isolated) and the 1950s (when the use of commercial IBV vaccines began), we examined 40 IBV field isolates from the 1940s. Thirty-eight of those isolates were recognized as Mass serotype viruses based on their reactivity to Mass-specific monoclonal antibody (Mab) and neutralization by Mass-specific chicken serum. The remaining two isolates, N-M24 and N-M39, that did not react with Mass-specific Mab, resisted neutralization by Mass-specific chicken serum, and were neutralized only by homologous chicken antibody were identified as non-Mass IBV. When the first 900 nucleotides (nt) from the 5′-end of the spike (S1) glycoprotein gene and their deduced amino acid (aa) sequences were compared, the two non-Mass isolates differed from each other by 24% and 28%, respectively. In a similar comparison, the non-Mass viruses N-M24 and N-M39 differed from M28, a Mass-type isolate from the 1940s, by 21% and 22% (nt) and 28% and 27% (aa), respectively. These data indicate that antigenic and genetic diversity among IBV isolates existed even in the 1940s. Interestingly, when the N-terminal region of the S1 of M28 was compared to that of M41, a prototype Mass virus that has undergone countless number of in vivo and in vitro host passages, the two viruses differed by only 2% (nt) and 4% (aa). This finding suggests that frequent genetic changes are not inherent in all IBV genomes.
Infectious bronchitis virus (IBV), a coronavirus, causes infectious bronchitis in chickens (2). IBV has a single-stranded RNA genome of approximately 27 kilobases. The genes for the structural proteins of IBV are located downstream of the viral polymerase gene and in order from 5′ to 3′ are, the spike (S) glycoprotein, small envelop protein, membrane protein, and nucleocapsid protein. The S protein of IBV is posttranslationally cleaved into a transmembrane domain (S2) and an outer domain (S1). The N terminus region of the S1 subunit encoded by the 5′ end of the S1 gene is believed to carry serotype-specific antigenic epitopes that induce virus-neutralizing (VN) antibodies (1,4). Based on the virus-neutralization test, many IBV serotypes and antigenic variants are currently recognized (2).
It is generally believed that between the early 1930s when IBV infection was first recognized (14) and the middle 1950s when widespread application of IBV vaccines began (7,15), only Massachusetts (Mass) serotype of IBV was prevalent in the United States. However, to our knowledge, there is no published report of a scientific verification of this assumption. Because such information could provide clues to the evolution of currently prevalent serotypes, we examined a significant number of IBV isolates from the 1940s in order to determine: 1) whether serotypes other than Mass were prevalent in that period, and 2) whether the genetic information obtained from these isolates would shed light on the evolution of the presently existing serotypes. The results indicate that although Mass was a predominant serotype in the 1940s, other serotypes were also present, and that there was considerable genetic and antigenic diversity among the IBV isolates even in the 1940s.
MATERIALS AND METHODS
Forty field isolates of IBV, recovered from the respiratory tracts of chickens raised in the northeastern United States in the late 1940s, were studied. Following isolation at Cornell University, the viruses were initially passed a minimum number of times (no more than three) in embryonating chicken eggs, and allantoic fluids from the embryos were lyophilized and stored at 4 C. For the present study, the lyophilized samples were reconstituted in phosphate-buffered saline and passed twice in specific-pathogen-free (SPF) chicken embryos, and allantoic fluids from the second passage were collected for use. The standard IBV serotypes used for comparison were from our departmental repository.
Monoclonal antibodies (Mabs).
The IBV-specific monoclonal antibodies (Mabs) used in this study were described previously (8). Two group-reactive Mabs, 919 and 94, which respectively recognize the membrane (M) and S2 glycoprotein of all IBV serotypes, were used to determine the presence of IBV. Mabs 1588, 940, and 1318, specific for the S1 glycoproteins of serotypes Mass, Connecticut (Conn), and Arkansas (Ark), respectively, were used for initial serotyping.
After the initial identification of the isolates from the 1940s into Mass-type and non-Mass-type IBV with the aid of Mabs, three isolates, including one Mass-type virus (M28) and two non-Mass-type viruses (N-M24 and N-M39) were used for antisera production. After the viruses were purified by the terminal-dilution procedure, approximately 1000 median embryo-infective doses (EID50) of each virus were inoculated via the ocular route into groups of five 6-wk-old chickens. Three weeks later, the chickens were bled. Sera from birds within each group were pooled and titrated against the homologous virus using virus neutralization in chicken embryos (3). Chicken antisera to Mass serotype strain M 41 were available in our department.
This was performed in two steps. First, the isolates were identified with the aid of the IBV-specific Mabs described above using enzyme-linked immunosorbent assay (ELISA) (13) and immunoperoxidase techniques (12), and in the second step they were subjected to a standard in vitro virus neutralization (VN) assay (3).
Reverse-transcription (RT)-polymerase chain reaction (PCR).
IBV genomic RNA was extracted from 250 µl of infected allantoic fluid using TRIzol LS reagent (Gibco BRL, Grand Island, NY) according to the manufacturer's instructions, and resuspended in 3 µl of RNAse-free water. Amplification of the S1 gene by reverse-transcription polymerase chain reaction (RT-PCR) was performed using the primer set NewS1oligo5′ (5′TGAAACTGAACAAAAGAC3′) and Degenerate3′ (5′CCATAAGTAACATAAGGRCRA3′) (5,11). The RT-PCR was performed using the GeneAmp RNA PCR kit (Perkin Elmer Cetus, Norwalk, CT) according to the manufacturer's instructions. Briefly, cDNA was synthesized from 3 µl of RNA using random hexamer primer at 42 C for 30 min, and the mixture was then heated for 5 min at 94 C to stop the reaction. Both the RT reaction and PCR were conducted in an MJ Research thermal cycler (PTC-100; MJ Research, Inc., Watertown, MA). For the PCR reaction, 30 pmol (2 µl) of each primer, NewS1oligo5′ and Degenerate3′, and 2.5 units of Taq DNA polymerase were added in a 100-µl reaction volume. The first cycle of PCR amplification was carried out for 90 sec at 94 C, 30 sec at 50 C, and 2 min at 72 C. The remaining 34 cycles were carried out for 30 sec at 94 C, 30 sec at 50 C, and 2 min at 72 C, with a final elongation step of 15 min at 72 C. PCR products were visualized by electrophoresis in 1% agarose gel, followed by staining with ethidium bromide (0.5 µg/ml).
PCR products were purified using the QIAquick gel extraction kit (QIAGIN Inc. Valencia, CA). Purified DNA was directly sequenced with the aid of BIG dye terminator cycle sequence kit (Perkin-Elmer, Branchburg, NJ) and run on an Applied Biosystem model ABI 377 automated DNA sequencing system at Cornell University's central DNA sequencing facility. A combination of flanking and internal primers was used to sequence both strands of cDNA in their entirety. Assembly of sequencing contigs, translation into amino acid sequence, and initial multiple sequence alignment were performed with the BioEdit software version 5.0 (NCSU, Raleigh, NC).
RESULTS AND DISCUSSION
From the 1930s, when IBV was first isolated in the United States, until the mid-1950s, Mass was believed to be the only IBV serotype prevalent in the U.S.A. (7,15). A second IBV serotype (Conn) was reported in the mid-1950s, and soon thereafter the use of commercially produced IBV vaccines began. A number of serotypes and antigenic variants were isolated in subsequent years (2). In order to determine whether serotypes other than Mass were present in the U.S.A. in the 1940s, and to assess the possible role of viruses from that period in the evolution of the present-day serotypes, we studied 40 IBV isolates that were recovered in the 1940s from naturally infected chickens raised in the northeastern United States.
In the initial screening by Mabs, all 40 virus isolates reacted with the two IBV group-reactive Mabs, 919 and 94. Of these, 38 reacted with Mass serotype-specific Mabs (1588), but not with those specific to Conn or Ark serotypes (940 and 1318). The remaining two isolates (N-M24 and N-M39) did not react with Mass, Conn, or Ark-specific Mabs (Table 1).
In the in vitro VN test, M28, which was initially selected at random as a representative of Mass serotype and was used for antiserum production in chickens, was replaced with M26, another Mass-type isolate from the 1940s. This was necessary because M28 induced only marginal embryo lesions that made the interpretation of VN test results difficult. The VN test results in Table 1 show that M26 and the prototype Mass strain M41 belong to the same serotype. On the other hand, since N-M24 and N-M39 were neutralized only by their homologous antisera, it is concluded that 1) they are distinct from Mass serotype, and 2) they are antigenically unrelated to each other.
For the genomic studies, we primarily focussed on the two non-Mass isolates and compared their genomes to 1) M28 virus, a Mass-type isolate from the 1940s, and 2) other IBV serotypes whose genome sequences are available through GenBank. We also compared M28 genome with that of M41 strain to determine the extent of genetic changes the latter virus might have accumulated during the past six decades of laboratory usage involving countless numbers of in vivo and in vitro passages.
We concentrated our studies on the first 900 nucleotides (nt) of the S1 gene from its 5′ end and their deduced amino acid sequences because this region is believed to encode IBV serotype-specific determinants (1). These studies revealed that the N-termini of N-M24 and N-M39 have higher sequence variability than the rest of the S1 subunit (Fig. 1) and that the majority of these differences are concentrated between amino acids 51 and 77, and 115 to 152, which correspond to the two hypervariable regions (HVRs) described earlier (9). Interestingly, the genome of N-M39 was found to have two short nucleotide sequences encoding amino acids 117 and 118, and 148–152 (Fig. 1), which have been found in the same location in Ark (6), Gray, and JMK serotypes (10), but not in Mass serotype viruses including M41.
A difference of 24% was observed between the 5′-end nucleotide sequences of N-M24 and N-M39, followed by that between N-M39 and M28 (22%), and N-M24 and M28 (21%) (Table 2). The deduced amino acid sequence from the same region of N-M24 genome differed from that of N-M39 by 28%, whereas those of N-M39 and N-M24 differed from M28 genome by approximately 27% and 28%, respectively. The two non-Mass isolates from the 1940s did not show close similarity to any of the IBV sequences available through GenBank, the nearest match being 80% between N-M24 and serotype Gray, and 80.5% between N-M39 and Conn (data not shown). There was neither evidence of crossover sites nor a fixed pattern of nucleotide change in the S1 genes of any of the isolates from the 1940s.
Interestingly, comparison of the S1 genes of M28 and M41 revealed a difference of only 2% in nucleotide sequence and 4% in amino acid sequence. These relatively minor differences between a Mass-type virus of the 1940s and the present-day Mass prototype M41 strain suggest that propensity for frequent mutation is not necessarily intrinsic to all IBV strains (1).
The results of this study present evidence for the first time that significant genetic and antigenic diversity in IBV existed during the 1940s, well before the introduction of IBV vaccines in the field. It shows that although Mass serotype viruses were predominant during the 1940s, non-Mass serotype viruses whose genomes significantly differ from each other and from that of the Mass-type viruses were also present. Based on these findings, we speculate that the high levels of genetic and antigenic diversity that exist in the present-day IBV serotypes have come from genetic changes both in the IBV populations that existed before the advent of vaccination and in the population of viruses that were introduced through live IBV vaccines.
We sincerely thank Dr. Julius Fabricant, Professor Emeritus, Department of Microbiology and Immunology, College of Veterinary Medicine, Cornell University, for making the IBV isolates from the 1940s available for this study. We are also thankful to Ms. Alice Andriguetto and Ms. Beverley Bowman for excellent technical assistance.
Characterization of four IBV isolates from the 1940s with the aid of Massachusetts (Mass)-specific monoclonal antibody (Mab) and virus neutralization (VN) by serotype-specific chicken sera.
Percent nucleotide (1-900/1-1611) and deduced amino acid (1-300/1-543) sequence differences between the S1 genes of IBV strain M41 and of IBV isolates from the 1940s.