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Virus Isolation (virus + isolation)
Selected AbstractsGenetic characterization of hantaviruses isolated from Guizhou, China: Evidence for spillover and reassortment in natureJOURNAL OF MEDICAL VIROLOGY, Issue 6 2008Yang Zou Abstract To gain more insights into the epidemiology of hantaviruses in the Guizhou province, China, rodents were captured in Guizhou during the period from 2001 to 2003. In addition, serum sample was collected from one patient. Virus isolation was attempted from human serum and rodent samples. Four hantaviruses were isolated successfully in cell culture from one human, two A. agrarius, and one R. norvegicus. The nucleotide sequences for the entire S and M and partial L segment were determined from these four isolates as well as six viruses isolated in 1980s. Phylogenetic analysis revealed that the S segment from all isolates belong to the Hantaan virus (HTNV) clade, regardless of the sources from which they were derived. According to the S sequences, these viruses could be divided into three distinct phylogroups, showing geographical clustering. Analysis of the entire M and the partial L segment sequences demonstrated that 8 out of the 10 isolates belong to the HTNV clade. However, two isolates (CGRn8316 and CGRn9415) isolated from R. norvegicus belong to the Seoul virus (SEOV) clade. In addition, these two isolates were distinct from other known members of SEOV clade. Together, the data suggest that at least three groups of HTNV are co-circulating and one new variant of SEOV may be present in Guizhou. Our results also suggest that HTNV from A. agrarius spilled over to R. norvegicus and natural reassortment between HTNV and SEOV occurred during or after the spillover. J. Med. Virol. 80:1033,1041, 2008. © 2008 Wiley-Liss, Inc. [source] Time course characteristics of human herpesvirus 6 specific cellular immune response and natural killer cell activity in patients with exanthema subitumJOURNAL OF MEDICAL VIROLOGY, Issue 6 2006Takuji Kumagai Abstract The time-course of cell-mediated immunity in exanthema subitum is not well documented. The lymphoproliferative response to purified human herpesvirus 6 (HHV-6) antigen and to phytohemagglutinin was measured and natural killer (NK) cell activities determined in three consecutive specimens obtained biweekly from 18 young children and infants with exanthema subitum. Virus isolation and PCR detection of virus DNA and determination of neutralization antibody to HHV-6 and -7 were also carried out. The magnitude of the HHV-6 specific lymphoproliferative response varied; however, in most cases the time course kinetics revealed a low response in the acute phase with a subsequent gradual increase. In contrast, NK cell activities were high in the acute phase and declined gradually during convalescence. The lymphoproliferative response to phytohemagglutinin did not show a consistent trend in kinetics of time; however, dynamic changes in activity were observed in patients during the acute and convalescent periods. The results suggest that NK cells play a major role in resolving acute phase infection while specific lymphocyte activity develops later. The cause of the delayed development of HHV-6 specific lymphoproliferative response is unknown. The lymphoproliferative response to phytohemagglutinin ratios implied that HHV-6 infection has some impact on host T-cell immunity during the course of exanthema subitum. J. Med. Virol. 78:792,799, 2006. © 2006 Wiley-Liss, Inc. [source] A serological and virological survey for evidence of infection with Newcastle disease virus in Australian chicken farmsAUSTRALIAN VETERINARY JOURNAL, Issue 6 2007VG Kite Objective, To determine the prevalence and distribution of antibodies to Newcastle disease virus on Australian chicken farms and to determine the pathotype and relationships of the Newcastle disease viruses present on those farms. Design, A cross-sectional survey of 753 commercial chicken farms. Procedure, The survey comprised a detailed questionnaire and collection of venous blood samples. The titre of antibodies to Newcastle disease virus was determined by haemagglutination inhibition. Virus isolation was conducted from cloacal and tracheal swabs taken from chickens in serologically positive flocks. Virus isolates were pathotyped on the basis of the deduced Fusion protein cleavage site determined by nucleotide sequencing of a 265 bp region of the genome in the region of the cleavage site. Results, Antibody evidence of Newcastle disease virus infection was found on 300 of the 753 surveyed farms throughout all 11 geographic regions of the survey. The highest prevalence occurred in the Sydney basin, New South Wales and Victoria east regions. Antibody titres were also highest in the regions where serologically positive flocks were most prevalent. The 259 virus isolates revealed nine different RNA sequences. Of the nine virus groups isolated, the most common group W was identical in sequence to the V4 vaccine strain. Five of the other groups had novel RNA sequences in the region of the F protein cleavage site. Conclusions, Antibodies to Newcastle disease virus are highly prevalent in the Australian chicken flock but all identified strains were avirulent in nature. [source] Epidemiology and control of Menangle virus in pigsAUSTRALIAN VETERINARY JOURNAL, Issue 3 2001PD KIRKLAND Objective To describe the epidemiology and eradication of Menangle virus infection in pigs. Design Field observations and interventions, structured and unstructured serological surveys, prospective and cross-sectional serological studies and laboratory investigations. Procedure Serum samples were collected from pigs at a 2600-sow intensive piggery in New South Wales that experienced an outbreak of reproductive disease in 1997. Serum samples were also collected from piggeries that received pigs from or supplied pigs to the affected piggery and from other piggeries in Australia. Serum and tissue samples were collected from pigs at piggeries experiencing reproductive disease in New South Wales. Sera and faeces were collected from grey-headed flying foxes (Pteropus poliocephalus) in the region of the affected piggery. Serum samples were tested for neutralising antibodies against Menangle virus. Virus isolation was attempted from faeces. Results Following the outbreak of reproductive disease, sera from 96% of adult pigs at the affected piggery, including sows that produced affected litters, contained neutralising antibodies against Menangle virus. Neutralising antibodies were also detected in sera from 88% of finisher pigs at two piggeries receiving weaned pigs from the affected piggery. No evidence of Menangle virus infection was found in other piggeries in Australia. In cross-sectional studies at the affected piggery, colostral antibodies were undetectable in most pigs by 14 to 15 weeks of age. By slaughter age or entry to the breeding herd, 95% of pigs developed high antibody titres (A 128) against Menangle virus in the virus neutralisation test. Menangle virus was eradicated from the affected piggery following a program of serological testing and segregation. Neutralising antibodies against Menangle virus were also detected in P poliocephalusfrom two colonies in the vicinity of the affected piggery. Two piggery workers were infected with Menangle virus. There was no evidence of infection in cattle, sheep, birds, rodents, feral cats and a dog at the affected piggery. Conclusions Serological evidence of infection with Menangle virus was detected in pigs at a piggery that had experienced reproductive disease, in pigs at two associated piggeries and in fruit bats in the region of the piggery. Two humans were infected. The mode of transmission between pigs is unknown, but spread by faecal or urinary excretion is postulated. This virus can be eradicated by the segregation of pigs into discrete age groups. [source] Synergistic effects of esfenvalerate and infectious hematopoietic necrosis virus on juvenile chinook salmon mortalityENVIRONMENTAL TOXICOLOGY & CHEMISTRY, Issue 7 2005Mark A. Clifford Abstract Sublethal concentrations of pollutants may compromise fish, resulting in increased susceptibility to endemic pathogens. To test this hypothesis, juvenile chinook salmon (Oncorhynchus tshawytscha) were exposed to sublethal levels of esfenvalerate or chlorpyrifos either alone or concurrently with infectious hematopoietic necrosis virus (IHNV). Three trials were performed with fish exposed to concentrations of IHNV between 0.8 × 102 and 2.7 × 106 plaque-forming units/ml and to 5.0 ,g/L of chlorpyrifos or 0.1 ,g/L of esfenvalerate. The presence and concentration of IHNV in dead fish were assayed by virus isolation and plaque assay techniques, respectively. Among groups exposed to both esfenvalerate and IHNV, 83% experienced highly significant (p < 0.001) mortality, ranging from 20 to 90% at 3 d post-virus exposure, and cumulatively died from 2.4 to 7.7 d sooner than fish exposed to IHNV alone. This trend was not seen in any other treatment group. Virus assays of dead fish indicate a lethal synergism of esfenvalerate and IHNV. Chlorpyrifos had no observed effect on total mortality or IHNV susceptibility. The present results suggest that accepted levels of pollutants may be seemingly nonlethal to fish but, in fact, be acting synergistically with endemic pathogens to compromise survivorship of wild fish populations through immunologic or physiologic disruption. [source] Experimental transmission of sleeping disease in one-year-old rainbow trout, Oncorhynchus mykiss (Walbaum), induced by sleeping disease virusJOURNAL OF FISH DISEASES, Issue 5 2006S Kerbart Boscher Abstract Sleeping disease (SD) is a serious disease of rainbow trout, Oncorhynchus mykiss, reared in fresh water caused by sleeping disease virus (SDV). In this study a detailed clinical, histological, virological and serological description of the experimental reproduction of SD in 1-year-old rainbow trout exposed to SDV was carried out. Two hundred disease-free fish were intraperitoneally inoculated with a SDV isolate and 100 fish were inoculated with an uninfected cell culture lysate as a negative control. Infected and control fish were randomly removed at days 4, 7, 14, 21, 42 and 70 post-infection. Blood and tissues were collected for virus isolation, histopathological examination and serum neutralization. SDV was detected in serum, kidney and brain of infected fish from 4 to 21 days post-infection (dpi). Characteristic pathological lesions were observed in infected fish as early as 7 dpi. Lesions were first detected in exocrine pancreas and subsequently observed in heart and skeletal muscle. Neutralizing antibodies to SDV were detected in infected fish from 14 to 70 dpi. Infected fish displayed typical signs of SD 1-month pi and the mortality reached 18.7% within 44 days. This study experimentally reproduced all the pathognomonic features of natural outbreaks of SD in 1-year-old rainbow trout. [source] Comparison of the efficiency and sensitivity of virus isolation and molecular methods for routine diagnosis of infectious haematopoietic necrosis virus and infectious pancreatic necrosis virusJOURNAL OF FISH DISEASES, Issue 2 2002-Maganja, D Barli Infectious haematopoietic necrosis virus (IHNV) and infectious pancreatic necrosis virus (IPNV) are widely distributed fish pathogens in Europe. A reverse transcriptase,polymerase chain reaction (RT,PCR) assay was developed for the detection of both viruses as an alternative method to virus assay in cell culture. Oligonucleotide primers corresponding to highly conserved regions of glycoprotein G-gene sequences were used for IHNV. For the detection of IPNV the VP2-coding region was selected for RT,PCR amplification. Products of the expected size were amplified from total ribonucleic acid (RNA) extracts of infected cells. The optimized RT,PCR methods successfully detected viral RNA from ovarian and seminal fluids and other organs. To enhance the sensitivity and specificity of RT,PCR, a semi-nested PCR assay was tested using additional specific inner primers for reamplification of products obtained by RT,PCR. Because of the possibility of template carry-over contamination, a closed one step RT,PCR method was tested. This technically simplified approach was then combined with the PCR,enzyme linked immunosorbent assay (ELISA) method for the detection of amplification products and verification using specific biotinylated probes. The test provides an additional tool for the detection of IHNV and IPNV which is rapidly and easily performed and is highly sensitive, especially for the detection of IHNV in fish samples coinfected with IPNV. The PCR,ELISA method for the detection of RT,PCR products enables the screening of large numbers of samples and offers the possibility for automatisation of diagnostic work. [source] Real-time PCR for the detection and quantitative analysis of IHNV in salmonidsJOURNAL OF FISH DISEASES, Issue 6 2001K Overturf The rapid identification and quantification of virus in diseased fish is a goal both conservationists and commercial aquaculturists have struggled to attain. Recently a technique for the detection of viral mRNA particles that uses fluorescent tagging and amplification has been developed. Utilizing primers and fluorescent labelled probes generated for the specific identification of the nucleocapsid (N) and glycoprotein (G) genes of infectious haematopoietic necrosis virus (IHNV), and an instrument that measures cyclic emittance of fluorescence, the presence or absence of virus can be easily and rapidly confirmed. This method is not only useful in confirming viral presence but is effective in measuring the relative or absolute quantity of virus present within the sample. This allows for the determination of the health status of a carrier fish by measuring the quantity of viral genomes or transcribed viral genes present. Because this method is based on sequence detection, instead of virus isolation in cell culture, it is also effective in determining the presence of pathogenic organisms from water, fish feeds, or other potential reservoirs of infection. [source] Naturally occurring fatal herpes simplex virus 1 infection in a family of white-faced saki monkeys (Pithecia pithecia pithecia)JOURNAL OF MEDICAL PRIMATOLOGY, Issue 1 2003M.D. Schrenzel Abstract: A family of three white-faced saki monkeys (Pithecia pithecia pithecia) died 48,96 hours after the onset of anorexia, nasal discharge, pyrexia and oral ulceration. One animal also had clonic seizures. Lesions found post-mortem consisted of oral and esophageal ulcers, hepatic and intestinal necrosis, meningoencephalitis and sporadic neuronal necrosis. Intranuclear inclusion bodies and syncytial cells were present in oral lesions and affected areas of liver. Herpes simplex virus 1 (HSV-1) was identified as the etiology of disease by virus isolation, polymerase chain reaction, or in situ hybridization in all three animals. Immunohistochemistry for detection of apoptotic DNA and activated caspase-3 showed significant levels of apoptosis in oral and liver lesions and occasional apoptotic neurons in the brain. These findings demonstrate the vulnerability of white-faced saki monkeys to HSV-1 and provide initial insight into the pathogenesis of fatal HSV-1-induced disease, indicating that apoptosis plays a significant role in cell death. [source] Epidemiologic aspects and laboratory features of enterovirus infections in Western Germany, 2000,2005JOURNAL OF MEDICAL VIROLOGY, Issue 7 2007Bernhard Roth Abstract From 2000 to 2005, a total of 1,096 enterovirus infections were diagnosed either by isolation of virus from cell culture or by RT-PCR (5,non-coding region (NCR)). Typing of viruses (n,=,674) was carried out by immunofluorescence with monoclonal antibodies, neutralization test or molecular methods. Seasons with high enterovirus activity were characterized by high prevalence of echovirus 30 (62.2% in 2000, 25.5% in 2001) and echovirus 13 (34.5% in 2001). In contrast, in the 2003 season, which had very low enterovirus activity, these types were rare. During this season, cell culture sensitivity (human colonic carcinoma cells and human embryonic lung fibroblasts (HEL)) was exceptionally low. In order to determine the type of "non-cultivable" enteroviruses, purified RNA from selected stool samples was subjected to direct molecular typing. VP1/2A-specific fragments were amplified by RT-PCR, cloned and sequenced. The predominant virus identified was coxsackie A. Consequently, rhabdomyosarcom cells were introduced into the daily routine, which improved the isolation of enteroviruses. Echovirus 30 was again most commonly isolated during seasons 2004 and 2005 with increasing enterovirus activity. In conclusion, high prevalence of echovirus 30 and 13 is indicative of seasons with high enterovirus activity. The type of circulating enteroviruses may influence isolation of enterovirus from cell culture. RT-PCR (VP1/2A) combined with cloning and sequencing of amplicons is a useful tool for viral typing directly from stool samples. In cases of severe enterovirus infection, virological diagnosis should not solely rely on virus isolation from cell culture. J. Med. Virol. 79:956-962, 2007. © 2007 Wiley-Liss, Inc. [source] Investigation of measles and rubella outbreaks in Tamil Nadu, India,2003JOURNAL OF MEDICAL VIROLOGY, Issue 4 2006Nalini Ramamurty Abstract The aims of the present study were to confirm measles outbreaks by detection of measles-specific IgM antibodies, isolation of measles virus, and genetic characterization to document the circulating genotypes in Tamil Nadu. Eight outbreaks were reported from six districts of Tamil Nadu, India during the period Jan,Dec 2003. Blood samples were collected for serology, urine, and throat swabs for virus isolation. Genotypic characterization of measles isolates was based on the sequence of the N gene. All the clinically suspected outbreaks (n,=,8) were confirmed by serology; six out of the eight as measles and two as combination of measles and rubella highlighting the need to carry out rubella serology on measles-negative samples. Genetic characterization of three isolates obtained revealed one as genotype D4 and two as D8. Measles genotypes D4 and D8 were found to circulate in three districts of Tamil Nadu. It is necessary to be aware of the circulating genotypes within the geographical area. The information would be valuable to evaluate control measures and identify viral transmission and importation. J. Med. Virol. 78:508,513, 2006. © 2006 Wiley-Liss, Inc. [source] Enterovirus meningitis in Brazil, 1998,2003,JOURNAL OF MEDICAL VIROLOGY, Issue 1 2006Gina P.L. dos Santos Abstract Acute viral infections of the central nervous system (CNS) such as acute flaccid paralysis, meningitis, and encephalitis, are responsible for a high morbidity, particularly in children. Non-Polio enteroviruses (NPEV) are known to be responsible for over 80% of viral meningitis in which the etiologic agent is identified. In the present study, we show the frequency of enterovirus meningitis in Brazil from December 1998 to December 2003. Enterovirus were isolated from 162 (15.8%), of a total of 1,022 cerebrospinal fluid (CSF) specimens analyzed. Echovirus 30 was identified in 139 of these isolates (139/162,85.2%). Other identified enteroviruses were: Coxsackievirus B5 (3.7%), Echovirus 13 (3.7%), Echovirus 18 (3%), Echovirus 6 (1.2%), Echovirus 25 (1.2%), Echovirus 1 (0.6%), and Echovirus 4 (0.6%). Patients's age ranged from 28 days to 68 years old. The most frequent symptoms were fever (77%), headache (69.5%), vomiting (71.3%), neck stiffness (41.3%), convulsion (7.1%), and diarrhea (3.7%). Although, the majority of the patients recovered without any complication or permanent squeal, five deaths occurred. Throughout the surveillance period, five viral meningitis outbreaks were confirmed: four in the Southern Brazil and one in the Northeast Brazil. Echovirus 30 was responsible for four out of the five outbreaks while Echovirus 13 caused the fifth one. Besides the outbreaks, 734 sporadic cases were also identified during the study period and 59 of these were positive for virus isolation (8%). Echovirus 30 accounted for 70% of the isolates. Our results showed that Echovirus 30 was the most prevalent etiological agent of viral meningitis in Brazil, causing both outbreaks and sporadic cases. J. Med. Virol. 78:98,104, 2006. © 2005 Wiley-Liss, inc. [source] Rapid and sensitive detection of mumps virus RNA directly from clinical samples by real-time PCRJOURNAL OF MEDICAL VIROLOGY, Issue 3 2005Kazue Uchida Abstract A rapid, sensitive, and specific assay to detect mumps virus RNA directly from clinical specimens using a real-time PCR assay was developed. The assay was capable of detecting five copies of standard plasmid containing cDNA from the mumps virus F gene. No cross-reactions were observed with other members of Paramyxoviridae, or with viruses or bacteria known to be meningitis pathogens. Seventy-three clinical samples consisting of throat swabs collected from patients with parotitis, and cerebrospinal fluid (CSF) collected from patients with aseptic meningitis, were examined with a real-time PCR assay developed by the authors, reverse-transcription nested-PCR (RT-n-PCR), and virus isolation using cell culture. Like the RT-n-PCR assay, the real-time PCR assay could detect mumps virus RNA in approximately 70% of both throat swabs and CSF samples, while, by tissue culture, mumps virus was isolated from only approximately 20% of CSF and 50% of throat swab samples. In addition, the real-time PCR assay could be developed easily into a quantitative assay for clinical specimens containing more than 1,800 copies of mumps virus RNA/ml by using serial dilutions of the standard plasmid. The results suggest that the real-time PCR assay is useful for identification of mumps virus infections, not only in typical cases, but also in suspected cases, which show only symptoms of meningitis or encephalitis. J. Med. Virol. 75:470,474, 2005. © 2005 Wiley-Liss, Inc. [source] Prevalence of tick-borne encephalitis virus in Ixodes ricinus ticks in FinlandJOURNAL OF MEDICAL VIROLOGY, Issue 1 2001Xiuqi Han Abstract Approximately 20 cases of tick-borne encephalitis (TBE) occur annually in Finland. The known endemic areas are situated mainly in the archipelago and coastal regions of Finland, with highest incidence in Åland islands. Ixodes ricinus panels collected in 1996,1997 from two endemic areas were screened for the presence of RNA. Two distinct RT-PCR methods were applied, and were shown to have an approximate detection limit of 10 focus forming doses (FFD)/100 ,l. One out of 20 pools (a total of 139 ticks) from Helsinki Isosaari Island and one out of 48 pools (a total of 450 ticks) from Åland were positive with both methods, whereas the remaining pools were negative. The observed overall frequency (0.34%) in ticks in endemic areas of Finland, was similar to the low incidence found by virus isolation in mice in the 1960s (0.5%). Viral RNA was detectable in a diluted sample representing 0.005% of a positive pool of ten nymphs suggesting that the viral RNA load within an infected tick pool was approximately equivalent to 20,000,200,000 FFD. Sequence analysis did not show geographical clustering of the Finnish strains, suggesting an independent emergence of different TBE foci from the south. TBE virus RNA positive ticks were not found in I. ricinus panels consisting of 130 pools (726 ticks) from Helsinki city parks or 41 pools (197 ticks) from Võrmsi Island in Estonia. J. Med. Virol. 64:21,28, 2001. © 2001 Wiley-Liss, Inc. [source] A Dengue Outbreak among Camp Participants in a Caribbean Island, 1995JOURNAL OF TRAVEL MEDICINE, Issue 2 2000Rob Lyerla Background: Dengue, a mosquito-transmitted viral disease, is a risk for visitors in tropical and subtropical areas. Several participants in a community-assistance program in Tortola, British Virgin Islands, in August, 1995, reported dengue-like symptoms either before or soon after leaving the island. Methods: We conducted a retrospective cohort study to determine the extent of the outbreak, risk factors for illness, and the proportion of inapparent infections. Program participants were interviewed by telephone or mail, and asked to submit a serum sample for dengue diagnosis. A clinically-diagnosed case of dengue was defined as a person with fever and two or more of the following: headache, retro-orbital pain, myalgia, arthralgia, rash, or hemorrhagic manifestations. Serum specimens were tested for virus isolation, polymerase chain reaction (PCR), plaque-reduction neutralization (PRNT) or anti-dengue IgM and IgG antibody. Results: Thirty-two (97%) of the 33 program participants responded; 21 of the 32 (66%) provided at least one serum sample for study. The median age was 17 years; 20 (62%) were women. Of 32 respondents, 22 (69%) met the clinical case definition for dengue: 15 of them (68%) had a positive IgM antibody response and 7 did not submit a serum sample. Dengue 1 virus (DEN-1) was identified by PCR in one case and all 11 positive PRNT results. No asymptomatic infections were identified. No respondent used effective mosquito repellent, and only 2 (6%) used bednets. Conclusion: A DEN-1 outbreak with a high attack rate (69%) occurred in a group of young short-term community aid workers. There were no asymptomatic infections documented. Participants' rare use of bednets or effective mosquito repellent highlights the importance of providing travelers to tropical areas with information about dengue fever and the recommended precautions to protect against infection. [source] Unique Susceptibility of the Fetal Thymus to Feline Immunodeficiency Virus Infection: An Animal Model for HIV Infection In Utero1AMERICAN JOURNAL OF REPRODUCTIVE IMMUNOLOGY, Issue 5 2001CALVIN M. JOHNSON PROBLEM: Human infants infected in utero with HIV develop thymus insufficiency and progress to AIDS sooner than infants infected peripartum. However, direct analysis of the thymus is difficult due to limited tissue access and variable timing of vertical transmission. METHOD OF STUDY: Fetal and neonatal cats were inoculated with feline immunodeficiency virus (FIV) at an equivalent infectious dose. The thymus, blood, and lymph nodes were harvested and compared at 23 and 46 days post-inoculation (p.i.) and also compared to sham-inoculated, age-matched controls. Lymphocyte phenotypes were analyzed by flow cytometry and virus burden was quantified in histologic sections and by virus isolation from plasma. RESULTS: Fetal cats inoculated with FIV had acute thymus atrophy at birth, which coincided with peak viremia. At 46 days p.i., thymus size and cell composition rebounded and supported increased productive infection. In contrast, neonatal cats inoculated with FIV developed chronic thymus atrophy and degeneration, which was associated with decreasing productive infection and low-level viremia. CONCLUSIONS: The fetal thymus is uniquely vulnerable to acute, transient depletion and high-level productive infection. The neonatal thymus is less vulnerable to acute changes, and responds through progressive atrophy and declining productive infection. Reduced immune competence, as reflected by the failure to control virus replication, may contribute to the accelerated progression of FIV and HIV infections in utero. [source] Experimental studies of the role of the little raven (Corvus mellori) in surveillance for West Nile virus in AustraliaAUSTRALIAN VETERINARY JOURNAL, Issue 6 2010J Bingham Objective To study the potential role of an Australian corvid, the little raven (Corvus mellori), in the surveillance for exotic West Nile virus (WNV) in Australia. Method In a series of trials, little ravens were infected with WNV (strain 4132 New York 1999) and Kunjin virus (strain K42886) by the intramuscular route. They were observed for 20 days during which blood and swab samples were taken for virus isolation. Tissue samples were taken from ravens humanely killed during the acute infection period, and at the termination of the trials, for virus isolation, histopathology and immunohistochemistry. Results Ravens infected with WNV became mildly ill, but all recovered and seroconverted. Blood virus titres peaked around 3 to 4 days after inoculation at levels between 103.0 to 107.5 plaque forming units/mL. Virus or viral antigen was detected in spleen, liver, lung, kidney, intestine, testis and ovary by virus isolation and/or immunohistochemistry. WNV was detected in oral and cloacal swabs from 2 to 7 days post inoculation. The molecular and pathogenic characteristics of the inocula were consistent with them being of high virulence, as expected for this isolate. Ravens infected with Kunjin virus developed viraemia and seroconverted, although they did not develop disease. Conclusions Little ravens do not develop severe disease in response to virulent WNV infection and for this reason may not be important sentinel hosts in the event of an outbreak of WNV, as in North America. However, as they have relatively high viraemias, they may be able to support virus cycles. [source] Detection of viruses in nasal swab samples from horses with acute, febrile, respiratory disease using virus isolation, polymerase chain reaction and serologyAUSTRALIAN VETERINARY JOURNAL, Issue 1-2 2007K Dynon Objective To examine the association of viruses with acute febrile respiratory disease in horses. Design Nasal swab and serum samples were collected from 20 horses with acute febrile upper respiratory disease that was clinically assessed to have a viral origin. Methods Each of the samples was inoculated onto equine fetal kidney, RK13 and Vero cell cultures, and viral nucleic acid was extracted for polymerase chain reaction (PCR) or reverse transcription PCR. PCR primers were designed to amplify nucleic acid from viruses known to cause or be associated with acute febrile respiratory disease in horses in Australia. A type specific ELISA was used to measure equine herpesvirus (EHV1 and EHV4) antibody, and serum neutralisation assays were used to measure equine rhinitis A virus (ERAV) and equine rhinitis B virus 1 and 2 (ERBV1 and ERBV2) antibody titres in serum samples. Results Virus was isolated from 4 of 20 nasal swab samples. There were three isolations of EHV4 and one of ERBV2. By PCR, virus was identified in the nasal swab samples of 12 of the 20 horses. Of those that were positive, 17 viruses were detected as follows: one triple positive (EHV4, EHV2, and EHV5), three double positives (EHV4, ERBV and EHV2, EHV5 in two horses) and eight single positives (EHV4 in two horses, EHV2 in one horse, EHV5 in six horses and ERBV in six horses). Conclusion By virus isolation and PCR, 17 viruses were identified in nasal swab samples from 12 of 20 horses that had acute febrile respiratory disease consistent with a diagnosis of virus infection. Initial PCR identification and subsequent virus isolation led to the isolation of ERBV2 for the first time in Australia and the second time anywhere of ERBV2. [source] Implementation in Australia of molecular diagnostic techniques for the rapid detection of foot and mouth disease virusAUSTRALIAN VETERINARY JOURNAL, Issue 7 2004DB BOYLE Objective To evaluate and implement rapid molecular diagnostic techniques for the detection of foot and mouth disease virus (FMDV) suitable for use in Australia. Design Two PCR TaqMan assays targeted to the FMDV internal ribosome entry site or the 3D polymerase coding region for the rapid detection of FMDV were evaluated using non-infectious materials to determine the test most appropriate for implementation as part of Australia's national preparedness for the rapid detection and diagnosis of FMD outbreaks. Results Two published tests (PCR TaqMan assays targeted to the FMDV IRES region or the FMDV 3D polymerase coding region) were evaluated for their ability to detect FMDV genetic material in non-infectious FMDV ELISA antigen stocks held at Australian Animal Health Laboratory. Both tests were able to detect FMDV genetic material from strains O1 Manisa, O-3039, A22, A24, A Malaysia, C, Asia 1 and SAT 1, 2 and 3. With the exception of Asia 1, the TaqMan assay targeted to the FMD 3D polymerase coding region had Ct values equal to or lower than for the TaqMan assay targeted to the IRES region suggesting that this test may provide broader serotype detection and sensitivity. However, the TaqMan assay directed to the FMDV IRES is the only one to date to have undergone substantial evaluation using clinical samples collected during an outbreak. The greatest differences observed were for O-3039, SAT 1, and 3. Conclusion Given the ease of setting up both tests, AAHL currently runs both tests on highly suspect FMD investigations to provide independent confirmation of the absence of FMDV because the tests are focused on two independent regions of the FMDV genome. These tests add substantially to Australia's preparedness for FMD diagnosis complementing the already well-established virus isolation and antigen capture ELISA tests for index case diagnosis of FMD in Australia. [source] | |||||||||||||