Spike S2 Subunit: Possible Target for Detecting Novel SARS-CoV-2 Variants with Multiple Mutations.

Novel SARS-CoV-2 variants have multiple mutations that may impact molecular diagnostics. The markedly conserved S2 subunit may be utilized to detect new variants. A comparison of 694 specimens (2019-2022) in Thailand using a commercial RT-PCR kit and the kit in combination with S2 primers and a probe was performed. Delayed amplification in ORF1ab was detected in one BA.4 omicron, whereas no amplification problem was encountered in the S2 target. There were no statistically significant differences in mean Ct value between the target genes (E, N, ORF1ab, and S2) and no significant differences in mean Ct value between the reagents. Furthermore, 230,821 nucleotide sequences submitted by 20 representative counties in each region (Jan-Oct 2022) have been checked for mutations in S2 primers and probe using PrimerChecker; there is a very low chance of encountering performance problems. The S2 primers and probe are still bound to the top five currently circulating variants in all countries and Thailand without mismatch recognition (Jun-Nov 2023). This study shows the possible benefits of detecting S2 in combination with simultaneously detecting three genes in a kit without affecting the Ct value of each target. The S2 subunit may be a promising target for the detection of SARS-CoV-2 variants with multiple mutations.

Implications of the antiviral drug favipiravir on rabies immunoglobulin for post-exposure prophylaxis of rabies in mice model with category III-like exposures.

Rabies is a fatal zoonotic disease caused by the rabies virus (RABV), with almost 100% mortality if proper post-exposure prophylaxis (PEP), consisting of rabies immunoglobulin (RIG) and rabies vaccine, is not applied in a timely manner. However, this is challenged by the limited availability of RIG, especially in resource-constrained countries. In this study, we assessed the scope of the antiviral drug favipiravir to treat rabies-infected mice as an alternative to RIG. Category III-like wounds were induced in RABV-challenged mice treated with favipiravir instead of RIG in the PEP regimen. The use of favipiravir followed by rabies vaccine provided complete protection against rabies-related death in 100% of mice, even after RABV propagated to the central nervous system during infection. Additionally, the virus-neutralizing antibody titer in the favipiravir and vaccine group was significantly higher than that of the RIG and vaccine recipients. The use of favipiravir with rabies vaccine seemingly prevents fatal outcomes and even rescues the cases that already express clinical symptoms. A clinical trial of this approach is warranted, especially in countries with low RIG availability.

Use of qRT-PCR for SARS-CoV-2 sgRNA leader for the therapeutic plan: a preliminary report on 10 patients.

INTRODUCTION: Duration of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) shedding is important for infection control. The presence of SARS-CoV-2 subgenomic RNA (sgRNA) leader indicates that the virus is replicative. This study examined the shedding duration of SARS-CoV-2 sgRNA leader and genomic RNA (gRNA) in diverse respiratory specimens. METHODOLOGY: One hundred and eleven respiratory specimens collected sequentially from 10 COVID-19 patients with real-time RT-PCR SARS-CoV-2 orf1ab gene confirmed positive admitted to King Chulalongkorn Memorial Hospital were examined for SARS-CoV-2 E sgRNA leader and E gRNA by using Real-time reverse transcription PCR (qRT-PCR). These specimens included nasopharyngeal swab and throat swabs, nasal swab and throat swabs, sputum, and endotracheal aspirate, and were collected from the first day of admission until the time of orf1ab real-time RT-PCR negative of at least 2-4 consecutive days. RESULTS: E sgRNA leader could only be detectable in specimens with ≥ 1E+05 virus E gene copies per ml within the first 15 days after hospitalization. SARS-CoV-2 sgRNA leader was undetectable from one to 15 days earlier than that of gRNA in all patients. Re-shedding of sgRNA was evident in 2 cases, both on a single occasion after being undetectable for 3-10 days. CONCLUSIONS: Assessment of the presence of sgRNA leader may be useful for therapeutic planning.

Increased pathogenicity of rabies virus due to modification of a non-coding region.

Sub-passaging of QS-05, a street rabies virus (RABV) isolate, in non-neuronal cells resulted in a virus with higher pathogenicity, QS-BHK-P7. Four full-length cDNA plasmids were constructed and the corresponding recombinant viruses were recovered: rQS-05, rQS-BHK-P7 and rQS05-2475G/rQS-BHK-P7-2475A (made by switching of intergenic P-M between these two backbones). rQS-BHK-P7-2475 A virus had eight instead of seven adenosines in its poly(A) sequence. Interestingly, mutant viruses with 6 or 8 adenosines infected more neuroblastoma cells than their parental ones. Mice that were infected intracerebrally and intramuscularly with rQS05-2475G and rQS-BHK-P7 exhibited highest mortality. However, mice infected with rQS-BHK-P7-2475AA had the shortest survival time. This study demonstrates that modifications in the non-coding region may play a role in determining the virulence of RABV.

Rabies vaccination at a virus-inoculated site as an alternative option to rabies immunoglobulin.

Combined active and passive immunization has been established to be an optimal strategy for rabies post-exposure prophylaxis (PEP). Prompt administration of vaccine and rabies immunoglobulin (RIG) can reliably prevent the disease. However, RIG is unavailable and unaffordable in the majority of cases. On the basis of a model experiment using hamsters, we demonstrated that vaccine injection at the wound site in the same manner as administration of RIG provided protective efficacy that was not inferior to the current optimal PEP, a combination of vaccination and RIG. Further study is needed to determine whether it can replace the use of RIG.

Molecular analysis of the mutational effects of Thai street rabies virus with increased virulence in mice after passages in the BHK cell line.

QS-BHK-P7, street rabies virus, after passages in the BHK cell line, had an in vitro phenotype that distinguished it from its parental virus. Both viruses caused lethal infection in mice by central nervous system inoculation; however, only QS-BHK-P7 killed mice by the intramuscular route. We found four mutations, S23R and H424P in ectodomain of the glycoprotein (G), I1711 V in the polymerase genes, and another at the non-coding region between the phosphoprotein and matrix protein genes of QS-BHK-P7. None of the mutations in the G gene occurred in previously reported pathogenic determinants. The roles of mutations in particular non-coding regions remain to be elucidated.