Attenuation and amino acid changes in infectious bronchitis virus Iranian 793/B serotype during serial passaging in embryonated chicken eggs.

The use of live attenuated vaccine (LAV) is the main method for controlling infectious bronchitis (IB). It is advisable to develop a LAV using a dominant serotype in the region in the case of vaccine failure. Since 793/B serotype is one of the most predominant circulating IB viruses in Iran, attenuation of three Iranian 793/B isolates (IR/773/2001, IR/794/2002 and IR/520/2002) was done by serial passaging in specific pathogen free (SPF) embryonated chicken eggs up to 90 passages to assess the degree of their attenuation to achieve a native LAV in the future. Virulence and pathogenicity of passage levels 15 and 90 of isolates 773 and 794 were compared using histopathology, ciliostasis and potency tests. The results showed a decrease in the virulence and pathogenicity of the isolates at passage 90 compared to passage 15, although this decrease in pathogenicity was very mild and viruses after passage 90 were not adequately attenuated. Each isolate underwent some amino acid changes at passage 90. In case of isolate 773 it was 5 aa changes, while in isolate 794 it was 19 aa changes. Some amino acid changes resulted in change into amino acid with different hydrophobicity characteristics. No amino acid change was found at passage level 15 compared to wild type viruses. Interestingly, we did not find previously reported change in amino acid 95 in passage levels 15 and 90. Keywords: infectious bronchitis; live attenuated vaccine; 793/B serotype; pathogenicity; attenuation; nucleotide sequencing.

Nucleotide Sequence Analysis of S1 Gene among Iranian Avian Infectious Bronchitis Viruses Isolated during 2001-2002.

Infectious bronchitis (IB) virus genome codes for four structural proteins, among which the S1 subunit of spike glycoprotein comprises the major epitopes to induce neutralizing antibodies. This study involved the comparison of the full S1 sequences of five IB viruses, namely two Massachusetts and three 793/B serotypes, isolated from IB outbreaks during 2001-2002, with all other Iranian and foreign 793/B isolates and 10 known serotypes. Analysis of S1 subunit showed three unique amino acid changes at positions 349 (V to L), 392 (T to N), and 393 (Q or R to T or K or S) for the Iranian 793/B isolates, compared to those of the foreign 793/B isolates reported before 2006 (onset of vaccination with 793/B vaccine in Iran). They were used as amino acid markers for the differentiation of Iranian 793/B isolates for years. Sequence alignment of the Iranian isolates with those of the foreign ones reported after 2006 demonstrated that amino acids 392 and 393 were no longer considered as amino acid markers, and only the change in amino acid 349 still remained specific to the Iranian 793/B isolates. Phylogenetic tree sequence analysis revealed that the Iranian 793/B isolates were closely related indicating that they came from a single source, more probably from France. There was a very close correlation between the first detection of 793/B serotype and the time of French chicken meat importation. Moreover, it was shown that one of the Massachusetts isolates was completely identical with the H120 vaccine strain. Furthermore, the other Massachusetts isolate with two amino acid changes at positions 64 (G to E) and 95 (S to R) was very similar to this vaccine strain. It seems that the latter isolate is a passaged chicken H120 vaccine strain.

Detection of 793/B serotype of infectious bronchitis virus in tissue sample by indirect immunoperoxidase assay.

The indirect immunoperoxidase (IIP) assay was compared with the reverse transcription-polymerase chain reaction (RT-PCR) for detection of 793/B serotype of infectious bronchitis virus in tissues samples collected from experimentally infected chickens. This technique was optimized in specific pathogen-free (SPF)-embryonated chicken eggs and broiler chickens inoculated with the Iranian IR/773/2001 strain of 793/B serotype The trachea, lung, kidney, and cecal tonsil tissue samples from experimentally infected chicken embryos and chickens were collected in order to prepare tissue sections in IIP assay and to detect in RT-PCR. The sensitivity and specificity values of IIP assay were, respectively, 83 and 84 %, and the positive and negative prediction values were 71 and 91 % when compared with RT-PCR.

Biological and molecular characterization of Avian influenza virus (H9N2) isolates from Iran.

Three Influenza A virus (H9N2) isolates obtained from three separate broiler flocks with variable mortality rates were cloned twice in embryonated SPF chicken eggs by limiting dilution. Biological properties of these isolates were examined in 4-week-old SPF chickens and chick embryo fibroblast (CEF) cultures. The isolates neither caused mortality in the inoculated chickens nor produced CPE in cell cultures, indicating low pathogenicity. PCR products of 486 bp containing the sequences for hemagglutinin (HA) cleavage site, which were generated from the isolates, were subjected to nucleotide sequencing. Sequence analysis of the HA region containing the cleavage site of the isolates showed a similar sequence motif (PARSSRG) but different flanking regions. Phylogenetic analysis of deduced amino acid sequences revealed that the isolates were closely related to those isolated earlier, indicating a common source. Moreover. the amino acid sequences of the recent isolates were very similar to those from Saudi Arabia, Germany and Pakistan. It is postulated that, except for some Chinese isolates, the pathogenicity of Iranian isolates seems to be similar to that of other Eurasian isolates. It is possible that an elevation in mortality rate under field condition could be caused by co-infection of recent isolates with the bacteria such as mycoplasma, Escherichia coli, and Ornithobacterium rhinotracheale rather than by an emerging a pathogenic H9N2 subtype of the virus.