Relating SARS-CoV-2 shedding rate in wastewater to daily positive tests data: A consistent model based approach.

During the COVID-19 pandemic, wastewater-based epidemiology (WBE) has been engaged to complement medical surveillance and in some cases to also act as an early diagnosis indicator of viral spreading in the community. Most efforts worldwide by the scientific community and commercial companies focus on the formulation of protocols for SARS-CoV-2 analysis in wastewater and approaches addressing the quantitative relationship between WBE and medical surveillance are lacking. In the present study, a mathematical model is developed which uses as input the number of daily positive medical tests together with the highly non-linear shedding rate curve of individuals to estimate the evolution of global virus shedding rate in wastewater along calendar days. A comprehensive parametric study by the model using as input actual medical surveillance and WBE data for the city of Thessaloniki (~700,000 inhabitants, North Greece) during the outbreak of November 2020 reveals the conditions under which WBE can be used as an early warning tool for predicting pandemic outbreaks. It is shown that early warning capacity is different along the days of an outbreak and depends strongly on the number of days apart between the day of maximum shedding rate of infected individuals in their disease cycle and the day of their medical testing. The present data indicate for Thessaloniki an average early warning capacity of around 2 days. Moreover, the data imply that there exists a proportion between unreported cases (asymptomatic persons with mild symptoms that do not seek medical advice) and reported cases. The proportion increases with the number of reported cases. The early detection capacity of WBE improves substantially in the presence of an increasing number of unreported cases. For Thessaloniki at the peak of the pandemic in mid-November 2020, the number of unreported cases reached a maximum around 4 times the number of reported cases.

Investigation on the incidence of adverse reactions, viraemia and haematological changes following field immunization of cattle using a live attenuated vaccine against lumpy skin disease.

The present study was performed to investigate the clinical impact and certain virological and haematological parameters following immunization of cattle against lumpy skin disease (LSD). The study was conducted in a dairy cattle farm (215 animals), immunized with a Neethling strain-based live vaccine. Twenty-seven animals (14 lactating cows, four dry cows and nine calves) were randomly selected for repetitive blood and saliva samplings. An EvaGreen-based real-time PCR was designed to differentiate vaccine from field LSDVs. Vaccinated animals underwent examination for adverse reactions. Nodule samples were collected from two representative cases for histopathological testing and virus identification. Milk yield was calculated based on bulk-tank measurements of all lactating cows (79). Viral DNA was detected between days 6-15 post-vaccination (p.v.) at 63% of the sampled animals (17/27). Saliva and bulk-tank milk samples were LSDV-negative. Pronounced swelling was observed at injection sites of 12% of the immunized animals (26/215), starting at day 6 p.v., and was resolved after 2-4 days. Small-sized (<0.5 cm) cutaneous lumps were developed between days 8-18 p.v. at 9% of the vaccinated animals (19/215). These were observed in adult cows and not in calves/heifers. Resolution was observable 10 days post-development. The vaccine virus was also identified in nodules and injection-site aspirates. Haematological changes (e.g., lower leucocyte counts) were observed in cows and not in calves. Daily milk production was being reduced during the first 12 days p.v. LSD immunization of cows resulted in nodules and low viraemia levels. The fact that nodules and haematological changes were not observed in calves, along with the low viraemia, supports the reduced virulence of the Neethling vaccine strain. The characteristic nodules in vaccinated animals could allow clinical differentiation from those observed in LSD. The developed real-time PCR efficiently differentiates infected from vaccinated cattle, and should be further validated as a tool in LSD surveillance.

Emergence of Equine West Nile Encephalitis in Central Macedonia, Greece, 2010.

During the summer of 2010, an outbreak of West Nile virus (WNV) infections attributed to a lineage 2 WNV strain was reported among humans and horses in Central Macedonia, Northern Greece. Here, the clinical and laboratory investigation of horses that showed severe neurological signs due to WNV infection is being described. Specifically, between August and September 2010, 17 horses with neurological signs were detected. WNV infection was confirmed in all 17 clinical cases by applying laboratory testing. The duration of WNV-specific IgM antibodies in sera obtained from seven of the clinically affected horses was relatively short (10-60 days; mean 44 days). In the regional unit of Thessaloniki, (i) seroprevalence of WNV and fatality rate in horses were high (33% and 30%, respectively), and (ii) the ratio of neurological manifestations-to-infections for this virus strain was high (19%). These observations indicate that the strain responsible for the massive human epidemic of 2010 in Greece was also highly pathogenic for horses. This is the first time that WNV infection has been documented in horses with clinical manifestations in Greece. WNV infection should be included in the differential diagnosis of horses with encephalitis in Greece.

Surveillance and Early Warning of West Nile Virus Lineage 2 Using Backyard Chickens and Correlation to Human Neuroinvasive Cases.

In 2010, a West Nile virus (WNV) epidemic was reported in Central Macedonia, Northern Greece, with 197 neuroinvasive disease (WNND) cases in humans. The following 3 years, WNV spreads to new areas of Greece and human cases reoccurred during the transmission periods. After the initial outbreak, a WNV surveillance system using juvenile backyard chickens was established in Central Macedonia (after the 2011 outbreak) and Eastern Macedonia-Thrace (after the 2012 outbreak). Sera were screened for the presence of antibodies against WNV using cELISA and serum neutralization test, to monitor the spread of WNV and to assess the correlation between the WNV point seroprevalence in chickens and the incidence rates of human WNND cases in the aforementioned areas. WNV seroprevalence in chickens was 10.4% (95% CI: 7-15) in Central Macedonia (2011) and 18.1% (95% CI: 14-23) in Eastern Macedonia-Thrace (2012). Seroprevalence in chickens and incidence rates of human WNND cases in Eastern Macedonia-Thrace were strongly positively correlated (ρ = 0.98, P = 0.005) at the regional unit level, with the incidence of WNND in humans increasing with increasing WNV point seroprevalence in chickens. In Central Macedonia, the correlation was weaker (ρ = 0.68, P = 0.20), apparently due to small number of reported human WNND cases. Another study was also conducted using juvenile backyard chickens in Central Macedonia, aiming to detect early WNV enzootic circulation, before the onset of human cases during 2011 and 2013. The first seroconverted chickens were detected about 1.5 months before the laboratory diagnosis of any human WNND cases in Central Macedonia, for both years. WNV surveillance, using juvenile backyard chickens, was reliable for the identification of areas with WNV enzootic and silent transmission, and for early warning. Timely diffusion of information to public health authorities facilitated the successful implementation of preparedness plans to protect public health.

Protective efficacy of Bluetongue virus-like and subvirus-like particles in sheep: presence of the serotype-specific VP2, independent of its geographic lineage, is essential for protection.

There have been multiple separate outbreaks of Bluetongue (BT) disease of ruminants in Europe since 1998, often entering via the Mediterranean countries of Italy, Spain and Greece. BT is caused by an orbivirus, Bluetongue virus (BTV), a member of the family Reoviridae. BTV is a non-enveloped double-capsid virus, which encodes 7 structural proteins (VP1-VP7) and several non-structural proteins (NS1, NS2, NS3/3a and NS4) from ten double-stranded RNA segments of the genome. In this report, we have prepared BTV virus-like particles (VLPs, composed of VP2, VP3, VP5 and VP7) and sub-viral, inner core-like particles (CLPs, VP3 and VP7) using a recombinant baculovirus expression system. We compared the protective efficacy of VLPs and CLPs in sheep and investigated the importance of geographical lineages of BTV in the development of vaccines. The Greek crossbred Karagouniko sheep, which display mild to sub-clinical BT, were vaccinated with VLPs or CLPs of BTV-1, derived from western lineage and were challenged with virulent BTV-1 from an eastern lineage. All VLP-vaccinated animals developed a neutralising antibody response to BTV-1 from both lineages prior to challenge. Moreover, post-challenged animals had no clinical manifestation or viraemia and the challenged virus replication was completely inhibited. In contrast, CLP-vaccinated animals did not induce any neutralising antibody response but developed the group specific VP7 antibodies. CLPs also failed to prevent the clinical manifestation and virus replication, but in comparison to controls, the severity of disease manifestation and viraemia was mitigated. The data demonstrated that the outer capsid was essential for complete protection, while the geographical origin of the BTV was not critical for development of a serotype specific vaccine.

Lactic acid bacteria from chicken carcasses with inhibitory activity against Salmonella spp. and Listeria monocytogenes.

This study was conducted to isolate psychrotrophic lactic acid bacteria (LAB) from chicken carcasses with inhibitory activity against strains of Salmonella spp. and Listeria monocytogenes. A total of 100 broiler samples were examined for the presence of LAB. Ninety-two LAB isolates that showed antimicrobial effects against Salmonella spp. and L. monocytogenes were further analysed to examine their LAB (Gram-positive, catalase negative, oxidase negative) and psychrotrophic characteristics (ability to grow at 7 °C). Fifty isolates were further selected and identified initially using standard biochemical tests in miniature (Micro-kits API CH 50) and then by sequencing of the 16s-23s rRNA gene boundary region (Intergenic Spacer Region). By molecular identification, these isolates were classified into 5 different LAB species: Lactobacillus salivarius, Lactobacillus reuteri, Lactobacillus johnsonii, Pediococcus acidilactici, and Lactobacillus paralimentarius. None of the isolates produced tyramine or histamine.

Characterization of the PRNP gene locus in Chios dairy sheep and its association with milk production and reproduction traits.

The objective of this study was to examine the prion protein gene locus (PRNP) in Chios sheep. PRNP is linked with scrapie resistance in small ruminants. Here, its impact on milk production (test-day and total lactation yield) and reproduction (age at first lambing, conception rate at first service, and prolificacy) was assessed. Genotyping at codons 136, 154 and 171 (classical scrapie) and 141 (atypical scrapie) was performed using DNA from milk somatic cells and PCR-RFLP analysis. A total of 1013 Chios ewes raised in 23 flocks were used. This constituted a random sample of the national breeding population. A total of 15 genotypes and 6 alleles linked to codons 136, 154 and 171 were detected. All animals were homozygous for the leucine allele at codon 141. Linear mixed models were used to assess the impact of PRNP genotypes and alleles on milk production and reproduction traits. The TRQ allele, whose association with such traits was assessed for the first time, had an adverse effect on age at first lambing. All other PRNP alleles, including ARR, which is associated with increased resistance to classical scrapie, had no significant effect on the traits studied. No significant associations of the PRNP genotypes with production and reproduction traits were observed. It was concluded that selection for scrapie-resistant sheep is not expected to affect the ongoing breeding programme that aims to enhance the milk yield and reproduction of the Chios breed.

Assessment of bluetongue viraemia in sheep by real-time PCR and correlation with viral infectivity.

Inoculation of embryonated chicken eggs is the standard method for the titration of infectious Bluetongue virus (BTV). Here, six RNA extraction methods coupled with optimised dsRNA denaturation and real-time RT-PCR were evaluated for the quantitation of BTV in blood samples from experimentally infected sheep and results were correlated to infectious virus titres. An exogenous dsRNA internal control (IC) from the closely related Epizootic hemorrhagic disease virus (EHDV) was used to assess the efficiency of BTV genome extraction, dsRNA denaturation, RT, and PCR amplification. Recovery rates of IC and BTV dsRNA copies from extracted blood samples were highly correlated. Adjustment of BTV concentrations according to the IC recovery reduced variation in sample analyses among the different extraction methods and improved the accuracy of BTV quantitation. The EID(50)/ml titre, determined in blood samples from sheep infected experimentally with BTV-1 or BTV-9, correlated highly with the assessed concentration of BTV dsRNA copies. However, this correlation was consistent only during the first 28 days post-infection. The optimised extraction methods and quantitative RT-PCR could be useful for experimental studies of BTV transmission, pathogenesis and vaccine efficacy, or adapted further for the detection and quantitation of EHDV, African horse sickness virus and other dsRNA viruses.

Detection and quantification of infectious avian influenza A (H5N1) virus in environmental water by using real-time reverse transcription-PCR.

Routes of avian influenza virus (AIV) dispersal among aquatic birds involve direct (bird-to-bird) and indirect (waterborne) transmission. The environmental persistence of H5N1 virus in natural water reservoirs can be assessed by isolation of virus in embryonated chicken eggs. Here we describe development and evaluation of a real-time quantitative reverse transcription (RT)-PCR (qRT-PCR) method for detection of H5N1 AIV in environmental water. This method is based on adsorption of virus particles to formalin-fixed erythrocytes, followed by qRT-PCR detection. The numbers of hemagglutinin RNA copies from H5N1 highly pathogenic AIV particles adsorbed to erythrocytes detected correlated highly with the infectious doses of the virus that were determined for three different types of artificially inoculated environmental water over a 17-day incubation period. The advantages of this method include detection and quantification of infectious H5N1 AIVs with a high level of sensitivity, a wide dynamic range, and reproducibility, as well as increased biosecurity. The lowest concentration of H5N1 virus that could be reproducibly detected was 0.91 50% egg infective dose per ml. In addition, a virus with high virion stability (Tobacco mosaic virus) was used as an internal control to accurately monitor the efficiency of RNA purification, cDNA synthesis, and PCR amplification for each individual sample. This detection system could be useful for rapid high-throughput monitoring for the presence of H5N1 AIVs in environmental water and in studies designed to explore the viability and epidemiology of these viruses in different waterfowl ecosystems. The proposed method may also be adapted for detection of other AIVs and for assessment of their prevalence and distribution in environmental reservoirs.

Complete genome analysis and immunodetection of a member of a novel virus species belonging to the genus Ampelovirus.

A new grapevine leafroll-associated virus isolate (GLRaV-Pr) from Greek grapevines was recently reported. This virus, along with the genetically related GLRaV-4, -5, -6 and -9, form a separate diverse lineage within the genus Ampelovirus. In this paper, the complete nucleotide sequence of GLRaV-Pr was determined, making it the first fully sequenced virus of this lineage. Its genome is 13,696 nt long and contains seven open reading frames, which potentially encode a 253-kDa polyprotein containing papain-like protease, methyltransferase, AlkB and helicase domains, a 58.2-kDa RNA-dependent RNA polymerase, a 5.2-kDa hydrophobic protein, a 58.5-kDa heat shock 70 protein homologue, a 60-kDa protein, a 30-kDa coat protein (CP) and a 23-kDa protein. A virus-specific antibody was raised against the recombinant CP of GLRaV-Pr and was applied in western blot analysis. The genomic, serological and phylogenetic data reported here confirm that GLRaV-Pr is a member of a distinct Ampelovirus species. Comparisons of GLRaV-Pr with the only available genetically related, fully sequenced virus, PMWaV-1, PBNSPaV and the partially sequenced GLRaV-9 revealed that this lineage, including GLRaV-4, -5, -6, -9 and -De, exhibits a high uniformity of genome organization and includes the smallest and simplest viruses within the family Closteroviridae.

Demarcation of ilarviruses based on the phylogeny of RNA2-encoded RdRp and a generic ramped annealing RT-PCR.

In this study, a generic ramped-annealing (RAN) nested RT-PCR was developed, allowing the simultaneous detection and fast characterization of ilarviruses. The method involves a one-step RT-PCR in which a pair of degenerate primers amplifies a 381-bp part of the polymerase gene (RNA2), followed by a nested PCR amplification that increases detection sensitivity. The sensitivity and detection range of the method were further increased by applying a ramped annealing thermocycling step both in the first RT-PCR and in the subsequent nested PCR. The 371-bp nested amplicons can be sequenced directly, without cloning, to obtain initial sequence information on ilarvirus genomes, or can undergo a restriction enzyme analysis for rapid identification of already known virus species. Phylogenetic relationships among different members of the family Bromoviridae were inferred with maximum likelihood and Bayesian analysis, using published homologous partial amino acid sequences corresponding to the nested amplicon and also to a longer residue data set (432-453 aa) comprising all possible positions of homology among the RNA2-encoded polymerases of members of the family Bromoviridae. The implications of these analyses on the taxonomy of ilarviruses are discussed. The specific partial polymerase sequence, corresponding to the polymerase core palm structure (motifs A-D), was verified as phylogenetically informative and can be used to separate ilarviruses from other members of the family Bromoviridae, providing initial information for ilarvirus species characterization. However, the phylogenetic signal of this region is not reliable for inferring relationships among distantly related ilarviruses.

Development of a semi-nested PCR using degenerate primers for the generic detection of small ruminant lentivirus proviral DNA.

A PCR assay was developed for the reliable detection of small ruminant lentivirus (SRLV) proviral DNA. The method involved the use of degenerate deoxyinosine-substituted primers and a second semi-nested PCR step that increased the polyvalency and sensitivity of the detection, respectively. Primers were designed from the pol gene conserved motifs of 85 SRLV isolates and were evaluated using different SRLV isolates together with Maedi-Visna virus (MVV) and caprine arthritis-encephalitis virus (CAEV) reference strains. The method successfully detected SRLV proviral DNA in total DNA extracts originating from whole blood samples, separated peripheral blood mononuclear cells (PBMCs) and tissue cultures. The semi-nested PCR was compared with the agar gel immunodiffusion test and proved to be highly sensitive, specific and capable of detecting many SRLV variants in infected or suspect animals. Therefore, it would be useful in the diagnosis of natural SRLV infections, in eradication programs and epidemiological studies. Whole blood samples can be used directly, thus alleviating the need for PBMC separation, and thereby enables a simple, fast and cost-effective analysis of a large number of samples.

A spot multiplex nested RT-PCR for the simultaneous and generic detection of viruses involved in the aetiology of grapevine leafroll and rugose wood of grapevine.

A spot nested RT-PCR assay using degenerate deoxyinosine-containing primers was developed, allowing rapid and simultaneous detection of Closterovirus sequences. Nested PCR amplification increased the specificity and sensitivity of detection. The sensitivity was also increased by a factor of 10 by using in addition to the deoxyinosine (dI)-containing primers, respective homologous primers in which dI was substituted by dG in the region of sequence homology. These homologous primers are shorter, having lower degeneracy and higher amplification efficiency than the dI-containing primers. This method was coupled to a similar nested RT-PCR detection method for Vitivirus and Foveavirus sequences. This permitted multiplex RT-PCR amplification of sequences belonging to the three genera in the same reaction tube and the two subsequent nested PCR amplifications (one for closteroviruses and one for viti- and foveaviruses) to run in parallel. Different primers and amplification parameters (additives and thermocycling conditions) were evaluated and optimised, respectively, in order to amplify efficiently all different templates. These improvements permitted the multiplex detection of fovea- and closteroviruses in petiole and cortical scraping preparations from 23 infected field-grown grapevines throughout the year, with the exception of GLRaV-1 in petioles that was only possible from June onwards. Preliminary results show that this method can detect reliably virus species from three genera in grapevine allowing simple, fast and cost-effective testing of a large number of samples in certification schemes.

A spot nested RT-PCR method for the simultaneous detection of members of the Vitivirus and Foveavirus genera in grapevine.

A reverse transcription (RT) polymerase chain reaction (PCR) assay was developed to allow rapid, and simultaneous detection of Vitivirus and Foveavirus sequences in two steps. The method involved a one-step RT-PCR, in which the combination of degenerate deoxyinosine-substituted primers amplified part of the polymerase region of both genera, followed by a nested PCR amplification that increased specificity and sensitivity of detection. The increase in sensitivity also permitted the use of a simple and rapid template preparation protocol, involving the spotting of plant sap extract on a nylon membrane. Consistent amplification with infected grapevine plants was possible after inclusion of additives for inhibiting polyphenolic compounds during template preparation. This spot nested RT-PCR method can reliably detect virus species of both genera in grapevine allowing simple, fast, and cost-effective analysis of a large number of samples in certification schemes.

Multiplex Detection of Criniviruses Associated with Epidemics of a Yellowing Disease of Tomato in Greece.

Since 1997, a yellowing disease has been observed in greenhouse tomato (Lycopersicon escu-lentum). By 2001, the disease was widespread, including open field tomato crops, and in most cases its incidence was 80 to 90% or even 100%. Epidemics in glasshouses were mainly associated with high populations of the whitefly Trialeurodes vaporariorum and Bemisia tabaci, the major whitefly pests in vegetable crops in Greece. The main leaf symptoms were severe yellowing, rolling, and brittleness. Samples from symptomatic plants were analyzed by polymerase chain reaction (PCR) and shown to be infected with Tomato infectious chlorosis virus (TICV) and Tomato chlorosis virus (ToCV) (family Closteroviridae, genus Crinivirus). TICV was found in 164 of 183 symptomatic samples, while ToCV was less representative (25/183). Sequence comparisons of the amplified 229-bp and 466-bp products revealed 99 and 100% identity with the reported sequences of TICV and ToCV, respectively. A reverse transcription (RT) multiplex PCR assay using a simple sample preparation procedure was developed to allow rapid, specific, and simultaneous detection of both ToCV and TICV sequences in two steps. The method involves a one-tube RT-PCR step in which the combination of primers amplifies part of the heat shock protein to homologue gene of both ToCV and TICV, followed by a multiplex nested PCR amplification. This is the first report of TICV and ToCV in Greece and, as far as we know, the first report of TICV in Europe.

Optimization of random-amplified polymorphic DNA producing amplicons up to 8500 bp and revealing intraspecies polymorphism in Leishmania infantum isolates.

In the case of investigation of polymorphism in closely related strains, the highest possible complexity of the patterns obtained by random-amplified polymorphic DNA PCR (RAPD-PCR) is required to assure revealing of limited polymorphism. In the present work, most parameters (reaction components concentration, additives, different polymerases, and thermal profiles) affecting RAPD-PCR were examined, in an effort to increase pattern complexity. A long PCR thermal profile, betaine as cosolvent, and Dynazyme EXT polymerase produced longer amplicons and higher pattern complexity, revealing polymorphism among Leishmania infantum isolates from infected dogs originating from Northern Greece (Macedonia).

First Report of Tomato yellow leaf curl virus on Tomato Crops in Greece.

In late summer 2000, tomato (Lycopersicon esculentum Mill.) grown in greenhouses in Ierapetra, Tympaki, and Chania (Crete) showed leaf curling, reduced leaf size, yellowing, shortened internodes, and a bushy appearance. More than 30 ha of tomato greenhouses were affected and the disease incidence ranged from 15 to 60% with estimated crop losses of over $500,000. Similar symptoms were observed in tomato samples from Marathon (Attiki) and Southern Peloponnese. All greenhouses with infected plants were infested with high populations of Bemisia tabaci (Gennadius), which were also observed outside the greenhouses on several weeds. Tomato symptoms were similar to those caused by Tomato yellow leaf curl virus (TYLCV). The assumed virus could not be transmitted mechanically but successful transmission was obtained by grafting onto healthy tomato plants. Over 100 samples of symptomatic tomato plants collected from Crete and southern Peloponnese gave positive reactions when tested by ELISA using monoclonal antibodies to TYLCV-European (Adgen Ltd). The serological results were confirmed by PCR using two pairs of primers, universal degenerate (1) and MA 13 and MA 17 (2), amplifying different parts of the virus genome. The restriction fragment length polymorphism (RFLP) analysis (AluI, HaeIII, and TaqI) of the 541 bp amplicon obtained with the degenerate primers showed patterns similar to TYLCV-Is (Israeli species). The second pair of primers gave the expected 348 bp product, which was sequenced. Sequence comparisons revealed 99% identity with TYLCV-Is (EMBL no. X15656, X76319). The resulting sequence was at least 97.7% identical to sequences of TYLCV isolates from the Dominician Republic (EMBL no. AF024715), Cuba (EMBL no. AJ223505), Portugal (EMBL no. AF105975), Iran (EMBL no. AJ13271), and Spain (EMBL no. AF071228). The disease appeared for the first time in 1992 in Tymbaki, but was limited to very few plants in one glasshouse. However, the cause was not determined. To our knowledge, this is the first report of TYLCV of the Begomovirus genus in Greece. References: (1) D. Deng et al. Ann. Appl. Biol. 125:327, 1994. (2) J. Navas-Castillo et al. J. Virol. Methods 75:195, 1998.

A New Ilarvirus Isolated from Grapevine in Greece.

In 1994, characteristic viruslike symptoms on grapevine were reported in the collection of the Grapevine Institute in Athens, Greece, on the hybrid Baresana × Baresana. The symptoms were sharp angular mosaic, leaf crinkle, and little leaf. The affected vines showed gradual decline and severe stunting or death. Such vines produced abortive flowers or very few berries with smaller, wrinkled, and nongerminating seeds. Serological testing, by enzyme-linked immunosorbent assay (ELISA), of the affected vines against the most common grapevine viruses Alfalfa mosaic, Arabis mosaic, Grapevine fanleaf, Grapevine fleck, Grapevine A, Rasberry ringspot, and grapevine leafroll-associated viruses gave negative results. A virus was isolated from affected grapevine young leaves by mechanical inoculation of Gomphrena globosa and single lesioned. The virus host range included G. globosa (local and systemic dark red or necrotic lesions), Chenopodium quinoa (necrotic local lesions and systemic mottle), and three tobacco cultivars (sharp necrotic local lesions, 1 to 3 mm in diameter). Pollination of C. quinoa with pollen from infected plant gave about 30% infected seedlings. The virus was purified from C. quinoa by differential centrifugation using 0.02 M phosphate buffer pH 8.0, containing 0.01 M DIECA and 0.01 M sodium thioglycolate as extraction buffers. In a purified preparation, quasisphaerical virus particles of about 29 nm were observed. Electrophoretic mobility of the viral coat protein showed a molecular weight of 30 kDa. Using purified preparations, an antiserum was obtained with a titer of 1:1024 in microprecipitin test and an optimum IgG dilution in ELISA of 1:10,000 for maximum absorption at OD405 nm Using degenerate primers designed from homologous regions in RNA-2 corresponding to a fragment of the polymerase gene of Ilarviruses, the expected 381-bp polymerase chain reaction product was obtained. This product was cloned and sequenced. Comparisons with sequence data from the homologous regions of RNA-2 of other known Ilarviruses, showed that the sequence of the above 381-bp amplicon shared 72% sequence similarity with Tobacco streak virus, 67% of Citrus variegation virus and Spinach latent virus, 66% of Asparagus virus 2 and Elm mottle virus, and 65% of Citrus leaf rugose virus. Based on the above data, it is concluded that the isolated virus is an Ilarvirus with closest similarity to Tobacco streak virus. From the relative bibliography (1-3) it appears that the virus reported here is different from Grapevine line pattern virus, a possible Ilarvirus, previously reported from Hungary. References: (1) J. Lehoczky et al. Kertgazdasag 19:61, 1987. (2) J. Lehoczky et al. Phytoparasitica 17:59, 1989. (3) J. Lehoczky et al. Phytopathol. Medit. 31:115, 1992.