The study aimed to determine the inhibition kinetics of Crocodylus mindorensis (Philippine crocodile) serum against recombinant HIV-1 Reverse Transcriptase (RT) using Enzyme-linked Immunosorbent Assay. The study utilized an experimental design where different concentrations of Philippine crocodile serum were used to determine its quantitative inhibitory effect and mechanism of inhibition against the HIV-1 reverse transcriptase activity. A colorimetric enzyme immunoassay that incorporates digoxigenin and biotin-labeled dUTP onto DNA were used for the assay (Roche Version 13.0, 2010). In addition, the kinetic activity of HIV-1 Reverse transcriptase was studied to determine the mechanism of enzyme inhibition by Philippine crocodile serum. A minimum of 2 mL serum sample from each of the seven (7) purposively selected Crocodylus mendoremsis were collected at the Animal clinic of the Davao Crocodile Park facility. The results showed that the HIV-1 Reverse transcriptase activity was inhibited by high as 92.93±0.72% (CM-07) at 0.5 vol/vol % and as low as 4.66±20.76 at 4 vol/vol % (CM-04) of Philippine crocodile serum. The average half maximal inhibitory concentration (IC 50) of the seven crocodile serum samples was 1.47% vol/ vol.The 92.93±0.72% inhibition of 0.5% vol/vol of Philippine crocodile serum has almost the same inhibition as the positive control Nevirapine (92.64±0.20%). The inhibition kinetics of the Philippine crocodile suggests a potential novel HIV-1 Reverse Transcriptase inhibitor.

Although drug-resistant HIV variants are considered to be less fit than drug-susceptible viruses, replication competence of these variants harbored by patients has not yet been elucidated in detail. We herein assessed the replication competence of strains obtained from individuals receiving antiretroviral therapy. Among 11 306 participants in a drug resistance surveillance in the Philippines, 2629 plasma samples were obtained from individuals after a 12-month treatment with zidovudine (ZDV)/lamivudine (3TC)/nevirapine (NVP). The replication competence of HIV isolates was then assessed by reinoculation into seronegative peripheral blood mononuclear cells in the absence of drugs in vitro. The drug resistance rate was estimated to be 9.2%. Drug-resistant strains were still a minority of closely related strains in a phylogenetic cluster. Among the available 295 samples, 37 HIV strains were successfully isolated. Progeny viruses were produced at a wide range (5.1 × 106 to 3.4 × 109 copies/mL) in primary culture of peripheral blood mononuclear cells. The viral yields were higher than the corresponding plasma viral load (1300 to 3.4 × 106 copies/mL) but correlated with those (r = 0.4). These results suggest that strains with higher intrinsic replication competence are one of the primary targets of newly selected drugs at the increasing phase of the plasma viral load during antiretroviral therapy.

In a previous study, we described the diverse growth capabilities of circulating seasonal influenza A viruses (IAVs) with low to high viral copy numbers in vitro. In this study, we analyzed the cause of differences in growth capability by evaluating pro-inflammatory cytokines (TNF-α, IL-6, IFN-β) and antiviral interferon-stimulated genes (ISG-15, IFIM1, and TRIM22). A549 cells (3.0 × 105 cells) were inoculated with circulating seasonal IAV strains and incubated for 6 and 24 h. In cells inoculated for 6 h, IAV production was assessed using IAV-RNA copies in the culture supernatant and cell pellets to evaluate gene expression. At 24 h post-infection, cells were collected for IFN-β and ISG-15 protein expression. A549 cells inoculated with seasonal IAV strains with a high growth capability expressed lower levels of IFN-β and ISGs than strains with low growth capabilities. Moreover, suppression of the JAK/STAT pathway enhanced the viral copies of seasonal IAV strains with a low growth capability. Our results suggest that the expression of ISG-15, IFIM1, and TRIM22 in seasonal IAV-inoculated A549 cells could influence the regulation of viral replication, indicating the existence of strains with high and low growth capability. Our results may contribute to the development of new and effective therapeutic strategies to reduce the risk of severe influenza infections.

The correlation of viral growth capability (n = 156) with the viral load in nasopharyngeal swabs (n = 76) was assessed. Epidemic influenza A/H1N1, A/H3N2, and B viruses showed a wide range of growth capability (10 4-10 11 copies/mL) in Madin-Darby canine kidney cells. The growth was correlated with the nasopharyngeal viral load (r = 0.53). Six selected strains showed growth-dependent cell death (r = 0.96) in a growth kinetics assay. Epidemic influenza viruses exhibit a wide range of growth capability. Growth capability should be considered one of the key factors in disease prognosis.

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