Reverse genetics (RG) represents an indispensable tool for elucidating the intricate characteristics of viruses both in vivo and in vitro. Initially implemented with DNA viruses and later expanded to encompass RNA viruses, the pioneering success of RG manipulation was achieved in a positive-sense RNA virus, namely poliovirus [8, 25].However, tackling negative-stranded RNA viruses posed daunting challenges, including the absence of genomic RNA, the stringent demands for exact genome length to enable replication and packaging, as well as the requirement for transient availability of viral RNA-dependent RNA polymerase [29]. Notwithstanding these formidable obstacles, the creation of a reporter IAV replicon was triumphantly accomplished using a helper virus [10]. A pivotal breakthrough emerged with the advent of plasmid-based systems, facilitating enhanced manipulation of negative-sense RNA viruses, marking a significant milestone in virology research [30, 31].

Recombinant virus generation techniques initially focused on DNA viruses, employing transfection with plasmids encoding the viral genome or heterologous recombination between plasmids and the viral genome with a helper virus [32]. Positive-sense RNA viral genomes, like poliovirus, were later manipulated through cell transfection with plasmid DNA or in vitro-transcribed RNA for recombinant virus production [29]. However, negative-sense RNA viruses, such as influenza, posed challenges due to their non-infectious nature without viral RdRps and vRNA [33]. The advent of reverse genetics and molecular engineering revolutionized influenza research, enabling exploration of viral replication, transcription, pathogenicity, host interactions, and vaccine development [32]. These technologies have also facilitated the creation of recombinant influenza viruses for vaccine vectors, expressing foreign proteins, or carrying reporter genes for easy infection tracking [29, 32, 34].

It’s a new era for the study of influenza virus with powerful reverse genetics technology, and the comparison of different methods used for the reverse genetic of IBV is shown in Table 3. These discoveries have enabled the study and resuscitation of extinct influenza viruses, quick characterization of new viral strains, production of conventional influenza vaccines, and development of state-of-the-art influenza vaccines. Its application has yielded significant benefits, contributing to the development of inactivated or live-attenuated influenza vaccines and the exploration of anti-influenza treatments with elucidated antiviral mechanisms [35]. The ability to manipulate viral genomes through reverse genetics has brought a transformative impact on influenza research. Researchers now have the capacity to work with infectious, recombinant, and genetically modified viruses, allowing them to target and address specific research concerns with precision and depth [35,36,37].

Table 3 Comparison of different methods used for the reverse genetic of influenza B virus

A major breakthrough occurred in 2002 when reverse genetics techniques successfully achieved the complete recovery of recombinant influenza B viruses from plasmid DNA [13]. This aided in the investigation of both the host and viral factors involved in influenza pathogenesis, transmissibility, host-range interactions and restrictions, and virulence [15]. The reverse genetics approaches allowed the researchers to determine the importance of the non-coding regions present in the genome of influenza B virus, generate novel vaccine strains, study the drug resistance mechanisms, and evaluate the function of viral proteins, which are analogous to influenza A virus proteins and uniquely present in influenza B viruses [10].

Helper virus-dependent methods

These were the first successful influenza virus reverse genetics methods. They relied on a helper virus and a selection system to obtain the desired recombinant/transfectant influenza virus [36]. During the era when helper virus-dependent systems were the sole accessible RG systems for influenza virus, selection systems for only six out of the eight genomic RNA segments of influenza A virus were documented [37]. Consequently, the genetic manipulation of two RNA segments, the first PA and the second PB1, was rendered impossible [36]. Many selection procedures differed in their level of strictness and, consequently, in their effectiveness. The quantity of mutated or reassortant influenza B viruses produced using this system was significantly less than the quantity produced [10, 38]. These technologies represent the initial advancements in reverse genetics for influenza viruses. Although they have demonstrated effectiveness and great significance in influenza virus research, their drawbacks on the helper influenza virus and their reliance consequently require the implementation of a selection method to enable the separation of necessary transfectant or recombinant influenza B viruses [35, 37].

Helper virus-independent methods

Helper virus-independent reverse genetics methods for influenza B virus revolutionized the study and manipulation of viral genomes. In this approach, individual plasmids containing cloned cDNA of the eight influenza B viral RNA segments, driven by RNA polymerase I or II promoters, are co-transfected into permissive cells [38]. The transfected cells serve as a host for the transcription, replication, and translation of the viral RNA segments, ultimately leading to the reconstitution of infectious influenza B virus [13, 39]. Most approaches use plasmids or vectors to produce all viral genomic RNA segments and required ‘helper’ proteins in cells, removing the requirement for selection procedures or helper virus elimination. Recent research proposed an alternate technique based on isolated RNPs from influenza virus preparations, but it has yet to gain attraction in the literature [40].

Plasmid-only reverse genetics systems

Viral RNA segments and necessary viral proteins are typically expressed by transfecting cells with specific plasmids (plasmid-based RG systems) [41]. These systems can be categorized based on factors like promoter types, plasmid numbers/types, transcription control elements, and cell species. Plasmid-based RG systems have been established for influenza A, B, C, and D viruses, along with the tick-transmitted Orthomyxoviruses, and Thogoto virus [41,42,43]. Jackson et al. described the use of cassette vector, pPRGCAT, cloned with the segments of influenza B/Panama/45/90 and flanked by human polymerase I promoter at the 5′ terminus and the hepatitis delta virus (HDV) antigenomic ribozyme at the 3′ terminus, so that the transcription resulted in the synthesis of negative-sense RNAs with exact viral-like termini [10].

Nogales et al. [15] described the generation of recombinant influenza B virus using an ambisense bidirectional plasmid pDP-2002 containing two transcription units in opposite direction. The influenza B vRNAs are expressed using the human polymerase I (hPol-I) promoter and a murine Pol-I transcription terminator (TI) while the mRNAs are expressed using polymerase II-driven cytomegalovirus promoter (pCMV) and the bovine growth hormone polyadenylation signal (aBGH). The synthesis of negative-sense vRNA from the hPol-I cassette, and positive-sense mRNA from the Pol-II unit, from one viral cDNA template is allowed by the orientation of both the polymerase units. The cloning of influenza B/Brisbane/60/2008 was carried out by inserting the influenza B viral cDNAs between the polymerase I transcription/terminator cassette flanked by an RNA polymerase II-dependent (Pol-II) cytomegalovirus promoter (pCMV) and a polyadenylation site (aBGH) [15].

Expression of viral RNA with the help of promotors

The initial reverse genetics systems employed plasmids that were free of helper viruses and had only the necessary genetic material. These plasmids utilized the human Pol I promoter to enable the production of viral RNAs [44]. The 3′ terminus of the viral RNAs was produced through the action of the ribozyme or the (murine) Pol I terminator sequence. Pol I promoters are typically regarded as species-specific, meaning that they function exclusively in the species where the promoter sequence originated or in closely related species [45]. Canine and chicken Pol I promoters were also utilized in specific cell types. Murine Pol I promoters were not widely used due to limited transfectable cell lines [46].

To overcome species-specific limitations, a universal plasmid-based reverse genetics system employed the T7 promoter, relying on T7 RNA polymerase expression within transfected cells. This system worked in human, canine, and quail cells. An alternative patent suggests using Pol II promoters with self-cleaving ribozymes at both ends of viral RNAs for reverse genetics [46, 47]. Nogales et al. shared their methods, which described the use of bidirectional plasmid pDP-2002 containing two promoters, human polymerase I (hPol-I) promoter, and polymerase II-driven cytomegalovirus promoter (pCMV) for influenza B/Brisbane/60/2008. This plasmid allowed the synthesis of vRNA and mRNA from the same vector, thereby, reducing the number of vectors used to eight for the efficient recovery of influenza B virus [15].

Human 293T and PER.C6 cells, as well as monkey Vero and COS-1 cells, have been employed in reverse genetics studies related to influenza virus. The cells have been cultivated either individually or in conjunction with more vulnerable cells, such as MDCK or chicken embryo cells [27, 39]. The rescue of recombinant influenza B viruses from the plasmid DNA have been reported using HEK293T and MDCK cell lines [15]. Another research has reported the efficient recovery of influenza B virus strains using human derived PER.C6 cell lines [45].

Reporter RNA and DNA polymerase-based cloning

Different techniques, such as using long-overhang primers and restriction enzymes, have been employed to create plasmids that produce RNA templates for influenza B virus reporters. Using these methods, the luciferase gene is added to target vectors, viral vectors are joined with UTRs, or a double-stranded DNA linker is used between terminator sequences and the Pol-I promoter [48]. It has been tried to create restriction enzyme-free methods for IBV reporter-based RNP activity assays using overlapping sequences and long overhang primers, but these methods cannot be standardized or established because no clear experimental protocols are currently present [49]. The difficulty is increased for influenza B viruses because their untranslated regions (UTRs) are greater in comparison to influenza A virus [48, 50].

Recent research shows a different way to make a firefly luciferase-based reporter plasmid for the influenza B/Brisbane/60/2008 virus that does not use restriction enzymes, specialized reagents or kits thus making the method fairly simple to be adopted. The cloning strategy developed by Kedia et al. utilized a single DNA polymerase, which was easily available due to its wide use in regular molecular biology work. The reporter RNA cassette with the reporter ORF which was flanked by the viral 5′- and 3′-UTR regions was generated by two consecutive PCR amplification reactions. It was then cloned into the selected vectors for the expression analysis to successfully establish a simple, adaptable, and user-friendly cloning of any other reporter RNA constructs [51].

Vaccine innovation and epidemic control

Epidemiologic studies have indicated the presence of selective pressure on influenza B viruses. This is certainly due to a phenomenon known as immunologic imprinting in which the individuals in the population exhibit the pre-existing immunity against the virus developed during the childhood by infection with the influenza strain circulating which then protect against unfamiliar HA or NA subtypes emerging from the same groups [52, 53]. The evolution of the influenza B virus is driven by the antigenic drift and reassortment mechanisms along with the mutations in the HA and NA genes allowing the evasion of pre-existing antibodies. Thus, to overcome the epidemic threats posed by rapidly evolving influenza B virus strains, it is crucial for the researchers to come up with vaccines developed from the updated strains [52, 54].

To mitigate the burden attributed to epidemics caused by influenza virus, a number of approaches including vaccines and antiviral drugs, are being developed. The high evolutionary rates of influenza B virus have constrained the production of a fully effective vaccine, making it difficult to prevent influenza completely. However, vaccination is deemed as an appropriate option to combat the viral attack [53]. The researchers have developed three types of vaccines (inactivated, live attenuated, and recombinant HA vaccines) with their advantages and disadvantages, respectively. The vaccine seed viruses for all these vaccines should be replaced periodically with respect to the antigenicity of the circulating viral strains which otherwise cause low vaccine efficacy. The epidemiologic information from individual countries, the genetic and antigenic characteristics of the circulating viruses are responsible for the selection of the correct influenza vaccine composition (Fig. 4) [55].

Fig. 4

IBV vaccine production: (1) Selection of IBV strain; (2) RNA extraction; (3) cDNA synthesis for gene manipulation; (4) Cloning of the targeted gene into a suitable plasmid vector; (5) Transfection of the selected mammalian cell lines by the cloned vector; (6) Recovery of the transfected viral strain from the cell lines; (7) Vaccine production

Previously, high uncertainty existed in the yield of influenza B virus for vaccine purposes based on the propensity of the selected antigenic variant to propagate in eggs. For example, with the change of recommended strain of influenza B virus to B/HK/330/2001, poor growth of the strain in the egg became apparent after a long time. Therefore, achieving the necessary antigen doses needed for the influenza B virus was difficult. Emergence of reverse genetics approaches eliminated the uncertainty of the reassortment process [52]. This was demonstrated by Hoffmann et al. (2002) who generated ‘6 + 2 reassortants’ by reverse genetics with internal genes from B/Yamanishi/166/98 and HA and NA genes from B/Victoria/504/2000, B/Hawaii/ 10/2001 and B/Hong Kong/330/2001 influenza strains. The recombinant viruses grew along with the wild-type virus in eggs, with the enhanced growth exhibited by B/Victoria recombinant virus [16].

Researchers further developed tissue culture cell-based approaches for vaccine production which might be fast, adaptable, and pose minimal risk of biological contamination. Cell lines including 293T, MDCK, and PER.C6 have been used for the recovery of influenza B viruses using reverse genetics procedures. MDCK and PER.C6 cell lines have been licensed for influenza vaccine production which are being widely used to generate vaccines [45, 56, 57]. Initially, trivalent vaccines were formulated which contained two influenza A (H1N1 and H3N2) and one lineage of influenza B viruses. This was to develop protection against three different influenza viruses irrespective of two different lineages of circulating B viruses. To provide wider protection, the second lineage of the influenza B virus was included to produce a quadrivalent influenza vaccine. The current approved vaccines for influenza virus are quantitatively standardized with respect to the antigenicity or HA quantity but not by the presence of neutralizing antibodies (NA) [58].