Vaccine-like Strains Research Articles

Unveiling the genetic landscape of infectious laryngotracheitis virus in Switzerland: Evidence for vaccine-like and wild-type strains

Infectious laryngotracheitis (ILT) is a respiratory disease affecting chickens worldwide. Unlike many countries, Switzerland does not vaccinate against ILT. This study analysed ILT samples from 21 natural outbreaks in Switzerland using restriction fragment length polymorphism (RFLP) and multiple gene sequencing. Chicken embryo origin (CEO) and tissue culture origin (TCO) vaccine strains were included as references. Both vaccine strains were distinguishable, and 14 out of 21 samples resembled the CEO vaccine. Additionally, four distinct non-vaccine-like groups were identified. Sequencing of three genes from selected Swiss samples and those from neighbouring countries revealed four phylogenetic clades. Notably, four Swiss field strains formed two unique clades, not closely related to vaccine strains or ILTV from neighbouring countries. Overall, RFLP results were supported by sequencing data. This study demonstrates the presence of both vaccine-like and wild-type ILT viruses in Switzerland, where vaccination is de facto prohibited.

Subclinical infection caused by a recombinant vaccine-like strain poses high risks of lumpy skin disease virus transmission.

Lumpy skin disease (LSD) is a transboundary viral infection, affecting cattle with characteristic manifestations involving multiple body systems. A distinctive characteristic of lumpy skin disease is the subclinical disease manifestation wherein animals have viremia and shed the virus through nasal and ocular discharges, while exhibiting no nodules but enlarged lymph nodes that are easily oversighted by inexperienced vets. Further research on the role of subclinically ill animals in the transmission of LSD virus (LSDV) can contribute to the development of more effective tools to control the disease worldwide. Thus, this study aims to determine the potential role of subclinical infection in virus transmission in a non-vector-borne manner. To achieve this, we inoculated animals with the recombinant vaccine-like strain (RVLS) Udmurtiya/2019 to cause clinical and subclinical LSDV infection. After the disease manifestation, we relocated the subclinically ill animals to a new clean facility followed by the introduction of another five animals to determine the role of RVLS-induced subclinical infection in the virus transmission via direct/indirect contact. After the introduction of the naïve animals to the relocated subclinically ill ones in a shared airspace, two introduced animals contracted the virus (clinically and subclinically), showing symptoms of fever, viremia, and seroconversion in one animal, while three other introduced animals remained healthy and PCR-negative until the end of the study. In general, the findings of this study suggest the importance of considering LSDV subclinical infection as a high-risk condition in disease management and outbreak investigations.

Genome sequence characterization of the unique recombinant vaccine-like lumpy skin disease virus strain Kurgan/2018.

In 2018, the molecular epidemiology of lumpy skin disease in Russia was characterized by a surge in novel recombinant vaccine-like strains causing outbreaks along the southern border, spreading in an easterly direction. Currently, five distinct novel recombinant vaccine-like lineages have been described, designated as clusters 2.1 to 2.5. Based on the complete genome sequence analysis of the causative lumpy skin disease virus (Kurgan/Russia/2018), obtained from an eponymous outbreak, the genome was shown to be composed of a Neethling vaccine strain virus as the dominant parental strain and KSGPO vaccine virus as its minor parental strain. These features are similar to those of Saratov/Russia/2017 and Tyumen/Russia/2018, representing clusters 2.1 and 2.4, respectively. However, Kurgan/Russia/2018 has 16 statistically significant recombination events unique to this sequence, contributing to the phylogenetic clustering of Kurgan/Russia/2018 in yet another cluster designed cluster 2.6, based on analysis involving the complete genome sequences.

Development and application of a real-time polymerase chain reaction assay to detect lumpy skin disease virus belonging to the Kenyan sheep and goat pox group

Lumpy skin disease (LSD) outbreaks in Southeast and South Asia are attributed to different lineages of LSD virus (LSDV). Variants belonging to the novel recombinant cluster 2.5 circulate in China and Thailand, while a Kenyan sheep and goat pox (KSGP) strain from cluster 1.1 circulates in India, Pakistan, and Bangladesh. The clusters representing these circulating strains are vastly different. However, if their distribution encroaches into each other’s ranges, it will be impossible to differentiate between them due to the lack of suitable molecular tools. Thus, fit-for-purpose molecular tools are in demand to effectively and timeously diagnose and investigate the epidemiology of LSDVs in a region. These could significantly contribute to the phylogenetic delineation of LSDVs and the development of preventive measures against transboundary spillovers. This work aimed to develop a real-time polymerase chain reaction assay targeting open reading frame LW032, capable of specifically detecting KSGP-related isolates and recombinant LSDV strains containing the KSGP backbone. The analytical specificity was proven against the widest possible panel of recombinant vaccine-like LSDV strains known to date. The amplification efficiency was 91.08%, and the assay repeatability had a cycle threshold variation of 0.56–1.1 over five repetitions across three runs. This KSGP-specific assay is reliable and fast and is recommended for use in LSDV epidemiological studies where the accurate detection of KSGP genetic signatures is a priority, particularly in regions where KSGP-like and other lineages are circulating.

Comparison of Gross Pathology between Classical and Recombinant Lumpy Skin Disease Viruses.

The pathology caused by three different isolates of lumpy skin disease virus, classical field cluster 1.2 strain Dagestan/2015, recombinant vaccine-like cluster 2.1 strain Saratov/2017, and cluster 2.2 strain Udmurtiya/2019, in cattle was compared from experimental infections. The infection of cattle was performed using intravenous administration of 2 mL of 105 TCID50/mL of each specific LSDV. Both classical and recombinant forms of LSDV cause pathological changes in the skin and lymph nodes, as well as the trachea and lungs. Due to circulatory disorders in the affected organs, multiple areas of tissue necrosis were observed, which, with the resurgence of secondary microflora, led to the development of purulent inflammation. Observed pathological changes caused by the recombinant vaccine-like strain Udmurtiya/2019 were characterized by a more pronounced manifestation of the pathoanatomical picture compared to the classical field strains Dagestan/2015 and Saratov/2017. Interestingly, Dagestan/2015 and Udmurtiya/2019 caused damage to the lymph nodes, characterized by serous inflammation and focal purulent lymphadenitis caused by purulent microflora. "Saratov/2017" did not cause pathology in the lymph nodes. All LSDVs were virulent and caused pathology, which was not distinguishable between viruses. This data set will serve as the experimentally validated basis for the comparative examination of novel LSDV strains in gross pathology.

Turkey adenovirus 3: ORF1 gene sequence comparison between vaccine-like and field strains

Haemorrhagic enteritis is an economically significant disease reported in the majority of the countries where turkeys are raised intensively; it is caused by Turkey adenovirus 3 (TAdV-3). The aim of this study was to analyse and compare the ORF1 gene 3′ region from turkey haemorrhagic enteritis virus (THEV) vaccine-like and field strains in order to develop a molecular diagnostic method to differentiate the strains from each other. Eighty samples were analysed by sequencing and phylogenetic analyses using a new set of polymerase chain reaction (PCR) primers targeting a genomic region spanning the partial ORF1, hyd and partial IVa2 gene sequences. A commercial live vaccine was also included in the analysis. The results showed that 56 of the 80 sequences obtained in this study showed ≥99.8% nucleotide identity with the homologous vaccine strain sequence. Three non-synonymous mutations – ntA1274G (aaI425V), ntA1420C (aaQ473H) and ntG1485A (aaR495Q) – were detected in the THEV field strains but not in the vaccine strain. Phylogenetic analysis confirmed the clustering of the field and vaccine-like strains in different phylogenetic branches. In conclusion, the method employed in this study could be a useful tool towards making a correct diagnosis. The data could contribute to the knowledge of field distribution of THEV strains and increase the limited existing information available on native isolates around the world.

The Characterization and Differentiation of Recombinant Lumpy Skin Disease Isolates Using a Region within ORF134

The recent description and characterization of several novel and unique lumpy skin disease virus (LSDV) strains have revealed the inadequacy of current techniques for differentiating between vaccine- and wild-type viruses. The lack of reliable sequencing targets for promptly distinguishing circulating recombinant vaccine-like strains (RVLSs) highlights the need to develop a single and simple differentiation tool. In this study, we analyzed the available LSDV whole-genome sequences and identified a 705-bp region in open reading frame (ORF) LW134. Based on a single run of nucleotide sequencing and phylogenetic analysis, the region with 13 informative single nucleotide polymorphisms (SNPs) was capable of accurately segregating the novel RVLSs into the same five clusters previously confirmed by whole-genome sequencing. In addition, archived RVLSs from Russia were analyzed for further characterization using the newly described single PCR and sequencing assay. The ORF LW134 assay identified one archived RVLS as a novel cluster distinct from the previously described five clusters, while clustering the remaining samples into previously designated lineages, demonstrating the reliability of the assay. The novel PCR and sequencing assays described in this study have great potential for accurately delineating the molecular and evolutionary affiliation of circulating RVLSs.

Genetic characterization and epidemiological analysis of the first lumpy skin disease virus outbreak in Mongolia, 2021.

Novel lumpy skin disease virus (LSDV) strains of recombinant origin are on the rise in South East Asia following the first emergence in 2017, and published evidence demonstrates that such genetic lineages currently dominate the circulation. Mongolia reported first LSD outbreaks in 2021 in a north-eastern region sharing borders with Russia and China. For each of 59 reported LSDV outbreaks, the number of susceptible animals ranged from 8 to 8600 with a median of 572, while the number of infected animals ranged from one to 355 with a median of 14. Phylogenetic inferences revealed a close relationship of LSDV Mongolia/2021 with recombinant vaccine-like LSDV strains from Russia, China, Taiwan, Thailand and Vietnam. These findings support the published data that the circulating strain of LSDV belongs to the dominant recombinant lineage recently established in the region.

A Recombinant Vaccine-like Strain of Lumpy Skin Disease Virus Causes Low-Level Infection of Cattle through Virus-Inoculated Feed.

Since 1989, lumpy skin disease of cattle (LSD) has spread out of Africa via the Middle East northwards and eastwards into Russia, the Far East and South-East Asia. It is now threatening to become a worldwide pandemic, with Australia possibly next in its path. One of the research gaps on the disease concerns its main mode of transmission, most likely via flying insect vectors such as biting flies or mosquitoes. Direct or indirect contact transmission is possible, but appears to be an inefficient route, although there is evidence to support the direct contact route for the newly detected recombinant strains first isolated in Russia. In this study, we used experimental bulls and fed them via virus-inoculated feed to evaluate the indirect contact route. To provide deeper insights, we ran two parallel experiments using the same design to discover differences that involved classical field strain Dagestan/2015 LSDV and recombinant vaccine-like Saratov/2017. Following the attempted indirect contact transmission of the virus from the inoculated feed via the alimentary canal, all bulls in the Dagestan/2015 group remained healthy and did not seroconvert by the end of the experiment, whereas for those in the Saratov/2017 recombinant virus group, of the five bulls fed on virus-inoculated feed, three remained clinically healthy, while two displayed evidence of a mild infection. These results provide support for recombinant virus transmission via the alimentary canal. In addition, of particular note, the negative control in-contact bull in this group exhibited a biphasic fever at days 10 and 20, developed lesions from day 13 onwards, and seroconverted by day 31. Two explanations are feasible here: one is the in-contact animal was somehow able to feed on some of the virus-inoculated bread left over from adjacent animals, but in the case here of the individual troughs being used, that was not likely; the other is the virus was transmitted from the virus-fed animals via an airborne route. Across the infected animals, the virus was detectable in blood from days 18 to 29 and in nasal discharge from days 20 to 42. Post-mortem and histological examinations were also indicative of LSDV infection, supporting further evidence for rapid, in F transmission of this virus. This is the first report of recombinant LSDV strain transmitting via the alimentary mode.

Molecular characterization of a novel subgenotype of lumpy skin disease virus strain isolated in Inner Mongolia of China

BackgroundThe outbreak of Lumpy skin disease (LSD) in cattle caused by LSD virus (LSDV) was first reported in August 2019 in China. Since then, several LSD outbreaks have been reported in seven different provinces of China. Until now, several Lumpy skin disease virus (LSDV) strains from China have been reported and sequenced including LSDV/Xinjiang/2019 (MN598005.1), China/GD01/2020 (MW355944.1), and LSDV/Hongkong/2021 (MW732649.1). In October 2020, more than 1,700 cattle imported from Chile arrived in Xilingol, Inner Mongolia, and were diagnosed with LSD. Currently, limited data on the origin of the virus is available.MethodsNucleotide sequences of the ORF11, ORF36, ORF74, ORF117, ORF126 genes and the complete genome of LSDV strains and isolates were downloaded from NCBI database. MEGA7.0 was used to perform phylogenetic analysis with Neighbor-Joining (NJ). DNASTAR software is used to analyze homologous comparison analysis with related genes of reference strains included in Genbank.ResultsCompared with other strains isolated from China, the results of full genome sequence analysis showed the LSDV/NMG/2020 strain belonged to the recombinant strains. The LSDV/NMG/2020 strain is different from the current LSDV field isolates in Africa, the Middle East, Europe, and the newly emerged LSDV Russia variants. Based on the identities of P32, RPO30, EEV, GPCR and LSDV117 genes (99.8%, 99%, 99.8%, 99% and 98.7%), the sub-cluster recombinant containing LSDV/NMG/2020 strain is phylogenetically closer to the Russia strain (Saratov/2017).ConclusionsIn this study, we reported a new isolated LSDV strain named LSDV/NMG/2020. The results of genomic characterization and phylogenetic analysis demonstrated that the LSDV/NMG/2020 isolate was a vaccine-like recombinant strain.

Recombinant LSDV Strains in Asia: Vaccine Spillover or Natural Emergence?

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.

Characterization of infectious laryngotracheitis virus isolates from laying hens during 2019-2020 outbreaks in Tamil Nadu, India.

Infectious laryngotracheitis (ILT) is an acute respiratory disease in chickens that is a serious threat to poultry-producing countries worldwide. In the present study, we isolated and characterized infectious laryngotracheitis (ILTV) virus isolates by sequencing and restriction fragment length polymorphism analysis of PCR-amplified products (PCR-RFLP). A total of 26 ILTV outbreaks were investigated that occurred between 2019 and 2020 in flocks that had not been vaccinated against ILTV. ILTV was isolated by cultivating tracheal samples in embryonated chicken eggs, which showed multiple opaque pock lesions and thickening of the chorioallantoic membrane after 120 hours of infection. The ILTV isolates were identified and characterized by PCR and sequencing a portion of the ICP4 and TK genes. Phylogenetic analysis based on the ICP4 region showed that the sequences clustered with chicken-embryo-origin vaccine-like strains. Sequence analysis of the ICP4 region differentiated chicken-embryo-origin (CEO), tissue-culture-origin (TCO), and field ILTV strains, with significant differences in nucleotide and amino acid sequences. Furthermore, PCR-RFLP analysis of the TK gene showed that the patterns were identical to those obtained with low-virulence and vaccine strains. In conclusion, sequencing of a portion of the ICP4 region of ILTV allowed differentiation of ILTV field, CEO, and TCO vaccine strains. In this study, CEO-vaccine-like strains were found to be the cause of ILTV outbreaks between 2019 and 2020 in Tamil Nadu in southern India.

Phylogenetic analysis of lumpy skin disease virus isolates in Russia in 2019-2021

Lumpy skin disease continues to pose a threat to countries in the East and Asia-Pacific regions. Although only occasional LSDV outbreaks have been reported recently in Russia, these have been mainly restricted to the Far East region of the country. An increase in the number of outbreaks in South East Asia has been attributed to recombinant vaccine-like LSDV strains. In this scenario, it is epidemiologically important to perform phylogenetic analysis to track the distribution of LSDV worldwide at the genetic level to understand routes of migration and molecular evolution patterns. In this study, we investigated the RPO30 and GPCR gene regions of LSDV isolates associated with outbreaks in 2019-2021 in Siberia and the Far East region of Russia. The inferred phylogeny confirms the recombinant origin of these sequenced isolates. Based on sequences of these selected loci, the isolates from 2019 differed from isolates detected in Russia in the past and from isolates from Asian countries, while the isolates from 2020 and 2021 exhibited a high degree of similarity to the Asian isolates. These findings indicate that recombinant LSDV strains continue to persist and additionally point to the establishment of a specific lineage of recombinant isolates in the region. Full genome sequencing is still needed to gain more information about how the circulating isolates are related to each other.

Phylogenetic analysis of porcine reproductive and respiratory syndrome virus in Vietnam, 2021.

The porcine reproductive and respiratory syndrome virus (PRRSV) causes more economic losses in the swine industry than any other virus. This study aimed to investigate the genetic diversity of PRRSV to assist in evaluating the effectiveness of PRRS vaccines. Twenty-eight samples from clinical cases were collected from 19 farms in seven provinces of Vietnam in 2021. Full-length PRRSV ORF5 genes from the 19 samples were amplified, sequenced, and compared to the corresponding sequences of referenced PRRSV strains from Genbank. The genetic analysis showed that 12 isolates were the highly pathogenic PRRSV subtype (HP—PRRSV) lineage 8, sublineage 8.7; six isolates were the classical North American PRRSV subtype (US-PRRSV), NADC-like group, lineage 1, sublineage 1.4, which were reported in Vietnam for the first time; and the final isolate was a vaccine-like strain. The field isolates of HP-PRRSV had relatively higher genetic diversity with US-PRRSV vaccine strains (84.0–94.5%) than HP-PRRSV vaccine strains (95.3–98.6%). Meanwhile, the six NADC-like isolates had low nucleotide similarity with US-PRRSV and HP-PRRSV vaccine strains (83.4–85.4% and 83.2–84.0%, respectively). Many amino acid substitutions were found in antigenic regions of GP5 involved in response to early antibody production, neutralizing antibodies, and viral immune evasion between these field strains and PRRSV vaccine strains. These findings provide insights into the molecular characteristics, genetic diversity, antigenicity, and evolution of PRRSV strains in Vietnam and postulate a compelling explanation for the limitations of current vaccination efforts.Supplementary InformationThe online version contains supplementary material available at 10.1007/s11262-022-01912-w.

Implementing a user-friendly format to analyze PRRSV next-generation sequencing results and associating breeding herd production performance with number of PRRSV strains and recombination events.

The open reading frames (ORF)5 represents approximately 4% of the porcine reproductive and respiratory syndrome virus (PRRSV)-2 genome (whole-PRRSV) and is often determined by the Sanger technique, which rarely detects>1 PRRSV strain if present in the sample. Next-generation sequencing (NGS) may provide a more appropriate method of detecting multiple PRRSV strains in one sample. This work assessed the effect of PRRSV genetic variability and recombination events, using NGS, on the time-to-low prevalence (TTLP) and total losses in breeding herds (n 20) that detected a PRRSV outbreak and adopted measures to eliminate PRRSV. Serum, lung or live virus inoculation material collected within 3-weeks of outbreak, and subsequently, processing fluids (PFs) were tested for PRRSV by RT-qPCR and NGS. Recovered whole-PRRSV or partial sequences were used to characterize within and between herd PRRSV genetic variability. Whole-PRRSV was recovered in five out of six (83.3%) lung, 16 out of 22 (72.73%) serum and in five out of 95 (5.26%) PF. Whole-PRRSV recovered from serum or lung were used as farm referent strains in 16 out of 20 (80%) farms. In four farms, only partial genome sequences were recovered and used as farm referent strains. At least two wild-type PRRSV strains (wt-PRRSV) were circulating simultaneously in 18 out of 20 (90%) and at least one vaccine-like strain co-circulating in eight out of 20 (40%) farms. PRRSV recombination events were detected in 12 farms (59%), been 10 out of 12 between wt-PRRSV and two out of 12 between wt-PRRSV and vaccine-like strains. Farms having ≥3 strains had a 12-week increase TTLP versus herds ≤2 strains detected. Farms with ≤2 strains (n 10) had 1837 and farms with no recombination events detected (n 8) had 1827 fewer piglet losses per 1000 sows versus farms with ≥3 PRRSV strains (n 8) or detected recombination (n 10), respectively. NGS outcomes and novel visualization methods provided more thorough insight into PRRSV dynamics, genetic variability, detection of multiple strains co-circulating in breeding herds and helped establish practical guidelines for using PRRSV NGS outputs.

Porcine reproductive and respiratory syndrome virus 2 (PRRSV-2) genetic diversity and occurrence of wild type and vaccine-like strains in the United States swine industry.

Porcine reproductive and respiratory syndrome virus genotype 2 (PRRSV-2) genetic diversity in the U.S. was assessed using a database comprising 10 years’ worth of sequence data obtained from swine production systems routine monitoring and outbreak investigations. A total of 26,831 ORF5 PRRSV-2 sequences from 34 production systems were included in this analysis. Within group mean genetic distance (i.e. mean proportion of nucleotide differences within ORF5) per year according to herd type was calculated for all PRRSV-2 sequences. The percent nucleotide difference between each sequence and the ORF5 sequences from four commercially available PRRSV-2 vaccines (Ingelvac PRRS MLV, Ingelvac PRRS ATP, Fostera PRRS, and Prevacent PRRS) within the same lineage over time was used to classify sequences in wild-type or vaccine-like. The mean ORF5 genetic distance fluctuated from 0.09 to 0.13, being generally smaller in years in which there was a relative higher frequency of dominant lineage. Vaccine-like sequences comprised about one fourth of sequences obtained through routine monitoring of PRRS. We found that lineage 5 sequences were mostly Ingelvac PRRS MLV-like. Lineage 8 sequences up to 2011 were 62.9% Ingelvac PRRS ATP-like while the remaining were wild-type viruses. From 2012 onwards, 51.9% of lineage 8 sequences were Ingelvac PRRS ATP-like, 45.0% were Fostera PRRS-like, and only 3.2% were wild-type. For lineage 1 sequences, 0.1% and 1.7% of the sequences were Prevacent PRRS-like in 2009–2018 and 2019, respectively. These results suggest that repeated introductions of vaccine-like viruses through use of modified live vaccines might decrease within-lineage viral diversity as vaccine-like strains become more prevalent. Overall, this compilation of private data from routine monitoring provides valuable information on PRRSV viral diversity.

Analysis of vaccine-like lumpy skin disease virus from flies near the western border of China.

Lumpy skin disease (LSD) is a devastating viral disease that occurs in cattle. In China, it was first detected in the Xin-Jiang autonomous region, near the border with Kazakhstan, in August 2019. As there were no new occurrences of LSD in either country following the first detection, the initial introduction of the virus remains unknown. Arthropod vectors were considered as potential vectors. Consequently, to identify the arthropod vectors involved in transmitting LSD virus (LSDV), an insect surveillance campaign was launched at four different sites scattered along the border, and samples from 22 flying insect species were collected and subjected to PCR assays. Following the Agianniotaki LSDV vaccine and Sprygin's general LSDV assays, two kinds of non-biting flies, namely, Musca domestica L and Muscina stabulans, were positive for LSDV. However, all the other insects tested negative. Viral DNA was only detected in wash fluid, implying body surface contamination of the virus. The negative test results suggest that non-biting flies are the dominant insects involved in the observed local epidemic. Three genomic regions encoding RPO30, GPCR, and LW126 were successfully sequenced and subjected to phylogenetic analysis. The sequences shared high homology with LSDV/Russia/Saratov/2017, a recombinant vaccine-like strain formerly identified in Russia, and clustered with LSDV vaccine strains in phylogenetic trees of RPO30 and LW126. However, the GPCR gene was seen to be solely clustered with LSDV field strains, implying differences in host affinity between these closely related vaccine-like strains. Despite this, there is no direct evidence to support cross-border transmission of the vaccine-like LSDV. To our knowledge, this is the first report of vaccine-like LSDV DNA detection in non-biting flies in China.

Differential expression and correlation analysis of miRNA\u2013mRNA profiles in swine testicular cells infected with porcine epidemic diarrhea virus

The variant virulent porcine epidemic diarrhea virus (PEDV) strain (YN15) can cause severe porcine epidemic diarrhea (PED); however, the attenuated vaccine-like PEDV strain (YN144) can induce immunity in piglets. To investigate the differences in pathogenesis and epigenetic mechanisms between the two strains, differential expression and correlation analyses of the microRNA (miRNA) and mRNA in swine testicular (ST) cells infected with YN15, YN144, and mock were performed on three comparison groups (YN15 vs Control, YN144 vs Control, and YN15 vs YN144). The mRNA and miRNA expression profiles were obtained using next-generation sequencing (NGS), and the differentially expressed (DE) (p-value < 0.05) mRNA and miRNA were obtained using DESeq R package. mRNAs targeted by DE miRNAs were predicted using the miRanda algortithm. 8039, 8631 and 3310 DE mRNAs, and 36, 36, and 22 DE miRNAs were identified in the three comparison groups, respectively. 14,140, 15,367 and 3771 DE miRNA–mRNA (targeted by DE miRNAs) interaction pairs with negatively correlated expression patterns were identified, and interaction networks were constructed using Cytoscape. Six DE miRNAs and six DE mRNAs were randomly selected to verify the sequencing data by real-time relative quantitative reverse transcription polymerase chain reaction (qRT-PCR). Based on bioinformatics analysis, we discovered the differences were mostly involved in host immune responses and viral pathogenicity, including NF-κB signaling pathway and bacterial invasion of epithelial cells, etc. This is the first comprehensive comparison of DE miRNA–mRNA pairs in YN15 and YN144 infection in vitro, which could provide novel strategies for the prevention and control of PED.

Development of a bead-based assay for detection and differentiation of field strains and four vaccine strains of type 2 porcine reproductive and respiratory syndrome virus (PRRSV-2) in the USA.

Porcine reproductive and respiratory syndrome (PRRS) remains one of the most economically devastating diseases in swine population in the United States of America. Due to high mutation rate of the PRRS virus (PRRSV) genome, it is difficult to develop an accurate diagnostic assay with high strain coverage. Differentiation of field strains from the four vaccines that have been used in the USA, namely Ingelvac PRRS MLV, Ingelvac ATP, Fostera PRRS and Prime Pac PRRS, adds an additional challenge. It is difficult to use current real-time PCR systems to detect and differentiate the field strains from the vaccine strains. Luminex xTAG technology allows us to detect more molecular targets in a single reaction with a cost similar to a single real-time PCR reaction. By analysing all available 678 type 2 PRRSV (PRRSV-2) complete genome sequences, including the 4 vaccine strains, two pairs of detection primers were designed targeting the conserved regions of ORF4-ORF7, with strain coverage of 98.8% (670/678) based on in silico analysis. The virus strains sharing ≥98% identity of the complete genomes with the vaccine strains were considered vaccine or vaccine-like strains. One pair of primers for each vaccine strain were designed targeting the nsp2 region. In silico analysis showed the assay matched 94.7% (54/57) of Ingelvac PRRS® MLV (MLV) strain and the MLV-like strains, and 100% of the other three vaccine strains. Analytical sensitivity of the Luminex assay was one to two logs lower than that of the reverse transcription real-time PCR assay. Evaluated with 417 PRRSV-2 positive clinical samples, 95% were detected by the Luminex assay. Compared to ORF5 sequencing results, the Luminex assay detected 92.4% (73/79) of MLV strains, 78.3% (18/23) of Fostera strains and 50% (2/4) of ATP strains. None of the 472 samples were the Prime Pac strain tested by either ORF5 sequencing or the Luminex assay.

Potential link of single nucleotide polymorphisms to virulence of vaccine-associated field strains of lumpy skin disease virus in South Africa.

South Africa is endemic for lumpy skin disease and is therefore reliant on various live attenuated vaccines for the control and prevention of the disease. In recent years, widespread outbreaks of vaccine‐like strains of lumpy skin disease virus (LSDV) were reported internationally, leading to an increase in the generation of full genome sequences from field isolates. In this study, the complete genomes of six LSDVs submitted during active outbreaks in the 1990s in South Africa were generated. Based on phylogenetic analysis, the six viruses clustered with vaccine strains in LSDV Subgroup 1.1 and are subsequently referred to as vaccine‐associated. The genetic differences between the phenotypically distinct vaccine and vaccine‐associated strains were 67 single nucleotide polymorphisms (SNPs). This study characterized the location and possible importance of each of these SNPs in their role during virulence and host specificity.