Evaluation of Two Real-Time, TaqMan Reverse Transcription-PCR Assays for Detection of Rabies Virus in Circulating Variants from Argentina: Influence of Sequence Variation

Abstract

In rabies diagnosis, it is essential to count on a rapid test to give a quick response. The combined sensitivity and robustness of the TaqMan RT-PCR assays (qRT-PCR) have made these methods a valuable alternative for rabies virus (RABV) detection. We conducted a study to compare the applicability of two widely used qRT-PCR assays targeting the nucleoprotein gene (LysGT1 assay) and leader sequences (LN34 qRT-PCR assay) of RABV genomes, in all variants circulating in Argentina. A total of 44 samples obtained from bats, dogs, cattle, and horses, that were previously tested for rabies by FAT and conventional RT-PCR, were used in the study. All variants were successfully detected by the pan-lyssavirus LN34 qRT-PCR assay. The LysGT1 assay failed to detect three bat-related variants. We further sequenced the region targeted by LysGT1 and demonstrated that the presence of three or more mismatches with respect to the primers and probe sequences precludes viral detection. We conclude that the LysGT1 assay is prone to yield variant-dependent false-negative test results, and in consequence, the LN34 assay would ensure more effective detection of RABV in Argentina.

Viral Metagenomic Data Analyses of Five New World Bat Species from Argentina: Identification of 35 Novel DNA Viruses

“…According to the database of bat-associated viruses (DbatVir, http://www.mgc.ac.cn/DBatVir/, accessed on 11 November 2021) [7], 13,059 viruses have been identified in bats globally, of which 1144 (8.8%) originated from South America, including 126 (0.96%) identified in Argentina. A total of 98.4% (124/126) of South American bat viruses belong to the family Rhabdoviridae (RNA viruses) and were identified by conventional molecular methods, mainly in arthropodophagous bat species during national rabies surveillance programs [8][9][10]. To date, only two DNA viruses have been identified in bats from Argentina, Tadarida brasiliensis papillomavirus type 1 (TbraPV1, Papillomaviridae) and Tadarida brasiliensis gemykibivirus 1 (Genomoviridae) [11].…”

Multiple detection and spread of novel strains of the SARS-CoV-2 B.1.177 (B.1.177.75) lineage that test negative by a commercially available nucleocapsid gene real-time RT-PCR

“…Thus, 28948C >T is the sole mutation characterizing the N-negative B.1.177.75 samples with respect to N-positive strains belonging to the same lineage. It is known that few mismatches in the oligonucleotide binding region can affect the amplification efficiency, with prevention of any amplification when located in the very 3' end of the primer(s) or on the middle of the TaqMan probe (25)(26)(27). In a TaqMan assay for detection of rabies virus RNA, the number of sequence mismatches between gene-specific oligonucleotides and the target sequence significantly affected amplification and point mutations at the center of the probe resulted in false-negative results through the prevention of probe binding and subsequent fluorescence (28).…”

Comparison of Pan-Lyssavirus RT-PCRs and Development of an Improved Protocol for Surveillance of Non-RABV Lyssaviruses

“…The LN34 assay recommended by the WHO [ 18 , 26 , 31 ] uses a combination of two forward and one reverse primer and two locked nucleic acid (LNA) probes designed in the same highly conserved region targeted by the JW12 primer. The LN34 probes demonstrated high tolerance to single-nucleotide polymorphisms (SNPs), showing optimal performances [ 31 ] and more inclusivity than JW12/N165-146, as noticed elsewhere [ 28 ]. Probes and primers were designed based on 13 lyssavirus species, excluding LLEBV and GBLV, in addition to others yet to be discovered [ 31 ].…”

“…The JW12/N165-146 assay was originally developed as a TaqMan lyssavirus assay using specific probes to differentiate among RABV, EBLV-1 and EBLV-2 lyssavirus species [ 23 , 24 ]. The RABV-specific protocol was evaluated using a large panel of field samples representative of the RABV diversity across the globe, displaying failure in detecting several RABV strains [ 27 , 28 ]. To overcome the low inclusivity of the probes, the original protocol was further adapted to the intercalating dye SYBR ® Green [ 29 ], and showed an ability to detect all lyssaviruses known at that time with better sensitivity than the hemi-nested RT-PCR.…”