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Oysters Crassostrea gigas were placed at water supply canals of three shrimp farms in Guasave, Mexico where WSSV outbreaks occur. Animals were sampled through April-August and September-December to detect WSSV DNA. By using three different PCR protocols, only oysters from a farm undergoing a WSSV outbreak were found WSSV-positive in gills and digestive gland. Two WSSV amplicons were sequenced and they corresponded over 99% to WSSV genome segments. Results showed that oysters can capture WSSV particles suspended in water. Susceptibility of oysters to WSSV infection and their role as a carrier remain to be determined., ((c) 2010 Elsevier Inc. All rights reserved.)

Iridoviruses, pathogens typically associated with fish and amphibians, have recently been shown to cause acute respiratory disease in chelonians including box turtles, red-eared sliders, gopher tortoises, and Burmese star tortoises. Case reports of natural infections in several chelonian species in the United States have been reported, however the prevalence remains unknown in susceptible populations of free-ranging chelonians. To determine the prevalence of iridovirus exposure in free-ranging gopher tortoises (Gopherus polyphemus) in the southeast United States, an indirect enzyme-linked immunosorbent assay (ELISA) was developed and used to evaluate plasma samples from wild gopher tortoises (G. polyphemus) from: Alabama (n=9); Florida (n=658); Georgia (n=225); Louisiana (n=12); Mississippi (n=28); and unknown locations (68) collected between 2001 and 2006. Eight (1.2%) seropositive tortoises were identified from Florida and seven (3.1%) from Georgia for an overall prevalence of 1.5%. Additionally, a population of eastern box turtles was sampled from a private nature sanctuary in Pennsylvania that experienced an outbreak of iridovirus the previous year, which killed 16 turtles. Only 1 turtle out of 55 survivors tested positive (1.8%). Results suggest a low exposure rate in chelonians to this pathogen; however, it is suspected that this is an underestimate of the true prevalence. Since experimental transmission studies and past outbreaks have shown a high rate of mortality in infected turtles, turtles may die before they develop an antibody response. Further, the duration of the antibody response is unknown and may also cause an underestimate of the true prevalence., (Copyright 2009 Elsevier B.V. All rights reserved.)

Loop-mediated isothermal amplification (LAMP) allows rapid amplification of nucleic acids under isothermal conditions. In this report, a 20-min LAMP amplification of the DPOL gene of infectious spleen and kidney necrosis virus (ISKNV) using a biotin-labeled primer was combined with lateral flow dipstick (LFD) chromatography for rapid and simple visual detection of ISKNV-specific amplicons. The LFD process involves a 5-min specific hybridization with an FITC-labeled DNA probe to confirm the presence of complement ISKNV amplicons that were biotinated in LAMP. The resulting DNA duplexes, consisting of labeled probes and amplicons, migrate along the LFD strip by chromatography for 5 min and are trapped at the test line and visualized by biotin labeling. The detection limit of ISKNV by LAMP-LFD was 10 copies. The results show that the LAMP-LFD method has the advantages of better sensitivity and speed and less dependence on equipment than the standard PCR for specifically detecting low levels of ISKNV DNA, and this can be useful in the field as a routine diagnostic tool.

A loop-mediated isothermal amplification (LAMP) assay was developed for the detection of lymphocystis disease virus (LCDV). A set of five specific primers, two inner and two outer primers and a loop primer, were designed on the basis of the major capsid protein gene of LCDV. The reaction time and temperatures were optimized for 60 min at 63 degrees C, respectively. LAMP amplification products were detected by a ladder-like appearance on agarose gel electrophoresis or a naked-eye inspection of a color change in the reaction tube by addition of SYBR Green I. The assay was specific for LCDV, and there was no cross-reactivity with white spot syndrome virus (WSSV) or six other Iridoviridae viruses (epizootic hematopoietic necrosis virus, EHNV; tiger frog virus, TFV; Bohle iridovirus, BIV; soft-shelled turtle iridovirus, STIV; infectious spleen and kidney necrosis virus, ISKNV; red sea bream iridovirus, RSIV). The detection limit of the LAMP assay was 15 fg, which was similar to that of real-time quantitative polymerase chain reaction (PCR) and 10-fold higher than the conventional PCR. The LAMP assay was evaluated using 109 clinical samples, and the results indicated the suitability and simplicity of the test as a rapid, field diagnostic tool for detection of LCDV. The LCDV LAMP assay has potential for early diagnosis of LCDV infection., (2009 Elsevier B.V. All rights reserved.)

Introduction: Myocardial inflammation often coexists with different types of autoimmune diseases. Our aim was to investigate the presence of myocarditis in these patients by Cardiovascular Magnetic Resonance (CMR) and endomyocardial biopsy., Patients-Methods: Twenty patients, aged 20-55 yrs with autoimmune diseases and cardiac symptoms (3 with Takayasu's arteritis, 3 with systemic lupus erythematosus, 5 with rheumatoid arthritis, 7 with autoimmune thyroid disease and 2 with systemic sclerosis) and 20 patients with the same autoimmune diseases but without cardiac symptoms (controls) were studied. The presence of myocarditis and LV function were evaluated by CMR. Myocarditis was documented using T2-weighted (T2-W), T1-weighted (T1-W) before and after contrast media injection and late enhanced images. In 10 patients (positive for myocarditis by CMR with either low LVEF or recent increase in troponin), endomyocardial biopsy was also performed. Myocardial specimens were evaluated by histology and polymerase chain reaction techniques (PCR)., Results: Myocarditis was identified in 18/20 patients by CMR. In the T2-W images the signal ratio of myocardium to skeletal muscle was 1.89+/-0.25 (control values 1.57+/-0.13, p<0.05). From the T1-W images the relative myocardial enhancement was 11.31+/-11.18 (control values 3.09+/-0.05, p<0.05). Epicardial late gadolinium enhanced areas were identified in 18/20. In myocardial specimens, histology revealed inflammation in 5/10 (50%) and PCR documented viral or microbial genomes in 8/10 (80%). Positive histology and PCR were in agreement with 50% and 80% of positive CMR examinations, respectively. Herpes virus was identified in 3/10, Adeno in 1/10, Coxsackie B6 in 1/10, echo in 1/10, Parvo-B19 in 3/10, CMV in 1/10 and Chlamydia trachomatis in 8/10., Conclusions: Myocardial inflammation is a common finding in patients with autoimmune diseases and cardiac symptoms. The diagnosis can be confirmed by CMR, which is a noninvasive and reliable tool for the investigation of these patients.

Background: In pediatric kidney transplant recipients, viral infections occur soon after transplant and may be transmitted from the graft., Methods: This study of 75 pediatric kidney transplants investigated whether genome sequences of parvovirus B19, Epstein-Barr virus (EBV), human cytomegalovirus (HCMV), and BK polyomavirus (BKV) could be detected in kidney graft samples (graft biopsy samples and preservation and washing solutions) collected before implantation and whether their presence was a risk factor for infections in the recipient., Results: B19 DNA was detected in approximately 30% of graft biopsy samples, preservation solutions, and washing solutions; EBV DNA was detected in approximately 20% of preservation and washing solutions but rarely in biopsy samples; and HCMV DNA and BKV DNA were rarely detected in graft biopsy samples. Seronegative recipients of B19 DNA-positive and EBV DNA-positive grafts had a significantly higher risk of infection during the early posttransplant period than did recipients of negative grafts. In particular, none of the B19-seronegative recipients of B19 DNA-negative grafts experienced infection soon after transplant, whereas most recipients of B19 DNA-positive grafts experienced infection within the first month after transplant., Conclusions: Molecular testing of donor grafts for viruses that infect circulating and resident cells in the graft-such as B19 in the kidney-could be useful (in association with donor/recipient serostatus) for identifying recipients at high risk for posttransplant infections.

Ranaviruses are known to cause mortality in a variety of anuran species and have the potential to significantly impact wild and captive frog populations. In this study, 16 captive frogs and toads from the Louisville Zoological Garden were examined for the presence of ranavirus; this group included 14 Cope's grey tree frogs (Hyla chrysoscelis), an American toad (Bufo americanus), and a southern toad (Bufo terrestris). All animals were wild caught and were evaluated via polymerase chain reaction (PCR), while animals that died were also assessed via histologic study to understand the role of ranaviral disease in these specimens. Of the animals that died, 82% were positive for ranavirus via PCR. Multiple swab samples collected over time from live tree frogs were positive for ranavirus via PCR. These findings reveal that ranaviral infection in captive adult anurans may occur without clinical signs or consistent histopathologic lesions.

Long-term analysis of infectious complication after high-dose immunosuppressive therapy with CD34-selected autologous hematopoietic stem cell transplantation for patients with severe autoimmune diseases (AD) was performed. Theoretically, CD34 selection can reduce the risk of reinfusion of autoreactive lymphocytes. However, it is also associated with a significant reduction in T cells, natural killer cells, and monocytes, which in turn may compromise immune reconstitution, thereby increasing the risk of infection. Moreover, AD compromises host immunity and causes organ damage resulting in dysfunction of the cutaneous or mucosal barrier. In this study, the incidence rate of infections is reported in 14 patients who underwent high-dose (200 mg/kg) cyclophosphamide therapy followed by reinfusion of CD34-selected autologous peripheral blood stem cells. Bacterial complication occurred in 3 of 14 (21%) patients. Cytomegalovirus reactivation and adenovirus hemorrhagic cystitis were observed in 9 (64%) and 2 (14%) patients, respectively. As for late infectious complications, 7 patients (50%) developed dermatomal varicella zoster virus infection. No infection-related mortality was seen in this case series. Because the risk for infections approaches that seen in allogeneic transplant recipients, infection surveillance, diagnostic workup, and prophylactic strategies similar to those applicable to allogeneic recipients are warranted.

Purpose: Opportunistic viral infections cause increased morbidity and mortality among human immunodeficiency virus (HIV) infected individuals, especially those who are not on antiretroviral treatment. Early diagnosis of these opportunistic viruses will be able to reduce the risk of disease progression with appropriate intervention., Materials and Methods: Multiplex PCR was attempted to detect the opportunistic herpes viruses (HSV-1, HSV-2, VZV, EBV, and CMV), adenovirus and polyoma viruses (JC and BK) in three cocktails of PCR reactions. Subsequently, all the viruses detected were quantitated by testing using monoplex real time PCR. Whole blood samples collected between 2006 and 2007 from 68 treatment naïve HIV-1 infected and 30 normal healthy individuals were tested for these eight viruses. Among the 68 HIV-1 infected individuals 35 had CD4+ T cell count less than or equal to 200 while the other 33 had greater than 200 CD4+ T cells., Results: Among the 68 HIV-1 infected individuals, 49 (72%) were positive for EBV, 5 (7%) samples were positive for CMV. All the five CMV positive individuals had CD4+ T cell count of less than or equal to 200 cells/microL. The mean EBV load among the individuals with a CD4+ T cells of less than or equal to 200 cells/microL was 3.88 log(10) while among those with greater than 200 CD4+ T cells it was 3.75 log(10) . The mean CMV load was 6.98 log(10). Three samples were positive for both CMV & EBV. None of the samples was positive for HSV-1, HSV-2, VZV, Adenovirus, JC and BK viruses., Conclusions: In our study, multiplex PCR based detection system was found useful in detecting opportunistic viruses in HIV infected individuals. Though EBV is the most prevalent opportunistic viral infection among HIV infected individuals, there was no significant association between EBV load, CD4+ T cell counts and HIV-1 virus load. CMV was seen in HIV infected individuals with low CD4+ T cell counts (less than 200 cells/microL).

Iridoviruses infect food and ornamental fish species from a wide range of freshwater to marine habitats across the globe. The objective of the current study was to characterize an iridovirus causing systemic infection of wild-caught Pterapogon kauderni Koumans 1933 (Banggai cardinalfish). Freshly frozen and fixed specimens were processed for histopathologic evaluation, transmission electron microscopic examination, virus culture, molecular virologic testing, microbiology, and in situ hybridization (ISH) using riboprobes. Basophilic granular cytoplasmic inclusions were identified in cytomegalic cells often found beneath endothelium, and hexagonal virus particles typical of iridovirus were identified in the cytoplasm of enlarged cells by transmission electron microscopy. Attempts at virus isolation in cell culture were unsuccessful; however, polymerase chain reaction (PCR)-based molecular testing resulted in amplification and sequencing of regions of the DNA polymerase and major capsid protein genes, along with the full-length ATPase gene of the putative iridovirus. Virus gene sequences were then used to infer phylogenetic relationships of the P. kauderni agent to other known systemic iridoviruses from fishes. Riboprobes, which were transcribed from a cloned PCR amplification product from the viral genome generated hybridization signals from inclusions within cytomegalic cells in histologic sections tested in ISH experiments. To the authors' knowledge, this is the first report of a systemic iridovirus from P. kauderni. The pathologic changes induced and the genomic sequence data confirm placement of the Banggai cardinalfish iridovirus in the genus Megalocytivirus family Iridoviridae. The ISH provides an additional molecular diagnostic technique for confirmation of presumptive infections detected in histologic sections from infected fish.

A panel of gastroenteritis outbreak samples was subjected to a virus purification and concentration algorithm followed by a sequence-independent amplification method devised to detect viral enteric pathogens. The application of these methods allowed the identification of torque teno virus (TTV) in one outbreak. The complete genome sequence of 3,260 nt was obtained through "genome walking", and four open reading frames were deduced from the genomic sequence. Phylogenetic analysis grouped this virus in TTV genetic group 3, clustering with genotype 27, with 85% similarity at the nt level with strain SAa-01.

Viral diseases of psittacine birds are detected presently by PCR. However, conventional PCR methods are not quantitative and the products can sometimes include non-specific products of the same size. To avoid these problems, real-time PCR assays based on the SYBR Green assay system were developed for the detection and quantitation of four virus diseases of psittacine birds: psittacine beak and feather disease, avian polyomavirus infection, psittacid herpesvirus infection, and psittacine adenovirus infection. Up to 1x10(2) copies of virus DNA were detected, indicating that these assays are as sensitive as conventional PCR assays. The assays are specific because they did not amplify any other pathogens including other viruses, bacteria, and fungi in psittacine birds. The assays measured successfully virus loads in clinical samples (blood, feathers, and tissues), showing that these specimens were suitable targets for the detection and quantitation of viral DNA in psittacine birds.

Few studies have been performed on the prevalence of Torque Teno Virus (TTV) infection in liver transplant (LT) recipients. The aim of this study was to assess the prevalence, viremia and genogroup pattern of TTV among LT patients and to ascertain whether TTV causes liver damage in liver transplanted patients with biochemical and histological changes of unknown origin. Twenty-five patients were evaluated before and after LT; 80 healthy subjects were considered as controls. Serum samples were serially obtained from all the patients before LT and thereafter at 3, 6 and 12 months post-transplant. Serum TTV-DNA and genogroups were assessed by PCR. Patients underwent protocol serial liver biopsies at 6 and 12 months after LT. Results were compared using the Chi-squared tests, McNemar's and Student's t-tests. TTV-DNA was found in 25/25 patients before LT and in 60/80 blood donors (P < 0.01). The TTV-DNA load increased significantly after LT (P < 0.001). TTV-DNA was significantly higher in patients on calcineurin inhibitors (CNI) and azathioprine or mycophenolate mofetil than in patients on CNI alone (P = 0.04) at 3 months after LT. Genogroup analysis showed a significant increase in genogroup 5 positivity after LT. No differences were seen in the viremia of patients compared according to their viral versus other etiologies of their liver disease before transplantation. Viremia and TTV genotype patterns did not correlate with the presence of hypertransaminasemia or histological liver damage of unknown etiology. The prevalence of TTV-DNA was significantly higher in patients with liver cirrhosis than in controls and the viral load was significantly higher after LT than beforehand. On the basis of our data, TTV does not seem to cause liver damage following LT, although larger studies with a long-term follow up are needed to confirm these findings.

Four species (Dendrobates auratus, Phyllobates terribilis, Pyxicephalus adspersus, and Rhacophorus dennysi) of captive anurans with a clinical history of lethargy and inappetence were found dead and were submitted for necropsy. Gross lesions included irregular patches of sloughed skin and rare dermal ulcerations. Histologic findings included epidermal proliferation that was most pronounced on the digits and that included intracytoplasmic chytrid organisms. Bacteria were often associated with the epidermal lesions. Intracytoplasmic inclusion bodies were observed in hepatocytes. Real-time polymerase chain reaction yielded positive results for both Ranavirus and Batrachochytrium dendrobatidis (Bd). Bacterial culture of internal organs yielded Aeromonas hydrophila. This is the first report of concurrent infections in anurans by Ranavirus and Bd and A. hydrophila.

Background: To commence proper antiviral treatment, timely knowledge of whether the infection is caused by DNA or RNA virus would be beneficial for the clinician., Objectives: Our objective was to develop a method for distinguishing between DNA and RNA virus infections., Study Design: In this prospective study, total and differential count of leukocytes, serum C-reactive protein level, erythrocyte sedimentation rate, and quantitative flow cytometric analysis of FcgammaRI (CD64) on neutrophils and monocytes were obtained from 289 hospitalized febrile patients. After microbiological confirmation, 89 patients (31%) were found to have either bacterial (n=46) or viral (n=43) infection. The patient data was compared to 60 healthy controls., Results: For the first time ever, it was noticed that in dsDNA virus infections (n=21) the average amount of CD64 on neutrophils was over five-fold compared to ssRNA virus infections (n=22)., Conclusions: DNA virus score (DNAVS) point, which incorporates quantitative analysis of CD64 on neutrophils and total and differential count of leukocytes, varied between 0 and 8, and displayed 95% sensitivity and 100% specificity in distinguishing between dsDNA and ssRNA virus infections [average (S.D.); DNAVS points: 5.4 (2.5) vs. 0.3 (0.4); p<0.001].

A biological terrorism event could have a large impact on the general population and health care system. The impact of an infectious disaster will most likely be great to emergency departments, and the collaboration between emergency and infectious disease specialists will be critical in developing an effective response. A bioterrorism event is a disaster that requires specific preparations beyond the usual medical disaster planning. An effective response would include attention to infection control issues and plans for large-scale vaccination or antimicrobial prophylaxis. This article addresses some general issues related to preparing an effective response to a biological terrorism event. It will also review organisms and toxins that could be used in biological terrorism, including clinical features, management, diagnostic testing, and infection control.

We recently identified a novel human virus classifiable into a third group in the genus Anellovirus, tentatively designated torque teno midi virus (TTMDV), with a circular DNA genome of 3.2 kb and genomic organization resembling those of torque teno virus (TTV) (3.8 to 3.9 kb) and torque teno mini virus (TTMV) (2.8 to 2.9 kb). TTMDV was characterized by extreme genetic diversity similar to the TTV and TTMV genomes. Taking advantage of universal and virus species-specific primers derived from a highly conserved area located just downstream of the TATA box of the TTV, TTMDV, and TTMV genomes, a PCR method with simultaneous amplification of the genomic DNAs of these three anelloviruses in the first round and subsequent differential amplifications of these viruses in the second round was developed. High prevalence of TTMDV viremia was seen in adults (75/100 [75%]), comparable with the prevalences of TTV viremia (100%) and TTMV viremia (82%). Although none of 10 cord blood samples had detectable TTV, TTMDV, and TTMV DNAs, the prevalences of these three anelloviruses increased with the number of months after birth of the individual and reached 100% for individuals at one year of age. Dual or triple infection of TTV, TTMDV, and/or TTMV was seen in 10 (47.6%) of 21 infants 9 to 180 days of age and more frequently among infants 181 to 364 days of age (20/23 [86.9%]), comparable with the 93.1% (243/261) prevalence among subjects 1 to 81 years of age, indicating early acquisition of dual or triple anellovirus infection during infancy.

The SEN virus has been tentatively linked to transfusion-associated non-A to E hepatitis. The aim of the present study was to 1) determine the prevalence of SEN virus among Egyptian patients with hepatitis C virus (HCV)-related chronic liver disease and patients undergoing hemodialysis and 2) demonstrate the clinical effect of SEN virus infection on coexistent hepatitis C in terms of severity and probability of developing hepatocellular carcinoma. Polymerase chain reaction was used to detect SEN virus-D and SEN virus-H DNA in serum samples of 74 patients with HCV-related chronic liver disease, 45 uremic patients undergoing maintenance hemodialysis, and 28 healthy controls. SEN virus-D/H DNA was detected in 13.5% of patients with chronic liver disease, 11.1% of patients undergoing hemodialysis, and 7.1% of healthy controls, with no significant differences between patients and the control group. Clinical and biochemical measures did not significantly differ between SEN virus-infected and noninfected patients in the chronic liver disease group or the hemodialysis group. The rate of SEN virus infection was significantly higher in patients with chronic liver disease and hepatocellular carcinoma (33.3%) than in those with chronic liver disease only (8.5%) (P < .05). In conclusion, SEN virus does not seem to be a common infection in Egyptian patients. It has no apparent influence on the severity of coexistent HCV-related chronic liver disease but could be a risk factor for hepatocellular carcinoma in such patients. Further studies are needed to define the etiopathogenic role of SEN virus infection in the development of hepatocellular carcinoma.

The use of quantitative polymerase chain reaction (QPCR) to test for largemouth bass virus (LMBV) was evaluated during a challenge experiment in which largemouth bass Micropterus salmoides were immersed in the type strain of LMBV. The real-time PCR and cell culture methods were both used to measure LMBV present in the inoculum. Additional samples tested by QPCR included gill, gonad, kidney, liver, mucus, spleen, and swim bladder. A plasmid clone containing a 248-base pair (bp) fragment of the major capsid protein gene (MCP*) was serially diluted and used as a standard to quantify the number of LMBV DNA copies present in the samples tested. A 62-bp fragment of DNA located in MCP* was amplified in the real-time PCR assay. This work has demonstrated the value of the QPCR assay in LMBV surveys.

Background: TT virus (TTV) infection is highly prevalent in general population and patients with hepatitis B virus (HBV) infection. The aim of the present study was to determine the distribution of the genotypes and genogroups of TTV in healthy and HBV-infected individuals in Korea., Methods: Distribution of TTV genotypes and genogroups was investigated in the serum samples of 69 healthy and 59 HBV-infected individuals. PCR products of N22 region were genotyped by sequence analysis. TTV genogroups were determined by 5 different genogroup-specific PCR assays., Results: Among the 20 sequenced isolates, 9 (45%) were genotype 2, 8 (40%) were genotype 1, 2 (10%) were genotype 3, and 1 (5%) was genotype 4. TTV genogroup 4 was found most frequently (52/128), followed by genogroup 3 (42/128), genogroup 1 (35/128), genogroup 5 (32/128), and genogroup 2 (1/128). Mixed infections with different genogroups were frequent., Conclusions: TTV genotype 2 and 1 are predominant genotypes. TTV genotype 3 was detected for the first time in Korea. TTV genogroups 4 and 3 were predominant genogroups. No significant difference was observed in the distribution of TTV genogroups between healthy and HBV-infected individuals.

Semi-nested polymerase chain reaction technique was used to detect Torquetenovirus (TTV) DNA in 234 healthy blood donors in northeast Thailand. The incidence of TTV was 28% in 101 healthy blood donors negative for HBsAg and anti-HCV antibody, 25% in 71 HBsAg carriers and 29% among 62 with anti-HCV antibody. No association of TTV infection was found with gender, age, and HBV or HCV infection.

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.

A protein chip based on surface plasmon resonance imaging (SPRI) was developed for detecting fish iridovirus antibody using a recombinant 50-kDa fragment of major capsid protein (MCP) as an antigen. The diagnostic potential of SPRI for measuring antibodies to the iridovirus MCP was compared with that of a conventional enzyme-linked immunosorbent assay (ELISA) using 40 juvenile rock bream (Oplegnathus fasciatus) serum samples in a nursery. There was a strong positive correlation between the SPRI and ELISA (n = 40, r = 0.939, P < 0.01). Therefore, this recombinant 50-kDa MCP can be used as an antigen for serological studies, and the SPRI, which is a label-free and high-throughput method, is potentially a valuable tool in the serodiagnosis of an iridoviral infection.

Aims: The development and evaluation of a protocol based on polymerase chain reaction (PCR) and nucleic acid hybridization techniques for the specific detection of lymphocystis disease virus (LCDV) in several marine fish species., Methods and Results: The pair of primers for PCR, OBL3 and OBL4, was designed based on published nucleotide sequence (LCDV-1) and amplifies a fragment within the major capsid protein. The sensitivity was evaluated using DNA from purified viral particles, as well as from cells inoculated with several viral concentrations. The PCR combined with slot blot was the most sensitive methodology, detecting 2.5 ng of viral DNA. Using this methodology LCDV was detected at 5 days postinoculation from SAF-1 cells initially inoculated with 10(-5) TCID(50) ml(-1). The combination of PCR with membrane hybridization has also been proved to be adequate to detect LCDV from apparently healthy carriers by means of caudal fin sample analysis. This asymptomatic infection was also demonstrated by classical virological methods (cell culture and immunoblot)., Conclusions: The protocol described in this study allows the specific detection of LCDV, both in cell cultures and in fin homogenates from asymptomatic fish., Significance and Impact of the Study: The detection of asymptomatic carriers by a rapid molecular method using caudal fin sampling, which does not imply animal killing, could be an important tool to control epizootics caused by LCDV, as fish could be analysed before their introduction and/or mobilization in farm facilities.

Pathogenesis of viral origin has long been suggested in autoimmune rheumatic diseases. Beside the well-defined virus induced transient or chronic rheumatic diseases often resembling systemic autoimmune disorders such as rheumatoid arthritis, viruses can contribute to disease pathogenesis by several different pathomechanisms. TT virus is a recently discovered virus of extremely high genetic diversity which commonly infects humans. Despite accumulated evidence on the biological characteristics of TTV, its pathogenicity is still in question; many consider TTV as a harmless endosymbiont. The recent paper overviews the biology of TT virus and investigates the hypothesis that TTV might have a causative role in human diseases with special attention to the possibility that TTV might trigger autoimmunity in rheumatic disorders.

Infections are considered to be an important cause of unexpected death in children. It has also been assumed that respiratory viruses are involved in the genesis of sudden infant death syndrome (SIDS). The Spanish National Institute of Toxicology and Forensic Sciences act as the forensic reference centre for Spain. We analyse the experience of this centre in the virological study of 64 cases of sudden children death where viral serology, virological cultures, herpesviruses polymerase chain reaction (PCR) and electron microscopy were performed. According to pathological findings, death could only be attributed to an adenovirus infection in one amygdalitis with upper airways stenosis and asphyxia. Human herpes virus 6 (HHV-6) was detected by PCR in one case with pathological findings characteristic of SIDS. Recent infection by respiratory syncytial virus (RSV), Epstein-Barr virus (EBV) and cytomegalovirus (CMV) were also detected. Meanwhile, 85.9% of the cases yielded negative viral results. Twenty-eight infants were finally categorised as SIDS. Pathological findings of infection were detected in 12 patients despite the negativity of viral analyses. Although viral infection is an uncommon cause of sudden children death, a complete microbiological investigation will help to solve the puzzle of SIDS. Definitive guidelines for microbiological analyses need to be updated whilst new pathogens are discovered or new techniques are implemented in order to clarify unsolved cases.

Anellovirus is a recently created, floating genus of viruses. Torque teno virus (TTV), the type species in the genus, was first discovered in a human patient with a post-transfusion hepatitis of unknown aetiology. Recently, TTV genetically related to but distinct from those discovered in humans have also been found in animals, including pigs. The aims of this study were to estimate the prevalence of swine TTV in Italian pig herds and some risk factors possibly associated with this infection. Serum samples from 179 healthy pigs from 10 farms located in north-central Italy were tested by polymerase chain reaction for the presence of swine TTV DNA. Viral DNA was found in the sera of 43 pigs (24.0%), coming from eight of the 10 farms examined. Prevalence was significantly higher in finishing herds (40.1%) than in farrow-to-finish herds (11.0%) and did not depend on the size of the herd. Within the finishing herds the prevalence was significantly higher in weaners (57.4%) than in fatteners (22.9%), but this difference was not observed in farrow-to-finish herds. No relationship was observed between the prevalence of swine TTV and the implementation of some general hygiene practices and biosecurity procedures within the herds.

Lymphocystis disease virus (LCDV) is the causative agent of lymphocystis disease. The viruses have been divided into three genotypes (genotype I for LCDV-1, II for Japanese flounder isolates, and III for rockfish isolates) on the basis of major capsid protein (MCP) gene sequences. In this study, we developed a multiplex PCR primer set in order to distinguish these genotypes. We also analyzed the MCP gene of a new LCDV isolate from the sea bass (SB98Yosu). Comparison of sequence identities between SB98Yosu and eight Japanese flounder isolates, revealed identity of more than 90.1% at nucleotide level and 96.5% at deduced amino acid level, respectively. Phylogenetic analyses based on the MCP gene showed that SB98Yosu belongs to genotype II, along with Japanese flounder isolates. Multiplex PCR based on the MCP gene allowed us to identify these genotypes in a simple and rapid manner, even in a sample that contained two genotypes, in this case genotypes II and III.

The diagnosis of viral infections is an important part of the daily work of a surgical and cytopathologist. Some viral infections, such as human papillomavirus infection of the lower genital tract, are seen commonly, whereas others, such as fatal enteroviral infection, cannot be diagnosed on routine histological examination and need to be addressed within the clinical context. In general, viral infections are best categorized for the surgical pathologist as low copy/RNA viruses and high copy/DNA viruses. In the latter, viral DNA enters the nucleus, undergoes rapid proliferation, and causes certain cytopathologic changes characteristic of the infection. Immunohistochemistry and/or in situ hybridization yields an intense signal, reflective of the productive infection. In comparison, RNA viruses typically do not show high copy numbers and, although they can induce characteristic cytopathologic changes such as inclusions, often times they do not. In such cases, immunohistochemistry and/or in situ-based hybridization methods, particularly in situ polymerase chain reaction amplification, may be required for a definitive diagnosis. A combination of routine histopathology, clinical information, and immunohistochemistry/in situ-based nucleic acid detection methodologies will allow the surgical pathologist to correctly diagnose viral infections.

An immunoblot technique for the detection of lymphocystis disease virus (LCDV) in naturally infected gilt-head seabream (Sparus aurata, L.) has been developed. A specific antiserum against a 60 kDa viral protein has been proven to be an appropriate tool for LCDV diagnosis either from inoculated cell cultures or from fish tissues using the immunoblot assay. The sensitivity of this technique varied between 10(-1) and 10(2) TCID50. LCDV has also been detected in fish tissues from both, diseased and asymptomatic gilt-head seabream. For the asymptomatic fish detection, a viral amplification step in cell culture and a subsequent viral concentration using polyethylene glycol (PEG) (600 wt) are required. On the contrary, immunoblot allowed the detection of LCDV antigens directly from tissue homogenates of diseased fish. The method described in this study shows higher sensitivity than classical detection techniques based on cell culture inoculation.

Background: Southern Taiwan is a hepatitis B and C viruses (HBV, HCV) endemic area. SEN virus (SENV) infection has been suggested as transfusion-related hepatitis. Two variants of SENV (SENV-D and SENV-H) have been studied in non-transfused children and transfusion-dependent thalassemia patients., Methods: Sera of 67 non-transfused children and 55 pediatric thalassemia patients with multiple transfusions were tested for SENV-D and SENV-H DNAs, liver function, iron status, HBV and HCV markers., Results: The prevalence of SENV (D or H), SENV-D, SENV-H infection, and SENV-D/H coinfection was significantly lower in nontransfused children than in thalassemia patients (22.4, 20.9, 5.0 and 1.5%, respectively, versus 67.3, 52.7, 40.0 and 25.5%, respectively, p < 0.001). The serum alanine aminotransferase (ALT) levels in thalassemia patients with SENV infection alone were significantly lower than levels in patients with SENV/HCV co-infection (p < 0.05), but not different when compared with those without SENV/HCV infection. SENV viremia was not associated with elevated ALT levels in thalassemia patients. SENV viremia did not increase the risk of HCV infection in thalassemia patients., Conclusions: SENV infection is high among non-transfused controls in Taiwan. Transfusion significantly increases the relevance of SENV infection. SENV viremia was not associated with the ALT levels in thalassemia patients., (2006 S. Karger AG, Basel)

The performance of the m1000 system (Abbott Laboratories, Illinois) as a front-end extraction system for high-throughput "in-house" quantitative real-time PCR assays was analyzed and compared to that of manual extraction of plasma and serum samples (hepatitis C virus [HCV] and hepatitis B virus [HBV]) and EDTA-blood samples (cytomegalovirus [CMV] and Epstein-Barr virus [EBV]). Linearity of extraction was tested on dilution series of HCV and HBV reference materials. The correlation coefficient for standard curves based on repeated extraction runs was 0.97 +/- 0.06 for HCV and 0.97 +/- 0.03 for HBV, indicating a linear extraction from 100 to 1.0 x 10(5) HCV IU/ml and from 100 to 1.0 x 10(6) HBV IU/ml. Intra- and interrun variability was below 0.23 log(10) IU/ml for 2.98 to 5.28 log(10) HCV IU/ml and 2.70 to 5.20 log(10) HBV IU/ml. Correlation between automated and manual extraction was very good. For HCV, the correlation coefficient was 0.91 and the mean difference in viral load was 0.13 log(10) HCV IU/ml. For HBV, the correlation coefficient was 0.98 and the mean difference in viral load 0.61 log(10) HBV IU/ml. For CMV and EBV, the correlation coefficient was 0.98 and the mean difference in viral load 0.33 log(10) copies/ml. Accuracy was confirmed with a reference panel (QCMD, Glasgow, Scotland) for all four assays. No cross-contamination was observed when extracting strongly positive polyomavirus samples (8.10 log(10) copies/ml) interspersed with polyomavirus-negative samples. Automated extraction via the m1000 system offers a high reliability of extraction and resulted in a strong reduction of the required extraction hands-on time for high-throughput PCR compared to manual extraction protocols.

The objective of this investigation was to determine whether or not herpesvirus (herpes-), adenovirus (adeno-), or canine parvovirus DNA is present in the brains of dogs with necrotizing meningoencephalitis (NME), necrotizing leukoencephalitis (NLE), and granulomatous meningoencephalitis (GME). Paraffin-embedded brain specimens from 12 histopathologically confirmed dogs with NME, 3 with NLE, and 7 with GME were screened for viral DNA with degenerate herpes- and adenovirus polymerase chain reaction (PCR) and a canine parvovirus-specific PCR. Positive-control specimens included genomic viral DNA and paraffin-embedded tissues from dogs with confirmed herpes-, adeno-, or canine parvovirus infections. Herpes-, adeno-, or canine parvovirus DNA was amplified by PCR from the corresponding positive-control specimens. Negative controls included 7 dogs with various brain disorders and produced no viral amplicons. The 22 dogs with NME, NLE, and GME were negative for viral DNA. Additional studies testing for other viruses or inherited genetic mutations are warranted to gain insight into the etiologies of NME, NLE, and GME. We discuss potential etiologies and provide a clinical and histopathologic overview of these common canine encephalitides.

A virus was isolated from the liver and stomach of a leaf-tailed gecko (Uroplatus fimbriatus) with granulomatous lesions on the tongue and hepatitis. The virus was identified as an iridovirus on the basis of morphology by electron microscopy, restriction endonuclease assay, and sequencing of a large portion of the major capsid protein gene. Comparative analysis revealed that this isolate is related to frog virus 3, the type species of the genus Ranavirus.

The first outbreak of red sea bream iridoviral disease caused by red sea bream iridovirus (RSIV) was recorded in cultured red sea bream Pagrus major in Shikoku Island, Japan in 1990. Since 1991, the disease has caused mass mortalities of cultured marine fishes not only red sea bream but also many other species. The affected fish were lethargic and exhibited severe anemia, petechiae of the gills, and enlargement of the spleen. The causative agent was a large, icosahedral, cytoplasmic DNA virus classified as a member of the family Iridoviridae and was designated as red sea bream iridovirus (RSIV). The genome of RSIV is liner dsDNA and considered to be circularly permitted and terminally redundant like other iridoviruses. The length of physical map of RSIV genome is 112,415bp. An indirect immunofluorescence test with a monoclonal antibody and PCR are commonly used for the rapid diagnosis of RSIV infected fish in the field. For the control of this disease, a formalin-killed vaccine against red sea bream iridoviral disease was developed and now commercially available.

Background/aims: This investigation compared the histological findings in the livers of chronic hepatitis C patients who were or were not co-infected with SEN virus (SEN-V) to determine the histological and clinical characteristics of SEN-V infection in Japan., Methods: Three hundred and ninety-two patients with hepatitis C virus-associated chronic hepatitis (CH) or liver cirrhosis (LC) were included in the study. Serum samples were tested for the presence of SEN-V DNA by nested polymerase chain reaction. The liver biopsy specimen of each patient was examined and scores were assigned to indicate the severity of each of the following features: inflammatory cell infiltration in the periportal, parenchymal, and portal areas; F stage; portal sclerotic change; perivenular fibrosis; pericellular fibrosis; damage to the bile ducts; steatosis and irregular regeneration of hepatocytes (IR)., Results: Of the 473 patients, 194 (41.0%) were positive for SEN-V DNA. The rate of progression of F stage correlated with SEN-V DNA positivity. The blood biochemical parameters did not differ significantly between the SEN-V DNA-positive and -negative patients. The histological features of the livers of SEN-V DNA-positive patients included more severe parenchymal inflammatory cell infiltration and more IR. In particular, among those at the F2, F3 and F4 stages, the degree of IR of the SEN-V DNA-positive patients was significantly greater than that of the SEN-V DNA-negative patients. The cumulative probability of hepatocellular carcinoma (HCC) incidence and survival rate did not differ between the SEN-V DNA-positive and-negative patients., Conclusions: SEN-V co-infection may influence the histopathological features of the livers of patients with type C CH and LC but does not affect the outcome of patients with type C chronic liver disease., (Copyright Blackwell Munksgaard 2005)

Mimivirus, the largest virus known to date, is an amebal pathogen-like Legionella sp. When Mimivirus was used as an antigen in a microimmunofluorescense assay, seroconversion was found in patients with both community- and hospital-acquired pneumonia. Mimivirus DNA was found in respiratory samples of a patient with hospital-acquired pneumonia.

TT virus (TTV) is a naked, single stranded DNA virus, which has been discovered in the serum of a patient with posttransfusion hepatitis of unknown etiology. TTV is widespread in the population, however, the mode of its transmission is unclear. This study was conducted to search for TTV-DNA positivity rates and its relationship with the clinical outcomes of recipients who underwent multiple blood or blood product transfusion, together with healthy children. TTV-DNA was investigated in 52 multitransfused pediatric patients (age range: 3 mnths - 17.5 yrs, mean age: 9.2 +/- 5.7 years) and 18 healthy children (age range: 1 mnth - 16.5 yrs, mean age: 8.1 +/- 4.9 years), by qualitative in-house semi-nested polymerase chain reaction (PCR) with the primers NG059, NG061 and NG063, generated from ORF1 region of the viral genome. TTV-DNA was found positive in 30.8% of multitransfused, and 16.7% of healthy children. The differences of TTV-DNA positivity rates between the multitransfused and control groups, and ALT values between the patients with positive and negative TTV-DNA, were statistically insignificant (p>0.05). As a result, no relationship was detected between TTV positivity and hepatitis, although there was a statistically insignificant increase of TTV-DNA positivity in multitransfused children. However, since the primers of ORF1 N22 region used in our PCR method did not have enough sensitivity for the detection of TTV-DNA, it has been concluded that more sensitive primers such as UTR primers, should be used for more reliable evaluation of the results.

TT virus (TTV) is the unique single stranded circular DNA virus, isolated from humans. Viral DNA is shown to be present not only in patients with hepatitis of unknown etiology but also in apparently healthy populations. TTV's role as a causative agent for non A-E hepatitis is widely questioned. In this study, 23 non A-E hepatitis patients (mean age: 46.8 yrs), 21 chronic hepatitis B (CHB) patients (mean age: 41.0 yrs), 28 chronic hepatitis C (CHC) patients (mean age: 48.1yrs) have been investigated, together with 90 healthy blood donors (mean age: 33.4 yrs), for the presence of TTV-DNA by nested polymerase chain reaction (PCR). Two sets of primers targeting different regions (N22 and NCR) of the viral genome were combined to enhance the detection sensitivity. TTV-DNA was detected in 10 (43.5%) of non A-E hepatitis, 10 (35.7%) of CHC, 4 (19.1%) of CHB patients, and 16 of studied 52 (30.8%) blood donors, by N22-PCR. Viral DNA positivity rates by using NCR-PCR were found as follows for the groups respectively; 65.2% (15/23), 50% (14/28), 42.9% (9/21) and 57.8% (52/90). The differences of TTV-DNA positivity rates between study groups were found statistically insignificant, when data from each set of primer were compared (p>0.05). However, the positivity rate of non A-E hepatitis group (91.3%) was significantly higher than CHC (64.2%), CHB (47.6%), and control (57.8%) groups, when the detection sensitivities of both sets were combined (p=0.002 and p=0.046, respectively). The study also revealed that the prevalence of TTV might be underestimated if appropriate molecular methods were not employed. DNA sequencing and genotyping are required to establish TTV's role as a causative agent of non A-E hepatitis and to identify certain genotypes that might have a role in the pathogenesis of hepatitis.

Background: This study was carried out to clarify differences in clinical characteristics between fatty liver and nonalcoholic steatohepatitis in a Japanese population, and to assess the significance of GB virus C (GBV-C) infection, SEN virus (SENV) infection, and HFE gene mutation in the pathophysiology of these conditions., Methods: Twenty patients with nonalcoholic steatohepatitis and 18 patients with simple steatosis were enrolled, and their clinical characteristics and histological findings were compared. Detection of GBV-C RNA and SENV DNA was performed by polymerase chain reaction (PCR). Mutational analysis of the HFE gene was performed by PCR-restriction fragment length polymorphism (RFLP)., Results: Serum aspartate aminotransferase (AST) and ferritin were significantly higher ( P < 0.05, for both) in NASH than in simple steatosis, and serum total cholesterol (T-Chol) was significantly lower ( P < 0.05) in NASH than in simple steatosis. While GBV-C was detectable in the serum of only one patient with NASH, SENV was detected in 50% (15/30) of the patients whose sera were tested for this virus, but the prevalence was not significantly different between the two groups (42% [8/19] in simple steatosis and 64% [7/11] in NASH). The sex ratio, body mass index (BMI), and age were not significantly different between the two groups, and mutation in the HFE gene was not detected in any patient., Conclusions: Higher serum AST and ferritin, and lower serum T-Chol are distinctive features in NASH when compared with simple steatosis. GBV-C infection, SENV infection, and HFE gene mutation were not considered to influence the development of NASH from simple fatty liver.

Background/aims: The prevalence of co-infections with hepatitis B virus (HBV) and novel hepatitis viruses GBV-C (Hepatitis G virus, HGV) and TT virus (TTV) in chronic hepatitis C (HCV) infection has been studied. In patients with chronic hepatitis C and in asymptomatic healthy HCV carriers, the influence of these agents on the course of HCV infection was assessed., Methods: a total of 110 HCV-positive individuals, among them 77 patients with chronic hepatitis C--50 of them treated with interferon (IFN)--and 33 HCV carriers with normal alanine aminotransferase have been investigated. HBV-DNA, HGV RNA and TTV DNA were detected by PCR, to determine HBsAg and anti-HBc ELISA technic has been used., Results: In the healthy population, the prevalence of anti-HCV was 0.3%, HBsAg 0.09%, anti-HBc 2.5%, HGV RNA 8.0% and TTV DNA 18.5%, respectively. In chronic hepatitis C HBsAg (accompanied with HBV-DNA) occurred in 1.29%, anti-HBc 25.97%, HGV RNA in 9.09% and TTV DNA in 40.25% of cases. In IFN-treated patients with sustained remission, the frequency of TTV was 20% vs. 45.7% found in non-responders. Among asymptomatic HCV-carriers, the prevalence of anti-HBc was 27.27%, HGV RNA 9.09% and TTV DNA 75.7% respectively., Conclusions: Neither previous HBV infection, nor HGV RNA and TTV DNA had apparent effect on the course of chronic HCV infection. TTV was detected with the lowest frequency in persons with sustained remission due to IFN, suggesting antiviral effect of IFN on TTV.

The aim of this study was to determine the transfusion transmitted Virus (TTV) prevalence in three groups of population from Abidjan, Côte d'Ivoire. The A group contained 39 multitransfused patients, the B group contained 10 blood donors supposed to be healthy persons which have never been transfused and the group C contained 43 patients with chronic liver pathology. In this last group, 33 patients had HBV positive serology and the 10 others, HCV positive serology. We used PCR to investigate TTV in patients serum. Detection rates were comprised between 67% and 82%. This is the first study to provide information about the high portage of TTV in ivorian population.

Background: Little is known about the pathogenic role and the endemic situation of transfusion transmitted virus (TTV)., Objectives: In this study, a molecular assay for detection of TTV based on automated nucleic acid extraction and real-time PCR was developed and evaluated. The new assay includes an internal control., Study Design: After optimization of the molecular assay, 103 clinical samples were studied retrospectively. All sera had been tested for anti-HCV and anti-HIV-1 antibodies earlier., Results: The amplification efficiency was found to be 102%. When clinical specimens were tested, 79 of 103 serum samples were found to be positive for TTV. There was no significant difference between various groups of patients. The internal control was detected in all negative and weak positive samples., Conclusions: This molecular assay proved to be suitable for routine detection of TTV in clinical samples. Moreover, a relative statement on the TTV serum load can be done.