Novel approach for detecting classical swine fever virus from swabs of wild boar cut tails using nested real-time PCR.

We devised a method to detect the classical swine fever virus (CSFV) in tail-wiped swabs from wild boars. The CSFV gene in swabs was detected with high sensitivity using nested real-time polymerase chain reaction (PCR), which is a combination of reverse transcription-PCR (RT-PCR) and real-time PCR. We compared CSFV gene detection from boar tissue using the conventional and our tail-wiped swab method. The tail-wiped swab method showed sensitivity and specificity of 100% (26/26) and 98.8% (172/174), respectively compared to the conventional method. Thus, the swab-based CSFV detection method was considered to have detection sensitivity comparable to that of conventional methods. Additionally, we conducted surveillance for CSFV in wild boars on Awaji Island. CSFV was detected in 10.7% (45/420) of samples.

Assessing the litter level agreement of RT-PCR results for porcine reproductive and respiratory syndrome virus in testicles, tails and udder wipes diagnostic samples relative to serum from piglets.

Porcine reproductive and respiratory syndrome (PRRS) is currently the most detrimental disease in the U.S swine industry. Clinical signs of PRRS virus (PRRSv) infection in breeding herds include reproductive failure with abortions, stillbirths, premature farrowings and increased pre-weaning mortality. Serum from due-to-wean piglets is considered the most suitable specimen to monitor PRRSv infection and stability in breeding herds. However, processing fluids (PF - the serosanguinous exudate resultant of the collection of tails and testicles during processing) are a new specimen proposed to monitor piglets at processing (3-5 days of age) and udder wipes (UW) of lactating sows is yet another specimen to monitor infection status of suckling piglets indirectly. Here, we assessed which specimen type (e.g. sera, testicles, tails or UW) should be used to accurately establish the PRRSv status of a litter. Twenty-four litters were conveniently selected on a farm at 10 weeks post PRRSv outbreak. Blood samples, tails and testicles from every piglet in a litter, and an udder skin wipe from the sow were collected at processing (3-5 days). Individual litter testicles and tails as well as the udder wipe were placed each in a reclosable bag to prevent cross-contamination. Sensitivity (Se), specificity (Sp), negative predictive value (NPV), positive predictive value (PPV) and global agreement at the litter level were calculated using the sera results of the litter as the gold standard. The optimum cycle threshold (Ct) value to classify a sample as negative was ≥35 for serum and ≥36 for the aggregated samples (testicles, tails, and UW) based on the ROC curve analysis. Using those thresholds, the fluid collected from the testicles showed the best overall performance (Se = 92 % [62-100]; Sp = 82 % [48-98], NPV = 90 % [55-100], PPV = 85 % [55-98], global agreement = 87 %) compared to tail fluid and UW. Sensitivity of the tail fluid was 62 % (32-86) and the UW was 23 % (5-54), both of which yielded a 100 % specificity and PPV. This study provides information on the contribution of each of the tissues collected at processing on the detection of PRRSv, which becomes relevant in countries were castration and/or tail docking is banned.

Quantum dot-fluorescence in situ hybridisation for Ectromelia virus detection based on biotin-streptavidin interactions.

Ectromelia virus (ECTV) is an pathogen that can lead to a lethal, acute toxic disease known as mousepox in mice. Prevention and control of ECTV infection requires the establishment of a rapid and sensitive diagnostic system for detecting the virus. In the present study, we developed a method of quantum-dot-fluorescence based in situ hybridisation for detecting ECTV genome DNA. Using biotin-dUTP to replace dTTP, biotin was incorporated into a DNA probe during polymerase chain reaction. High sensitivity and specificity of ECTV DNA detection were displayed by fluorescent quantum dots based on biotin-streptavidin interactions. ECTV DNA was then detected by streptavidin-conjugated quantum dots that bound the biotin-labelled probe. Results indicated that the established method can visualise ECTV genomic DNA in both infected cells and mouse tissues. To our knowledge, this is the first study reporting quantum-dot-fluorescence based in situ hybridisation for the detection of viral nucleic acids, providing a reference for the identification and detection of other viruses.

Adverse events post smallpox-vaccination: insights from tail scarification infection in mice with Vaccinia virus.

Adverse events upon smallpox vaccination with fully-replicative strains of Vaccinia virus (VACV) comprise an array of clinical manifestations that occur primarily in immunocompromised patients leading to significant host morbidity/mortality. The expansion of immune-suppressed populations and the possible release of Variola virus as a bioterrorist act have given rise to concerns over vaccination complications should more widespread vaccination be reinitiated. Our goal was to evaluate the components of the host immune system that are sufficient to prevent morbidity/mortality in a murine model of tail scarification, which mimics immunological and clinical features of smallpox vaccination in humans. Infection of C57BL/6 wild-type mice led to a strictly localized infection, with complete viral clearance by day 28 p.i. On the other hand, infection of T and B-cell deficient mice (Rag1(-/-)) produced a severe disease, with uncontrolled viral replication at the inoculation site and dissemination to internal organs. Infection of B-cell deficient animals (µMT) produced no mortality. However, viral clearance in µMT animals was delayed compared to WT animals, with detectable viral titers in tail and internal organs late in infection. Treatment of Rag1(-/-) with rabbit hyperimmune anti-vaccinia serum had a subtle effect on the morbidity/mortality of this strain, but it was effective in reduce viral titers in ovaries. Finally, NUDE athymic mice showed a similar outcome of infection as Rag1(-/-), and passive transfer of WT T cells to Rag1(-/-) animals proved fully effective in preventing morbidity/mortality. These results strongly suggest that both T and B cells are important in the immune response to primary VACV infection in mice, and that T-cells are required to control the infection at the inoculation site and providing help for B-cells to produce antibodies, which help to prevent viral dissemination. These insights might prove helpful to better identify individuals with higher risk of complications after infection with poxvirus.

Mosquitoes inoculate high doses of West Nile virus as they probe and feed on live hosts.

West Nile virus (WNV) is transmitted to vertebrate hosts by mosquitoes as they take a blood meal. The amount of WNV inoculated by mosquitoes as they feed on a live host is not known. Previous estimates of the amount of WNV inoculated by mosquitoes (10(1.2)-10(4.3) PFU) were based on in vitro assays that do not allow mosquitoes to probe or feed naturally. Here, we developed an in vivo assay to determine the amount of WNV inoculated by mosquitoes as they probe and feed on peripheral tissues of a mouse or chick. Using our assay, we recovered approximately one-third of a known amount of virus inoculated into mouse tissues. Accounting for unrecovered virus, mean and median doses of WNV inoculated by four mosquito species were 10(4.3) PFU and 10(5.0) PFU for Culex tarsalis, 10(5.9) PFU and 10(6.1) PFU for Cx. pipiens, 10(4.7) PFU and 10(4.7) PFU for Aedes japonicus, and 10(3.6) PFU and 10(3.4) PFU for Ae. triseriatus. In a direct comparison, in vivo estimates of the viral dose inoculated by Cx. tarsalis were approximately 600 times greater than estimates obtained by an in vitro capillary tube transmission assay. Virus did not disperse rapidly, as >99% of the virus was recovered from the section fed or probed upon by the mosquito. Furthermore, 76% (22/29) of mosquitoes inoculated a small amount of virus ( approximately 10(2) PFU) directly into the blood while feeding. Direct introduction of virus into the blood may alter viral tropism, lead to earlier development of viremia, and cause low rates of infection in co-feeding mosquitoes. Our data demonstrate that mosquitoes inoculate high doses of WNV extravascularly and low doses intravascularly while probing and feeding on a live host. Accurate estimates of the viral dose inoculated by mosquitoes are critical in order to administer appropriate inoculation doses to animals in vaccine, host competence, and pathogenesis studies.

Sensitivity of a diagnostic test for amphibian Ranavirus varies with sampling protocol.

Field samples are commonly used to estimate disease prevalence in wild populations. Our confidence in these estimates requires understanding the sensitivity and specificity of the diagnostic tests. We assessed the sensitivity of the most commonly used diagnostic tests for amphibian Ranavirus by infecting salamanders (Ambystoma tigrinum; Amphibia, Caudata) with Ambystoma tigrinum virus (ATV) and then sampling euthanized animals (whole animal) and noneuthanized animals (tail clip) at five time intervals after exposure. We used a standard polymerase chain reaction (PCR) protocol to screen for ATV. Agreement between test results from whole-animal and tail-clip samples increased with time postexposure. This indicates that the ability to identify infected animals increases following exposure, leading to a more accurate estimate of prevalence in a population. Our results indicate that tail-clip sampling can underestimate the true prevalence of ATV in wild amphibian populations.

Biological activity of an intravenous preparation of human vaccinia immune globulin in mouse models of vaccinia virus infection.

The biological activity of a new intravenous (i.v.) preparation of human vaccinia immune globulin (VIGIV) was evaluated in two mouse models of vaccinia virus (VV) infection. In a mouse tail lesion model, female CD-1 mice were inoculated i.v. with 7 x 10(4) PFU of VV to produce >10 lesions per tail 8 days later. In a mouse lethality model, female severe combined immunodeficient (SCID) mice were inoculated i.v. with 3 x 10(4) PFU of VV to produce 100% mortality within 45 days. The ability of VIGIV to reduce tail lesion formation in CD-1 mice and mortality in SCID mice was determined by (i) pretreatment of a lethal VV dose with VIGIV prior to i.v. inoculation into SCID mice and (ii) i.v. administration of VIGIV to CD-1 and SCID mice the day before and up to 8 days after VV infection. VIGIV reduced the proportion of CD-1 mice with >10 tail lesions in a dose-related manner when VIGIV was given 1 day before and up to 1 day after VV inoculation. The pretreatment of VV with VIGIV prolonged survival and decreased mortality. VIGIV (100 and 400 mg/kg) prolonged survival when given up to 4 days after VV inoculation, and the 400-mg/kg dose reduced the mortality rate by 80% when given the day before or immediately after VV inoculation. The biological activity of VIGIV was demonstrated in both the immunocompetent and immunocompromised murine models. The timing of treatment relative to VV inoculation appeared to be important for the demonstration of VIGIV's biological activity.

Low rates of proviral integration in SWR/J-RF/J hybrid mice.

A high frequency of proviral acquisition has previously been reported in the offspring of SWR/J-RF/J hybrid mice. In the present study, it was investigated whether this proviral acquisition would be useful for large-scale insertional mutagenesis studies. A population of SWR/J-RF/J hybrid mice with a predominantly SWR/J background was created. Lines of mice with such a background and partially congenic for two active proviruses from the RF/J strain were generated (the insert lines). Control lines were derived from mice which had no proviral loci but had an otherwise similar genetic background. DNA samples of mice in the insert lines were screened for the appearance of new proviral loci by Southern hybridization. The rate of proviral acquisition, calculated from the observed number of new proviral loci was 0.023 new proviruses per mouse. This rate is lower than found in previous studies and too low for large-scale insertional mutagenesis studies. A sensitivity experiment indicated that there was adequate detection of new proviral loci. The number of segregating proviruses was consistent with the number of newly acquired proviruses actually detected. Two additional crosses between mice in the insert lines and SWR/J mice were performed. The rate of proviral acquisition was greatly increased when SWR/J females were initially mated to insert mice, but remained unchanged when SWR/J males were used. This suggested that mice in the insert lines had acquired a maternally transmitted factor, which was suppressing viral expression and thus reducing the rate of proviral acquisition.