RESUMO
The current epidemic in Asia, driven by LSDV recombinants, poses difficulties to existing DIVA PCR tests, as these do not differentiate between homologous vaccine strains and the recombinant strains. We, therefore, developed and validated a new duplex real-time PCR capable of differentiating Neethling-based vaccine strains from classical and recombinant wild-type strains that are currently circulating in Asia. The DIVA potential of this new assay, seen in the in silico evaluation, was confirmed on samples from LSDV infected and vaccinated animals and on isolates of LSDV recombinants (n = 12), vaccine (n = 5), and classic wild-type strains (n = 6). No cross-reactivity or a-specificity with other capripox viruses was observed under field conditions in non-capripox viral stocks and negative animals. The high analytical sensitivity is translated into a high diagnostic specificity as more than 70 samples were all correctly detected with Ct values very similar to those of a published first-line pan capripox real-time PCR. Finally, the low inter- and intra-run variability observed shows that the new DIVA PCR is very robust which facilitates its implementation in the lab. All validation parameters that are mentioned above indicate the potential of the newly developed test as a promising diagnostic tool which could help to control the current LSDV epidemic in Asia.
Assuntos
Capripoxvirus , Doença Nodular Cutânea , Vírus da Doença Nodular Cutânea , Vacinas Virais , Animais , Bovinos , Vírus da Doença Nodular Cutânea/genética , Reação em Cadeia da Polimerase em Tempo Real , Doença Nodular Cutânea/diagnóstico , Doença Nodular Cutânea/prevenção & controle , Vacinas Virais/genética , Vacinas Atenuadas , Capripoxvirus/genéticaRESUMO
Lumpy Skin Disease virus is a poxvirus from the genus Capripox that mainly affects bovines and it causes severe economic losses to livestock holders. The Lumpy Skin Disease virus is currently dispersing in Asia, but little is known about detailed phylogenetic relations between the strains and genome evolution. We reconstructed a whole-genome-sequence (WGS)-based phylogeny and compared it with single-gene-based phylogenies. To study population and spatiotemporal patterns in greater detail, we reconstructed networks. We determined that there are strains from multiple clades within the previously defined cluster 1.2 that correspond with recorded outbreaks across Eurasia and South Asia (Indian subcontinent), while strains from cluster 2.5 spread in Southeast Asia. We concluded that using only a single gene (cheap, fast and easy to routinely use) for sequencing lacks phylogenetic and spatiotemporal resolution and we recommend to create at least one WGS whenever possible. We also found that there are three gene regions, highly variable, across the genome of LSDV. These gene regions are located in the 5' and 3' flanking regions of the LSDV genome and they encode genes that are involved in immune evasion strategies of the virus. These may provide a starting point to further investigate the evolution of the virus.
Assuntos
Doença Nodular Cutânea , Vírus da Doença Nodular Cutânea , Humanos , Bovinos , Animais , Vírus da Doença Nodular Cutânea/genética , Doença Nodular Cutânea/epidemiologia , Filogenia , Genômica , Surtos de Doenças , Evolução MolecularRESUMO
Vaccines have proven themselves as an efficient way to control and eradicate lumpy skin disease (LSD). In addition to the safety and efficacy aspects, it is important to know the duration for which the vaccines confer protective immunity, as this impacts the design of an efficient control and eradication program. We evaluated the duration of immunity induced by a live attenuated vaccine (LSDV LAV) and an inactivated vaccine (LSDV Inac), both based on LSDV. Cattle were vaccinated and challenged after 6, 12 and 18 months for LSDV LAV or after 6 and 12 months for the LSDV Inac. The LSDV LAV elicited a strong immune response and protection for up to 18 months, as no clinical signs or viremia could be observed after a viral LSDV challenge in any of the vaccinated animals. A good immune response and protection were similarly seen for the LSDV Inac after 6 months. However, two animals developed clinical signs and viremia when challenged after 12 months. In conclusion, our data support the annual booster vaccination when using the live attenuated vaccine, as recommended by the manufacturer, which could potentially even be prolonged. In contrast, a bi-annual vaccination seems necessary when using the inactivated vaccine.
RESUMO
Lumpy skin disease virus (LSDV) is a vector-transmitted capripox virus that causes disease in cattle. Stomoxys calcitrans flies are considered to be important vectors as they are able to transmit viruses from cattle with the typical LSDV skin nodules to naive cattle. No conclusive data are, however, available concerning the role of subclinically or preclinically infected cattle in virus transmission. Therefore, an in vivo transmission study with 13 donors, experimentally inoculated with LSDV, and 13 naïve acceptor bulls was performed whereby S. calcitrans flies were fed on either subclinical- or preclinical-infected donor animals. Transmission of LSDV from subclinical donors showing proof of productive virus replication but without formation of skin nodules was demonstrated in two out of five acceptor animals, while no transmission was seen from preclinical donors that developed nodules after Stomoxys calcitrans flies had fed. Interestingly, one of the acceptor animals which became infected developed a subclinical form of the disease. Our results show that subclinical animals can contribute to virus transmission. Therefore, stamping out only clinically diseased LSDV-infected cattle could be insufficient to completely halt the spread and control of the disease.
Assuntos
Capripoxvirus , Doenças dos Bovinos , Doença Nodular Cutânea , Vírus da Doença Nodular Cutânea , Muscidae , Bovinos , Animais , Masculino , Insetos VetoresRESUMO
From 2017 to 2019, several vaccine-like recombinant strains of lumpy skin disease virus (LSDV) were discovered in Kazakhstan and neighbouring regions of Russia and China. Shortly before their emergence, the authorities in Kazakhstan launched a mass vaccination campaign with the Neethling-based Lumpivax vaccine. Since none of the other countries in the affected region had used a homologous LSDV vaccine, it was soon suspected that the Lumpivax vaccine was the cause of these unusual LSDV strains. In this study, we performed a genome-wide molecular analysis to investigate the composition of two Lumpivax vaccine batches and to establish a possible link between the vaccine and the recent outbreaks. Although labelled as a pure Neethling-based LSDV vaccine, the Lumpivax vaccine appears to be a complex mixture of multiple CaPVs. Using an iterative enrichment/assembly strategy, we obtained the complete genomes of a Neethling-like LSDV vaccine strain, a KSGP-like LSDV vaccine strain and a Sudan-like GTPV strain. The same analysis also revealed the presence of several recombinant LSDV strains that were (almost) identical to the recently described vaccine-like LSDV strains. Based on their InDel/SNP signatures, the vaccine-like recombinant strains can be divided into four groups. Each group has a distinct breakpoint pattern resulting from multiple recombination events, with the number of genetic exchanges ranging from 126 to 146. The enormous divergence of the recombinant strains suggests that they arose during seed production. The recent emergence of vaccine-like LSDV strains in large parts of Asia is, therefore, most likely the result of a spillover from animals vaccinated with the Lumpivax vaccine.
Assuntos
Doença Nodular Cutânea , Vírus da Doença Nodular Cutânea , Vacinas Virais , Animais , Ásia/epidemiologia , Bovinos , Vacinas Atenuadas , Vacinas Virais/genéticaRESUMO
Vaccination is an effective approach to prevent, control and eradicate diseases, including lumpy skin disease (LSD). One of the measures to address farmer hesitation to vaccinate is guaranteeing the quality of vaccine batches. The purpose of this study was to demonstrate the importance of a quality procedure via the evaluation of the LSD vaccine, Lumpivax (Kevevapi). The initial PCR screening revealed the presence of wild type LSD virus (LSDV) and goatpox virus (GTPV), in addition to vaccine LSDV. New phylogenetic PCRs were developed to characterize in detail the genomic content and a vaccination/challenge trial was conducted to evaluate the impact on efficacy and diagnostics. The characterization confirmed the presence of LSDV wild-, vaccine- and GTPV-like sequences in the vaccine vial and also in samples taken from the vaccinated animals. The analysis was also suggestive for the presence of GTPV-LSDV (vaccine/wild) recombinants. In addition, the LSDV status of some of the animal samples was greatly influenced by the differentiating real-PCR used and could result in misinterpretation. Although the vaccine was clinically protective, the viral genomic content of the vaccine (being it multiple Capripox viruses and/or recombinants) and the impact on the diagnostics casts serious doubts of its use in the field.
RESUMO
Lumpy skin disease (LSD) diagnosis is primarily based on clinical surveillance complemented by PCR of lesion crusts or nodule biopsies. Since LSD can be subclinical, the sensitivity of clinical surveillance could be lower than expected. Furthermore, real-time PCR for the detection of LSD viral DNA in blood samples from subclinical animals is only intermittently positive. Therefore, this study aimed to investigate an acceptable, easily applicable and more sensitive testing method for the detection of clinical and subclinical LSD. An animal experiment was conducted to investigate ear notches and biopsies from unaffected skin taken from the neck and dorsal back as alternatives to blood samples. It was concluded that for early LSD confirmation, normal skin biopsies and ear notches are less fit for purpose, as LSDV DNA is only detectable in these samples several days after it is detectable in blood samples. On the other hand, blood samples are less advisable for the detection of subclinical animals, while ear notches and biopsies were positive for LSD viral DNA in all subclinically infected animals by 16 days post infection. In conclusion, ear notches could be used for surveillance to detect subclinical animals after removing the clinical animals from a herd, to regain trade by substantiating the freedom of disease or to support research on LSDV transmission from subclinical animals.
RESUMO
Lumpy skin disease virus (LSDV) causes a severe, systemic, and economically important disease in cattle. Here, we report coding-complete sequences of recombinant LSDVs from four outbreaks in October and November 2020 in northeastern Vietnam.