Development and evaluation of real-time RT-PCR using ear hair for specific detection of sheep persistently infected with border disease virus (BDV).

The aim of this study was to develop an improved border disease virus (BDV) specific real time RT-PCR and to evaluate its performance on manually plucked hairs from sheep persistently infected with BDV that may act as a non-invasive alternate sample. The BDV real time RT-PCR assay reported here showed a high analytical sensitivity (100.6 TCID50/ml), specificity (no reactivity with BVDV-1, BVDV-2, HoBi-like pestivirus and CSFV) and reproducibility. When the assay was validated on 210 samples from BDV-infected and uninfected sheep, it showed a 100% diagnostic sensitivity and specificity with virus isolation. Further evaluation of the assay on manually plucked hair follicles from ear (mid-lateral, mid-medial) and tail tip from sheep persistently infected with BDV showed that a minimum of 20 hair follicles need to be tested for correct diagnosis of BDV. The BDV load was comparatively higher in hairs from mid-medial ear than those from other tested locations. Evaluation on other samples from PI sheep demonstrated that the test performance was similar to that of pestivirus generic real-time RT-PCR, but improved than the currently available BDV specific real-time RT-PCR. Although more number of PI animals need to be evaluated, the results of the study showed that manually plucked hairs from mid-medial ear pinna is a suitable alternative sample in real-time RT-PCR for detection of BDV persistently infected sheep. Use of the non-invasive ear hair samples and the improved BDV specific real-time RT-PCR reported here may be useful for BDV surveillance in several sheep rearing countries.

[The detection of the human papilloma virus during hyperplastic processes in the nose, ears and throat]. / Obnaruzhenie virusa papillomy cheloveka pri giperplasticheskikh protsessakh v LOR-organakh.

The objective of the present work was to carry out the virological and histological studies of various neoplastic and hyperplastic processes in the nose, ears, and throat with a view to identifying the presence of human papilloma virus and Epstein-Barr virus. The brush biopsies and remote neoplasms obtained from 18 patients (including 2 children and 16 adults) presenting with various ENT diseases and tumours were available for the virological investigation with the use of the polymerase chain reaction (PCR) and a system MY09-MY11 degenerate primers . The histological study of biopsies and remote neoplasms was carried out by means of conventional light microscopy. The virological and histological studies conducted in parallel confirmed the diagnostic significance of morphological changes at the tissue and cellular levels caused by the human papilloma virus.

Dermal-resident versus recruited γδ T cell response to cutaneous vaccinia virus infection.

The study of T cell immunity at barrier surfaces has largely focused on T cells bearing the αß TCR. However, T cells that express the γδ TCR are disproportionately represented in peripheral tissues of mice and humans, suggesting they too may play an important role responding to external stimuli. In this article, we report that, in a murine model of cutaneous infection with vaccinia virus, dermal γδ T cell numbers increased 10-fold in the infected ear and resulted in a novel γδ T cell population not found in naive skin. Circulating γδ T cells were specifically recruited to the site of inflammation and differentially contributed to dermal populations based on their CD27 expression. Recruited γδ T cells, the majority of which were CD27(+), were granzyme B(+) and made up about half of the dermal population at the peak of the response. In contrast, recruited and resident γδ T cell populations that made IL-17 were CD27(-). Using a double-chimera model that can discriminate between the resident dermal and recruited γδ T cell populations, we demonstrated their divergent functions and contributions to early stages of tissue inflammation. Specifically, the loss of the perinatal thymus-derived resident dermal population resulted in decreased cellularity and collateral damage in the tissue during viral infection. These findings have important implications for our understanding of immune coordination at barrier surfaces and the contribution of innate-like lymphocytes on the front lines of immune defense.

Comparison of serum, ear notches, and nasal and saliva swabs for Bovine viral diarrhea virus antigen detection in colostrum-fed persistently infected (PI) calves and non-PI calves.

The diagnosis of neonatal and young calves persistently infected (PI) with Bovine viral diarrhea virus (BVDV) by antigen-capture enzyme-linked immunosorbent assay (ACE) may be complicated by interference from colostrum-derived specific antibodies. Ten calves, with 3 calves identified as PI and 7 as non-PI were used in the current study. All non-PI calves were shown to be seropositive for BVDV-specific antibodies by antibody enzyme-linked immunosorbent assay (Ab-ELISA) on serum. Serum samples, ear notch samples, and nasal and saliva swabs were collected from each calf from birth until 12 weeks of age and tested by ELISA for BVDV-specific antigen and antibodies. Following colostrum ingestion, Ab-ELISA sample-to-positive (S/P) ratios rose by a mean of 0.95 (95% confidence interval [CI] = 0.64-1.25) and 1.72 (95% CI = 1.55-1.89) in seropositive, non-PI calves and in PI calves, respectively. The mean S/P ratios then declined to approximately 1.1 in non-PI calves and 0.5 in PI calves at between 60 and 80 days of age. In PI calves, testing for antigen in serum and nasal and saliva swabs was subject to interference by colostrum-derived antibodies in calves up to 3 weeks of age. Nasal swabs were less affected than serum and saliva swabs. Ear notches maintained positive ACE corrected optical densities at all sample times, despite a drop in the signal following the ingestion of colostrum.

Generation of calves persistently infected with HoBi-like pestivirus and comparison of methods for detection of these persistent infections.

The identification and elimination of persistently infected (PI) cattle are the most effective measures for controlling bovine pestiviruses, including bovine viral diarrhea virus (BVDV) and the emerging HoBi-like viruses. Here, colostrum-deprived calves persistently infected with HoBi-like pestivirus (HoBi-like PI calves) were generated and sampled (serum, buffy coat, and ear notches) on the day of birth (DOB) and weekly for 5 consecutive weeks. The samples were subjected to diagnostic tests for BVDV--two reverse transcriptase PCR (RT-PCR) assays, two commercial real-time RT quantitative PCR (RT-qPCR), two antigen capture enzyme-linked immunosorbent assays (ACE), and immunohistochemistry (IHC)--and to HoBi-like virus-specific RT-PCR and RT-qPCR assays. The rate of false negatives varied among the calves. The HoBi-like virus-specific RT-PCR detected HoBi-like virus in 83%, 75%, and 87% of the serum, buffy coat, and ear notch samples, respectively, while the HoBi-like RT-qPCR detected the virus in 83%, 96%, and 62%, respectively. In comparison, the BVDV RT-PCR test had a higher rate of false negatives in all tissue types, especially for the ear notch samples (missing detection in at least 68% of the samples). The commercial BVDV RT-qPCRs and IHC detected 100% of the ear notch samples as positive. While ACE based on the BVDV glycoprotein E(rns) detected infection in at least 87% of ear notches, no infections were detected using NS3-based ACE. The BVDV RT-qPCR, ACE, and IHC yielded higher levels of detection than the HoBi-like virus-specific assays, although the lack of differentiation between BVDV and HoBi-like viruses would make these tests of limited use for the control and/or surveillance of persistent HoBi-like virus infection. An improvement in HoBi-like virus tests is required before a reliable HoBi-like PI surveillance program can be designed.

[Two years BVD ear notch samples diagnostics--results from 16 districts of Lower Saxony]. / Zwei Jahre Bovine Virusdiarrhoe Virus- Ohrstanzprobendiagnostik--Ergebnisse aus 16 Landkreisen Niedersachsens.

Since June 1st 2010 all calves in Lower Saxony are tested by ear notch samples for the presence of Bovine Virus Diarrhea (BVD) Virus based on the Lower Saxony BVDV-regulation. Since January 1st 2011 the new German BVDV-act requires an examination of the calves in the first 6 months of their life. In the Institute for Animal Health of LUFA Nord-West 1000-2000 ear notch samples originating from 16 rural districts are tested daily. In the period from June 1st 2010 to May 31st 2012 a total of 524,214 tissue samples were examined by an antigen ERNS ELISA. In case of low positive results the tests were verified by PCR. 2454 ear notch samples (0.47%) were from persistently with BVDV infected calves (PI-calves) coming from 763 farms (10.2% of the participating farms). In the first seven months of the eradication program 0.75% of the tested samples were positive. This number decreased in the year 2011 to 0.52%. In the first 5 months of 2012, only 0.18% of the ear notch samples tested positive.

Porcine ear necrosis syndrome: a preliminary investigation of putative infectious agents in piglets and mycotoxins in feed.

The aim of this study was to identify the causative factors of porcine ear necrosis syndrome (PENS) in 72 pigs, 5.5-10 weeks in age housed on nine farms. Biopsy samples of ear pinnae were collected from all piglets for bacteriology, histopathology and in situ hybridization for porcine circovirus type 2 (PCV2). At the same time, serum samples were taken for serological analysis and viral PCR, and feed was sampled for mycotoxin analysis. The initial lesion of PENS seemed to be a focal epidermal necrosis. Streptococci were isolated from 44 and staphylococci from 36 pinnae. PCV2 could not be detected by in situ hybridization or qPCR. Seven piglets were positive for porcine reproductive and respiratory syndrome virus, and one for Mycoplasma suis. One piglet had antibodies against Sarcoptes scabiei var. suis. No infectious agents were found in 15 samples. Positive virology and parasitology were often found alongside positive bacteriology. Deoxynivalenol, zearalenone and ergot alkaloids were detected in feed. The findings suggest that PENS is multifactorial in origin and that although infectious agents can be involved in the development of the syndrome they are not the exclusive triggering factor.

Evaluation of the effects of long-term storage of bovine ear notch samples on the ability of 2 diagnostic assays to identify calves persistently infected with bovine viral diarrhoea virus.

Research aimed at optimising diagnostic laboratory procedures is central to the development of effective bovine viral diarrhoea virus (BVDV) control programmes. BVDV is a single-stranded RNA virus that crosses the placenta to infect foetuses, resulting in reproductive losses due to foetal death or persistently infected calves that die early in life. Persistently infected animals are widely accepted to be the primary reservoir of BVDV and the largest source of infection. This poses important challenges to overall animal/herd health and can cause major losses to the cattle industry. Long-term storage of bovine ear notch samples from calves persistently infected with BVDV may adversely affect the ability of diagnostic assays to detect the virus efficiently. In order to test this hypothesis, ear notch samples from 7 animals were divided into 2 groups. One set was subjected to prompt formalin fixation and the other set stored either as fresh samples without preservatives at -2 degrees C, or soaked overnight in phosphate buffered saline followed by freezing of the supernatant fluid at -2 degrees C. Frozen ear notches and ear notch supernatant yielded positive results with an antigen-capture, enzyme linked immunosorbent assay (AC-ELISA) for the duration of the study (6 months) and optical density (OD) values remained significantly within range. There was no significant difference between storing fresh ear notch samples or PBS at -2 degrees C. However, positive immunohistochemistry (IHC) staining on formalin fixed ear notches started to fade between Day 17 and Day 29 when stored at room temperature. It was concluded that fresh ear notches could safely be stored at -2 degrees C for a period of 6 months prior to testing for BVD viral antigens.

Development of an indirect immunofluorescence assay for diagnosis of bovine viral diarrhoea virus on ear notch tissue samples in cattle infected persistently.

Bovine viral diarrhoea virus (BVDV) causes a disease that has a wide range of clinical symptoms in domestic and wild ruminants. It is a major problem in cattle and causes significant economic losses in the cattle industry. The virus infects bovines of all ages and causes both immunosuppression and reproductive, respiratory and digestive disorders. Cattle infected persistently, as a continuing source of the virus and the main factor in transmission of the disease between and among herds, are the main source of BVDV and a primary factor in the epidemiology of the disease. To determine whether a BVDV infection is persistent, two samples should be taken at 3-4 week intervals and tested for the virus antigen. Animal sera, whole blood, organ and ear notch tissue samples can be used for BVDV diagnosis. In ear notch tissue, viral antigen can be detected by an antigen enzyme-linked immunosorbent assay (antigen ELISA), immunohistochemistry (IHC) and reverse-transcription polymerase chain reaction (RT-PCR). This paper describes the development and implementation of an indirect immunofluorescence (IF) method using ear notch tissue samples for diagnosis of cattle infected persistently. Results obtained by this method show that IF is a good alternative to RT-PCR and antigen ELISA and can be a quick and accurate method in diagnosis of BVDV in cattle infected persistently with this virus.

Strong foreign promoters contribute to innate inflammatory responses induced by adenovirus transducing vectors.

E1-deleted adenovirus (FG Ad) transducing vectors are limited for use in vivo by their induction of strong innate and adaptive inflammatory responses. We have examined the contribution of the transgene cassette, particularly the foreign promoter driving transgene expression, in the induction of innate inflammation using a mouse ear model in which swelling is measured as a sensitive surrogate marker of the total innate inflammatory response. The commonly used cytomegalovirus major immediate early (CMV) promoter led to high-level swelling that was independent of transgene expression, while the Rous sarcoma virus and human ubiquitin C promoters led to intermediate levels of swelling and the Ad E1A promoter or no promoter led to equally low levels of swelling. Significant swelling was induced by a virus in which the E1A promoter directed pIX expression, supporting the possibility that activation of expression of Ad genes retained in the vector plays an important role in the inflammatory response. Taken together, our findings support the idea that strong foreign promoters likely play the limiting role in the induction of innate and adaptive immune responses that limit the duration of transgene expression after transduction by FG Ad vectors.

Insertion of CTCF-binding sites into a first-generation adenovirus vector reduces the innate inflammatory response and prolongs transgene expression.

We have made improvements to E1-deleted adenovirus (Ad) transducing vectors that both substantially reduce the innate inflammatory response provoked by the virus in BALB/c mouse ears and increase the duration of expression of the GFP transgene in BALB/c mouse liver. These improvements result from testing the hypothesis that induction of strong innate responses is primarily a result of the powerful enhancer contained within the strong CMV promoter activating expression of Ad genes retained within the vector. A DNA fragment containing four CTCF-binding sites, which was expected to act as a chromatin insulator, was introduced 5', 3', or both 5' and 3' of a CMV-GFP cassette in an attempt to reduce activation of Ad gene expression by the enhancer. The presence of this sequence in any of the configurations led to reduction of the innate immune response, as assayed by mouse ear swelling, to the low level induced by a virus deleted for the E1 region and carrying no introduced sequence. In addition, the duration of GFP expression in the liver more than doubled. The prolonged GFP expression indicates that GFP does not play the limiting role in shutting down vector expression. The CTCF-binding sequence introduced appears to act as a chromatin insulator in Ad DNA, but position-independence of the elements in reducing the innate immune response indicate unanticipated complexities in the mechanism by which Ad vectors induce innate immune responses.

Transmissible infection of human 293T cells with porcine endogenous retroviruses subgroup a from NIH-miniature pig.

In pig-to-human xenotransplantation, zoonotic infections have been an important barrier. The risk of zoonosis has been emphasized in xenotransplantation after finding that porcine endogenous retroviruses (PERVs) can infect human cells in vitro. Until now, transmissions of PERVs from PK15 cells have been studied in vitro and in vivo, but transmission of PERVs originating from miniature pigs have not been extensively reported. Peripheral blood mononuclear cells from miniature swine showed PERV transmission to human cells. In contrast, specific pathogen-free (SPF) pig islet cells showed no PERV transmission when co-incubated with 293T cells. To evaluate the risk of zoonosis with our experimental mini pigs, we tested the infectivity of PERVs from NIH-miniature pig primary ear cells for human 293T cells. As a result, all subgroups of infectious PERV virion (PERV-A, -B, and -C) were detected in the primary cell culture media. Unlike PERV-C, PERV-A and -B infected human 293T cells. Interestingly, only proviral PERV-A replicated in 293T cells to produce virions after infection. Our results suggested that a prevention study of PERV xenotransmission from experimental miniature pigs should concentrate on PERV-A control.

Bovine viral diarrhea virus multiorgan infection in two white-tailed deer in southeastern South Dakota.

The susceptibility of wild ruminants, especially cervids, to bovine viral diarrhea virus (BVDV) has remained an enigma. Two white-tailed deer (Odocoileus virginianus) were submitted to the Animal Disease Research and Diagnostic Laboratory (ADRDL) in the fall of 2003 by the South Dakota Game Fish and Parks for chronic wasting disease (CWD) testing. Both animals were CWD negative. The animals were necropsied and histopathology, viral antigen detection, and virus isolation were performed. A noncytopathic (NCP) BVDV was isolated from the lungs and several other tissues of both animals. Formalin-fixed ear notches from both animals were positive for BVDV antigen by immunohistochemistry. The BVDV isolates were typed with the use of polymerase chain reaction in 5' untranslated region (UTR) and one isolate was typed a Type 2a and the other a Type 1b. Future field surveys to determine the incidence of BVDV along with experimental studies to determine if white-tailed deer fawns can be persistently infected with BVDV are needed.

Prevalence and characterization of bovine viral diarrhea virus in the white-tailed deer population in Indiana.

Bovine viral diarrhea (BVD) is one of the economically important diseases of cattle. For many years, different types of vaccines have been commercially available, yet this disease is hard to control in high-density population areas. Detection and isolation of bovine viral diarrhea virus (BVDV) from any potential reservoir is vital, especially when considering virus eradication from a herd or locale. One potential source is wild ruminants. Ear notches and lymph nodes were collected from the wild population of white-tailed deer (Odocoileus virginianus) during deer hunting season in Indiana and tested for BVDV with a commercial BVD antigen capture enzyme-linked immunosorbent assay. Two samples out of 745 collected samples were positive, and subsequently cp and ncp BVDV was isolated from 1 ear notch and 1 lymph node. These isolates were genotyped as type 1a and 1b based on sequence analysis of the 5' untranslated region (UTR). The results of the present study indicate that the prevalence of BVDV in the white-tailed deer population of Indiana is about 0.3%. Wild ruminants infected with BVDV should be taken into consideration during an eradication program of BVDV from the livestock population.

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.

Genital herpes due to acyclovir-sensitive herpes simplex virus caused secondary and recurrent herpetic whitlows due to thymidine kinase-deficient/temperature-sensitive virus.

Herpes simplex virus (HSV)-2 caused a genital ulcer in a 40-year-old allogenic stem cell recipient, and a secondary herpetic whitlow appeared during 2 months of acyclovir (ACV) therapy. Both genital ulcer, and whitlow were cured 3 months later, but 6 months after recovery the whitlow alone recurred. DNA of the genital, first, and recurrent whitlow isolates showed similar endonuclease digestion fragment profiles. The genital virus was ACV-sensitive, and the two whitlow isolates were ACV-resistant/thymidine kinase (TK)-deficient. The TK gene of the whitlow isolates had the same frame shift from the 274th amino acid and termination at the 347th amino acid due to the deletion of a cytosine at the 819th nucleotide. Because the temperature of the thumb is 33/34 degrees C or lower, the temperature sensitivity of the isolates were compared, and both whitlow isolates were significantly more temperature-sensitive (ts) at 39 degrees C than the genital isolate. The two whitlow isolates showed cutaneous pathogenicity in mouse ear pinna but not midflank, while the genital isolate was pathogenic at both sites, suggesting that temperature adaptation was an important element of pathogenicity in the whitlow. The virus populations of isolates of the genital, and first whitlow were examined by 31, and 82 clones, respectively, and the clones from genital, and whitlow isolates were ACV-sensitive, and -resistant, respectively, showing their homogeneity. The acyclovir-sensitive genital lesion had spread as a TK-deficient/ts herpetic whitlow during ACV treatment, and an apparently TK-deficient virus adapted to the local temperature might have caused the whitlow recurrence.

[Identification of human papilloma viruses (HPV) in inflammatory states and ear neoplasms]. / Identyfikacja wirusów ludzkich brodawczaków (HPV) w stanach zapalnych i nowotworach uszu.

INTRODUCTION: Human Papilloma Virus has a strong relation to oropharyngeal mucosa and is considered to be responsible for a wide range of upper respiratory tract pathologies, like laryngeal papilloma. There's a hypothesis, that it plays a significant role in middle ear chronic inflammations and neoplasm's. MATERIAL AND METHODIC. The examination was carried on a group of 53 patients, 39 of which was suffering from granulation tissue chronic otitis media, 7-cholesteatomatous otitis media, 6--middle ear malignant neoplasm, and 1 middle and/or external ear benign neoplasm. The control group consisted of 5 patients operated on: otosclerosis--4 cases and post-traumatic tympanic membrane perforation--1 case. The material was postoperative tissue, like polyps, inflammatory granulation tissue, cholesteatoma masses and malignant neoplasm's tissue. RESULTS: In the whole group of 53 examined cases, HPV DNA was confirmed in 22 cases (41.5%), in that group oncogenic types 16 or 18 in 12 cases (22.6%), and in 14 cases (26.4%) types 6 or 11. In a group of chronic granulomatous otitis media DNA characteristic for Papilloma was identified in 12 cases (25.6%), in it in 9 cases DNA HPV type 6 or 11 was confirmed, and in 7 cases type 16 or 18. Among cholesteatomatous chronic otitis media HPV DNA types 6 or 11 was identified in 70%. In every case of middle ear malignant neoplasm a presence of high-risk DNA Papilloma types 16 or 18 was confirmed. In any case of control group HPV DNA was detected. CONCLUSIONS: The results has been compared with other authors examinations and it is claimed that they confirm the observation, that Human Papilloma Viruses may be a factor, that might play an important role in pathology of chronic otitis media and ear neoplasm's. It is concluded, that differences in percentages of HPV presence in chronic inflammations (70%) and ear neoplasm's may be explained by viral co-infection during bacterial c. o. m. Viral infection probably evolves carcinogenesis, which leads to a neoplastic growth.

Failure to detect bovine viral diarrhea virus in necropsied farm-raised white-tailed deer (Odocoileus virginianus) in Pennsylvania.

Between January 1 and December 31, 2005 gross and histologic examinations were performed on carcasses of 61 farm-raised white-tailed deer originating from Pennsylvania. Single-tube real-time reverse transcription polymerase chain reaction (real-time RT-PCR) for the detection of bovine viral diarrhea virus type 1 (BVDV-1) and type 2 (BVDV-2) was performed on each animal. Virus isolation was performed on tissue samples from 25 of 61 animals. Immunohistochemical (IHC) staining of ear-notch skin to identify BVDV antigen was performed on each animal. All tissues samples tested negative for both BVDV-1 and BVDV-2 by real-time RT-PCR, virus isolation, and IHC. Gross or histopathologic lesions suggestive of BVDV infection were not detected. Results of this study suggest that BVD is not a common cause of mortality in farm-raised white-tailed deer in Pennsylvania.