Paper Example on IAVs: The Growing Threat of Zoonotic Diseases

The world today has witnessed a surge in zoonotic diseases. According to Dadonaite et al. (2019), zoonotic maladies are those viruses commonly transmitted from animals to humans. In most cases, these ailments are usually highly contagious. Influenza A viruses (IAVs) are one of the zoonotic illnesses transmitted from animals and could yield devastating effects and pandemics among the human populations. People can be affected by IAVs through their interactions and consumption of animal products. The year 1918 witnessed a global epidemic that led to the death of over 50 million people in the world due to the rise and spread of IAVs (Bolte et al., 2019). For there to be a pandemic, it is worth noting that the IAVs present in animals cross over to the human species. In humans, the viruses acquire a new gene variant (Bolte et al., 2019). In addition to that, Dadonaite et al. (2019) described the IAV genome by indicating that it contains eight strands of viral RNA segments usually present in different viral ribonucleoprotein (vRNP) complexes that are typically packaged to becoming a single virus particle. Dadonaite et al. (2019) further indicated that the makeup of the RNA of the IAVs plays a substantial role in the assemblage of vRNPs into mature virions. Bolte et al. (2019) argued that the segmented genome of the IAVs enhances the evolution of the virus and a complicated packaging mechanism for the genome, as described by Dadonaite et al. (2019), is required. Besides, they encourage the re-assortment of IAVs. The re-assortment between the IAVs found in humans and that harbored in animals could result in the emergence of the pandemic strains of the influenza A virus.

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Consequently, during the process of incorporation of the genomes into viral particles, the vRNPs, which formed from the viral RNA segments as described initially, adopt an arrangement where seven vRNPs surround the particle located at the center (Bolte et al., 2019). The genome packaging process is quite selective, having in mind only a copy of each of the vRNA segments is available in most of the viral particles. The majority of the research pieces in existence show that the interactions between two strands of RNA among the eight vRNPs synchronize the viral packaging of genomes. All the eight portions of vRNA have unique sequences of packaging that facilitate the integration of complete vRNP into a set of viral particles. In addition to that, recombinant mutants of the IAVs that contain nucleotide changes in one sequence of packaging develop some defects that include the lack of creation of virions that lack a particular aperture of vRNA segments or an entire genome. In most cases, such errors are restorable through mutations, which is proof that specific RNA-to-RNA associations are critical in the process of genome packaging.

Further, amino acids also play a significant role in the process of genome packaging. The amalgamation of seven amino acid substitutes in the nucleoprotein (NP7) head domain harms the assimilation of four of the eight vRNPS into virions. According to Dadonaite et al. (2019), the defects of genome packaging are restored through the addition of amino acid exchanges. Primarily, this suggests that nucleoprotein amino acids support connections among the vRNPS by enhancing RNA interactions. However, limited information is known about the intricacy, plasticity, and redundancy of the genome packaging process. Therefore, the article analyzed in this section is, 'Packaging of the Influenza Virus Genome is Governed by a Plastic Network of RNA- and Nucleoprotein-Mediated Interactions' by Bolte et al. (2019). In the article, the researchers analyze the outcome of one and several metamorphosed casing sequences situated in the vRNA segments. The authors show that a mutated packaging series in a vRNA segment has no significant impact on the process. Contrary to that, researchers show how specific groupings of the mutated sections of vRNA could lead to the production of non-infectious virus elements that do not have subsets of vRNA segments. The evidence presented in the article shows that a sophisticated vRNP interaction network that contains packaging progression of vRNA and amino acids coordinate the process of genome packaging of the IAV virus.

Material and Methods

The cell lines used in this study were 293T cells and MDCK-II cells that were maintained at a temperature of 37 degrees Celsius and in 5% carbon dioxide (Bolte et al., 2019). The cells were placed in the Dulbecco Modified Eagle Medium (DMEM) that contained 10% of serum acquired from a fetal calf and penicillin-streptomycin (Bolte et al., 2019). In addition to that, the researchers generated recombinant influenza A viruses using the eight-plasmid transfection system. The subconfluent of the 293T cells were transfected with both the plasmids and lipofectamine. About six hours later, the cells were upheld in DMEM that contained serum albumin and penicillin-streptomycin. After a period of 48hours, the supernatants of the cell structures were collected and cleared through means of centrifugation for five minutes and 500 grams (Bolte et al., 2019). The recovery of the contagion viruses was evaluated through the plaque assay on MDCK-II cells. In a bid to produce clonal virus stocks, the isolated plaques underwent propagation for two days on confluent cells of the MDCK-II (Bolte et al., 2019). Mutations of the modified segments of vRNA were confirmed using the Sanger sequencing process.

The Sanger sequencing process refers to the technique that involves the incorporation of chain-termination deoxynucleotides by DNA polymerase during the replication of in vitro DNA. Further, researchers mixed the virus stock with a reagent referred to as peqGOLD TriFAST, and the process of removal of RNA was conducted using a Direct-zol RNA MiniPrep kit (Bolte et al., 2019). In addition to that, some RNA was reverse transcribed and augmented using the OneStep RT-PCR kit together with segment-specific primer sets. GATC Biotech was then used to sequence the extracted DNA products (Bolte et al., 2019).

Consequently, the researchers, as indicated in the article, conducted multicycle replication kinetics where confluent MDCK-II cells were placed in plates and infected with recombinant wild type and mutant viruses (Bolte et al., 2019). The cell structures were collected, and after 48hours, they were cleared through the processes of centrifugation for about five minutes. In addition to that, the plaque assays on the MDCK-II cells facilitated the quantification processes of the infectious viruses. Asides from that, the researchers transformed the titers of the contagious viruses to log10 to enhance statistical computations and analysis. The researchers compared the wild type and mutant viruses in a bid to understand their genome packaging processes. As such, different analytical tools of computation were used that include the one-way analysis of variance (ANOVA), Microsoft excel, and Tukey's multiple-comparison test (Bolte et al., 2019).

In a bid to determine the quantification of viral RNA segments per PFU (Plaque Forming Unit), the researchers placed MDCK-II cells on a plate. They infected them with recombinant wild-type and mutant viruses. PFU refers to the number of particles that have the capability of forming plaques for every unit of volume. After a day, the researchers collected the cell structure and cleared through centrifugation for about five minutes. The plaque assays of the MDCK-II cells were used in the quantification of the PFU titers of the virion preparations. Researchers also performed RT-qPCRs tests to quantify the amounts of each of the vRNA segments in the development of virions. The peqGOLD TriFAST and RNA were withdrawn using the Direct-zol RNA MiniPrep kit and eluted in RNase-free water (Bolte et al., 2019). The purified RNA was attenuated using the free water, and some amount of it transcribed using the Revert Air first-strand cDNA synthesis kit (Bolte et al., 2019). The researchers diluted the cDNA products using milli-Q water and quantified PCR using a sensiFAST SYBR-Bi-ROX kit and some primers. The comparison between the wild type and mutant virion was made through a calculation of the log2-fold change of all of the vRNA segments for every PFU.

Lastly, the quantification of hemagglutination units for every PFU was later conducted. The procedure involved the infection of confluent MDCK-II cells with wild-type and mutant viruses. After 24 hours or 36 hours, the researchers collected the cell structure and cleared them using the centrifugation process for five minutes. The plaque assays were used on the MDCK-II cells as a process of quantification of the PFU titers of the virion preparations. An estimation of the total quantity of virus elements in virion preparations was done, and the hemagglutination units were quantified using the assays that have been described previously.

Results

The Single Mutated Chain Cause No or Only Minor Defects of Genome Packaging

The first result derived from this study is that a single packaging chain of mutation does not yield any defects of genome packaging. If any, the error is likely to be minimal. The researchers compared the individual and several metamorphosed packaging progressions on the viral genome, a prototype of the H7N7 influenza strain A virus (SC35M) was used and introduced into the vRNA segment (Bolte et al., 2019). The results are summarized in figure one below.

From the figure above, A to C shows the chart representation of the vRNA portions, 1, 2, and 3. In addition to that, the black color from the figure signifies the wild type, while the red shows mutated nucleotide sequences. D to F shows the multicycle duplication kinetics of the altered viruses contrasted to the wild type. G to I shows the quantification of the levels of the vRNA parts between both mutant and wild type viruses using RT-qPCR kits. The researchers normalized the RNA levels to similar PFU numbers. J, from the figure, shows the quantification of HAU (Hemagglutination unit)-to-PFU ratios between mutant and wild type virus elements.

Consequently, the authors chose sets of mutations that cause defects of genome packaging when introduced to the vRNA portions of the H1N1 influenza strain. Bolte et al. (2019) generated recombinant viruses that contained mutated parts of the genome and labeled as rS1mut, rS2mut, and rS3mut. In a bid to observe the creation of contagion elements of the virus with time, a multicycle replication process was conducted in the MDCK-II cell cultures. The results gathered included the fact that the proliferation of rS2mut was damaged due to the wild type virus, while rS1mut and rS3mut were not affected in any way. The researchers progressed to testing the capacity of genome packaging of the mutant viruses through the quantification process of all the levels of the vRNA parts acquired from the virion preparations that had similar numbers of the PFU. The analysis revealed similar levels of vRNA segments hence showing deficient incorporation of vRNA. The researchers progressed to estimating the extent to which the mutants produced non-infectious viruses by identifying the ratio of hemagglutination units per PFU. The results were that rS1mut and rS3mut showed a similar rate of hemagglutination, and rS2mut generate non-infectious viral particles that lack the vRNA segment (Bolte et al., 2019). The genome packaging for single vRNA mutants was not affected.

Combination of Mutated Packaging Series in Various Portions of vRNA Cause Divergent Packaging Defects of the Genome

The next process of the research involved the...