key: cord-0004836-u3cnsdl8 authors: Lin, Z.; Kato, A.; Kudou, Y.; Umeda, K.; Ueda, S. title: Typing of recent infectious bronchitis virus isolates causing nephritis in chicken date: 1991 journal: Arch Virol DOI: 10.1007/bf01310957 sha: 97c8ea419efa5c8113bf3ff804b7ed14d4d2f5c3 doc_id: 4836 cord_uid: u3cnsdl8 Four isolates of infectious bronchitis viruses (IBV) from chickens with nephritis, were characterized by polymerase chain reaction (PCR) and restriction enzyme fragment length polymorphism (RFLP), and were found to be genetically different from the other twelve strains which we previously studied. . Amplification of cDNA from four isolates. The strains listed at the top of the lanes were amplified by PCR in 25 cycles. IBV M 41 was also amplified as a standard strain. Amplified cDNAs were analyzed by electrophoresis on 1.5% agarose gel in Tris-borate buffer containing 0.5 ~tg/ml of ethidium bromide. HinfI digested pUC 19 DNA was used as the molecular size markers (3//), and their sizes are indicated in base pairs (bp) on the left In this study, essentially the same procedure was used in an attempt to characterize four IBV isolates F-88, Y-4, M-l, and NI-1 from chickens with nephritis in different endemic areas of Japan between 1988 and 1989. Three of them, F-88, M-l, and NI-1, were obtained from affected kidneys, while Y-4 was from a gizzard, cDNA fragments of the four isolates were amplified by PCR. The amplified DNAs comigrated exactly in a 1.5% agarose gel, giving an identical size approximately 400 bp corresponding to the length between the two primers ( Fig. 1 ). This indicates that the amplified region was well conserved without apparent deletion or insertion. To see the RFLP of these amplified DNAs, they were cleaved by each of 9 restriction enzymes (HpaII, MaeIII, XhoII, Hinfl, ScaI, DdeI, HincII, HaeIII, and PstI) under the conditions recommended by the enzyme suppliers, and the digestants were run on 6% polyacrylamide gels. Figure 2 shows the representative cleavage patterns of the amplified DNA of M-1 and Y-4 isolates. The DNA of M-1 was cleaved at one site by all of the restriction enzymes, except HpaII and PstI (Fig. 2 a) . The DNA of Y-4 DNA was cleaved at two sites by ScaI and at one site by MaelII, XhoII, DdeI, and HincII (Fig. 2 b) . The differences in cleavage sites between the two isolates are shown in HinfI, ScaI, and HaeIII digestion. The DNA from the other two isolates, F-88 and NI-1, gave an identical cleavage pattern with that of the M-1 isolate, indicating that these three isolates are closely related to each other, and may be derived from a common origin (data not shown). To facilitate the classification of strains, a pairwise comparison of cleavage sites was performed. There are 17 cleavage sites by 9 restriction enzymes as shown in Fig. 3 . For each pair of strains, the difference in restriction sites was counted (0-17). The results with four new isolates and previously defined 12 strains were schematically illustrated in Fig. 4 . The four new isolates are obviously closer with each other than with the other 12 strains which previously classified (group I-V) [6] , and therefore classified into a new distinct group (VI). Serological data based on the cross-neutralization test measured by plaque reduction in chicken kidney (CK) cell culture showed that the M-1 strain was not neutralized by the antiserum against any other previous strains (Y. Kudou, unpubl, data). This suggests that our PCR and RFLP data are consistent with serological relationships. The results of the present study, indicate that the four recently obtained IBV isolates causing nephritis are, by our genetic criteria, similar to each other but different from previous isolates, hence are classified together into a new subtype (group VI). Because there is no comparison between our Japanese isolates and the Australian T strain, it is not clear, despite their similar ne-148 Z. Lin et al. The grouping from I to VI is essentially according to Lin et al. [6] phrotropic features, if there is any serological or genetic relationship between them. To control IBV infection, it is necessary to survey whether the prevalent IBV strains are similar to or different from current vaccine strains. Several investigators have characterized the isolates obtained from IBV outbreaks, and showed that they had a strong relationship with the vaccine strain, suggesting that the prevalent strains may have originated by recombination with live vaccine strains [1, 5] . Our present observation that the four new isolates used were different from the reference strains including current vaccine strains suggests that they were not derived from the live attenuated vaccine. However, it is totally u n k n o w n and remains to be clariEed how these new strains evolved. Oligonucleotide fingerprinting of ribonucleic acids of infectious bronchitis virus strain Infectious avian nephrosis (ureamia) in Australia Occurrence and significance of infectious bronchitis virus variant strains in egg and broiler production in the Netherlands Serological comparisons of strains of infectious bronchitis virus using plaque-purified isolants Molecular epidemiology of infectious bronchitis virus in the Netherlands A new typing method for the avian infectious bronchitis virus using polymerase chain reaction and restriction enzyme fragment length polymorphism Etiology of an infectious nephritis-nephrosis syndrome of chickens