Genetic and antigenic diversity in avian infectious bronchitis virus isolates of the 1940s

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.     

Molecular and serologic characterization, pathogenicity, and protection studies with infectious bronchitis virus field isolates from California

In this study, we characterized three variant infectious bronchitis virus (IBV) strains isolated in 2003 and 2004 from broiler chickens in California and compared them to previously isolated California variant viruses and to common vaccine serotypes used in the United States. We conducted genetic, serologic, and pathogenicity studies on all three isolates, then tested different vaccines against one of the viruses. Genetically the three variant IBV strains, designated CA557/03, CA706/03, and CA1737/04, were not related to each other. GenBank BLAST database search and phylogenetic analysis of the hypervariable region of the S1 subunit of the spike gene to determine the most closely related viruses to the three variants showed the CA557/03 variant to be 81.8% similar to the CAV/CA56b/91 whereas the CA706/03 and CA1737/04 variant viruses were only distantly related to Dutch/D1466/81 (72.2%), a vaccine strain used in Europe, and Korea/K142/02 (72.7%), a Korean field isolate, respectively. Cross virus-neutralization testing showed that none of the 2003-04 California IBV variant viruses were serologically related to each other or to Ark, Conn, or Mass vaccine strains. In addition the CA1737/04 isolate was also tested against DE072 and found not to be serologically related. All three variant viruses were pathogenic in 1-wk-old broilers and vaccination with Mass/Conn followed by Holland/Conn provided 80% protection against the CA1737/04 virus. The 2003-04 California variant viruses were not compared with variants isolated in California during 1970s and 1980s because, to our knowledge, no genetic information is available and those viruses are no longer obtainable. This study shows that the CA557/03 virus was distantly related to the CAV-type viruses isolated in California in the early 1990s, but that none of the 2003-04 viruses were similar genetically or serologically to the CAL99-type viruses, indicating that new IBV variants continue to emerge and cause disease in commercial chickens in California.     

Identification of recent infectious bronchitis virus isolates that are serologically different from current vaccine strains

Three 1986 infectious bronchitis virus (IBV) isolates were serotyped by a hemagglutination-inhibition method and were found to be serologically different from the seven strains currently used in regular and specially licensed vaccines in the United States (Mass 41, H52, H120, Conn 46, Fla 18288, JMK, and Ark 99) and from 13 other reference IBV strains. The recent isolates were from layer flocks that had histories of egg-production declines and shell-quality problems. Two of the isolates, one each from Maine and Ohio, were serologically related to a 1983 IBV isolate from layer replacements in Pennsylvania. The third isolate, from Korea, was serologically different from any of the reference strains and the other new isolates.     

Evaluation of hemagglutinin subtype 1 swine influenza viruses from the United States

Swine influenza viruses (SIV) of the hemagglutinin subtype 1 (H1) isolated from the United States (U.S.) have not been well-characterized in the natural host. An increase in the rate of mutation and reassortment has occurred in SIV isolates from the U.S. since 1998, including viruses belonging to the H1 subtype. Two independent animal studies were done to evaluate and compare the pathogenesis of 10 SIV isolates dating from 1930 to currently circulating isolates. In addition, the hemagglutinin and neuraminidase genes of each isolate were sequenced for genetic comparison, and serological cross-reactivity was evaluated using all sera and virus combinations in hemagglutination inhibition and serum neutralization assays. Statistically significant differences in percentage of pneumonia and virus titers in the lung were detected between isolates, with modern isolates tending to produce more severe disease, have more virus shedding and higher viral titers. However, nasal shedding and virus titers in the lung were not always correlated with one another or lung lesions. Serologically, the classic historical H1N1 viruses tended to have better cross-reaction between historical sera and antigens, with moderate to good cross-reactivity with modern viral antigens. However, the modern sera were less reactive with historical viruses. Modern viruses tended to have less consistent cross-reactivity within the modern group. Overall, H1 isolates collected over the last 75 years from the U.S. pig population exhibit considerable variability in pathogenicity. There appears to be an increase in genetic and antigenic diversity coincident with the emergence of the swine triple reassortant H3N2 in 1998.     

Identification and analysis of the Georgia 98 serotype, a new serotype of infectious bronchitis virus

Twenty-five field infectious bronchitis viruses (IBVs) similar to, but genetically distinct from, the DE072 serotype were isolated from several states in the United States from 1990 through 1999 and were examined molecularly and antigenically. A 421-bp sequence in the hypervariable region of the S1 gene was examined, and phylogenetic analysis on that region indicated that these viruses are closely related but fall into unique groups. Cross-virus neutralization testing and entire S1 sequence analysis on selected isolates further confirmed that fact, and we divided the viruses into the DE072 serotype and two other unique groups. In a vaccine protection trial, the commercially available DE072 vaccine showed less than 50% protection against viruses in one of the groups. The majority of the recent isolates belong to that group and share very low antigenic relatedness to the DE072 strain as well as other serotypes of IBV. Consequently, we designated this group as a new serotype, Georgia 98. We developed a restriction fragment length polymorphism analysis that can differentiate this new serotype from all other serotypes of IBV.     

Genetic analysis of vesicular stomatitis virus-New Jersey from the 1995 outbreak in the western United States

OBJECTIVE: To compare molecular associations between the vesicular stomatitis virus (VSV)-New Jersey isolates of the 1995 outbreak with those from previous outbreaks between 1982 and 1985 in the western United States. SAMPLE POPULATION: 23 virus isolates considered representative of the 1995 outbreak of vesicular stomatitis. PROCEDURE: Viral gene coding for surface-envelope protein G was evaluated by use of nucleotide sequencing and phylogenetic analysis. RESULTS: Changes in up to 0.77% of the nucleotide bases and 1.35% of the amino acids were detected among the 1995 viral isolates, whereas changes in up to 3.2 and 2.9% of the nucleotides and amino acids, respectively, were found, compared with the 1982 to 1985 viruses. Insertions or deletions were not found in the entire gene, which spanned 1,554 nucleotide bases. CONCLUSIONS AND CLINICAL RELEVANCE: Phylogenetic analysis indicated that the 1995 VSV-New Jersey belongs to a lineage distinct from that of the 1982 to 1985 viruses that caused previous outbreaks in the western United States. Furthermore, it also is distinct from strains from Central America and from the Georgian Hazelhurst strain.     

Antigenic relationship of turkey coronavirus isolates from different geographic locations in the United States

The purpose of the present study was to examine the antigenicity of turkey coronavirus (TCV) isolates from various geographic areas with antibodies to different viruses. Seventeen isolates of TCV were recovered from intestinal samples submitted to Animal Disease Diagnostic Laboratory, Purdue University, from turkey farms located in different geographic areas. The prototype TCV Minnesota isolate (TCV-ATCC) was obtained from the American Type Culture Collection. Intestinal sections were prepared from turkey embryos infected with different TCV isolates and reacted with polyclonal or monoclonal antibodies to TCV, infectious bronchitis virus (IBV), bovine coronavirus (BCV), transmissible gastroenteritis virus (TGEV), reovirus, rotavirus, adenovirus, or enterovirus in immunofluorescent antibody staining. All 18 TCV isolates have the same antigenic reactivity pattern with the same panel of antibodies. Positive reactivity was seen with polyclonal antibodies to the TCV Indiana isolate, the TCV Virginia isolate, TCV-ATCC, and the IBV Massachusetts strain as well as monoclonal antibodies to the TCV North Carolina isolate or the membrane protein of IBV. Antibodies to BCV or TGEV were not reactive with any of the TCV isolates. Reactivity of antibodies to unrelated virus, rotavirus, reovirus, adenovirus, or enterovirus with different TCV isolates was all negative, except positive response was seen between enterovirus antibody and a TCV western North Carolina isolate, suggesting coinfection of turkeys with TCV and enterovirus in that particular case. The results indicated that the TCV isolates from these geographic locations in the U.S. shared close antigenicity and were antigenically related to IBV.     

1994—2009年我国部分地区37株鸡传染性支气管炎病毒S蛋白基因序列分析

对自1994—2009年从我国5省区免疫鸡群中分离到的37株传染性支气管炎病毒(IBV)的S蛋白基因序列进行分析,发现S1基因序列存在广泛的氨基酸替换、缺失和插入现象,大部分IBV分离株S1基因的推导氨基酸序列变异主要集中在60～63、73～74、97、128、282～299位等。S2基因较为保守,主要在裂解位点后的2～47、122～152位发生氨基酸的替换,可见IBV S基因的不断变异可能是造成本试验所调查的5省区免疫鸡群IB频发的重要原因。遗传进化分析发现本试验所调查地区近十多年来肾型毒株仍是主要流行株,没有或少有4/91型毒株流行。所调研地区鸡的腺胃炎持续广泛地发生,但是从临床腺胃病料中很少分离到IBV,可见IBV不太可能是引起腺胃炎的主要病原。     

Sequence analysis of related low-pathogenic and highly pathogenic H5N2 avian influenza isolates from United States live bird markets and poultry farms from 1983 to 1989

The last highly pathogenic outbreak of avian influenza in the United States was caused by an H5N2 influenza virus in Pennsylvania and New Jersey in 1983-84. Through a combined federal and state eradication effort, the outbreak was controlled. However, in 1986-89, multiple H5N2 viruses were isolated from poultry farms and the live bird markets (LBMs) in the United States. To determine the epidemiologic relationships of these viruses, the complete coding sequence of the nonstructural gene and the hemagglutinin protein subunit 1 of the hemagglutinin gene was determined for 11 H5N2 viruses and compared with previously available influenza sequences. The H5N2 isolates from 1986-89 were all closely related to the isolates from the 1983-84 Pennsylvania outbreak by nucleotide and amino acid sequence analysis for both genes, providing additional evidence that the Pennsylvania/83 (PA/83) virus lineage was not completely eradicated. The PA/83 lineage also had a large number of unique amino acid changes not found in other avian influenza viruses, which was suggestive that this lineage of virus had been circulating in poultry for an extended period of time before the first isolation of virus in 1983. High substitution and evolutionary rates were measured by examining the number of nucleotide or amino acid substitutions over time as compared with the index case, CK/PA/21525/83. These rates, however, were similar to other outbreaks of avian influenza in poultry. This study provides another example of the long-term maintenance and evolution of influenza viruses in the U.S. LBMs and provides further evidence of the connection of the LBMs and the Pennsylvania 1983 H5N2 outbreak.     

Characterization of infectious bronchitis virus isolates by slot blot hybridization

We used slot blot hybridization of the hypervariable regions of the S1 subunit of spike peplomer gene to identify and characterize infectious bronchitis virus (IBV) strains. Template DNA was created from six reference strain IBVs of different serotypes and immobilized on a nitrocellulose membrane. We synthesized digoxigenin-labeled probes from reference and unknown field viruses and hybridized them to template DNA. All reference strains could be distinguished and isolates identified by serotype if they were at least 95% identical to a reference strain. This slot blot hybridization procedure was specific and reproducible, and strain typing was consistent with the S1 sequencing of the IBV genome. This study thus provides a simple and rapid method for typing of IBV.     

Molecular characterization of avian infectious bronchitis virus strains isolated in Colombia during 2003

Sixteen infectious bronchitis virus (IBV) isolates were recovered from broilers and layers from five geographic poultry regions in Colombia. The viruses were isolated from tracheas, lungs, and cecal tonsils of birds, previously vaccinated with the Massachusetts strain, that were showing respiratory signs. Further analysis of the IBV isolates was achieved by phylogenetic analysis comparing their deduced amino acid sequences in the hypervariable region 1 of the S1 gene with reference strains. Four unique genotype clusters containing isolates with indigenous genotypes were observed. One isolate was found to be the Connecticut genotype and three isolates were found to be the Massachusetts genotype.     

Diversity of genome segment B from infectious bursal disease viruses in the United States

Several phylogenetic lineages of the infectious bursal disease virus (IBDV) genome segment B have been identified. Although this genome segment has been shown to contribute to virulence, little is known about the genetic lineages that exist in the United States. The nucleotide genome segment B sequences of 67 IBDV strains collected from 2002 to 2011 in the United States were examined. Although they were from nine different states, a majority (47) of these viruses were from California. A 722-base pair region near the 5' end of genome segment B, starting at nucleotide 168 and ending at 889, was examined and compared to sequences available in GenBank. The nucleotide sequence alignment revealed that mutations were frequently observed and that they were uniformly spaced throughout the region. When the predicted amino acids were aligned, amino acids at positions 145, 146, and 147 were found to change frequently. Six different amino acid triplets were observed and the very virulent (vv) IBDV strains (based on presence of vvIBDV genome segment A sequence) all had the triplet T145, D146, and N147. None of the non-vvIBDV strains had this TDN triplet. Phylogenetic analysis of the 67 nucleotide sequences revealed four significant genome segment B lineages among the U.S. viruses. One of these included the genome segment B typically found in vvIBDV and three contained non-vvIBDV genome segment B sequences. When the available sequences in GenBank were added to the analysis, two additional lineages were observed that did not contain U.S. viruses; one included viruses from China and the other contained viruses from the Ivory Coast. Although the samples tested do not represent all poultry producing regions in the United States, serotype 1 viruses from states outside California all belonged to one genome segment B lineage. The other three lineages observed in the United States were populated with viruses exclusively found in California, except the serotype 2 lineage, where the type strain was a serotype 2 virus from Ohio. The data provide further evidence for the importance of genome segment B identification during routine molecular diagnosis of all IBDV strains.     

Serotypes of previously unclassified isolates of Erysipelothrix rhusiopathiae from swine in the United States and Puerto Rico

Serotypes of 46 previously unclassified isolates of Erysipelothrix rhusiopathiae from porcine tissues in the United States and serotypes of 31 isolates of the organism from porcine tissues received from Puerto Rico were determined. The 46 isolates from the United States were classified in serotype 21. Four isolates (from Georgia, Minnesota, Ohio, and Oklahoma) were tested and found to be pathogenic for swine. Serotypes 1 (subtypes 1a and 1b), 2, 5, 6, and 21 were found in porcine tissues from Puerto Rico. The relative frequency of the various serotypes was similar to that previously reported in the United States.     

Characterization of three infectious bronchitis virus isolates from China associated with proventriculus in vaccinated chickens.

Outbreaks of an avian disease in infectious bronchitis-vaccinated chickens in China have led to the characterization of coronaviral isolates Q1, J2, and T3, which were isolated from proventricular tissues of the affected young layer flocks. Serologic analysis revealed that they could induce high titers of infectious bronchitis virus (IBV) antibodies in inoculated specific-pathogen-free (SPF) chickens in indirect enzyme-linked immunosorbent assay but were not neutralized by antisera specific to the IBV serotype M41 and the Australian T strain. In a pathogenicity experiment, the clinical signs and related gross lesions resembling those of field outbreaks were reproduced in SPF chickens, and viruses were reisolated from the damaged tissues, including trachea, proventriculus, duodenum, and cecal tonsil. Sequence data demonstrated the complete S1 amino acid sequences of these isolates were almost identical despite recovery from geographically different areas in China and had 47.3%-82.3% similarity in comparison with the 47 published S1 sequences. On the basis of genotyping and limited serology, the three isolates, which were responsible for field outbreaks of the disease, might be a new IBV variant.