Results Of Virus Isolation Research Articles

Emergence of lumpy skin disease virus (LSDV) infection in domestic Himalayan yaks (Bos grunniens) in Himachal Pradesh, India.

In this study, we investigated and confirmed natural lumpy skin disease virus (LSDV) infection in Himalayan yaks (Bos grunniens) in Himachal Pradesh, India, based on clinical manifestations and results of genome detection, antibody detection, virus isolation, and nucleotide sequencing. Subsequent phylogenetic analysis based on complete GPCR, RPO30, and EEV gene sequences revealed that the LSDV isolates from these yaks and local cattle belonged to LSDV subcluster 1.2.1 rather than the dominant subcluster 1.2.2, which is currently circulating in India, suggesting a separate recent introduction. This is the first report of natural LSDV infection in yaks in India, expanding the known host range of LSDV. Further investigations are needed to assess the impact of LSDV infection in yaks.

Analyses of the Relationship Between the Results of Virus Isolation and Rapid Antigen Detection Tests of SARS-CoV-2 RT-PCR Test-Positive Specimens

Analyses of the Relationship Between the Results of Virus Isolation and Rapid Antigen Detection Tests of SARS-CoV-2 RT-PCR Test-Positive Specimens

81 Reproductive Dysfunction in Cattle Following Vaccination Not a Result of Viral Infection of the Reproductive Tract

Abstract Impairment of bovine reproductive function and decreased pregnancy rates have been reported in modified-live vaccinated females. The objective of this study was to determine if virus could be found in various areas of the bovine reproductive tract following vaccination. Previously vaccinated beef females (n = 50) were administered a single dose of one of three treatments on d0 of the estrous cycle; BoviShield (n = 20), Titanium 5 (n = 20), or ViraShield (n = 10) based on estrus expression. Blood samples were collected on d0, 15, and 30 to evaluate Infectious Bovine-Rhinotracheitis (IBR) and Bovine Viral Diarrhea Virus (BVDV) titers and progesterone concentrations. On d15/16, half the animals from each treatment were non-surgically flushed with sterile saline (Uterine Fluid), and uterine biopsies (Uterine Tissue) were collected. On d17, follicular fluid and granulosa cells were collected by follicle aspirations. Remaining animals had uteri flushed and uterine tissue collected on d29, and follicles aspirated on d31. All samples were evaluated for presence of virus. Data were analyzed using the MIXED procedure of SAS. Sub-luteal progesterone concentrations were observed in 15% of the BoviShield and Titanium treatments on d15 and d30, versus 0% in the ViraShield treatment. Virus isolation and RT-PCR results were negative in all samples for all animals. Titers for BVDV-1 did not differ between treatments (P = 0.99) or treatment by time (P = 0.48). Titers for IBR tended to differ by treatment (P = 0.09) and treatment by time (P = 0.06), while BVDV-2 titers were affected by treatment (P < 0.01) and treatment x time (P < 0.01). Sub-luteal progesterone animals had a rapid rise in IBR and BVDV-2 titers from d0 to d15 and then tended (P < 0.07) to decrease to d30, compared to animals that had normal cycles where titers increased from d0 to d15 and remained elevated on d30. Collectively, these results indicate an alternative mechanism of impacting reproductive efficiency, possibly through an immune response.

Comparative Susceptibility of Madin-Darby Canine Kidney (MDCK) Derived Cell Lines for Isolation of Swine Origin Influenza A Viruses from Different Clinical Specimens.

Madin–Darby canine kidney (MDCK) cells are commonly used for the isolation of mammalian influenza A viruses. The goal of this study was to compare the sensitivity and suitability of the original MDCK cell line in comparison with MDCK-derived cell lines, MDCK.2, MDCK SIAT-1 and MDCK-London for isolation of swine-origin influenza A viruses (IAV-S) from clinical specimens. One-hundred thirty clinical specimens collected from pigs in the form of nasal swabs, lung tissue and oral fluids that were positive by PCR for the presence of IAV-S RNA were inoculated in the cell cultures listed above. MDCK-SIAT1 cells yielded the highest proportion of positive IAV-S isolations from all specimen types. For nasal swabs, 58.62% of the specimens were IAV-S positive in MDCK-SIAT1 cells, followed by MDCK-London (36.21%), and conventional MDCK and MDCK.2 cells (27.5%). For lung specimens, 59.38% were IAV-S positive in MDCK-SIAT1 cells, followed by MDCK-London (40.63%), and conventional MDCK and MDCK.2 cells (18.75–31.25%). Oral fluids yielded the lowest number of positive virus isolation results, but MDCK-SIAT1 cells were still had the highest rate (35%) of IAV-S isolation, whereas the isolation rate in other cells ranged from 5–7.5%. Samples with lower IAV-S PCR cycle threshold (Ct) values were more suitable for culturing and isolation. The isolated IAV-S represented H1N1-β, H1N2-α, H1N1pdm and H3N2 cluster IV and cluster IVB viruses. The result of the current study demonstrated the importance of using the most appropriate MDCK cells when isolating IAV-S from clinical samples.

Host Associations of Culex pipiens: A Two-Year Analysis of Bloodmeal Sources and Implications for Arboviral Transmission in Southeastern Virginia.

Understanding vector-host interactions is crucial for evaluating the role of mosquito species in enzootic cycling and epidemic/epizootic transmission of arboviruses, as well as assessing vertebrate host contributions to maintenance and amplification in different virus foci. To investigate blood-feeding pattern of Culex pipiens, engorged mosquitoes were collected on a weekly basis at 50 sites throughout Suffolk, Virginia, using Centers for Disease Control and Prevention miniature light traps, BG-Sentinel traps, and modified Reiter gravid traps. Vertebrate hosts of mosquitoes were identified by amplifying and sequencing portions of the mitochondrial cytochrome b gene. Of 281 Cx. pipiens bloodmeals successfully identified to species, 255 (90.7%) contained solely avian blood, 13 (4.6%) mammalian, 1 (0.4%) reptilian, and 12 (4.3%) both avian and mammalian blood. Nineteen avian species were identified as hosts for Cx. pipiens with American robin (n = 141, 55.3% of avian hosts) and northern cardinal (n = 57, 22.4%) as the most common hosts. More American robin feedings took place in areas of higher development. Three mammalian species were also identified as hosts for Cx. pipiens with Virginia opossum and domestic cat as the most common hosts in this class (each n = 6, 46.2% of mammalian hosts). There was no significant seasonal difference in the proportion of bloodmeals obtained from avian hosts, but there was a decrease in the proportion of bloodmeals from mammalian hosts from spring to fall. One engorged specimen of Cx. pipiens with Virginia opossum-derived bloodmeal tested positive for West Nile virus (WNV), and another with black-and-white warbler-derived bloodmeal tested positive for eastern equine encephalitis virus. Our findings, in conjunction with the results of vector competence studies and virus isolation from field-collected mosquitoes, lend additional support that Cx. pipiens serves as the principal enzootic vector and potential epizootic/epidemic vector of WNV in southeastern Virginia.

English

Equine herpesvirus 1 (EHV-1) causes considerable economic loss to the equine industry and is spread among susceptible animals during the cycles of latency and reactivation, causing rhinopneumonitis, abortion, and neurological disease. Nucleotide polymorphisms within ORF30 and ORF68 sequences of the viral genome are associated with strain neuropathogenicity and geographical origin. A total of 142 tissue and nasal swab samples from apparently healthy unvaccinated horses were examined to ascertain EHV-1 distribution, diversity, and clinical significance considering the results of virus isolation, sequence analysis, and anamnestic data. The ORF30 and ORF68 molecular study of these circulating strains and archival isolates from abortion storms aimed to contribute to the perception of strain pathogenicity and origin. EHV-1 was detected by PCR and virus isolation in 81 and 45.1% of the analyzed samples, respectively, and 82.1% of the representative samples were neuropathogenic strains. The ORF68-based grouping was restricted by the pronounced polymorphism of Balkan EHV-1 strains, and only two isolates were assigned to group 4. The cases of abortion were caused by neuropathogenic strains that also circulate within the horse population with no documented outbreaks of disease. It was evident that strain virulence is not solely accountable for the development of clinical symptoms in affected animals. Neural tissue is significant for virus latency and reactivation, considering the number of EHV-1 isolates from apparently healthy stressed horses. Special care must be taken when accommodating together immunologically naive and latently infected horses since asymptomatic carriers silently shed EHV-1.

Development of a novel reverse transcription PCR and its application to field sample testing for feline calicivirus prevalence in healthy stray cats in Korea.

BackgroundFeline calicivirus (FCV) is a major and highly infectious pathogen in cats worldwide. However, there have been limited studies about the status of FCV infections in Korea.ObjectivesTo investigate the current status of FCV infections in stray cats in Korea.MethodsA novel reverse transcription polymerase chain reaction (RT-PCR) assay was developed based on the conserved nucleotide sequences of reported FCV strains. Field swab samples were collected from 122 cats (2 hospital admitted cats and 120 stray cats) in 2016 and 2017. All the samples were tested by virus isolation and 2 different RT-PCRs, including the novel RT-PCR, for the detection of FCV.ResultsThe novel RT-PCR assay showed no cross-reactivity to the nucleic acids of the other feline pathogens tested, and the limit of detection was calculated as 100 TCID50/mL based on an in vitro assessment. The novel RT-PCR assay detected 5 positive samples from the 122 field samples, which showed perfect agreement with the results of the virus isolation method. In contrast, another RT-PCR assay used in a previous study in Korea detected no positive samples. The prevalence of FCV infection in stray cats was 2.5% (3/120) based on the results of virus isolation and the novel RT-PCR assays.ConclusionsThe current study is the first report of the detection and prevalence of FCV in stray cats in Korea. The novel RT-PCR assay developed in this study showed high sensitivity and specificity, which indicates a useful diagnostic assay to identify FCV infection in cats.

Concurrent detection of bovine viral diarrhoea virus and bovine herpesvirus-1 in bulls’ semen and their effect on semen quality

ABSTRACT Reproductive diseases may have destructive effects on the fertility of cattle. Bovine viral diarrhoea virus (BVDV) and bovine herpes virus-1 (BoHV-1) are potent viral pathogens linked to reproduction. Thus, the aim of this study was to utilize raw semen samples for conventional and molecular detection of BVDV and BoHV-1, simultaneously. Additionally, the effect of virus infection on the semen quality of naturally infected bulls has been investigated. Therefore, 40 bulls were employed for semen collection, evaluation and testing for both viruses by virus isolation, direct fluorescent antibody technique (FAT) and SYBR Green real-time PCR assay. In virus isolation results, no cytopathic effect (CPE) was observed for BVDV on cell culture whereas, eight (20%) samples displayed characteristic grape-like clusters of cells for BoHV-1. By direct FAT, 12 (30%) positive BVDV and 8 (20%) positive BoHV-1 samples were confirmed. SYBR Green real-time PCR analysis using 48 h inoculated semen samples revealed 14 (35%) and 8 (20%) positive samples for BVDV and BoHV-1, respectively. Statistical analysis of semen evaluation parameters showed a significant difference between viral-infected and free groups represented by increased sperm abnormalities and decreased sperm motility, liveability and concentration. However, there was no significant difference among BVDV, BoHV-1 and mixed-infected groups. The study concluded that BVDV and/or BoHV- 1 infected bulls expressed low semen quality. Real-time PCR was confirmed to be the ideal laboratory assay for detection of both viruses in semen.

Isolation of Swine Influenza A Virus in Cell Cultures and Embryonated Chicken Eggs.

Influenza virus isolation is a procedure to obtain a live and infectious virus that can be used for antigenic characterization, pathogenesis investigation, vaccine production, and so on. Embryonated chicken egg inoculation is traditionally considered the "gold standard" method for influenza virus isolation and propagation. However, many primary cells and continuous cell lines have also been examined or developed for influenza virus isolation and replication. Specifically, influenza A virus in swine (IAV-S) isolation and propagation has been attempted and compared in embryonated chicken eggs, some primary porcine cells, and a number of continuous cell lines. Currently, Madin-Darby canine kidney (MDCK) cells remain the most commonly used cell line for the isolation, propagation, and titration of IAV-S. Virus isolation in embryonated chicken eggs or in different cell lines offers alternative approaches when IAV-S isolation in MDCK cells is unsuccessful. Optimal specimens for IAV-S isolation includes nasal swabs, nasopharyngeal swabs, oral fluids, bronchoalveolar lavage, lung tissues, and so on. In this chapter, we describe the procedures of sample processing, IAV-S isolation in MDCK cells and in embryonated chicken eggs, as well as the methods used for confirming the virus isolation results.

Surveillance and Molecular Characterization of Newcastle Disease Virus From Chickens in North Egypt During 2015-2017

In the present study, fifty broiler chickens flocks suspected to be infected with Newcastle disease virus (NDv) in 5 Egyptian provinces Gharbia (7/50), El Behera(21/50), Kafr EL sheikh (8/50) and Matrouh(7/50) and Alexendaria(7/50) during the period (from september 2015 to February 2017) were investigated for clinical and postmortem examination as well as virus isolation and molecular identification. The results of virus isolation on embryonated chicken SPF eggs (ECE) revealed that 16/50 samples were haemagglutinatig agents by slide haemagglutination test.These haemagglutinating agents were confirmed as NDv by real time PCR .The deduced amino acid sequences of the fusion protein of selected 8 positive isolates revealed that (7/8) isolates possessed the motif 112RRQKRI117at the cleavage site,indicating that these isolates are velogenic ,whereas the other (1/8) isolates carries the motif 112GRQGRL117indicating that is lentogenic . Phylogenetic analysis demonstrated that the 7 strains belong to the NDv genotype VIId, and the other strain was of genotype II.These isolates were detected even in vaccinated flocks that indicate the possible causes of those outbreaks may be not only useless of vaccines ,but also immunesuppression,use of heterogenous vaccines ,improper application of biosecurity ,moreover,may be arole in spreading of velogenic strain of NDv in Egypt .

Experimental infection of BALB/c mice with a caprine Pestivirus H isolate

To data, small animal Pestivirus H infection models have not been established. In order to develop a new infection model, BALB/c mice were inoculated with Pestivirus H strain HN1507. The virus-inoculated mice displayed nasal discharge and fever clinical signs. Histopathological changes in Pestivirus H-infected mice included alveolar septa thickening and alveolar atrophy in the lungs from 1 to 11 days post-inoculation (PI). Furthermore, we observed tracheal epithelial cell abscission and inflammatory cell infiltration in the tracheas from 1 to 9 days PI, infiltration of eosinophils in the spleens from 1 to 9 days PI, intestinal villi abscission and lysis of epithelial cells in the intestines from 1 to 11 days PI. The results of virus isolation showed that Pestivirus H replicated well in the lungs, tracheas, spleens, and intestines of infected BALB/c mice, and peak viral titers were observed 3 days PI. RT-PCR and immunofluorescence results were in agreement with the virus isolation results; however, the hearts of infected mice from 1 to 3 days PI were positive while virus isolation results were negative. To the best of our knowledge, this is the first study reporting Pestivirus H detection in BALB/c mice. Our findings indicated that Pestivirus H strain HN1507 was pathogenic to BALB/c mice and caused clinical signs and histopathological lesions in Pestivirus H-infected BALB/c mice.

Establishment and characterization of a new cell line from koi brain (Cyprinus carpio L.).

A novel permanently growing brain cell line from koi (Cyprinus carpio L.) (KB cell line) was established, and its suitability for detection of koi herpesvirus (KHV) was demonstrated in this study. The KB cell line was optimally maintained at 27°C in Leibovitz's L-15 medium supplemented with 10% foetal bovine serum (FBS). It was subcultured more than 100 times, and chromosome analysis revealed that 51.54% of KB cells at passage 80 maintained the abnormal diploid chromosome number 2n=96 while the modal chromosome number was 2n=100. The cell line was cryopreserved in liquid nitrogen at -196°C and was recovered from storage after 1year with good cell viability and vitality. The results of virus isolation demonstrated that KB cells were susceptible to KHV, which was shown by the presence of an obvious cytopathic effect and abundant virus particles. The viral titres of KHV in KB reached 105.73 TCID50 /0.1ml within 7days. Immunofluorescence and Western blot assays confirmed that KB replicated KHV. The newly established KB cell line will serve as a useful tool to elucidate KHV disease (KHVD) pathogenesis.

Possible over-wintering of bluetongue virus in Culicoides populations in the Onderstepoort area, Gauteng, South Africa.

Several studies have demonstrated the ability of certain viruses to overwinter in arthropod vectors. The over-wintering mechanism of bluetongue virus (BTV) is unknown. One hypothesis is over-wintering within adult Culicoides midges (Diptera; Ceratopogonidae) that survive mild winters where temperatures seldom drop below 10 °C. The reduced activity of midges and the absence of outbreaks during winter may create the impression that the virus has disappeared from an area. Light traps were used in close association with horses to collect Culicoides midges from July 2010 to September 2011 in the Onderstepoort area, in Gauteng Province, South Africa. More than 500 000 Culicoides midges were collected from 88 collections and sorted to species level, revealing 26 different Culicoides species. Culicoides midges were present throughout the 15 month study. Nine Culicoides species potentially capable of transmitting BTV were present during the winter months. Midges were screened for the presence of BTV ribonucleic acid (RNA) with the aid of a real-time quantitative reverse transcription polymerase chain reaction (RT-qPCR) assay. In total 91.2% of midge pools tested positive for BTV RNA. PCR results were compared with previous virus isolation results (VI) that demonstrated the presence of viruses in summer and autumn months. The results indicate that BTV-infected Culicoides vectors are present throughout the year in the study area. Viral RNA-positive midges were also found throughout the year with VI positive midge pools only in summer and early autumn. Midges that survive mild winter temperatures could therefore harbour BTV but with a decreased vector capacity. When the population size, biting rate and viral replication decrease, it could stop BTV transmission. Over-wintering of BTV in the Onderstepoort region could therefore result in re-emergence because of increased vector activity rather than reintroduction from outside the region.

Evaluation of rapid diagnostic test kits for feline leukemia virus infection using samples from naturally infected cats.

ObjectivesFeline leukemia virus (FeLV) is a potentially life-threatening oncogenic retrovirus. The p27 viral core protein is produced by the virus in infected feline cells, is found in the cytoplasm in several blood cells and can be free in the serum and plasma. ELISA or particle-based immunoassay are commonly used to detect the presence of the p27 core protein in samples obtained from blood. The objective of this study was to compare the performance of several in-clinic tests: the SNAP Feline Triple Test (IDEXX Laboratories), the WITNESS FeLV-FIV Test (Zoetis) and the VetScan Feline FeLV/FIV Rapid Test (Abaxis).MethodsThe sample population (100 positive, 105 negative samples) consisted of serum and plasma samples submitted to IDEXX’s worldwide reference laboratory for feline retrovirus testing. Virus isolation and reverse transcriptase PCR results were not available and so samples were judged to be positive or negative based on the results of the ViraCHEK FeLV (Zoetis) microtiter plate assay.ResultsThe percentage of samples positive and negative for FeLV p27 antigen using the three in-clinic tests compared with the ViraCHEK method were as follows: IDEXX Feline Triple (positive 98.0%, negative 100%); Zoetis WITNESS (positive 79.0%, negative 97.1%); Abaxis VetScan (positive 73.0%, negative 97.1%).Conclusions and relevanceThe SNAP Feline Triple Test demonstrated a high level of agreement for FeLV-positive and FeLV-negative samples when assessed in this model. Results of FeLV assays can vary among tests.

Prolonged intermittent virus shedding during an outbreak of canine influenza A H3N2 virus infection in dogs in three Chicago area shelters: 16 cases (March to May 2015).

OBJECTIVE To estimate an appropriate isolation period for dogs infected with canine influenza A H3N2 virus on the basis of the duration of virus shedding. DESIGN Retrospective case series. ANIMALS 16 dogs, from 3 Chicago area shelters, naturally infected with canine influenza A H3N2 virus. PROCEDURES Medical records of 16 affected dogs were reviewed. Nasal swab specimens from each dog had been tested periodically for a minimum of 15 days following an initial positive real-time reverse transcriptase PCR (rRT-PCR) assay result for influenza A virus shedding. Amplicons were purified, quantified, and sequenced by the Sanger DNA sequencing technique. Virus isolation and sequence results of canine influenza A H3N2 virus from nasal swab specimens were obtained in conjunction with signalment, description of clinical signs, type of treatment, and outcome. RESULTS Viruses from each dog were identified as canine influenza A H3N2 virus on the basis of DNA sequencing. The interval between first and last positive rRT-PCR assay results ranged from 13 to 24 days, whereas the time interval from first reported clinical signs to last positive assay results ranged from 15 to 26 days. Isolation of canine influenza A H3N2 virus was successful in the late shedding period from nasal swab specimens of 4 dogs at 15 and 20 days after the first positive rRT-PCR assay result and 18 to 20 days after the first clinical signs. Clinical signs resolved for all dogs that remained in the shelters during the testing period. CONCLUSIONS AND CLINICAL RELEVANCE Dogs infected with H3N2 virus should be isolated for a period of ≥ 21 days following onset of illness. Even when resolution of clinical signs occurs sooner than 21 days, shedding of H3N2 virus may persist.

Efficacy Evaluation of an Inactivated Duck Tembusu Virus Vaccine.

To evaluate the potential use of an inactivated virus-based vaccine for the control and prevention of the newly emerged duck Tembusu virus infection in China, a duck Tembusu virus isolate, Tembusu-HB, was propagated in 12-day-old duck embryos and inactivated by treatment with formaldehyde. The inactivated viral antigen was emulsified with mineral oil, and five batches of the vaccine were manufactured. The immunogenicity and protection efficacy of the vaccine were evaluated in Beijing ducks and Beijing white geese. Results showed that more than 80% of immunized ducks were protected against virulent virus challenge after two intramuscular or subcutaneous injections of the inactivated vaccine, as evidenced by the negative virus isolation results. The protection is also correlated with a positive virus-specific antibody response as detected by ELISA. In contrast, none of the control ducks and geese had any detectable antibody response. Virus was isolated from all control ducks and geese after virulent virus challenge. Interestingly, a variable level of protection (20%-80%) was observed in Beijing white geese immunized twice with the same batches of vaccine, suggesting a species-specific effect of the vaccine. Overall, the results clearly suggest that the inactivated duck Tembusu virus vaccine is immunogenic and provides protection against virulent virus challenge.

Effect of time at temperature on wild poliovirus titers in stool specimens

BackgroundThe effect of transport temperature on the viability of poliovirus in stool specimens from paralyzed cases has not been tested. Quality assurance of programmatic indicators will be necessary in the final phase of polio eradication. ObjectiveTo estimate the effect of time at elevated temperatures on wild poliovirus titers in stool specimens. MethodsWe exposed aliquots of pooled wild poliovirus type 1 specimens to elevated temperatures (27°C, 31°C, and 35°C) for varying time periods up to 14 days. We determined the virus titer of these aliquots and created decay curves at each temperature to estimate the relationship between time at temperature and virus titer. ResultsWe found significantly different slopes of decay at each temperature. The negative slopes increased as the temperature increased. ConclusionsWhile poliovirus in stool remains relatively stable at moderately elevated temperature, transport at higher temperatures could impact sample integrity and virus isolation results.

Development and evaluation of a polydiacetylene based biosensor for the detection of H5 influenza virus

H5N1 avian influenza has caused serious economic losses as well as posed significant threats to public health, agriculture and wildlife. It is important to develop a rapid, sensitive and specific detection platform suitable for disease surveillance and control. In this study, a highly sensitive, specific and rapid biosensor based on polydiacetylene was developed for detecting H5 influenza virus. The polydiacetylene based biosensor was produced from an optimized ratio of 10,12-pentacosadiynoic acid and 1,2-dimyristoyl-sn-glycero-3-phosphocholine, with the anti-H5 influenza antibody embedded onto the vesicle surface. The optimized polydiacetylene vesicle could detect H5 influenza virus sensitively with a detection limit of 0.53copies/μL, showing a dramatic blue-to-red color change that can be observed directly by the naked eye and recorded by a UV-vis spectrometer. The sensitivity, specificity and accuracy of the biosensor were also evaluated. The sensor could specifically differentiate H5 influenza virus from H3 influenza virus, Newcastle disease virus and porcine reproductive and respiratory syndrome virus. Detection using tracheal swabs was in accord with virus isolation results, and comparable to the RT-PCR method. These results offer the possibility and potential of simple polydiacetylene based bio-analytical method for influenza surveillance.

Bovine Arboviruses in Culicoides Biting Midges and Sentinel Cattle in Southern Japan from 2003 to 2013.

Epizootic congenital abnormalities, encephalomyelitis and febrile illnesses in cattle caused by arthropod-borne viruses (arboviruses) are prevalent in Japan. Causative viruses including orthobunyaviruses, orbiviruses and rhabdovirus are thought to be transmitted by Culicoides biting midges. Recently, the incursions of several arboviruses, potentially Culicoides-borne, were newly confirmed in Japan. However, their spread pattern and exact vector species are currently uncertain. Attempts to isolate arboviruses from Culicoides biting midges and sentinel cattle were conducted in Kagoshima, located at the southernmost end of the main islands of Japan, a potentially high-risk area for incursion of arboviral diseases and outbreak of endemic ones. Seventy-eight isolates comprising Akabane, Peaton and Sathuperi viruses of the genus Orthobunyavirus of the family Bunyaviridae, bluetongue virus serotype 16, D'Aguilar virus, Bunyip Creek virus and epizootic haemorrhagic disease virus serotype 1 of the genus Orbivirus of the family Reoviridae, a potentially novel rhabdovirus of the genus Ephemerovirus and unidentified orbivirus-like viruses were obtained from Culicoides biting midges and sentinel cattle between 2003 and 2013. Akabane, Sathuperi, D'Aguilar and Bunyip Creek viruses were selectively isolated from Culicoides oxystoma, suggesting this vector's responsibility for these arbovirus outbreaks. The results of virus isolation also implied that C.tainanus, C.jacobsoni and C.punctatus are competent for the transmission of bluetongue virus serotype 16, Peaton virus and epizootic haemorrhagic disease virus serotype 1, respectively. Our monitoring in Culicoides biting midges and sentinel cattle detected the circulation of Akabane virus just prior to the accumulations of bovine congenital abnormalities and encephalomyelitis by it around study sites in 2003, 2006, 2008 and 2013. Silent circulations of the other arboviruses, including potentially new viruses, were also detected during the study period.

Development and characterization of a cell line from the snout of koi (Cyprinus carpio L.) for detection of koi herpesvirus

A permanent growing cell line, designated as koi snout (KS), was established from snout tissues of koi utilizing the trypsin method. The KS cell line was maintained in Leibovitz's L-15 medium supplemented with 10–20% fetal bovine serum (FBS) within incubation temperatures ranging from 22 to 27 °C. Maximum cell proliferation rates occurred at 27 °C in Leibovitz's L-15 medium containing 10% FBS. The KS cell line was subcultured more than 115 times, and karyotyping analysis indicated the modal chromosome number was 2n = 100. The results of virus isolation demonstrated that KS cells were susceptible to koi herpesvirus (KHV), which was demonstrated by the presence of obvious cytopathic effects and abundant virus particles with high virus titer of 106.98 TCID50/mL. Immunofluorescence and Western blot assays provided confirmation that KS could replicate KHV. The newly established KS cell line will provide a useful tool for KHV detection.