Discovery of a novel bat lyssavirus in a Long-fingered bat (Myotis capaccinii) from Slovenia.

Lyssaviruses are the causative agents of rabies, a zoonotic, fatal disease that is thought to be ancestral to bats. In the last decade, the detection of bat associated lyssaviruses is increasing also in Europe. Within a retrospective bat associated lyssavirus surveillance study a total of 225 dead bats of 21 bat species were collected in Slovenia between 2012 and 2019 and tested by specific real-time RT-PCR method. The first lyssavirus positive sample in bats in Slovenia was detected using the real-time RT-PCR, the fluorescent antibody test, and next generation sequencing, while the rabies tissue culture inoculation test was unsuccessful due to sample degradation and storage conditions. The nearly complete genome of Divaca bat lyssavirus from Slovenia consists of 11,871 nucleotides and reflects the characteristic gene organization known for lyssaviruses, encoding the five viral proteins. Phylogenetic analysis of Divaca bat lyssavirus revealed that it belongs to phylogroup I lyssaviruses and is most closely related to Kotalahti bat lyssavirus (KBLV) with 87.20% nucleotide and 99.22% amino acid identity. Together with KBLV, Khujand virus, European bat lyssavirus 2, Bakeloh bat lyssavirus, and Aravan virus, Divaca bat lyssavirus was detected in the genus Myotis suggesting its key role in the transmission and maintenance of certain lyssaviruses.

The Principles of the Voluntary Programme for the Control and Elimination of Bovine Viral Diarrhoea Virus (BVDV) From Infected Herds in Slovenia.

In Slovenia, the control of bovine viral diarrhoea virus (BVDV) infections started in 1994. Since 2014, a voluntary programme has been running according to the national rules that prescribe the conditions for recognising, acquiring, and maintaining a BVDV-free status for an individual herd. The principle is based on periodical laboratory testing and preventive measures that need to be strictly implemented in a herd. Between 2014 and 2020, a total of 348 herds were included in BVDV antibody testing, and 25.0% of tested herds were detected to be BVDV antibody positive. To recognise the BVDV-free status of the herd, the breeder should provide two consecutive tests with intervals of at least 6 months in all animals in the age from 7 to 13 months, with negative results for BVDV antibodies in ELISA. The BVDV-free status of the herd can be maintained by implementing preventive measures and can be renewed each year with one laboratory test in the age group of animals from 7 to 13 months for antibodies in ELISA. During the 7 years of the voluntary programme, 236 herds were included in the detection of BVDV in individual herds by real-time RT-PCR method and the elimination of positive animals from herds. In 71 (31.3%) herds, at least one BVDV-positive animal was detected, with the identification of a total of 267 persistently infected (PI) animals, representing an average of 2.9% of tested animals. The cost of testing for an average herd, recognised as BVDV-negative, and maintaining its BVDV-free status within the implemented voluntary programme, was €97.64/year, while for the average positive herd, the laboratory costs for elimination of BVDV were €189.59/year. Only limited progress towards eradication at the national level has been achieved in Slovenia since 2014.

Review of Infections With Bovine Herpesvirus 1 in Slovenia.

In the 1950s, infectious bovine rhinotracheitis/infectious pustular vulvovaginitis (IBR/IPV) disease was clinically detected and documented in cattle for the first time in Slovenia. The bovine herpes virus 1 (BoHV-1) was confirmed several times from infected herds by virus isolation on cell cultures. To keep the IC virus-free, high biosecurity measures were introduced. Before entering the IC, all calves are serologically tested and quarantined. Bulls in Slovenian insemination centres (IC) have been negative for IBR /IPV infection since 1979. From 1985 to 1991, few large-scale studies of the prevalence of IBR/IPV were carried out. In 1985, a high percentage (56.9%) of serologically positive animals were found in large state farms with Holstein Friesian cattle. Epidemiological studies in farm with bulls' mother herds were also carried out in the farms with Simmental and Brown cows. Antibodies against BoHV-1 were detected in the serum of 2.3% of Brown cattle and 3.5% of Simmental cattle. In the year 2000, 3.4% of bulk tank milk samples from 13,349 dairy farms were detected BoHV-1 antibodies positive. The highest percentage of positive animals was found in regions with an intensive grazing system (6.2% positive) and the lowest percentage in the east part of Slovenia (0.9% positive) on farms with mostly Simmental cattle. In 2006, a total 204,662 sera of cattle older than 24 months were tested for the presence of BoHV-1 antibodies and positive cattle were detected in 3.6% of tested farms. These farms kept 34,537 animals that were potential carriers of the BoHV-1. Most of the positive farms kept Holstein Friesian cattle, descendants from the state-owned farms, which were privatised or closed after 1990. In 2015, the Administration of the Republic of Slovenia for Food Safety, Veterinary and Plant Protection issued a rule that describes the conditions for granting and maintaining the status of BoHV-1 free holdings. The rule provides a voluntary control programme for breeders who want to obtain BoHV-1 free status and are willing to cover all the cost of acquiring and maintaining that status. There has been very little response from breeders.

Overview of Slovenian Control Programmes for Cattle Diseases Not Regulated by the European Union.

The European Union (EU) regulates the control of cattle diseases listed in categories A and B of the Animal Health Law (AHL). However, the control of other cattle diseases that have no, or limited EU regulation, is left to each member state. Slovenia has five control programmes (CPs) for non-EU regulated cattle diseases: bovine viral diarrhoea (BVD), infectious bovine rhinotracheitis (IBR), enzootic bovine leukosis (EBL), bluetongue and anthrax. Two (IBR and BVD) are voluntary and the others (EBL, anthrax and bluetongue) are compulsory. The three compulsory CPs are funded by the government. All the CPs are run by the government and laboratory tests are performed by the National Veterinary Institute. The rules for the CPs are laid down in Slovenian legislation. In addition, there is a national directive for the control of salmonellosis. Both BVD and IBR are endemic and have CPs based on increased biosecurity, testing and culling or vaccination, financed by the animal owners. Slovenia has been officially free of EBL since 2005 and carries out surveillance based on serological testing of a representative number of herds and inspection of carcasses at slaughter or necropsy. Vaccination is the main disease control measure for anthrax (sporadic) and bluetongue (currently perceived free-vaccination since 2017). Lack of motivation of farmers to participate in voluntary disease CPs and to implement and follow strict biosecurity measures are the most pressing issues in improving the health status of Slovenian cattle. An overview of the existing CPs and the circumstances leading to their implementation are presented.

The Successful Elimination of Sylvatic Rabies Using Oral Vaccination of Foxes in Slovenia.

Sylvatic rabies was present in Slovenia between 1973 and 2013, with the red fox as the main reservoir of the rabies virus. The first oral rabies vaccination (ORV) control program in foxes started in 1988, using the manual distribution of baits. Significant improvement of fox vaccination was achieved with the aerial distribution of baits, starting in 1995 and successfully finished with the final, fifty-ninth vaccination campaign in 2019. Between 1979 and 2019, a total of 86,471 samples were tested, and 10,975 (12.69%) rabies-positive animals were identified. Within the ORV, two different vaccines were used, containing modified live virus strain Street Alabama Dufferin (SAD) B19 and SAD Bern, while the last ORV campaigns were completed in 2019, with a vaccine containing a genetically modified strain of SPBN GASGAS. Molecular epidemiological studies of 95 rabies-positive samples, originating from red foxes, badgers, cattle, dogs, martens, cats, and horses, revealed a low genetic diversity of circulating strains and high similarity to strains from neighboring countries. During the elimination program, few vaccine-induced rabies cases were detected: three in red foxes and one case in a marten, with no epidemiological relevance. Slovenia has been officially declared a country free of rabies since 2016.

Corrigendum: Overview of Slovenian Control Programmes for Selected Cattle Diseases, Listed Under Category C, D or E of the European Animal Health Law.

[This corrects the article DOI: 10.3389/fvets.2021.674515.].

Genetic and spatial characterization of the red fox (Vulpes vulpes) population in the area stretching between the Eastern and Dinaric Alps and its relationship with rabies and canine distemper dynamics.

Information on the population dynamics of a reservoir species have been increasingly adopted to understand and eventually predict the dispersal patterns of infectious diseases throughout an area. Although potentially relevant, to date there are no studies which have investigated the genetic structure of the red fox population in relation to infectious disease dynamics. Therefore, we genetically and spatially characterised the red fox population in the area stretching between the Eastern and Dinaric Alps, which has been affected by both distemper and rabies at different time intervals. Red foxes collected from north-eastern Italy, Austria, Slovenia and Croatia between 2006-2012, were studied using a set of 21 microsatellite markers. We confirmed a weak genetic differentiation within the fox population using Bayesian clustering analyses, and we were able to differentiate the fox population into geographically segregated groups. Our finding might be due to the presence of geographical barriers that have likely influenced the distribution of the fox population, limiting in turn gene flow and spread of infectious diseases. Focusing on the Italian red fox population, we observed interesting variations in the prevalence of both diseases among distinct fox clusters, with the previously identified Italy 1 and Italy 2 rabies as well as distemper viruses preferentially affecting different sub-groups identified in the study. Knowledge of the regional-scale population structure can improve understanding of the epidemiology and spread of diseases. Our study paves the way for an integrated approach for disease control coupling pathogen, host and environmental data to inform targeted control programs in the future.

In-depth genome analyses of viruses from vaccine-derived rabies cases and corresponding live-attenuated oral rabies vaccines.

Live-attenuated rabies virus strains such as those derived from the field isolate Street Alabama Dufferin (SAD) have been used extensively and very effectively as oral rabies vaccines for the control of fox rabies in both Europe and Canada. Although these vaccines are safe, some cases of vaccine-derived rabies have been detected during rabies surveillance accompanying these campaigns. In recent analysis it was shown that some commercial SAD vaccines consist of diverse viral populations, rather than clonal genotypes. For cases of vaccine-derived rabies, only consensus sequence data have been available to date and information concerning their population diversity was thus lacking. In our study, we used high-throughput sequencing to analyze 11 cases of vaccine-derived rabies, and compared their viral population diversity to the related oral rabies vaccines using pairwise Manhattan distances. This extensive deep sequencing analysis of vaccine-derived rabies cases observed during oral vaccination programs provided deeper insights into the effect of accidental in vivo replication of genetically diverse vaccine strains in the central nervous system of target and non-target species under field conditions. The viral population in vaccine-derived cases appeared to be clonal in contrast to their parental vaccines. The change from a state of high population diversity present in the vaccine batches to a clonal genotype in the affected animal may indicate the presence of a strong bottleneck during infection. In conclusion, it is very likely that these few cases are the consequence of host factors and not the result of the selection of a more virulent genotype. Furthermore, this type of vaccine-derived rabies leads to the selection of clonal genotypes and the selected variants were genetically very similar to potent SAD vaccines that have undergone a history of in vitro selection.

Identification of novel reassortant mammalian orthoreoviruses from bats in Slovenia.

BACKGROUND: Recently, mammalian orthoreoviruses (MRVs) were detected for the first time in European bats, and the closely related strain SI-MRV01 was isolated from a child with severe diarrhoea in Slovenia. Genetically similar strains have also been reported from other mammals, which reveals their wide host distribution. The aim of this study was to retrospectively investigate the occurrence and genetic diversity of MRVs in bats in Slovenia, from samples obtained throughout the country in 2008 to 2010, and in 2012 and to investigate the occurrence of the novel SI-MRV01 MRV variant in Slovenian bats. RESULTS: The detection of MRVs in bat guano was based on broad-range RT-PCR and specific bat MRV real-time RT-PCR. Subsequently, MRV isolates were obtained from cell culture propagation, with detailed molecular characterisation through whole-genome sequencing. Overall, bat MRVs were detected in 1.9% to 3.8% of bats in 2008, 2009 and 2012. However, in 2010 the prevalence was 33.0%, which defined an outbreak of the single SI-MRV01 strain. Here, we report on the identification of five MRV isolates of different serotypes that are designated as SI-MRV02, SI-MRV03, SI-MRV04, SI-MRV05 and SI-MRV06. There is high genetic variability between these characterised isolates, with evident genome reassortment seen across their genome segments. CONCLUSIONS: In conclusion, we have confirmed the presence of the SI-MRV01 strain in a Slovenian bat population. Moreover, according to genetic characterisation of S1 genome segment, all three MRV serotypes were present in the bat population. In this study, five independent MRV isolates were obtained and detailed whole genome analysis revealed high diversity between them. This study generates new information about the epidemiology and molecular characteristics of emerging bat MRV variants, and provides important molecular data for further studies of their pathogenesis and evolution.

Evaluation of ELISA for the detection of rabies virus antibodies from the thoracic liquid and muscle extract samples in the monitoring of fox oral vaccination campaigns.

BACKGROUND: The main goal of oral vaccination of foxes is eradication of rabies in the red fox population as rabies reservoirs. To evaluate the success of vaccination a serological testing is conducted as a part of monitoring program. Two different methods are used regarding rabies serology: virus neutralisation test and ELISA. METHODS: In this study the reliability of BioPro ELISA was evaluated for testing haemolytic thoracic liquids and muscle extracts originated from 147 foxes in comparison to mFAVN. Also, the influence of heat treatment of samples on test results was investigated. RESULTS: The specificity of the test for not-heat treated samples was 92.98% and sensitivity 79.20%. Diagnostic validity of the ELISA compared to the mFAVN test when not-heat treated samples were used was 89.16%. The specificity of the test for heat treated samples was 79.10% and sensitivity 96.36%. Diagnostic validity of the BioPro ELISA compared to the mFAVN test for heat treated samples was 94.30%. CONCLUSION: According to this study, the BioPro ELISA is reliable tool for detection of rabies specific antibodies in the context of evaluation of oral vaccination of foxes from poor quality samples as a substitution for virus neutralisation tests.

Vaccine-induced rabies in a red fox (Vulpes vulpes): isolation of vaccine virus in brain tissue and salivary glands.

Oral vaccination campaigns to eliminate fox rabies were initiated in Slovenia in 1995. In May 2012, a young fox (Vulpes vulpes) with typical rabies signs was captured. Its brain and salivary gland tissues were found to contain vaccine strain SAD B19. The Basic Logical Alignment Search Tool alignment of 589 nucleotides determined from the N gene of the virus isolated from the brain and salivary glands of the affected fox was 100% identical to the GenBank reference SAD B19 strain. Sequence analysis of the N and M genes (4,351 nucleotides) showed two nucleotide modifications at position 1335 (N gene) and 3114 (M gene) in the KC522613 isolate identified in the fox compared to SAD B19.

A step forward in molecular diagnostics of lyssaviruses--results of a ring trial among European laboratories.

Rabies is a lethal and notifiable zoonotic disease for which diagnostics have to meet the highest standards. In recent years, an evolution was especially seen in molecular diagnostics with a wide variety of different detection methods published. Therefore, a first international ring trial specifically designed on the use of reverse transcription polymerase chain reaction (RT-PCR) for detection of lyssavirus genomic RNA was organized. The trial focussed on assessment and comparison of the performance of conventional and real-time assays. In total, 16 European laboratories participated. All participants were asked to investigate a panel of defined lyssavirus RNAs, consisting of Rabies virus (RABV) and European bat lyssavirus 1 and 2 (EBLV-1 and -2) RNA samples, with systems available in their laboratory. The ring trial allowed the important conclusion that conventional RT-PCR assays were really robust assays tested with a high concordance between different laboratories and assays. The real-time RT-PCR system by Wakeley et al. (2005) in combination with an intercalating dye, and the combined version by Hoffmann and co-workers (2010) showed good sensitivity for the detection of all RABV samples included in this test panel. Furthermore, all used EBLV-specific assays, real-time RT-PCRs as well as conventional RT-PCR systems, were shown to be suitable for a reliable detection of EBLVs. It has to be mentioned that differences were seen in the performance between both the individual RT-PCR systems and the laboratories. Laboratories which used more than one molecular assay for testing the sample panel always concluded a correct sample result. Due to the markedly high genetic diversity of lyssaviruses, the application of different assays in diagnostics is needed to achieve a maximum of diagnostic accuracy. To improve the knowledge about the diagnostic performance proficiency testing at an international level is recommended before using lyssavirus molecular diagnostics e.g. for confirmatory testing.

Modification of the fluorescent antibody virus neutralisation test--elimination of the cytotoxic effect for the detection of rabies virus neutralising antibodies.

The virus neutralisation test is used for the quantitation of specific antibodies in serum samples. However, the success of the test depends on the quality of samples. In the case of poor quality samples, a cytotoxic effect can be observed and the results of the test can be compromised. Additionally, the cytotoxic effect limits the use of different substances, such as muscle extract or liquid from thoracic cavity (thoracic liquid), as a sample for the detection of rabies virus neutralising antibodies in the follow-up of fox oral vaccination campaigns. To eliminate the cytotoxic effect, a modified fluorescent antibody virus neutralisation (mFAVN) test was developed and evaluated. In the mFAVN test, inocula were removed after a 1h and the cytotoxic effect was prevented. According to the results obtained, the specificity of the mFAVN test compared to the FAVN test was 88.8% and the sensitivity was 94.4%. The diagnostic validity of the test was 0.99 (CI=0.98-1.00). To evaluate the possibility of using muscle extract and thoracic liquid as samples for the virus neutralisation test, 102 sera, muscle extract and thoracic liquid samples of dog origin were tested with the mFAVN test. The correlation between sera and muscle extracts was 87.9% (r=0.88, p<0.001). The correlation between sera and thoracic liquid was 94.2% (r=0.94, p<0.001). These findings indicated that both muscle extract and thoracic liquid could be used as samples for detection of rabies virus neutralising antibodies in the follow-up of oral vaccination campaigns. To evaluate the level of elimination of the cytotoxic effect, the 102 samples of sera, muscle extracts and thoracic liquid of dog origin were also tested in parallel using the mFAVN and FAVN tests. In the mFAVN test, no instance of cytotoxic effect was observed in the cells. In the FAVN test, two sera (1.9%), 35 muscle extracts (34.3%) and 56 thoracic liquid samples (54.9%) showed cytotoxic effect. The results of this study strongly suggest that cytotoxic effect can be eliminated completely from the rabies virus neutralising antibody detection tests used in the follow-up of oral vaccination campaigns and that very poor quality samples, such as muscle extract and thoracic liquid, can be used.

Presence of antibodies against rabies in wild boars.

Serum samples of 746 shot wild boars collected throughout Slovenia during the hunting season of 2005/2006 were examined for the presence of antibodies against rabies virus: 541 samples were collected in areas subjected to yearly antirabies vaccination, and 205 samples were collected in areas where preventive antirabies vaccination was not practised. Using a modified enzyme-linked immunosorbent assay (ELISA), in 209 out of 746 sera (28%) the levels of antibodies against rabies virus were higher than 0.5 IU/ml and deemed positive. A total of 173/541 (32%) and 36/205 (18%) samples were positive in the vaccinated and nonvaccinated areas, respectively. Further analysis of 191 out of the 746 samples using the fluorescent antibody virus neutralisation (FAVN) test revealed the presence of antibodies against rabies virus in 122/191 (64%) samples. This is the first extended research reporting that antibodies against rabies virus that originate from preventive oral vaccination targeting the fox population are present in wild boar.

First isolation and genotyping of viruses from recent outbreaks of viral haemorrhagic septicaemia (VHS) in Slovenia.

In November and December 2007, the virus causing viral haemorrhagic septicaemia (VHS) was detected in rainbow trout Oncorhynchus mykiss from 2 fish farms in Slovenia. During 2008 and 2009 the infection spread only among rainbow trout farms and 4 new outbreaks were confirmed. High mortality and clinical signs of VHS were observed among the diseased fish. VHSV was confirmed by virus isolation, immunoperoxidase test, reverse transcriptase polymerase chain reaction (RT-PCR) and phylogenetic analysis. Based on 1 complete (1524 nucleotides [nt]) and 9 partial (600 nt) glycoprotein gene nucleotide sequences, 9 VHSV isolates from the 6 VHS outbreaks were genetically closely related (99 to 100% identity), and were classified into the Subgroup I-a of Genotype I, most closely related to the German isolates Dstg21-07, Dstg36-06, and Dstg54-1-07 (99 to 100% identity). Phylogenetic analysis and epidemiological investigations confirmed that the VHS virus had been (re)introduced with imported live fish, and that subsequent outbreaks were linked to the initial infection. Our study shows that direct nucleotide sequencing of RT-PCR products, amplified from the tissue of VHSV-infected fish, represents a reliable tool for fast routine genotyping in diagnostic laboratories. This is the first report of a natural epidemic associated with VHSV infection in Slovenia since the eradication of the disease in 1977.

Identification of SARS-like coronaviruses in horseshoe bats (Rhinolophus hipposideros) in Slovenia.

Bats have been identified as a natural reservoir for an increasing number of emerging zoonotic viruses, such as Hendra virus, Nipah virus, Ebola virus, Marburg virus, rabies and other lyssaviruses. Recently, a large number of viruses closely related to members of the genus Coronavirus have been associated with severe acute respiratory syndrome (SARS) and detected in bat species. In this study, samples were collected from 106 live bats of seven different bat species from 27 different locations in Slovenia. Coronaviruses were detected by RT-PCR in 14 out of 36 horseshoe bat (Rhinolophus hipposideros) fecal samples, with 38.8% virus prevalence. Sequence analysis of a 405-nucleotide region of the highly conserved RNA polymerase gene (pol) showed that all coronaviruses detected in this study are genetically closely related, with 99.5-100% nucleotide identity, and belong to group 2 of the coronaviruses. The most closely related virus sequence in GenBank was SARS bat isolate Rp3/2004 (DQ071615) within the SARS-like CoV cluster, sharing 85% nucleotide identity and 95.6% amino acid identity. The potential risk of a new group of bat coronaviruses as a reservoir for human infections is highly suspected, and further molecular epidemiologic studies of these bat coronaviruses are needed.

Control of rabies in Slovenia.

Red foxes (Vulpes vulpes) are the main reservoir of rabies in Slovenia, whereas cases of rabies in other wildlife species occur sporadically. In 1995, a program of oral vaccination of wildlife in Slovenia was initiated; baits with oral vaccine were distributed by air at a density of 20 baits/km(2). During 1995, when the oral vaccination program was started, 1,089 cases of rabies (including both wild and domestic animals) were reported. Five years later (1999), only six positive animals were detected among 1,195 tested (0.5%). Despite an increase in bait density (25 baits/km(2)) during the years 2000 and 2001, reported rabies cases increased to 115 and 135, respectively. In 2003, following initiation of a new bait-dropping strategy, which incorporated perpendicular rather than parallel flight lines, the number of rabies cases decreased to eight.

Biological and molecular characterization of chicken anemia virus isolates from Slovenia.

The presence of chicken anemia virus (CAV) in Slovenia was confirmed by inoculation of 1-day-old chickens without antibodies against CAV and isolation of the virus on the Marek's disease chicken cell-MSB1 line and by polymerase chain reaction (PCR). Experimental inoculation of 1-day-old chickens resulted in lower hematocrit values, atrophy of the thymus, and atrophy of bone marrow. CAV was confirmed by PCR in the thymus, bone marrow, bursa of Fabricius, liver, spleen, ileocecal tonsils, duodenum, and proventriculus. The nucleotide sequence of the whole viral protein (VP)1 gene was determined by direct sequencing. Alignment of VP1 nucleotide sequences of Slovenian CAV isolates (CAV-69/00, CAV-469/01, and CAV-130/03) showed 99.4% to 99.9% homology. The VP1 nucleotide sequence alignment of Slovenian isolates with 19 other CAV strains demonstrated 94.4% to 99.4% homology. Slovenian isolates shared highest homology with the BD-3 isolate from Bangladesh. Alignment of the deduced VP1 amino acids showed that the Slovenian isolates shared 100% homology and had an amino acid sequence most similar to the BD-3 strain from Bangladesh (99.6%) and were 99.1% similar to the G6 strain from Japan and the L-028 strain from the United States. The Slovenian isolates were least similar (96.6%) to the 82-2 strain from Japan. A phylogeneric analysis on the basis of the alignment of the VP1 amino acids showed that CAV isolates used in the study formed three groups that indicated the possible existence of genetic groups among CAV strains. The CAV isolates were grouped together independent of their geographic origin and pathogenicity.

Molecular detection of BHV-1 in artificially inoculated semen and in the semen of a latently infected bull treated with dexamethasone.

Two polymerase chain reaction (PCR) assays specific for glycoprotein B (gB) and glycoprotein E (gE) gene detection, respectively, were adopted for the detection of bovine herpesvirus-1 (BHV-1) in naturally infected bulls. The methods were tested on bovine semen artificially inoculated with BHV-1 and were compared with an optimised virus isolation method. Raw and extended semen samples were diluted in minimal essential medium (MEM) and spiked with equal dose of BHV-1. The extended semen was found to be more toxic for the cells than the raw semen, while the viral DNA could be detected by the PCR method in all tested dilutions of raw and extended semen samples. The sensitivity of both methods was compared also for BHV-1 detection in semen, nasal swabs and leucocytes of a seropositive bull in a different time period after virus reactivation with dexamethasone treatment. The sensitivity of virus detection by the PCR method was equivalent to that of virus isolation in cell culture. However, PCR was shown to be faster and easier to perform and may be a good alternative to virus isolation especially when bovine semen has to be screened for BHV-1 prior to artificial insemination.

Influence of storage temperature on infectious hematopoietic necrosis virus detection by cell culture isolation and RT-PCR methods.

The detection of infectious hematopoietic necrosis virus (IHNV) in infected rainbow trout Oncorhynchus mykiss and in cell culture supernatants stored under different conditions was studied. IHNV-positive fish visceral organ homogenates and cell culture supernatants were incubated at 4 and 25 degrees C. Virus titre was measured by virus isolation on epithelioma papulosum cyprini (EPC) cells and the IHNV RNA was detected by RT-PCR and semi-nested RT-PCR. The influence of repeated freezing and thawing on the virus isolation from organ homogenates and from cell culture supernatants was studied as well. It was possible to isolate the virus from IHNV-positive organ material during the 3 d of incubation at 4 degrees C but, only on the first day of incubation at 25 degrees C. Viral RNA could be amplified during the incubation period of 35 d at 4 degrees C but only during 8 d of incubation at 25 degrees C. In IHNV-infected cell culture supernatant stored at 4 degrees C, it was possible to detect virus for 36 and 16 d in supernatant stored at 25 degrees C. Viral RNA could be followed by using molecular methods during the entire experimental period of 123 d. Each cycle of freezing and thawing of samples resulted in a reduction of IHNV titre in the suspension of visceral organs, while the virus titre in cell culture supernatant remained almost the same following 33 freezing-thawing cycles. The present results show that rapid laboratory processing and storage of potentially virus-containing tissue samples as well as the use of different detection methods are very important for efficient IHNV diagnosis.