Pooled Tissue Samples Research Articles

Localization of Iris Yellow Spot Virus in Naturally Infected Onion Plants: A Sampling Approach for Reliable Diagnosis

Iris yellow spot virus (IYSV) from the genus Orthotospovirus of the family Tospoviridae can cause considerable yield reduction and crop loss in allium crops, especially dry bulb onion. While infected plants may or may not express symptoms of iris yellow spot disease, these onion plants can test negative for the virus because not all tissues contain detectable levels of IYSV due to its uneven distribution. The purpose of this study was to identify tissue locations of symptomatic onion plants that have the highest likelihood of testing positive for IYSV in DAS-ELISA using specific antibodies. Results revealed that the greatest likelihood of detecting IYSV from a single symptomatic plant occurred with tissue near foliar lesions located on the distal portions of older leaves (range: 42-71%). These low to moderate probability detection levels can predictably be overcome by testing pooled tissue samples from the same plant or from multiple plants. Indeed, the likelihood of distal and proximal youngest leaf tissue testing positive for IYSV from a single plant to a four-plant composite sample would increase from 44% to 90% and from 50% to 94%, respectively. Implications of these results for IYSV detection and surveillance in onion production systems are discussed.

Monitoring of Astroviruses, Brno-Hantaviruses, Coronaviruses, Influenza Viruses, Bornaviruses, Morbilliviruses, Lyssaviruses and Pestiviruses in Austrian Bats.

Here, we report the results of a monitoring study of bat viruses in Austria to strengthen the knowledge of circulating viruses in Austrian bat populations. In this study, we analyzed 618 oropharyngeal and rectal swab samples from 309 bats and 155 pooled tissue samples from dead bats. Samples were collected from 18 different bat species from multiple locations in Austria, from November 2015 to April 2018, and examined for astroviruses, bornaviruses, coronaviruses, hantaviruses, morbilliviruses, orthomyxoviruses (influenza A/C/D viruses), pestiviruses and rhabdoviruses (lyssaviruses) using molecular techniques and sequencing. Using RT-qPCR, 36 samples revealed positive or suspicious results for astroviruses, Brno-hantaviruses, and coronaviruses in nine different bat species. Further sequencing revealed correspondent sequences in five samples. In contrast, none of the tested samples was positive for influenza viruses A/C/D, bornaviruses, morbilliviruses, lyssaviruses, or pestiviruses.

Duck Tembusu virus in North Vietnam: epidemiological and genetic analysis reveals novel virus strains.

Tembusu virus (TMUV) is an important infectious disease, causing economic losses in duck production. Since the first report of TMUV infection in Vietnam in 2020, the disease has persisted and affected poultry production in the country. This study conducted epidemiological and genetic characterization of the viral strains circulating in north Vietnam based on 130 pooled tissue samples collected in six provinces/cities during 2021. The TMUV genome was examined using conventional PCR. The results indicated that 21 (16.15%) samples and 9 (23.68%) farms were positive for the viral genome. The positive rate was 59.26% for ducks at ages 2-4 weeks, which was significantly higher than for ducks at ages >4 weeks and < 2 weeks. Genetic analysis of the partial envelope gene (891 bp) sequences indicated that the five Vietnamese TMUVs shared 99.55-100% nucleotide identity, while the rates were in the range 99.59-100% based on the pre-membrane gene sequences (498 bp). The five Vietnamese TMUV strains obtained formed a novel single subcluster. These strains were closely related to Chinese strains and differed from the vaccine strain, suggesting that Vietnamese TMUV strains were field viruses. It needs to be further studied on vaccine development to prevent effects of TMUV infection on poultry production across Vietnam.

Porcine circovirus 2d associated with stillborn mummified foetus of pig

Recurrent occurrence of stillborn piglets was noticed along with live births during farrowing in pigs in an organized pig farm of Uttar Pradesh. On detailed post mortem examination, externally, the foetus was found to be partially dehydrated and dark brown in colour with shrunken eyes. Internally, visceral organs including lungs, heart, kidneys, liver, spleen and intestine were partially dehydrated and dark brown in colour. Upon molecular investigation, the pooled tissue samples were found positive for porcine circovirus 2 (PCV2) infection using PCR technique and further confirmed the PCV2d genotype by DNA sequencing. Other most possible infections including porcine parvoviral infection (PPV), PCV3 and porcine respiratory and reproductive syndrome virus infection (PRRS) were found negative using PCR and RT-PCR techniques, respectively. This result is suggestive of role of PCV2d as a possible etiological agent to cause stillbirth and mummification of porcine foetuses.

Ovine adenoviruses infecting sheep and goats in Türkiye: detection and molecular characterization of three different types.

Adenoviruses (AdVs) have been detected in a wide variety of animals. To date, eight types of AdVs in sheep and two types in goats have been identified, which belong to two distinct genera, Mastadenovirus and Atadenovirus. Typically, the term pneumo-enteritis is used to describe adenovirus-induced disease in small ruminants, which has been associated with both enteric and respiratory symptoms of varying severity. The aim of this study was to detect and identify AdVs of small ruminants belonging to the genera Mastadenovirus and Atadenovirus. For this purpose, diagnostic samples (47 lung, 27 intestine, and two pooled tissue samples including intestine and lung) from 49 small ruminants (39 sheep and 10 goats) were used. Following the viral DNA extraction, PCR was carried out by using the primers targeting the hexon gene in order to detect both mast- and atadenoviruses. Sequencing the amplified fragments revealed the presence of three types of ovine adenovirus (OAdV): OAdV-3, OAdV-4, and OAdV-8. Specifically, OAdV-3 was detected in two sheep and a goat while OAdV-4 and OAdV-8 were found in only one sheep each. There is still limited data on the interaction between the viruses in different adenovirus genera and the detected disease, as well as the genetic diversity of adenoviruses, especially in small ruminants. In conclusion, the detection of AdVs in lung and intestinal tissues of small ruminants in this study suggests that these viruses may have contributed to the disease and/or predisposed to other agents.

TMT-Based Comparative Peptidomics Analysis of Rice Seedlings under Salt Stress: An Accessible Method to Explore Plant Stress-Tolerance Processing.

Rice (Oryza sativa L.) is an important staple crop, particularly in Asia, and abiotic stress conditions easily reduce its yields. Salt stress is one of the critical factors affecting rice growth and yield. In this study, a tandem mass tag (TMT)-based comparative peptidomics analysis of rice seedlings under salt stress was conducted. Rice seedlings were exposed to 50 and 150 mM NaCl for 24 and 72 h, respectively, and the root and shoot tissues of different treatment groups were collected separately for peptidomics analysis. A total of 911 and 1263 nonredundant peptides were identified in two pooled shoot tissue samples, while there were 770 and 672 nonredundant peptides in two pooled root tissue samples, respectively. Compared with the control groups, dozens to hundreds of differentially expressed peptides (DEPs) were characterized in all treatment groups. To explore the potential functions of these DEPs, we analyzed the basic characteristics of DEPs and further analyzed the annotated Gene Ontology terms according to their precursor proteins. Several DEP precursor proteins were closely related to the response to salt stress, and some were derived from the functional domains of their corresponding precursors. The germination rate and cotyledon greening rate of transgenic Arabidopsis expressing two DEPs, OsSTPE2 and OsSTPE3, were significantly enhanced under salt stress. The described workflow enables the discovery of a functional pipeline for the characterization of the plant peptidome and reveals two new plant peptides that confer salinity tolerance to plants. Data are available via ProteomeXchange with identifier PXD037574.

Tembusu virus infection in laying chickens: Evidence for a distinct genetic cluster with significant antigenic variation.

Tembusu virus (TMUV) associated disease is a growing cause of egg production decrease and encephalitis in domestic waterfowl, with expanding distribution. In previous studies, TMUV isolates were phylogenetically classified into two genetic lineages and different clusters with varied pathogenicity. However, little is known about the phenotypic and virulence characteristics of cluster 3 isolates within the duck TMUV lineage. In this study, the etiological agent causing egg drop in a laying chicken farm in southern China was investigated and a TMUV was isolated from pooled tissue samples. Genome sequencing and phylogenetic analysis grouped the isolate into TMUV cluster 3 with closest relation to the mosquito-origin TMUV YN12193. Cross-neutralization testing using convalescent sera revealed significant antigenic variation between the isolate and a representative strain of cluster 2.2. The experimental infection of SPF hens confirmed the ability of the isolate to replicate in multiple tissues and led to ovary damage. Additionally, high seroconversion rates (95.83%-100%) were detected in the three flocks following retrospective investigation. Our study demonstrates the occurrence of cluster 3 TMUV infection in laying chickens and that the virus exhibits significant antigenic variation compared with cluster 2 TMUV.

Rapid and specific detection of Mycoplasma gallisepticum and Mycoplasma synoviae infection in poultry using single and duplex PCR assays

Avian mycoplasmosis, mainly caused by Mycoplasma gallisepticum (MG) and Mycoplasma synoviae (MS), is an economically important disease of poultry industry. The present study was aimed to develop duplex PCR as a rapid, specific and economical method for accurate detection of MG and MS in poultry and its comparison with single (monoplex) MG/MS PCR. During present investigation, a total of 146 poultry flocks having clinical history of respiratory disease were screened. Pooled tissue samples (trachea, lungs and air sacs) from 4-5 birds of each flock were collected during necropsy at disease investigation laboratories, Hisar, Haryana, India. The single and duplex PCR assays were standardized using primers of intergenic spacer region (IGSR; 16S-23S rRNA) for MG and hemagglutinin vlhA gene for MS, with expected amplicon size of 812 bp and 1200 bp products, respectively. In single PCR, 6.85%, 2.74% and 2.74% tissue samples were found positive for MG, MS and both MG and MS, respectively. However, duplex PCR showed, 7.53%, 2.74% and 1.37% positivity for MG, MS and both MG and MS, respectively. Taking the results of monoplex PCR as a gold standard, sensitivity and specificity of the developed duplex PCR was found to be 94.44% and 100%, respectively. Moreover, Cohen's kappa statistic (k = 0.97) measured a ‘perfect’ agreement between monoplex and duplex PCR assays. The positive and negative predictive values of duplex PCR was found to be 1.0 and 0.9922, respectively at 95% confidence interval (CI), as compared to monoplex PCR. The simultaneous use of two genes in a duplex PCR was more rapid and economical than two separate single PCR reactions.

Isolation and Phylogenetic Analysis of Avian Mycoplasmas from Poultry Affected with Respiratory Infections in India

Mycoplasma gallisepticum (MG) and Mycoplasma synoviae (MS) are the two most pathogenic avian mycoplasmas. In the present study, examination of 92 pooled tissue samples from broiler chicken of 92 different poultry flocks of Haryana (India) exhibiting respiratory infections resulted in isolation of 13 (14%) Mollicutes. Based on biochemical reactions, growth inhibition test, PCR and/or sequencing, 8 (8.6%) isolates could be characterized as MG, 1 (1.08%) as MS, 3 (3.24%) as M. gallinarum and 1 (1.08%) as Acholeplasma laidlawii. The phylogenetic analysis using Intergenic spacer region (IGSR) of these MG isolates revealed that they clustered with USA strain whereas the vaccine strains were in different clade. Single locus sequence typing (SLST) revealed considerable nucleotides variation between 8 MG isolates and vaccine strains. Conclusively, Sequencing of IGSR region of MG can be used as a valuable epidemiological investigation tool for the differentiation of wild-type MG strains from vaccine strains.

A Multiplexed Microarray Platform for Targeted Proteomics

A Multiplexed Microarray Platform for Targeted Proteomics

Orthologous nuclear markers and new transcriptomes that broadly cover the phylogenetic diversity of Acanthaceae.

PremiseInformation on orthologous groups of genes, their sequence variability, and annotation is required for project design in phylogenetic reconstruction. This resource is unavailable for the flowering plant family Acanthaceae (>4000 species).MethodsWe compared transcriptome sequences spanning the extant diversity of Acanthaceae in order to provide a set of orthologous low‐copy nuclear genes and assess their utility for reconstructing phylogenetic relationships within this group of plants.ResultsWe present new transcriptome assemblies for eight species representing all major clades of Acanthaceae. The assemblies of five of these species are entirely based on new sequence data. Of these five species, three are from subfamilies for which no genomic resources were previously available (Nelsonioideae and Thunbergioideae). These five new transcriptomes are more complete than all others from public databases. Furthermore, we provide alignments with sequence information, annotation, and statistics for potential phylogenetic utility of 1619 orthologous low‐copy nuclear markers.DiscussionOur method of inferring assemblies from multiple pooled tissue samples delivers more complete transcriptomes than any available ones from Acanthaceae. We make available to the community new resources (e.g., sequence information, variability, and annotation of orthologous low‐copy nuclear genes) that will help phylogenetic reconstruction in Acanthaceae.

Occurrence of infectious bronchitis in layer birds in Plateau state, north central Nigeria.

A flock of 54 wk-old layer birds exhibiting signs of respiratory distress, greenish diarrhea, and drop in egg production was investigated. A marked drop in egg production (55%) was recorded with eggs appearing white and soft-shelled. Mortality was in the range of 1%–2% with post-mortem lesions revealing cloudy air sacs, frothy, and congested lungs. Viral RNA was extracted from pooled tissue samples (trachea, lungs, spleen, and liver) and tested for Avian influenza virus (AIV), Newcastle disease virus (NDV), and infectious bronchitis virus (IBV) by reverse transcriptase-polymerase chain reaction (RT-PCR). In addition, virus isolation was attempted in 9–11 day-old embryonating chicken eggs (ECE). In order to determine the prevalence of IBV serotype(s) in the flock, serum samples were screened by hemagglutination-inhibition (HI) test using IBV antigens and antisera (Arkansas, Connecticut, and Massachusetts). Neither AIV nor NDV but IBV was detected in the tissue samples by RT-PCR. In addition, virus isolate obtained after four serial passages in ECE produced dwarfed, stunted, and hemorrhagic embryos, and the isolate was confirmed by RT-PCR to be IBV. The serum samples were 100% seropositive for three serotypes with HI titres ranging from 5 to 12 Log2. In this study, IBV was confirmed as the causative agent of the observed respiratory distress and drop in egg production. Also, the evidence of co-circulation of multiple IBV serotypes was established, this to the best of our knowledge is the first of such report in Nigeria. We recommend extensive molecular and sero-epidemiology of circulating IBV genotypes and serotypes in Nigeria with the aim of developing better control strategies, including vaccination.

Concurrent Infection of Infectious Bronchitis (IB) and Sub-Clinical Infectious Bursal Disease (IBD) in an Organised Poultry Farm

Four carcasses of 4-5 days old chicks were submitted for post-mortem examination to the Department of Veterinary Pathology, College of Veterinary and Animal Sciences, Mannuthy, Thrissur. The owner reported 60% mortality in the flock. Grossly, chalky deposits could be observed on the pericardium, liver and in mesonephric tubules of the kidneys. Histopathology revealed focal pneumonia and congestion, uric acid pericarditis, fatty degeneration with capsular thickening in liver, degeneration and uric acid crystals in the renal tubules. Spleen and bursa revealed lymphoid depletion. Reverse-transcriptase polymerase chain reaction carried out with the pooled tissue samples gave positive results for both IB and IBD. The nephrogenic form of IB would have resulted in nephrosis, uric acid pericarditis and perihepatitis. Though the bursa did not reveal any striking gross pathology, the histopathological changes in the bursa and positive PCR results for IBD suggest a sub-clinical infection.

Duplex PCR for Direct Detection of Mycoplasma gallisepticum and Mycoplasma synoviae from Tissues of Poultry Affected with Respiratory Infections

In the present study, 92 pooled tissue samples including trachea, lungs and air sacs were collected from 92 different poultry flocks affected with respiratory infections and were subjected to mycoplasmological examination by duplex PCR. Result of duplex PCR revealed, 25/92 (27%) MG, 2/92 (2.1%) MS and 1 (1.08%) sample was positive for both MG and MS. In conclusion, duplex PCR was found to be a better assay to detect two most pathogenic avian mycoplasmas (M. gallisepticum and M. synoviae) simultaneously directly from poultry tissue sample and also could serve as a sensitive, specific and rapid tool in monitoring and surveillance programmes on poultry farms in Haryana. It seems to be the first study of this type in India.

Outbreak of encephalitic listeriosis in red-legged partridges (Alectoris rufa)

An outbreak of neurological disease was investigated in red-legged partridges between 8 and 28 days of age. Clinical signs included torticollis, head tilt and incoordination and over an initial eight day period approximately 30–40 fatalities occurred per day. No significant gross post mortem findings were detected. Histopathological examination of the brain and bacterial cultures followed by partial sequencing confirmed a diagnosis of encephalitis due to Listeria monocytogenes. Further isolates were obtained from follow-up carcasses, environmental samples and pooled tissue samples of newly imported day-old chicks prior to placement on farm. These isolates had the same antibiotic resistance pattern as the isolate of the initial post mortem submission and belonged to the same fluorescent amplified fragment length polymorphism (fAFLP) subtype. This suggested that the isolates were very closely related or identical and that the pathogen had entered the farm with the imported day-old chicks, resulting in disease manifestation in partridges between 8 and 28 days of age. Reports of outbreaks of encephalitic listeriosis in avian species are rare and this is to the best of our knowledge the first reported outbreak in red-legged partridges.

Isolation and molecular characterization of nephropathic infectious bronchitis virus isolates of Gujarat state, India.

Infectious bronchitis (IB) is a common, highly contagious, acute, and economically important viral disease of chickens caused by Infectious bronchitis virus (IBV, sp. Avian coronavirus). Five pooled tissue suspensions of 50 layer birds and one reference Massachusetts vaccine strain were inoculated into specific pathogen free (SPF) chicken egg for isolation of IBV. Reverse-transcription polymerase chain reaction (RT-PCR) was carried out using post inoculated allontoic fluid to amplify the spike (S) glycoprotein of S1 subunit of IBV. All the eggs inoculated with five pooled tissue samples and vaccine sample showed dwarfing and curling of SPF embryos indicative of IBV. All the five samples and the vaccine sample produced the expected amplicons of 466 bp by RT-PCR. The sequencing of five isolates revealed that all the five sequences were 99.09–100 % similar among themselves and showed 99.10–100 % nucleotide identity with the vaccine strain. On multiple sequence alignment it was found that our isolates were more similar at S1 subunit nucleotide level with the reference Ma5 and H120 vaccine strains than the reference Mass41 strain. The sequences of Anand isolates revealed further genetic changes in the circulating IBV in comparison to previous isolate of Gujarat as well as higher differences with the strains isolated in other states showing substantial changes at genetic level in Indian IBV isolates, which may partially explain the increasing incidences of IB in the country in spite of the vaccination.Electronic supplementary materialThe online version of this article (doi:10.1007/s13337-015-0248-x) contains supplementary material, which is available to authorized users.

Validation of Armadillo officinalis Dumèril, 1816 (Crustacea, Isopoda, Oniscidea) as a bioindicator: In vivo study of air benzene exposure

This study tests the potential for using Armadillo officinalis as a bioindicator of exposure to and activation of benzene metabolic pathways using an in vivo model.A. officinalis specimens collected in a natural reserve were divided into a control and three test groups exposed to 2.00, 5.32 or 9.09µg/m3 benzene for 24h. Three independent tests were performed to assess model reproducibility. Animals were dissected to obtain three pooled tissue samples per group: hepatopancreas (HEP), other organs and tissues (OOT), and exoskeleton (EXO). Muconic acid (MA), S-phenylmercapturic acid (S-PMA), two human metabolites of benzene, and changes in mtDNA copy number, a human biomarker of benzene exposure, were determined in each sample; benzene was determined only in EXO. MA was measured by high-performance liquid chromatography (HPLC) with ultraviolet (UV) detection, S-PMA by triple quadrupole mass spectrometer liquid chromatography with electro spray ionization (LC-MS-ESI-TQD), mtDNA by real-time quantitative PCR and end-point PCR, and benzene by quadrupole mass spectrometer head-space gas chromatography (HSGC-MS).MA and S-PMA levels rose both in HEP and OOT; EXO exhibited increasing benzene concentrations; and mtDNA copy number rose in HEP but not in OOT samples. Overall, our findings demonstrate that A. officinalis is a sensitive bioindicator of air benzene exposure and show for the first time its ability to reproduce human metabolic dynamics.

The presence of six potentially pathogenic viruses in pigs suffering from post-weaning multisystemic wasting syndrome.

BackgroundPorcine circovirus type 2 (PCV2) is an etiological agent of porcine circovirus diseases (PCVDs). Post-weaning multisystemic wasting syndrome (PMWS) as the most important PCVD is considered a multifactorial disease. It was demonstrated that not only PCV2 but several viruses are associated with PMWS. Studies of viral co-infections in PMWS pigs led often to controversial results. The aim of this work was to determine the presence of emerging (PRRSV), re-emerging (PTV) and newly-emerging (TTSuV1, TTSuV2, PBoV1) viruses in samples of dead pigs suffering from PMWS. The impact of vaccination against PCV2 and the influence of age on the occurrence of single and multiple viral infections in pigs were also investigated.ResultsViruses were detected by PCR, RT-PCR and real-time PCR in the pooled tissue samples (lymph nodes, liver and spleen) of pigs with PMWS (n = 56) which were divided into three groups: suckling piglets, post-weaning pigs and fattening pigs. In addition, lymph node samples were collected from apparently healthy fattening pigs (n = 59). The effect of vaccination against PCV2 with Ingelvac CircoFlex vaccine was also investigated. Between non-vaccinated pigs, the highest prevalence of individual viruses and multiple viral infections were found in diseased post-weaning and fattening animals with PMWS. Severe clinical disease was observed in swine co-infected with PCV2 and PRRSV. The prevalence of TTSuV1 and TTSuV2 was high in all groups of pigs and did not appear to have a significant effect on the syndrome. Simultaneous infection with TTSuV1 and PBoV1 was frequently confirmed in pigs with PMWS. No healthy pig was found to be infected with PRRSV, PTV or PBoV1. Vaccination against PCV2 did not influence the prevalence of TTSuVs, but significantly protected pigs against multiple viral infections.ConclusionsPost-weaning PMWS pigs were more often co-infected with viral pathogens than suckling or fattening pigs. Co-infection with PRRSV enforces clinical signs of PMWS, the influence of other viral co-infections is not clear. Vaccination against PCV2 significantly reduced viral co-infections in pigs.

Analysis of the Skin Transcriptome in Two Oujiang Color Varieties of Common Carp

BackgroundBody color and coloration patterns are important phenotypic traits to maintain survival and reproduction activities. The Oujiang color varieties of common carp (Cyprinus carpio var. color), with a narrow distribution in Zhejiang Province of China and a history of aquaculture for over 1,200 years, consistently exhibit a variety of body color patterns. The molecular mechanism underlying diverse color patterns in these variants is unknown. To the practical end, it is essential to develop molecular markers that can distinguish different phenotypes and assist selective breeding.Methodology/Principal FindingsIn this exploratory study, we conducted Roche 454 transcriptome sequencing of two pooled skin tissue samples of Oujiang common carp, which correspond to distinct color patterns, red with big black spots (RB) and whole white (WW), and a total of 737,525 sequence reads were generated. The reads obtained in this study were co-assembled jointly with common carp Roche 454 sequencing reads downloaded from NCBI SRA database, resulting in 43,923 isotigs and 546,676 singletons. Over 31 thousand (31,445; 71.6%) isotigs were found with significant BLAST matches (E<1e-10) to the nr protein database, which corresponds to 12,597 annotated zebrafish genes. A total of 70,947 isotigs and singletons (transcripts) were annotated with Gene Ontology, and 60,221 transcripts were found with corresponding EC numbers. Out of 145 zebrafish pigmentation genes, orthologs for 117 were recovered in Oujiang color carp transcriptome, including 18 found only among singletons. Our transcriptome analysis revealed over 52,902 SNPs in Oujiang common carp, and identified 63 SNP markers that are putatively unique either for RB or WW.ConclusionsThe transcriptome of Oujiang color varieties of common carp obtained through this study, along with the pigmentation genes recovered and the color pattern-specific molecular markers developed, will facilitate future research on the molecular mechanism of color patterns and promote aquaculture of Oujiang color varieties of common carp through molecular marker assisted-selective breeding.

Monitoring West Nile virus (WNV) infection in wild birds in Serbia during 2012: first isolation and characterisation of WNV strains from Serbia

West Nile virus (WNV), a neurovirulent mosquito-transmissible zoonotic virus, has caused recent outbreaks in Europe, including Serbia from August until October 2012. Although humans can be infected, birds are the main natural WNV reservoir. To assess WNV circulation in northern Serbia, 133 wild birds were investigated. These comprised resident and migratory birds, collected between January and September 2012 in the Vojvodina province. The birds belonged to 45 species within 27 families. Blood sera (n=92) and pooled tissues from respective birds (n=81) were tested by enzyme-linked immunosorbent assay (ELISA), plaque reduction neutralisation test (PRNT) and real-time reverse transcription-polymerase chain reaction (RT-qPCR). WNV antibodies were detected in seven (8%) sera: four from Mute Swans (Cygnus olor), two from White-tailed Eagles (Haliaeetus albicillas), and one from a Common Pheasant (Phasianus colchicus). Five sera neutralised WNV but not Usutu virus. For the first time in Serbia, WNV RNA was detected by RT-qPCR in pooled tissue samples of eight respective birds. WNV RNA was also derived from an additional bird, after a serum sample resulted infective in cell culture. The total nine WNV RNA positive birds included three Northern Goshawks (Accipiter gentilis), two White-tailed Eagles, one Legged Gull (Larus michahelis), one Hooded Crow (Corvus cornix), one Bearded Parrot-bill (Panarus biramicus), and one Common Pheasant. Phylogenetic analysis of partial E region sequences showed the presence of, at least, two lineage 2 Serbian clusters closely related to those responsible for recent human and animal outbreaks in Greece, Hungary and Italy. Full genomic sequence from a goshawk isolate corroborated this data. These results confirm WNV circulation in Serbia and highlight the risk of infection for humans and horses, pointing to the need for implementing WNV surveillance programmes.