Pathogenicity and genomic characterization of a novel avian orthoreovius variant isolated from a vaccinated broiler flock in China.

Avian orthoreovirus (ARV) infections of broiler flocks cause arthritis/tenosynovitis syndrome and significant economic losses. ARV variants were detected in the USA and Canada. Viral arthritis/tenosynovitis syndrome has occurred frequently in China in recent years. In this study, a variant ARV strain associated with viral arthritis/tenosynovitis syndrome was isolated from broilers and designated as LY383. Genomic sequence and phylogenetic analysis of the σC nucleic acid and amino acid sequences revealed that the isolate was closely related to ARV field strains Reo/PA/Layer/01224B/14, Reo/PA/Broiler/1551/13, GA/14602/2014, GA/13569/2013 and GA/13542/2013, in cluster V, but distinct from most Chinese field strains or commercial vaccine strains. Experimental challenge showed that the isolate could cause arthritis/tenosynovitis syndrome in broilers, which possessed a high level of maternal antibodies induced by commercial ARV vaccines (S1133, 1733 and T98). Furthermore, viral nucleic acid could be detected in cloacal swabs of all challenged birds throughout the entire test from 5 dpi onward. These results suggest that a novel ARV genotype emerges and might become prevalent in broiler flocks in China. RESEARCH HIGHLIGHTS A variant avian orthoreovirus was isolated from a vaccinated broiler flock in North China. The ARV field strain was distinct from previous China-origin ARV isolates and vaccine strains. The current commercial ARV vaccine could not provide effective protection of broilers against the field isolate infection. These findings indicated that variant ARV field strains might become frequent in broiler flocks in China and effective measures should be conducted to prevent and control the disease.

A Newly Emergent Turkey Arthritis Reovirus Shows Dominant Enteric Tropism and Induces Significantly Elevated Innate Antiviral and T Helper-1 Cytokine Responses.

Newly emergent turkey arthritis reoviruses (TARV) were isolated from tendons of lame 15-week-old tom turkeys that occasionally had ruptured leg tendons. Experimentally, these TARVs induced remarkable tenosynovitis in gastrocnemius tendons of turkey poults. The current study aimed to characterize the location and the extent of virus replication as well as the cytokine response induced by TARV during the first two weeks of infection. One-week-old male turkeys were inoculated orally with TARV (O'Neil strain). Copy numbers of viral genes were estimated in intestines, internal organs and tendons at ½, 1, 2, 3, 4, 7, 14 days Post inoculation (dpi). Cytokine profile was measured in intestines, spleen and leg tendons at 0, 4, 7 and 14 dpi. Viral copy number peaked in jejunum, cecum and bursa of Fabricius at 4 dpi. Copy numbers increased dramatically in leg tendons at 7 and 14 dpi while minimal copies were detected in internal organs and blood during the same period. Virus was detected in cloacal swabs at 1-2 dpi, and peaked at 14 dpi indicating enterotropism of the virus and its early shedding in feces. Elevation of IFN-α and IFN-ß was observed in intestines at 7 dpi as well as a prominent T helper-1 response (IFN-γ) at 7 and 14 dpi. IFN-γ and IL-6 were elevated in gastrocnemius tendons at 14 dpi. Elevation of antiviral cytokines in intestines occurred at 7dpi when a significant decline of viral replication in intestines was observed. T helper-1 response in intestines and leg tendons was the dominant T-helper response. These results suggest the possible correlation between viral replication and cytokine response in early infection of TARV in turkeys. Our findings provide novel insights which help elucidate viral pathogenesis in turkey tendons infected with TARV.

Infectious feline herpesvirus detected in distant bone and tendon following mucosal inoculation of specific pathogen-free cats.

Emerging evidence suggests that cats infected with feline herpesvirus-1 (FHV-1) may experience a brief viremic phase. The objective of this study was to determine whether natural routes of FHV-1 inoculation could result in viremic transmission of infectious virus to connective tissues (cortical bone, tendon). Three specific pathogen-free cats were experimentally inoculated with FHV-1 via a combined mucosal (oronasal, ocular) route. Cats were euthanized at the peak of clinical signs to aseptically harvest tissues (cortical bone, tendon, trachea/tongue) for co-culture with a susceptible cell line to promote spread of infectious virus. Viral infection of Crandall-Rees feline kidney cells was microscopically visualized by cytopathic effect (CPE). Additionally, co-culture DNA was extracted either at the point of CPE or 16 days of culture without evidence of CPE, to amplify FHV-1 glycoprotein B gene using real-time PCR. Infectious virus was detected in distant cortical bone (two cats, moderate to severe clinical signs) and tendon (one cat, severe clinical signs). Direct infection of mucosal (trachea, tongue) tissues also was confirmed in these two cats. In contrast, all co-cultured tissues from the third cat (mild clinical signs) were negative for FHV-1 by CPE and PCR. Results of this study demonstrated that early primary FHV-1 viremia may be distributed to distant connective tissues.

Inactivation of enveloped and non-enveloped viruses on seeded human tissues by gamma irradiation.

Human tissue allografts are widely used in a variety of clinical applications with over 1.5 million implants annually in the US alone. Since the 1990s, most clinically available allografts have been disinfected to minimize risk of disease transmission. Additional safety assurance can be provided by terminal sterilization using low dose gamma irradiation. The impact of such irradiation processing at low temperatures on viruses was the subject of this study. In particular, both human tendon and cortical bone samples were seeded with a designed array of viruses and the ability of gamma irradiation to inactivate those viruses was tested. The irradiation exposures for the samples packed in dry ice were 11.6-12.9 kGy for tendon and 11.6-12.3 kGy for bone, respectively. The viruses, virus types, and log reductions on seeded tendon and bone tissue, respectively, were as follows: Human Immunodeficiency Virus (RNA, enveloped), >2.90 and >3.20; Porcine Parvovirus (DNA, non-enveloped), 1.90 and 1.58; Pseudorabies Virus (DNA, enveloped), 3.80 and 3.79; Bovine Viral Diarrhea Virus (RNA, enveloped), 2.57 and 4.56; and Hepatitis A Virus (RNA, non-enveloped), 2.54 and 2.49, respectively. While proper donor screening, aseptic technique, and current disinfection practices all help reduce the risk of viral transmission from human allograft tissues, data presented here indicate that terminal sterilization using a low temperature, low dose gamma irradiation process inactivates both enveloped and non-enveloped viruses containing either DNA or RNA, thus providing additional assurance of safety from viral transmission.

Reovirus tenosynovitis in a flock of layer breeders.

The present paper describes a reovirus infection with clinical course in a flock of layer breeders. Lameness and tenosynovitis of flexor tendons were observed in approximately 15% of the cockerels and 3% of the hens from 17 weeks of age onwards. Affected birds did not die; on the contrary, most of them recovered clinically within a period of 8 weeks. Two other breeds of layer parents that were housed in close contact with the affected flock did not develop clinical signs, although serology indicated that infection with reovirus had taken place. These field observations constitute the first report of clinical reovirus tenosynovitis in layer parents and indicate different susceptibilities of layer parent breeds in developing clinical signs following reovirus infection.

Avian reovirus-induced apoptosis related to tissue injury.

Apoptosis plays an important role in pathogenesis of many viral infections. Infection of chicken with avian reovirus S1133 causes tissue injury related to virus-induced apoptosis. To determine whether avian reovirus (ARV) induced apoptosis in chicken tissues, six 3-week-old specific pathogen free White Leghorn chicks were inoculated with ARV S1133. Tissues were dual-labelled for the simultaneous detection of viral antigen containing and apoptotic cells. DNA laddering was detected in ARV-infected but not mock-infected chicken tissues. Dual-labelling assay revealed that the majority of antigen-expressing cells were not apoptotic. Surprisingly, some apoptotic but non-antigen-expressing cells were frequently located in the vicinity of antigen-expressing cells. Syncytium formation in ARV-infected chicken tissues undergoing apoptosis was apparent, suggesting a correlation between virus replication and apoptosis in chicken tissues.

Hand surgery on patients who are "high risk" for blood-borne viruses.

There is a risk of transmission of blood-borne viruses (BBV) to health-care workers when performing hand surgery on intravenous drug abusers and other patients known to have BBV. This review summarises methods and procedures that may be employed to help reduce this risk to a minimum. High-risk patients should be identified early and a non-invasive procedure considered. Only experienced staff should scrub and appropriate clothing should be worn. Sharp instrument use should be kept to a minimum and only instrument retraction and suturing should be employed. When possible, wounds should be closed with staples, glue or absorbable sutures. Appropriate steps must be taken to reduce the risk of injuries from sharp bone ends, K-wires and splash exposure during irrigation.

Lyophilization does not inactivate infectious retrovirus in systemically infected bone and tendon allografts.

BACKGROUND: A review of multiple transplantations of human immunodeficiency virus-infected musculoskeletal allografts found that recipients of lyophilized (freeze-dried) bone or tendon from an infected donor all tested negative for human immunodeficiency virus. The finding that 75% of the recipients of fresh-frozen bone from the same donor contracted human immunodeficiency virus has led to speculation that freeze-drying may render retroviral-infected musculoskeletal allografts noninfectious. HYPOTHESIS: Lyophilization does not inactivate retrovirus in systemically infected bone and tendon. STUDY DESIGN: Controlled laboratory study. METHODS: Tendons and cortical bone segments from cats systemically infected with feline leukemia virus were used in this study. Feline embryonic fibroblast cells were cultured in the presence of fresh-frozen or freeze-dried cortical bone or tendon segments. At each passage, feline leukemia virus p27 antigen was measured in media by enzyme-linked immunosorbent assay, and feline leukemia virus (pro)viral nucleic acids were quantified by real-time quantitative polymerase chain reaction in the DNA extracted from cells. RESULTS: Enzyme-linked immunosorbent assay results and quantitative polymerase chain reaction results demonstrated retroviral antigen and proviral DNA in all cultured cell replicates after exposure to fresh-frozen or freeze-dried bones or tendons. CONCLUSION: Freeze-drying (lyophilization) of retroviral-infected cortical bone and tendon does not inactivate retrovirus. CLINICAL RELEVANCE: These results conclusively demonstrate that freeze-drying should not be relied on to inactivate infectious retrovirus in systemically infected musculoskeletal allografts.

Characterization of monoclonal antibodies against avian reovirus S1133 protein sigmaA synthesized in Escherichia coli.

Monoclonal antibodies (MAbs) were prepared against avian reovirus S1133 protein sigmaA (esigmaA) synthesized in Escherichia coli. MAbs were characterized and used to develop a diagnostic test. Ten MAbs were selected for competitive binding assay following coupling with horseradish peroxidase. The results indicated that these MAbs delineated two epitopes I and II of esigmaA. An immuno-dot binding assay was used to detect the effect of denaturation on antibody recognition of the epitopes. All MAbs bound to esigmaA in its native form. After denaturation by boiling in SDS and 2-mercaptoethanol, the binding of MAbs recognizing epitope I was fully abolished. However, the reactivity of MAbs recognizing epitope II was not affected. MAbs 31 and 32, recognizing epitopes I and II, respectively, were selected for the cross-reactivity to heterologous reovirus strains. The results suggest that the two epitopes are highly conserved among these virus strains. A MAb capture enzyme-linked immunosorbent assay (ELISA) procedure was developed using MAbs 32 and 31 to detect reovirus protein sigmaA in samples from tendon tissues of infected bird and chicken embryo fibroblast (CEF) cell cultures. Avian reovirus sigmaA antigens in tendon specimens were detected from the inoculated birds as early as 2 days post-inoculation (PI), approximated a peak at 7 days PI, and maintained this until 16 days PI, then decreased gradually. A clear difference in absorbance values between the tendon samples of the avian reovirus- and mock-infected birds is obtained. Positive results were also obtained from avian reovirus-infected CEF and from the tendon tissues of naturally infected broilers. These results indicated that the MAb capture ELISA is a useful methods for the detection of avian reovirus from chickens suspected to have avian reovirus infections.

A reovirus challenge model applicable in commercial broilers after live vaccination.

The efficacy of live reovirus vaccines may be determined by challenge via the foot pad route 3 to 4 weeks after vaccination. Swelling and discoloration in the foot pad and shank are scored for a period of 14 days. The major disadvantages of this challenge model are the subjective judgement of gross foot pad and/or shank lesions, that it is very difficult to induce lesions in broilers, and that it causes animal suffering. Other reovirus challenge models are based on reisolation of the virus from different tissues or on scoring microscopic lesions in the tendons. Some disadvantages of these models are that they either cannot be used after vaccination with live reovirus because they cannot discriminate between vaccine and challenge virus or that the microscopic lesions scored need not necessarily be related to the challenge virus but may have been induced by other factors. Therefore, we have attempted to develop a reovirus challenge model that was an improvement on the existing ones, using isolation of reovirus from different organs followed by specific detection of the challenge virus with a monoclonal antibody that can discriminate between challenge and vaccine virus. The reovirus challenge model was examined in specific pathogen free (SPF) White Leghorn chickens and commercial broilers. In vivo studies were conducted to examine the efficacy of an attenuated reovirus vaccine in SPF White Leghorn chickens and commercial broilers with maternal immunity against reovirus. No challenge virus could be detected in any of the organs of the vaccinated chickens 3 and 10 days after challenge. In contrast, challenge virus could be isolated from the unvaccinated control group. At an increased challenge dose all unvaccinated challenge control birds were positive, while the vaccinated chickens were protected. It was shown that 1-day-old vaccination in the presence of maternal immunity was effective. It seemed that protection induced in broilers by the attenuated reovirus vaccine may not have been entirely humoral because in protected birds no antibodies against reovirus were detected by enzyme-linked immunosorbent at the time of challenge. Protection in these birds might therefore have been induced by cellular immunity.

The use of monoclonal antibody probes for the detection of avian reovirus antigens.

Two monoclonal antibodies (MAb), E9 and H3, prepared against avian reovirus (ARV) S1133, were used in an immuno-dot assay to detect ARV antigens from cell culture and from tendon tissue samples of chickens. The limit of viral antigens detected was 8 ng using both MAb probes. The probes detected 10 ARV isolates representing at least two serotypes or pathotypes. The results indicated that these probes had broad specificity. The probes, however, did not cross-react with viral antigens prepared from six unrelated avian viruses. The ARV antigens in tendon tissue samples were detected by both probes, and it is possible, therefore, to use either of the two MAb probes for detection of ARV infections.

Ethanol treatment of tendon allografts: a potential HIV inactivating procedure.

The penetration rate of ethanol in human tendons was studied to in order to define the conditions which were necessary to achieve an inactivating concentration against the Human Immunodeficiency Virus (HIV) within the tendon. The rate of alcohol penetration was found to be slow and did not differ with different types of tendons. An average ethanol concentration of 14% (v/v) was measured in human tendons after they had been immersed for 2 h in 70% (v/v) ethanol, and a concentration of 19% (v/v) was reached after 3 h. Ethanol immersion of human tendons may represent an additional safety procedure in inactivating the HIV virus provided the duration is at least 3 h.

Production and characterization of monoclonal antibodies against avian reovirus strain S1133.

Monoclonal antibodies (MAbs) were produced against the avian reovirus strain S1133. MAbs were isotyped and used to develop diagnostic tests. Splenocytes from immunized mice were screened by enzyme-linked immunosorbent assay (ELISA). Two hybridomas secreted MAbs against avian reovirus S1133. One MAb secreted IgG1 and the other secreted IgG2a. All MAb light chains were kappa Specificity of MAbs was tested against four avian reovirus strains: S1133, 1733, CO8, and 2408. Strains S1133, 1733, and 2408 viruses were in the same subtype; the CO8 virus belonged to a different subtype. The MAbs reacted with all reovirus strains by ELISA, dot blot, immunofluorescence assay, and immunoblotting. No MAb had neutralizing activity against the tested reoviruses. Immunoblot analysis showed the one MAb bound to protein sigma A with molecular weight of 39,000 Daltons for all reovirus strains. Another MAb bound to the protein sigma C with an approximate molecular mass of 32,000 Daltons. An indirect immunoperoxidase (IP) procedure was developed using a MAb to detect reovirus in formalin-fixed, paraffin-embedded tissues from infected chickens and chicken embryo fibroblast cell cultures. The IP test was simple, fast, and economical and enabled simultaneous evaluation of viral antigen-producing cells with tissue pathologic changes confirming that the reovirus caused the lesions.

Restriction of avian reovirus in primary chicken embryo tendon cells.

Upon infection of the gastrointestinal tract, some avian reovirus strains spread to multiple organs of their natural hosts, chickens, and establish persistent infections. One manifestation of chronic infection is the development of arthritis in the tendons of the chickens. In order to study events associated with persistent infections in the tendon, primary cultures of chick embryo tendon (CET) cells were infected with avian reovirus. The CET cells supported a noncytopathic infection for at least 16 days, shedding lower amounts of progeny virus than a permissive cell culture, chick embryo fibroblasts (CEF). The virus from the CET cells was predominantly cell-associated unlike virus from the CEF cells. Initiation of infection was much slower in CET cells as indicated by fewer cells expressing antigen or exporting virus over the first 48 hr. However, most CET cells gained these abilities over the course of infection. Initial events in virus infection, binding, and penetration were not impaired in tendon cells but transcription of single-strand RNA was delayed and double-strand RNA production was clearly inhibited. The CET cells supported efficient replication of another avian virus, Newcastle disease virus, suggesting that restriction for avian reovirus is virus specific, in addition to being tissue specific.