An expression cassette containing overlapping open reading frames and an application thereof are provided. The overlapping open reading frames are overlapping open reading frames of a first ORF and a second ORF and include in sequence from a 5 end to a 3 end: a first promoter at least used to drive gene transcription of the first ORF; a 5 part of a gene of the first ORF; an intron; and a 3 part of a gene of the second ORF, the intron including a second promoter used only to drive gene transcription of the second ORF. By arranging two promoters in a single expression cassette in the disclosure, the two promoters are used to drive the expression of proteins of the overlapping reading frames and regulate the relative expression time and expression intensity of different proteins.

A baculovirus vector and a use thereof in the preparation of a recombinant adeno-associated virus (rAAV) in an insect cell are provided. The baculovirus vector includes an exogenous gene expression cassette and a stable sequence. The stable sequence is located at a site 5 kb or less from the exogenous gene expression cassette, and the stable sequence is a conserved noncoding element (CNE) sequence or a nucleocapsid assembly-essential element (NAE) sequence. When an insect cell is infected with a recombinant baculovirus (rBV) constructed in this way, after multiple continuous passages, production levels of the rBV and the rAAV still remain relatively stable.

Embodiments provide systems and methods for transfecting cells to produce a viral vector or other selected virus. One embodiment of a method for transfecting cells to produce a selected virus includes providing a first solution comprising plasmids or other extrachromosomal DNA encoding the virus and a second solution comprising a transfection agent; the two solutions kept separate. The two solutions are then mixed to produce a transfection solution (TS) to be delivered to a cell culture medium (CCM) for transfection of cells in the medium with the plasmid DNA encoding the virus wherein the mixing is initiated responsive to a trigger event. The TS is then incubated to form transfection complexes. Subsequently, the TS is delivered to the CCM to transfect the cells in the medium wherein a viral production parameter resulting from transfection is optimized by initiating mixing of the solutions responsive to the trigger event.

An expression cassette containing overlapping open reading frames and an application thereof are provided. The overlapping open reading frames are overlapping open reading frames of a first ORF and a second ORF and include in sequence from a 5 end to a 3 end: a first promoter at least used to drive gene transcription of the first ORF; a 5 part of a gene of the first ORF; an intron; and a 3 part of a gene of the second ORF, the intron including a second promoter used only to drive gene transcription of the second ORF. By arranging two promoters in a single expression cassette in the disclosure, the two promoters are used to drive the expression of proteins of the overlapping reading frames and regulate the relative expression time and expression intensity of different proteins.

Provided herein are recombinant viruses and/or insect cells suitable for detecting the infection of a pathogen in a biological sample of a test subject. The information derived from the detection may also be used to render a diagnosis on whether the test subject is infected with the pathogen or not, so that proper course of treatment may be assigned to the subject. Also provided herein is a vaccine for the prophylaxis and/or treatment of infection caused by the pathogen.

A baculovirus vector and a use thereof in the preparation of a recombinant adeno-associated virus (rAAV) in an insect cell are provided. The baculovirus vector includes an exogenous gene expression cassette and a stable sequence. The stable sequence is located at a site 5 kb or less from the exogenous gene expression cassette, and the stable sequence is a conserved noncoding element (CNE) sequence or a nucleocapsid assembly-essential element (NAE) sequence. When an insect cell is infected with a recombinant baculovirus (rBV) constructed in this way, after multiple continuous passages, production levels of the rBV and the rAAV still remain relatively stable.

Materials for efficient production of recombinant baculovirus seed stocks containing a nucleic acid sequence that encodes a gene of interest and methods of producing recombinant baculovirus seed stocks containing a nucleic acid sequence that encodes a gene of interest are provided. Also provided are rapid methods of producing rAAV comprising a nucleic acid having a nucleic acid sequence that encodes a gene of interest; the method allows production of rAAV at a high titer.