Development of multiplex PCR based NGS protocol for whole genome sequencing of West Nile virus lineage 2 directly from biological samples using Oxford Nanopore platform.

West Nile virus (WNV) can affect humans, birds, horses and another mammals, causing asymptomatic infection, mild febrile disease, neurological and systematic disease and death. In order to gain insight into the prevalence of WNV, a monitoring program has been established in the Republic of Serbia. Whole genome sequencing is essential for the molecular epizootiological analysis of virus entry and transmission routes, especially in high-risk regions. This paper describes the development of a multiplex PCR based NGS protocol for whole genome sequencing of WNV lineage 2 directly from biological samples using Oxford Nanopore (ONT) platform. The results obtained using this platform, confirmed by Sanger sequencing, indicate that this protocol can be applied to obtain whole sequences of the WNV genome, even when the virus concentration in the sample is medium, Ct value is approximately 30. The use of this protocol does not require prior virus isolation on cell culture nor the depletion of host nucleic acids.

Spatiotemporal Analysis of West Nile Virus Epidemic in South Banat District, Serbia, 2017-2019.

West Nile virus (WNV) is an arthropod-born pathogen, which is transmitted from wild birds through mosquitoes to humans and animals. At the end of the 20th century, the first West Nile fever (WNF) outbreaks among humans in urban environments in Eastern Europe and the United States were reported. The disease continued to spread to other parts of the continents. In Serbia, the largest number of WNV-infected people was recorded in 2018. This research used spatial statistics to identify clusters of WNV infection in humans and animals in South Banat County, Serbia. The occurrence of WNV infection and risk factors were analyzed using a negative binomial regression model. Our research indicated that climatic factors were the main determinant of WNV distribution and were predictors of endemicity. Precipitation and water levels of rivers had an important influence on mosquito abundance and affected the habitats of wild birds, which are important for maintaining the virus in nature. We found that the maximum temperature of the warmest part of the year and the annual temperature range; and hydrographic variables, e.g., the presence of rivers and water streams were the best environmental predictors of WNF outbreaks in South Banat County.

The detection and phylogenetic analysis of equine herpesviruses 1, 4 and 5 identified in nasal swab samples of asymptomatic horses from Serbia and Bosnia and Herzegovina.

Nasal swabs originating from 112 apparently clinically healthy and unvaccinated horses of different age, breed and from diverse rearing conditions from Serbia and Bosnia and Herzegovina were examined for the presence of equine herpesviruses 1, 4 and 5 using multiplex nested PCR (Mn­PCR) and virus isolation. The detected viruses were subsequently characterised by gB gene nucleotide sequencing and their phylogenetic analysis was performed. The infections with EHV­1, EHV­4, and EHV­5 in the examined horse populations are apparently chronic, subclinical and persistent, whilst the shedding of EHV­1 and EHV­5 was confirmed by their successful isolation. A connection was established between the finding of EHVs and rearing conditions since horses kept together in stables were positive for at least one EHV in contrast to animals held free grazing or individually. EHV­5 was found most often in younger horses, however descending in frequency in animals up to 10 years of age. The phylogenetic analysis showed that the identified EHV strains group mostly with Turkish and German strains of respective viruses. A certain degree of genetic heterogeneity was determined regarding the identified EHV­5 strains in contrast to EHV­1 and EHV­4.

Supercritical Fluid Extraction of Celery and Parsley Fruit-Chemical Composition and Antibacterial Activity.

Supercritical fluid extraction as an environmentally friendly technology was applied to isolate biologically active extracts from celery and parsley fruits for potential applications in the food industry. The extractions were performed under mild temperature conditions of 39.85 °C and at pressures of 10 and 30 MPa. The extracts were analyzed regarding their chemical composition, antibacterial activity, and cytotoxic effect. Sedanolide was the dominant component of the celery fruit extracts, comprising more than 70% of the obtained fraction, while the content of apiole in the parsley fruit SC CO2 extracts exceeded 85%. The celery fruit extracts showed strong and moderately strong antibacterial activity against tested Staphylococcus aureus, Bacillus (B.) cereus, B. subtilis, B. circulans, Listeria (L.) greyi, L. seeligeri and L. welshimeri, with minimal inhibitory concentration (MIC) values between 160 and 640 µg/mL, and weak activity against the selected Salmonella isolates with a MIC of 2560 µg/mL. The parsley extract obtained at 10 MPa showed strong and moderately strong antibacterial effects against Bacillus strains with obtained MICs of 160-640 µg/mL, and weak activity against Staphylococcus, Listeria, and Salmonella with a MIC of 2560 µg/mL. Cytotoxicity investigation showed that the extracts with proven antibacterial activity had no cytotoxic effect on rabbit kidney cells at concentrations of up to 640 µg/mL.

Epizootic features and control measures for lumpy skin disease in south-east Serbia in 2016.

Lumpy skin disease (LSD) is an infectious disease of cattle caused by virus of the Capripoxvirus genus (LSDV), family Poxviridae. Until 2015, it had not appeared in the Balkans. In June 2016, LSD spread throughout Serbia. This study analyses the first LSD occurrence, epizootic features, applied diagnostic procedures and control measures in five districts in south-east Serbia (Pcinja, Jablanica, Pirot, Toplica and Nisava). In total, there were 225 LSD outbreaks reported in Serbia, out of which 189 (84%) were located in the study area. The highest number of outbreaks was registered in Pcinja district (169), where LSD was first registered. The median distance and time between the nearest previous outbreak sites were calculated (4.32 km and 9 days). The median altitude of outbreak locations was 992 m with more than 90% above 500 m (p ≤ 0.001). The average herd morbidity rate in the study area was 13.6% and the herd mortality rate was recorded only in Pcinja (0.5%) and Jablanica (1.6%) districts. Samples taken from the cattle suspected to LSD were subjected to real time PCR analysis. Out of 233 samples tested for LSDV 132 (56.7%) were positive. The LSDV genome was identified in skin nodules (85.4%), blood (72.7%) and nasal swabs (62.5%). Phylogenetic analysis indicated that the LSDV strain circulating in Serbia fell within the cluster of field LSDV found worldwide. In response to the LSD epizootic, animal trade and movement were prohibited, complete stamping out, disinfection, disinsection and an entire bovine population vaccination using the homologous Neethling live attenuated vaccine (OBP, South Africa) were conducted. A month and a half after the completion of the vaccination campaign, the LSD epizootic was stopped, and no new cases have been reported since.

Characterization of velogenic Newcastle disease viruses isolated from dead wild birds in Serbia during 2007.

Avian paramyxoviruses type 1 or Newcastle disease viruses (NDV) are frequently recovered from wild birds and such isolates are most frequently of low virulence. Velogenic NDV are usually recovered from poultry and only occasionally from wild birds. Five NDV isolates were obtained from carcasses of four wild bird species during 2007 in Serbia: Mallard (Anas platyrhynchos), Eurasian Sparrowhawk (Accipiter nisus), feral Rock Pigeon (Columba livia), and Eurasian Collared Dove (Streptopelia decaocto). All the isolates have a typical fusion protein cleavage site motif of velogenic viruses ((112)R-R-Q-K-R-F(117)). The highest homology (99%) for the nucleotide sequences spanning the M and F gene of the studied isolates was with the genotype VII NDV isolate Muscovy duck/China(Fujian)/FP1/02. Phylogenetic analysis based on a partial F gene sequence showed that the isolates from wild birds cluster together with concurrent isolates from poultry in Serbia within the subgenotype VIId, which is the predominant pathogen involved currently in Newcastle disease outbreaks in poultry worldwide. It is unlikely that the wild birds played an important role in primary introduction or consequent spread of the velogenic NDV to domestic poultry in Serbia, and they probably contracted the virus from locally infected poultry.