Unravelling the genomic origins of lumpy skin disease virus in recent outbreaks

Abstract Lumpy skin disease virus (LSDV) belongs to the genus Capripoxvirus and family Poxviridae. LSDV was endemic in most of Africa, the Middle East and Turkey, but since 2015, several outbreaks have been reported in other countries. In this study, we used whole genome sequencing approach to investigate the origin of the outbreak and understand the genomic landscape of the virus. Our study showed that the LSDV strain of 2022 outbreak exhibited many genetic variations compared to the Reference Neethling strain sequence and the previous field strains. A total of 1819 variations were found in 22 genome sequences, which includes 399 extragenic mutations, 153 insertion frameshift mutations, 234 deletion frameshift mutations, 271 Single nucleotide polymorphisms (SNPs) and 762 silent SNPs. Thirty-eight genes have more than 2 variations per gene, and these genes belong to viral-core proteins, viral binding proteins, replication, and RNA polymerase proteins. We highlight the importance of several SNPs in various genes, which may play an essential role in the pathogenesis of LSDV. Phylogenetic analysis performed on all whole genome sequences of LSDV showed two types of variants in India. One group of the variant with fewer mutations was found to lie closer to the LSDV 2019 strain from Ranchi while the other group clustered with previous Russian outbreaks from 2015. Our study highlights the importance of genomic characterization of viral outbreaks to not only monitor the frequency of mutations but also address its role in pathogenesis of LSDV as the outbreak continues

Multi-site evaluation of the LN34 pan-lyssavirus real-time RT-PCR assay for post-mortem rabies diagnostics

<div><p>Rabies is a fatal zoonotic disease that requires fast, accurate diagnosis to prevent disease in an exposed individual. The current gold standard for post-mortem diagnosis of human and animal rabies is the direct fluorescent antibody (DFA) test. While the DFA test has proven sensitive and reliable, it requires high quality antibody conjugates, a skilled technician, a fluorescence microscope and diagnostic specimen of sufficient quality. The LN34 pan-lyssavirus real-time RT-PCR assay represents a strong candidate for rabies post-mortem diagnostics due to its ability to detect RNA across the diverse <i>Lyssavirus</i> genus, its high sensitivity, its potential for use with deteriorated tissues, and its simple, easy to implement design. Here, we present data from a multi-site evaluation of the LN34 assay in 14 laboratories. A total of 2,978 samples (1,049 DFA positive) from Africa, the Americas, Asia, Europe, and the Middle East were tested. The LN34 assay exhibited low variability in repeatability and reproducibility studies and was capable of detecting viral RNA in fresh, frozen, archived, deteriorated and formalin-fixed brain tissue. The LN34 assay displayed high diagnostic specificity (99.68%) and sensitivity (99.90%) when compared to the DFA test, and no DFA positive samples were negative by the LN34 assay. The LN34 assay produced definitive findings for 80 samples that were inconclusive or untestable by DFA; 29 were positive. Five samples were inconclusive by the LN34 assay, and only one sample was inconclusive by both tests. Furthermore, use of the LN34 assay led to the identification of one false negative and 11 false positive DFA results. Together, these results demonstrate the reliability and robustness of the LN34 assay and support a role for the LN34 assay in improving rabies diagnostics and surveillance.</p></div

LN34 and DFA test results for 2,978 samples tested in all laboratories during the LN34 assay evaluation.

<p>LN34 and DFA test results for 2,978 samples tested in all laboratories during the LN34 assay evaluation.</p

Primers and probes used in the LN34 real-time RT-PCR assay.

<p>Primers and probes used in the LN34 real-time RT-PCR assay.</p

List of the laboratories that participated in the LN34 assay evaluation and the country or region of sample origin.

<p>List of the laboratories that participated in the LN34 assay evaluation and the country or region of sample origin.</p

LN34 assay diagnostic algorithm for post-mortem brain stem samples.

<p>LN34 assay diagnostic algorithm for post-mortem brain stem samples.</p

LN34 assay repeatability and reproducibility.

<p>A–B. Comparison of replicate Ct values for the same RNA sample tested in the same assay run for LN34 (A) and β-actin (B) assays. Ct value for replicate 1 is plotted against replicate 2. Gray line indicates identity (y = x). Results of linear regression analysis is shown in the upper left corner. Vertical red lines indicate the diagnostic cut-off values for positive samples for each assay. Points are transparent; darker color indicates overlapping points. Samples that failed to amplify are plotted at Ct 0. C. LN34 Ct values reported for positive control RNA tested in 12 laboratories shown as a beeswarm plot. Each dot represents the average value for one assay run; points are plotted according to Ct value (y-axis), then offset along the x-axis to show the distribution of points at each Ct value. Orange dots indicate Ct values observed in one laboratory using a PCR machine with decreased sensitivity, and green dots indicate Ct values reported from the same laboratory using a different PCR machine. D. Comparison of LN34 Ct value for a panel of 14 samples tested in three real-time PCR machines. Machine 2 was determined to produce significantly higher Ct values than either Machine 1 or 3, for the same sample. Boxplots show median (thick line) and 25^th and 75^th quartiles. Whiskers extend to 1.5×(inter-quartile range); data outside whiskers are plotted individually. ** p < 0.01.</p

A multi-laboratory international evaluation of the LN34 assay.

<p>A. Proportion of samples originating and tested in the United States (US), originating and tested outside the U.S. (INT), or originating outside the U.S. and tested in the U.S. (US-INT). B. Frequency of LN34 Ct values for DFA positive samples. C. Frequency of β-actin Ct values for all 2,978 samples tested. Diagnostic cut-off values for positive samples (Ct 35 for LN34 and Ct 33 for β-actin) are highlighted by red vertical lines.</p

Variation between replicate, extraction, assay run, and laboratory observed during repeatability and reproducibility studies.

<p>Variation between replicate, extraction, assay run, and laboratory observed during repeatability and reproducibility studies.</p