Use of reverse transcriptase-polymerase chain reaction-restriction fragment length polymorphism to examine the interaction between infectious bronchitis virus strains Massachusetts 41 and JMK in ovo

Author： Wang Xiuqing Khan Mazhar I.

Publisher： Taylor & Francis Ltd

ISSN： 1465-3338

Source： Avian Pathology, Vol.29, Iss.5, 2000-10, pp. : 441-448

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Abstract

The reverse transcriptase-polymerase chain reaction-restriction fragment length polymorphism (RTPCR-RFLP) technique has been developed and used in the serotype diagnosis of infectious bronchitis virus (IBV) infection. In this report, we first demonstrate that the RT-PCR-RFLP was sensitive in detecting both Massachusetts 41 (Mass 41) and JMK strains of IBV singly; the detection limit for both was approximately 0.15ng viral RNA using gel electrophoresis. Subsequently, the ability of the technique to semi-quantify the relative amounts of Mass 41 and JMK RNA in their mixture was shown by performing RT-PCR-RFLP on various combinations of serially diluted known amounts of Mass 41 and JMK RNA. Results indicated that both Mass 41 and JMK can be detected and semi-quantified when there is less than 103-fold difference between the two viral RNA template inputs, which are above the lower detection limit. Based on this result, the interaction between Mass 41 and JMK in ovo was examined by inoculating 10-day-old embryos with various combinations of different EID₅₀ titers of Mass 41 and JMK. RT-PCR-RFLP results showed that Mass 41 dominates over JMK in an EID₅₀ titerdependent fashion, but not the reverse. To further investigate the underlying mechanism, replication efficiency of Mass 41 and JMK was compared at 6, 12, 18, 24, 36 and 48h after inoculation of embryos with 105.3 EID₅₀ of individual Mass 41 and JMK. No significant difference ( P > 0.05) on replication efficiency between these two serotypes was found based on the results of total RNA concentration and RT-PCR results of 10-fold dilution of RNA at the indicated time points. By inoculating heat-inactivated Mass 41 with live JMK into embryos, only JMK was detected by RT-PCR-RFLP and no dominating interference was observed. Data suggest that the dominance is not due to the replication efficiency of Mass 41 and JMK; a receptor-mediated mechanism might be responsible for it.