Compositions and methods for preventing influenza infection in canines, felines and equines
A composition, a technology for canine influenza, applied in the field of respiratory diseases of cats or horses, and the treatment of dogs, which can solve problems such as use limitations
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[0036] The preparation of such a transcription cassette and its insertion into the MV viral genome involves only conventional molecular biology techniques, as exemplified in International Patent Application PCT / US07 / 64046 and US Patent Application 60 / 783,194. In particular, techniques such as site-directed mutagenesis and PCR mutagenesis can be used for this purpose. More particularly, recombinant MV viral vectors according to the present invention can be prepared by the well-established "reverse genetics" approach, which enables genetic modification of unsegmented, negative-strand RNA viruses of MV order, as described in U.S. Patent 6,033,886, which is hereby incorporated by reference in its entirety.
[0037] In this method, under conditions sufficient to allow the transcription and co-expression of the MV (anti)genome and supporting proteins, and the production of recombinant MV vectors, by containing nucleosides encoding the full-length genome or, preferably, the antigenom...
Embodiment 1
[0072] Example 1. Construction of recombinant NDV expressing canine influenza hemagglutinin gene
[0073] A. Preparation of DNA encoding influenza hemagglutinin (HA)
[0074] Influenza HA inserts were obtained by reverse transcriptase polymerase chain reaction (RT-PCR) using the Access RT-PCR System Kit (Promega Corporation, Madison, WI). exist On a microprocessor-controlled robotic thermocycler (Stratagene, Santa Clara, California), using Tf1 DNA polymerase from the kit, virus isolate A / canine / Jacksonville / 05 (also known as canine / Jax / 05, Accession No. PTA-7941, American Type Culture Collection (ATCC), 10801 University Blvd, Manassas, VA 20110-2209), using hemagglutinin-specific primers (5'-acggcaatcgATGAAGACAACCATTATTTTGA-3') and (5'-acggcagcgcgcTCAAATGCAAATGTTGCATC -3') to amplify the HA insert.
[0075] The amplified HA sequence was separated and separated from the other components of the PCR mixture on an electrophoretic gel. According to the manufacturer's instructi...
Embodiment 2
[0090] Example 2. Efficacy Study of Canine Influenza Vaccine
[0091] In the following study, a method of protecting dogs against respiratory infections involving the administration of a recombinant single-stranded negative-strand virus with a heterologous respiratory virus nucleotide sequence was investigated.
[0092] Recombinant viruses were passaged in eggs a total of seven times, and allantoic fluid was collected. EID was determined by inoculating 200 μl of 10-fold serial dilutions of P7 allantoic fluid into 10 embryonated 10-day-old eggs / dilution 50 titer. Five days after inoculation, the allantoic fluid was tested for coagulation. Calculate EID 50 titer. Dilute the allantoic fluid 10-fold in sterile phosphate-buffered saline to an appropriate 10 8 Virus / ml titers were used to prepare vaccines, aliquoted and frozen.
[0093] Each dose of the vaccine contains 10 8 EID 50 And enough PBS was added to bring the total dose volume to 1 ml.
[0094] Fifteen female beag...
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