The distribution of different virulence grass carp reovirus strains in some neglected tissues.

Grass carp reovirus (GCRV) is the causative agent of hemorrhagic disease in infected grass carp. During an outbreak, a mortality rate of up to 85% can be experienced, thus leading to substantial economic losses. The current understanding of disease pathogenesis is limited, with the distribution and dynamics of replication amongst different GCRV strains in vivo largely unknown. We determined distribution of different GCRV strains in infected grass carp, especially in some neglected tissues, such as the gill, brain, blood and so on. The results showed elevated viral RNA copy numbers in the blood, with some tissues such as the kidney, heart, brain, and bladder exhibiting even higher viral loads following infection with the virulent GCRV-CL strain. Even more interesting is that the brain exhibited the highest viral load, with a copy number of 800,000 following GCRV-CL infection. Overall, this study provides further insight into GCRV viral load distributions following infection and potentially identified some new viral tropism sites to provide a foundation for further studies aimed at characterizing GCRV viral pathogenesis.

Virulence potential of fusogenic orthoreoviruses.

Several severe respiratory virus infections that have emerged during the past decade originated in animals, including bats. In Indonesia, exposure to bats has been associated with increased risk of acquiring orthoreovirus infection. Although orthoreovirus infections are mild and self-limiting, we explored their potential for evolution into a more virulent form. We used conventional virus culture, electron microscopy, and molecular sequencing to isolate and identify orthoreoviruses from 3 patients in whom respiratory tract infection developed after travel to Indonesia. Virus characterization by plaque-reduction neutralization testing showed antigenic similarity, but sequencing of the small segment genes suggested virus reassortment, which could lead to increased virulence. Bats as a reservoir might contribute to virus evolution and genetic diversity, giving orthoreoviruses the potential to become more virulent. Evolution of this virus should be closely monitored so that prevention and control measures can be taken should it become more virulent.

A Duplex Fluorescent Microsphere Immunoassay for Detection of Bluetongue and Epizootic Hemorrhagic Disease Virus Antibodies in Cattle Sera.

Bluetongue virus (BTV) causes internationally reportable hemorrhagic disease in cattle, sheep, and white-tailed deer. The closely related, and often co-circulating, epizootic hemorrhagic disease virus causes a clinically similar devastating disease in white-tailed deer, with increasing levels of disease in cattle in the past 10 years. Transmitted by Culicoides biting midges, together, they constitute constant disease threats to the livelihood of livestock owners. In cattle, serious economic impacts result from decreased animal production, but most significantly from trade regulations. For effective disease surveillance and accurate trade regulation implementation, rapid, sensitive assays that can detect exposure of cattle to BTV and/or EHDV are needed. We describe the development and validation of a duplex fluorescent microsphere immunoassay (FMIA) to simultaneously detect and differentiate antibodies to BTV and EHDV in a single bovine serum sample. Performance of the duplex FMIA for detection and differentiation of BTV and EHDV serogroup antibodies was comparable, with higher sensitivity than commercially available single-plex competitive enzyme-linked immunosorbent assays (cELISA) for detection of each virus antibody separately. The FMIA adds to the currently available diagnostic tools for hemorrhagic orbiviral diseases in cattle as a sensitive, specific assay, with the benefits of serogroup differentiation in a single serum sample, and multiplexing flexibility in a high-throughput platform.

Isolation and characterization of novel reassortant mammalian orthoreovirus from pigs in the United States.

Mammalian orthoreovirus (MRV) infects multiple mammalian species including humans. A United States Midwest swine farm with approximately one thousand 3-month-old pigs experienced an event, in which more than 300 pigs showed neurological signs, like "down and peddling", with approximately 40% mortality. A novel MRV was isolated from the diseased pigs. Sequence and phylogenetic analysis revealed that the isolate was a reassortant virus containing viral gene segments from three MRV serotypes that infect human, bovine and swine. The M2 and S1 segment of the isolate showed 94% and 92% nucleotide similarity to the M2 of the MRV2 D5/Jones and the S1 of the MRV1 C/bovine/Indiana/MRV00304/2014, respectively; the remaining eight segments displayed 93%-95% nucleotide similarity to those of the MRV3 FS-03/Porcine/USA/2014. Pig studies showed that both MRV-infected and native contact pigs displayed fever, diarrhoea and nasal discharge. MRV RNA was detected in different intestinal locations of both infected and contact pigs, indicating that the MRV isolate is pathogenic and transmissible in pigs. Seroconversion was also observed in experimentally infected pigs. A prevalence study on more than 180 swine serum samples collected from two states without disease revealed 40%-52% positive to MRV. All results warrant the necessity to monitor MRV epidemiology and reassortment as the MRV could be an important pathogen for the swine industry and a novel MRV might emerge to threaten animal and public health.

Development and validation of an indirect enzyme-linked immunosorbent assay for the detection of antibodies against duck swollen head hemorrhagic disease virus.

An indirect enzyme-linked immunosorbent assay (iELISA) was developed for detecting antibody to duck swollen head hemorrhagic disease virus (DSHDV) using purified whole virus by sucrose density gradient ultracentrifugation as a coating antigen. Antiserum against DSHDV strains HY-99 (hyperimmume serum) was prepared in 30-day-old ducks by vaccination with inactivated DSHDV and used as positive sera. The iELISA test was optimized with different reagents or dilutions. The validation results showed that this assay was only specific for antibodies against duck viral swollen head hemorrhagic disease. The OD450 value for positive serum diluted 1:800 was also determined to be greater than the positive threshold. The highest coefficient of variation value was 3.66% for the intra-assay and 5.79% for the interassay, which were all less than 10%. This assay has been successfully applied to the examination of the duck sera clinically. These results in this study indicate that the newly-developed iELISA offers a precise, specific, sensitive, and reproducible means of measuring DSHDV antibodies in duck sera.

Serological evidence and experimental infection of cynomolgus macaques with pteropine orthoreovirus reveal monkeys as potential hosts for transmission to humans.

Pteropine orthoreoviruses (PRV) are emerging bat-borne viruses with proven zoonotic transmission. We recently demonstrated human exposure to PRV in Singapore, which together with previous reports from Malaysia and Vietnam suggest that human infection of PRV may occur periodically in the region. This raises the question whether bats are the only sources of human infection. In this study, we screened 517 cynomolgus macaques caught in Singapore for evidence of exposure to PRV3M (also known as Melaka virus), which was first isolated from human patients in Melaka, Malaysia. We found that 67 serum samples were PRV3M positive by ELISA and 34 were also positive by virus neutralization assay. To investigate whether monkeys could act as hosts for PRV transmission, we experimentally infected cynomolgus macaques with PRV3M and housed these animals with uninfected monkeys. Although no clinical signs of infection were observed in infected animals, viral RNA was detected in nasal and rectal swabs and all infected macaques seroconverted. Additionally, one of the uninfected animals seroconverted, implying active shedding and transmission of PRV3M. We provide evidence that PRV exposure in the macaque population in Singapore occurs at a relatively high prevalence and this study suggests that cynomolgus macaques may be an intermediate or reservoir host for PRVs.

Lethal murine infection model for human respiratory disease-associated Pteropine orthoreovirus.

Pteropine orthoreovirus (PRV) is an emerging bat-borne human pathogen causing severe respiratory illness. To date, however, the evaluation of PRV virulence has largely depended on the limited numbers of clinical cases owing to the lack of animal models. To develop an in vivo model of PRV infection, an inbred C3H mouse strain was infected intranasally with pathogenic PRV strain Miyazaki-Bali/2007. C3H mice suffered severe lung infection with significant body weight reduction and died within 7 days after intranasal infection. Infectious viruses were isolated mainly from the lungs and trachea. Histopathological examination revealed interstitial pneumonia with monocytes infiltration. Following repeated intranasal infection, mice developed antibodies to particular structural and non-structural proteins of PRV. The results of these immunological assays will help to develop laboratory protocols for sero-epidemiological studies. Our small rodent model of lethal respiratory infection will further allow investigation of the molecular mechanisms underlying the high pathogenicity of PRV.

Viral Protein Kinetics of Piscine Orthoreovirus Infection in Atlantic Salmon Blood Cells.

Piscine orthoreovirus (PRV) is ubiquitous in farmed Atlantic salmon (Salmo salar) and the cause of heart and skeletal muscle inflammation. Erythrocytes are important target cells for PRV. We have investigated the kinetics of PRV infection in salmon blood cells. The findings indicate that PRV causes an acute infection of blood cells lasting 1-2 weeks, before it subsides into persistence. A high production of viral proteins occurred initially in the acute phase which significantly correlated with antiviral gene transcription. Globular viral factories organized by the non-structural protein µNS were also observed initially, but were not evident at later stages. Interactions between µNS and the PRV structural proteins λ1, µ1, σ1 and σ3 were demonstrated. Different size variants of µNS and the outer capsid protein µ1 appeared at specific time points during infection. Maximal viral protein load was observed five weeks post cohabitant challenge and was undetectable from seven weeks post challenge. In contrast, viral RNA at a high level could be detected throughout the eight-week trial. A proteolytic cleavage fragment of the µ1 protein was the only viral protein detectable after seven weeks post challenge, indicating that this µ1 fragment may be involved in the mechanisms of persistent infection.

Testicular lesions and antler abnormalities in Colorado, USA mule deer (Odocoileus hemionus): a possible role for epizootic hemorrhagic disease virus.

Antler abnormalities of deer and other cervids often result from testicular lesions and decreased levels of testosterone, inhibiting normal cycles of antler growth. Affected males have antlers with retained velvet, numerous short, misshapen points ("cactus bucks"), and failure to shed these abnormal antlers annually. In Colorado, US, we observed a high occurrence of "cactus bucks" in mule deer (Odocoileus hemionus) populations after management efforts to increase the number of mature male deer in the state. Affected males consistently had antibody to epizootic hemorrhagic disease virus serotype 2 (EHDV-2), and examination of the testes of these animals demonstrated nonspecific end-stage lesions of chronic inflammation, fibrosis, and mineralization. To examine more acute stages of testicular lesions, and to screen for EHDV specifically within the testes, we sampled 16 male mule deer from affected herds, but with essentially normal antlers (n = 14) or retained velvet only (n = 2). Testicular and epididymal lesions identified from these samples included necrotizing vasculitis (n = 2), hemorrhage (n = 6), edema (n = 2), seminiferous tubular necrosis (n = 5), orchitis (n = 5), epididymitis (n = 10), hypospermia (n = 6), and end-stage lesions of seminiferous tubular loss (n = 2), fibrosis (n = 2), and mineralization (n = 2). Each of the 16 cases was blindly scored on the basis of number of histologic lesions, with a median score of two. Five of seven (71%) testes that were PCR positive for EHDV had lesion scores above the median, whereas none of the nine (0%) EHDV PCR-negative testes had lesion scores above the median, suggesting an association between testicular lesions and detection of EHDV RNA in the testes (P = 0.003). Although the role of EHDV infection remains unconfirmed, the association between testicular and epididymal lesions and presence of EHDV RNA in the affected tissues suggests that cactus buck antlers may be a sequela of EHDV infection.

Development of an ELISA for detection of antibodies to avian reovirus in chickens.

An enzyme-linked immunosorbent assay (ELISA) using the expressed sigmaC and sigmaB proteins which induce neutralizing antibodies as the coating antigen (sigmaC-sigmaB-ELISA) for the detection of antibodies to avian reovirus in chickens was developed and compared with serum neutralization and conventional ELISA tests. These assays were used to examine the sera from chickens vaccinated experimentally and farm chickens. The correlation rate between serum neutralization and a sigmaC-sigmaB-ELISA was 100% (156/156), and that between serum neutralization and conventional ELISA was 89.1% (139/156). The results revealed that preparation of an ELISA by using sigmaC and sigmaB of ARV as the coating antigen in detecting the field chicken sera in comparison with the conventional ELISA gave a titer more correlated to the serum neutralization test. The sigmaC-sigmaB-ELISA showed a higher correlation with the serum neutralization-positive and -negative sera than that obtained with conventional ELISA. This combination antigen may thus be the best suited for preparing an ELISA for improving the determination of the immune status of chicken flocks or for detection of chicken infections with avian reovirus.

A rapid quantitative method for detecting infectious bursal disease virus using polystyrene latex microspheres.

A monoclonal antibody (mAb) to infectious bursal disease virus (IBDV) was bound to polystyrene latex microspheres. The microspheres agglutinated with extracts of bursae and sera from chickens infected with all strains or isolates of IBDV tested. Agglutination appeared within a 10-min reaction time. The assay could detect a 10(3.7) to 10(4.5) mean embryo infective dose (EID50) of the virus in 0.01 ml and the titer of the assay was 10- to 40-times higher than that of the agar gel precipitin test.

A group-specific, indirect sandwich ELISA for the detection of equine encephalosis virus antigen.

A polyclonal antibody-based, group-specific, indirect, sandwich ELISA (S-ELISA) for the detection of equine encephalosis virus (EEV) antigen was developed. Purified EEV particles were titrated in the S-ELISA and the limit of detection was determined to be approximately 9.0 ng of antigen/ml (0.45 ng/well). Positive S-ELISA reactions were recorded with seven serologically distinct EEV serotypes. No cross-reactions were recorded with other arboviruses including African horse sickness virus (AHSV) serotypes 1-9, bluetongue serotypes 1-24, epizootic haemorrhagic disease serotypes 1-8 and isolate 318, and selected isolates of Palyam, Eubenangee, Corriparta, Warrego, Akabane and bovine ephemeral fever viruses. The assay proved to be sensitive and specific for the rapid detection of EEV in cell cultures and in homogenated suckling mouse brain (MB). The data generated in this study suggest that the ELISA will be valuable for epidemiological studies of EE and will assist in making a reliable differential diagnosis between EEV and AHSV infections.

Effect of exogenous corticosteroids on circulating virus and neutralizing antibodies in striped bass (Morone saxatilis) infected with infectious pancreatic necrosis virus.

Corticosteroids have been reported to induce immunosuppression in fish exposed to many types of bacterial antigens. We document a similar phenomenon in fish exposed to infectious pancreatic necrosis virus (IPNV). Fingerling striped bass that were injected with the steroid triamcinolone acetonide (100 mg/kg body weight) 24 hours before receiving intraperitoneal inoculation with IPNV became viremic 3 days post inoculation (dpi) and virus was still detected in the buffy coat cells 14 dpi. In contrast, viremia could not be detected after 7 dpi in fish that received virus but not steroids. Circulating virus neutralizing antibodies were first detected in steroid treated fish at 10 dpi compared to 7 dpi for the virus injected fish and titers were consistently lower in the steroid group. Steroid treatment of chronic IPNV-carriers did not induce detectable viremia nor alter circulating antibody levels in chronic IPNV-carriers. None of the striped bass demonstrated clinical signs of viral disease.

Group-reactive ELISAs for detecting antibodies to African horsesickness and equine encephalosis viruses in horse, donkey, and zebra sera.

Group-reactive enzyme-linked immunosorbent assays (ELISAs) were developed to selectively detect antibodies to African horsesickness virus (AHSV) and equine encephalosis virus (EEV), 2 orbiviruses that infect equids. In indirect ELISA, guinea pig antisera to all known AHSV or EEV serotypes recognized immobilized AHSV serotype 3 or EEV Cascara, respectively. Antisera from naturally infected animals did not cross-react with their respective heterologous viruses. The ELISA was used in parallel with the complement fixation (CF) and agar gel immunodiffusion tests to detect antibodies in sera from animals in the field. The ELISA distinguished among those that contained antibodies to AHSV, EEV, or both viruses and was useful with sera that did not yield results in CF tests because of anticomplementary activity. Zebra and donkeys, both potential subclinical carrier animals in Africa, contained AHSV or EEV antibodies. Some sera reacted with 1 of the 2 orbiviruses, whereas others reacted with both. The ELISA can be used in projected epidemiological studies in which many serum samples must be assayed.

Experimental induction of hemorrhagic-aplastic anemia in chickens. II. Serum protein changes.

The serologic response of chickens to infectious bursal disease virus (IBDV) and inclusion body hepatitis virus (IBHV) was analyzed. Inoculation at one day old with either IBDV or IBHV significantly (P less than 0.05) reduced levels of serum gamma-globulins at 4 weeks postinoculation. This response was not elicited by inoculation of IBDV together with IBHV. Birds with experimentally induced or naturally occurring hemorrhagic anemia syndrome (HAS) had serum proteins quantitatively and qualitatively changed from those of controls. Serum protein profiles did not coincide, however, in experimentally infected and naturally infected chickens. Among naturally infected chickens, those that were IBHV-positive upon culture had significantly (P less than 0.05) lower hematocrit values.

Reverse transcription-polymerase chain reaction detection and nucleic acid sequence confirmation of reovirus infection in laboratory mice with discordant serologic indirect immunofluorescence assay and enzyme-linked immunosorbent assay results.

Reovirus infections are typically subclinical in weaned mice, and are best detected using serologic tests. After exposure to the soiled bedding of some mice obtained from various sources, numerous sentinel mice tested reovirus seropositive by use of indirect immunofluorescence assays (IIFA) in our institution. A major commercial rodent pathogen testing laboratory verified our IIFA results, but since the same samples were reovirus seronegative using their "more specific" enzyme-linked immunosorbent assay (ELISA), the IIFA results were reported as "false positives." As past in-house observations suggested transmission of the virus to sentinel and other animals, we sought to determine whether the IIFA results were always "false positives." An opportunity to test this notion arose after receipt of reovirus IIFA-positive transgenic mice from an academic source. Using reverse transcriptase-polymerase chain reaction (RT-PCR) assays, the presence of reovirus RNA was detected in fecal specimens taken from some sentinel animals that subsequently seroconverted from IIFA-negative to IIFA-positive for reovirus. The virus could not be isolated by use of tissue culturing methods. Nucleotide sequence analysis established the presence of unique reovirus sequences. These results indicate that contemporary reovirus infections may not be detected by use of some serologic tests, and that RT-PCR analysis may be useful for confirmation of active reovirus infection in certain situations.

Plasmid encoding interferon-gamma exacerbates reovirus type-2-induced diabetes in DBA/1 suckling mice.

This study was designed to examine the effects of administration of a plasmid encoding interferon(IFN)-gamma on reovirus type-2(Reo-2)-induced autoimmune insulitis in suckling DBA/1 mice. Cumulative incidences of diabetes and insulitis at 17 days post-infection in the mice treated with IFN-gamma-encoding plasmid were higher than those in control mice treated with "empty" plasmid. These results suggested that the IFN-gamma-encoding plasmid promoted autoimmune insulitis in Reo-2-induced diabetes.

Haematological values and changes in blood chemistry in chickens with infectious bursal disease.

Haematological and blood serum chemical changes were studied in two groups of specific pathogen free chickens infected at five weeks old with different field isolates of infectious bursal disease virus. Blood and serum components were determined five days after infection. There were significant decreases (P less than 0.05) in the total erythrocyte count, packed cell volume, haemoglobin concentration, albumin, albumin: globulin ratio, uric acid and glucose. Serum components which increased significantly (P less than 0.05) were globulins and cholesterol.

An age-related coagulation disorder associated with experimental infection with infectious bursal disease virus.

Specific-pathogen-free White Leghorn chickens were inoculated with a field strain of infectious bursal disease virus. One group (A) was inoculated at 17 days after the chicks were hatched, and the other groups (C and E) were inoculated at posthatch day 42. Blood samples were obtained for determination of clotting times (whole blood recalcification, prothrombin, and activated partial thromboplastin times), virus-neutralizing antibody, and total hemolytic complement. There were significant increases in clotting times for groups C and E at 3 and 5 days after they were inoculated. There were no significant increases in clotting times at 3 days after inoculation in the group A chickens (inoculated at 17 days after hatching). There were no significant decreases in total complement activity in any of these chickens (groups A, C, and E). This study indicates that the mortality and clinical symptoms observed in chickens experimentally infected with infectious bursal disease virus may be associated with a clotting abnormality because it was noted only in chickens that developed severe clinical disease (inoculated at 42 days after hatching) and was not noted in chickens that remained clinically normal (inoculated at 17 days).

In vitro replication of epizootic hemorrhagic disease and bluetongue viruses in white-tailed deer peripheral blood mononuclear cells and virus-cell association during in vivo infections.

In vitro and in vivo infections were conducted to determine if the epizootic hemorrhagic disease (EHD) and bluetongue (BT) viruses would replicate in peripheral blood mononuclear (PBM) cells of white-tailed deer (Odocoileus virginianus). All of the North American EHD and BT viruses (EHD virus serotypes 1 and 2, and BT virus serotypes 2, 10, 11, 13, and 17) replicated in vitro in cultures of white-tailed deer PBM cells. However, this replication appeared to be monocyte-dependent and was not enhanced by lymphocyte blastogenesis induced by the addition of concanavalin A. In white-tailed deer infected with either EHD virus serotype 2 or BT virus serotype 10, virus could be isolated consistently from PBM cells only from post-infection day 4 through 8, although they remained viremic through post-infection day 21. In deer, highest viral titers were associated with the erythrocyte fraction, and in no cases did viral titers detected in the platelet, PBM cell or polymorphonuclear cell fractions approach titers observed in whole blood. In the in vitro infections of white-tailed deer erythrocytes, the EHD and BT viruses were associated with pits in the erythrocyte membrane. This association may be important in the long-term viremia observed in deer.