Turkey adenovirus 3, a siadenovirus, uses sialic acid on N-linked glycoproteins as a cellular receptor.

Turkey adenovirus 3 (TAdV-3) is the causative agent of an immune-mediated disease in turkeys, haemorrhagic enteritis, through targeting B lymphocytes. In the present study, we investigated the role of sialic acid in TAdV-3 entry and characterized the structural components of TAdV-3 receptor(s) on RP19, B lymphoblastoid cells. Removal of the cell-surface sialic acids by neuraminidases or blocking of sialic acids by wheat germ agglutinin lectin reduced virus infection. Pre-incubation of cells with Maackia amurensis lectin or Sambucus nigra agglutinin resulted in virus reduction, suggesting that TAdV-3 uses both α2,3-linked and α2,6-linked sialic acids as attachment receptor. Virus infectivity data from RP19 cells treated with sodium periodate, proteases (trypsin or bromelain) or metabolic inhibitors (dl-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol, tunicamycin, or benzyl N-acetyl-α-d-galactosaminide) indicated that N-linked, but not O-linked, carbohydrates are part of the sialylated receptor and they are likely based on a membrane glycoprotein, rather than a glycolipid. Furthermore, our data, in conjunction with previous findings, implies that the secondary receptor for TAdV-3 is a protein molecule since the inhibition of glycolipid biosynthesis did not affect the virus infection, which was rather reduced by protease treatment. We can conclude that terminal sialic acids attached to N-linked membrane glycoproteins on B cells are used for virus attachment and are essential for successful virus infection.

In vitro growth kinetics and gene expression analysis of the turkey adenovirus 3, a siadenovirus.

Turkey adenovirus 3 (TAdV-3) belongs to the genus Siadenovirus, family Adenoviridae. Previously, nucleotide sequencing and annotation of the Virginia avirulent strain (VAS) of TAdV-3 genome, isolated in our laboratory, indicated the presence of a total of 23 genes and open reading frames (ORFs). The goals of this study were 1) to delineate the growth kinetics of the virus using a qPCR-based infectivity assay, and 2) to determine the virus gene expression profile during the early and late phases of infection in target B lymphocytes. The one-step growth curve experiment demonstrated 3 phases of virus replication cycle: a lag phase lasted for 12-18 h post-infection (h.p.i.), in which the virus titer declined; a log phase from 18 to 120 h.p.i., in which the number of infectious virus particles increased over 20,000 folds, and a brief decline phase thereafter. Southern blot analysis indicated that the synthesis of new viral DNA started by 8 h.p.i. Gene-specific RT-PCR analysis revealed the expression of mRNAs from the 23 TAdV-3 genes/ORFs. According to the temporal transcriptional profiling of TAdV-3 genome, genes could be divided into 3 groups based on the time of transcription initiation: group 1 showed detectable levels of transcription at 2 h.p.i and included 7 genes, i.e., hyd, III, pX, pVI, II, 100 K, and 33 K; group 2 included 12 genes whose mRNAs were detected for the first time at 4 h.p.i., i.e., ORF1, IVa2, pol, pTP, pIIIa, EP, DBP, E3, U exon, IV, ORF7, and ORF8; group 3 of transcripts were detectable starting 8 h.p.i. and included only 4 genes, i.e., 52 K, 22 K, pVII, and pVIII. Our data suggest that the transcriptional kinetics of genus Siadenovirus differ from that observed in other adenoviral genera; however, a few TAdV-3 genes showed similar expression patterns to their adenoviral homologs.

Effect of Different Instructional Methods on Contamination and Personal Protective Equipment Protocol Adherence among Veterinary Students.

Proper use of personal protective equipment (PPE) is crucial to prevent disease spread. Recent studies in human medicine have shown disconcerting inconsistencies in the use of PPE in hospital wards. In this study, we compared the effect of three instructional methods for PPE use on contamination and protocol adherence among veterinary students. Students were divided into three groups according to the instructional method to which they had access (instructional video, wall chart, or both). They underwent an isolation exercise consisting of donning, patient examination (mock patient prepared with contamination marker), and doffing. Student contamination after the exercise was evaluated using UV light. Videos of student performance were reviewed for errors committed. Results showed that the number of students with contamination was higher in the group who only had access to video instruction than in the two other groups. The number of students with contamination on forearms, hands, and wrists was higher in the group who only had access to charts. Disinfecting gloves between doffing steps was the most frequently omitted step. The number of students who touched the environment with unprotected areas of their bodies was higher in the group who only had access to video instruction than in the other two groups. In conclusion, video instruction was less effective in achieving PPE protocol adherence among veterinary students than was instruction with a chart or chart-video combination. Incorporating video instruction as part of the instructions may be valuable to reinforce individual steps of donning and doffing.

Advancing the Sustainability of US Agriculture through Long-Term Research.

Agriculture in the United States must respond to escalating demands for productivity and efficiency, as well as pressures to improve its stewardship of natural resources. Growing global population and changing diets, combined with a greater societal awareness of agriculture's role in delivering ecosystem services beyond food, feed, fiber, and energy production, require a comprehensive perspective on where and how US agriculture can be sustainably intensified, that is, made more productive without exacerbating local and off-site environmental concerns. The USDA's Long-Term Agroecosystem Research (LTAR) network is composed of 18 locations distributed across the contiguous United States working together to integrate national and local agricultural priorities and advance the sustainable intensification of US agriculture. We explore here the concept of sustainable intensification as a framework for defining strategies to enhance production, environmental, and rural prosperity outcomes from agricultural systems. We also elucidate the diversity of factors that have shaped the past and present conditions of cropland, rangeland, and pastureland agroecosystems represented by the LTAR network and identify priorities for research in the areas of production, resource conservation and environmental quality, and rural prosperity. Ultimately, integrated long-term research on sustainable intensification at the national scale is critical to developing practices and programs that can anticipate and address challenges before they become crises.

Biofilm formation and avian immune response following experimental acute and chronic avian cholera due to Pasteurella multocida.

Pasteurella multocida is the causative agent of avian cholera, an important economic and ecological disease that can present as a peracute, acute, chronic, or asymptomatic infection. Acute avian cholera is associated with encapsulated P. multocida, while chronic and asymptomatic cases of avian cholera may be associated with capsule-deficient P. multocida isolates. We hypothesize that biofilm formation is also associated with chronic and asymptomatic avian cholera. Experimental infections of chickens with encapsulated, biofilm-deficient P. multocida strain X73, proficient biofilm forming P. multocida strain X73ΔhyaD, and proficient biofilm forming clinical strains 775 and 756 showed that virulence was inversely correlated with biofilm formation. Biofilm-proficient isolates induced chronic avian cholera in the chicken host. Histopathological analysis was used to show that biofilm-proficient isolates induced little inflammation in the lungs, heart, and liver, while biofilm-deficient isolates induced greater inflammation and induced the recruitment of heterophil granulocytes. Putative biofilm matrix material and exopolysaccharide was detected in pulmonary tissue of chickens diagnosed with chronic avian cholera using scanning electron microscopy and a fluorescently-tagged lectin, respectively, supporting a role for biofilm in chronic avian cholera. P. multocida induced Th1 and Th17 immune responses during acute and chronic avian cholera, as determined by quantitative real-time PCR of splenic cytokine genes. Chickens that succumbed to acute avian cholera after experimental challenge with strain X73 had high levels of INF-γ, IL-1ß, IL-6, IL-12A, IL-22, IL-17A, and IL-17RA expressed in the spleen compared to all other experimental groups. Birds infected with capsule-deficient strains had chronic infections lasting 7 days or longer, and had increased levels of IL-17RA, CCR6, and IL-16 compared to non-infected control chickens. However, specific antibody titers increased only transiently to capsule-deficient strains and were low, indicating that antibodies are less important in managing and clearing P. multocida infections.

Review of Nonfoodborne Zoonotic and Potentially Zoonotic Poultry Diseases.

Emerging and re-emerging diseases are continuously diagnosed in poultry species. A few of these diseases are known to cross the species barrier, thus posing a public health risk and an economic burden. We identified and synthesized global evidence for poultry nonfoodborne zoonoses to better understand these diseases in people who were exposed to different poultry-related characteristics (e.g., occupational or nonoccupational, operational types, poultry species, outbreak conditions, health status of flocks). This review builds on current knowledge on poultry zoonoses/potentially zoonotic agents transmitted via the nonfoodborne route. It also identifies research gaps and potential intervention points within the poultry industry to reduce zoonotic transmission by using various knowledge synthesis tools such as systematic review (SR) and qualitative (descriptive) and quantitative synthesis methods (i.e., meta-analysis). Overall, 1663 abstracts were screened and 156 relevant articles were selected for further review. Full articles (in English) were retrieved and critically appraised using routine SR methods. In total, eight known zoonotic diseases were reviewed: avian influenza (AI) virus (n = 85 articles), Newcastle disease virus (n = 8), West Nile virus (WNV, n = 2), avian Chlamydia (n = 24), Erysipelothrix rhusiopathiae (n = 3), methicillin-resistant Staphylococcus aureus (MRSA, n = 15), Ornithonyssus sylvarium (n = 4), and Microsporum gallinae (n = 3). In addition, articles on other viral poultry pathogens (n = 5) and poultry respiratory allergens derived from mites and fungi (n = 7) were reviewed. The level of investigations (e.g., exposure history, risk factor, clinical disease in epidemiologically linked poultry, molecular studies) to establish zoonotic linkages varied across disease agents and across studies. Based on the multiple outcome measures captured in this review, AI virus seems to be the poultry zoonotic pathogen that may have considerable and significant public health consequences; however, epidemiologic reports have only documented severe human cases clustered in Asia and not in North America. In contrast, avian Chlamydia and MRSA reports clustered mainly in Europe and less so in North America and other regions. Knowledge gaps in other zoonoses or other agents were identified, including potential direct (i.e., nonmosquito-borne) transmission of WNV from flocks to poultry workers, the public health and clinical significance of poultry-derived (livestock-associated) MRSA, the zoonotic significance of other viruses, and the role of poultry allergens in the pathophysiology of respiratory diseases of poultry workers. Across all pathogens reviewed, the use of personal protective equipment was commonly cited as the most important preventive measure to reduce the zoonotic spread of these diseases and the use of biosecurity measures to reduce horizontal transmission in flock populations. The studies also emphasized the need for flock monitoring and an integrated approach to prevention (i.e., veterinary-public health coordination with regard to diagnosis, and knowledge translation and education in the general population) to reduce zoonotic transmission.

Preharvest Salmonella Detection for Evaluation of Fresh Ground Poultry Product Contamination.

Salmonella is an important economic and public health concern for the poultry industry. Fresh ground product has been linked with multiple salmonellosis outbreaks in humans. Exposure can be controlled by proper handling and preparation by consumers; however, the industry desires to minimize carriage levels in the final product. A substantial obstacle in reducing product contamination stems from limitations in diagnostic methodologies. Detection of Salmonella contamination currently requires extended incubation periods, and by the time test results are available, the fresh product has reached retail shelves. The goal of this study was to develop a preharvest diagnostic protocol for the evaluation of ground product contamination. The turkey processing plant where this research was conducted had previously established Salmonella screening (BAX system) of ground product, thus providing an opportunity for preharvest sample comparison. Drag swabs were collected from live-haul trailers entering the processing plant over a 12-month period. The swabs were added to modified buffered peptone water and incubated at 40°C. After incubation for 6 h or overnight, samples were tested for the presence of Salmonella with the DNAble assay and related to ground turkey samples from corresponding lots. The linear relationship for the percentage of Salmonella-positive live-haul trailers was significant for both the 6-h (slope = 1.02, R(2) = 0.96, and P < 0.0001) and overnight (slope = 0.35, R(2) = 0.93, and P = 0.0015) incubations, with the percentage of Salmonella-positive ground turkey samples. These data indicate that preharvest screening provides a meaningful evaluation of product contamination. Additionally, the 6-h incubation protocol is rapid enough to allow for product mitigation and could potentially aid in the reduction of future salmonellosis outbreaks.

RNA transcripts of full-length cDNA clones of rabbit hepatitis E virus are infectious in rabbits.

Hepatitis E virus (HEV), the causative agent of hepatitis E, is a single-stranded positive-sense RNA virus belonging to the family Hepeviridae. At least four genotypes of the family infect humans: genotypes 1 and 2 are transmitted to humans through contaminated water, while genotypes 3 and 4 are zoonotic and have animal reservoirs. A novel strain of HEV recently identified in rabbits is a distant member of genotype 3, and thus poses a potential risk of zoonotic transmission to humans. The objective of this study was to construct and characterize an infectious cDNA clone of the rabbit HEV. Two full-length cDNA clones of rabbit HEV, pT7g-rabHEV and pT7-rabHEV, were constructed and their infectivity was tested by in vitro transfection of Huh7 human liver cells and by direct intrahepatic inoculation of rabbits with capped RNA transcripts. Results showed that positive signal for rabbit HEV protein was detected by an immunofluorescence assay with a HEV-specific antibody in Huh7 human liver cells transfected with capped RNA transcripts from the two full-length cDNA clones. Rabbits intrahepatically inoculated with capped RNA transcripts from each of the two clones developed active HEV infection as evidenced by seroconversion to anti-HEV antibodies, and detection of rabbit HEV RNA in sera and feces of inoculated animals. The availability of a rabbit HEV infectious cDNA clone now affords us the ability to delineate the mechanism of HEV replication and cross-species infection in a small animal model.

Experimental comparison of hemolytic and nonhemolytic Ornithobacterium rhinotracheale field isolates in vivo.

Ornithobacterium rhinotracheale (ORT) is a nonhemolytic, gram-negative, pleomorphic, rod-shaped bacterium that causes upper and lower respiratory tract disease in poultry. Recently, hemolytic strains of ORT have been isolated with increasing frequency from field outbreaks. A study was conducted to determine whether the hemolytic phenotype is associated with any change in virulence. Briefly, 225 turkey poults, vaccinated against hemorrhagic enteritis at 4 wk of age, were randomly divided into nine replicates housed in separate rooms: three sham treatment controls (25 poults/replicate), three challenged with a nonhemolytic (NH) field isolate (24 poults/replicate), and three challenged with a hemolytic (H) field isolate (24 poults/replicate). Nine days postvaccination, poults were inoculated intratracheally with either 0.2 ml sterile phosphate-buffered saline (PBS), 2 x 10(8) colony-forming units (CFU) of the NH isolate in 0.2 ml PBS, or 2 x 10(8) CFU of the H isolate in 0.2 ml PBS. Serum and body weights were obtained at 0, 7, 14, and 21 days postinoculation (dpi). Tissues were taken for culture and histopathology from five randomly selected poults/replicates at 7, 14, and 21 dpi. When compared with poults inoculated with the H isolate or controls, those inoculated with the NH isolate showed a highly significant depression in weight gain at 7 dpi. NH poults also had significantly higher levels of antibody against ORT at 14 and 21 dpi. Reisolations decreased over time and, by 21 dpi, only the NH phenotype could be found. Based on a Likert-type scale, poults inoculated with the NH isolate had significantly higher histopathologic lesion scores in lung tissue at 7, 14, and 21 dpi. Results suggest that nonhemolytic field isolates are more virulent then hemolytic ones. These findings are unusual because hemolytic phenotypes are often more virulent in other bacterial species.

Egg quality traits differ in hens selected for high as compared with low antibody response to sheep red blood cells.

White Leghorn chickens were selected for 36 generations for high (HAS) or low (LAS) antibody response to SRBC 5 d after an intravenous challenge. Our objective was to determine differences in egg quality resulting from that selection. In total, eggs from 45 hens from each line were assessed for shape index (SI), weight (WT, g), albumen height (AH, mm), Haugh units (HU), yolk color (YC), and eggshell weight (ESW, g) and thickness (EST, mm). Three cycles representing early, middle, and late stages of production were examined. Eggs from HAS hens had higher SI scores (4.12 ± 0.55; P < 0.001) and greater AH (0.27 ± 0.12; P < 0.001) and HU (1.89 ± 0.91; P = 0.04) than LAS hens; conversely, eggs from LAS hens had greater EST (0.03 ± 0.01 g; P < 0.001) and heavier ESW (0.66 ± 0.09 g; P < 0.001) than HAS hens. Lines were similar for WT and YC (P > 0.52). Albumen height and HU decreased (P < 0.001), whereas WT, ESW, and EST increased (P < 0.001) over cycles for both lines. However, SI decreased in LAS hens, yet increased in HAS hens, across cycles (P < 0.001). An interaction between line and cycle was observed in WT, SI, ESW, and EST (P < 0.001), but only for WT did the interaction cause re-ranking across cycles. Egg quality was, generally, superior in HAS compared with LAS hens, suggesting that higher antibody response may maintain overall fitness.

Reproductive soundness is higher in chickens selected for low as compared with high antibody response.

White Leghorn chickens were selected for 36 generations for high (HAS) or low (LAS) antibody response to SRBC 5 d after an intravenous challenge. The aim of this study was to investigate possible changes in reproductive soundness resulting from that selection. Age and BW at onset of lay (first egg), along with weight of the first egg, were recorded on 45 hens from each line. Intensity of lay was measured as the number of ovulations within a 15-d period over 15 sequential intervals (total 225 d). Three cycles of fertility also were assessed, coinciding with early, middle, and late production stages. For fertility of males and females within a line to be independently evaluated, roosters and hens were mated by artificial insemination to an unrelated control line of White Plymouth Rocks. Twenty roosters from each antibody line were considered, as well as the 45 hens. Pooled semen from the control line was used for mating the hens from the antibody lines. Hens from the LAS line commenced lay at a younger age (11.67±3.53 d; P<0.001), lighter BW (-169.46±40.20 g; P<0.001), and with greater intensity (2.68±0.25%; P=0.001) than those from the HAS line. Any differences in intensity thereafter were trivial between lines (P=0.42), with intensity decreasing sharply toward the end of the 7-mo production period in both lines. Length of fertility differed between hens of the antibody lines during the first cycle (3.35±0.85 d; P=0.002) and between roosters during the first (3.58±1.06 d; P=0.02) and second (3.38±1.07 d; P=0.03) cycles, with chickens from the LAS line having the longer length of fertility in both sexes. A correlated response in reproductive soundness to divergent selection for antibody response was observed. This may in part be due to differences in resource allocations, with particular impact on duration of fertility.

The PSAP motif within the ORF3 protein of an avian strain of the hepatitis E virus is not critical for viral infectivity in vivo but plays a role in virus release.

The ORF3 protein of hepatitis E virus (HEV) is a multifunctional protein important for virus replication. The ORF3 proteins from human, swine, and avian strains of HEV contain a conserved PXXP amino acid motif, resembling either Src homology 3 (SH3) cell signaling interaction motifs or "late domains" involved in host cell interactions aiding in particle release. Using an avian strain of HEV, we determined the roles of the conserved prolines within the PREPSAPP motif in HEV replication and infectivity in Leghorn male hepatoma (LMH) chicken liver cells and in chickens. Each proline was changed to alanine to produce 8 avian HEV mutants containing single mutations (P64, P67, P70, and P71 to A), double mutations (P64/67A, P64/70A, and P67/70A), and triple mutations (P64/67/70A). The results showed that avian HEV mutants are replication competent in vitro, and none of the prolines in the PXXPXXPP motif are essential for infectivity in vivo; however, the second and third prolines appear to aid in fecal virus shedding, suggesting that the PSAP motif, but not the PREP motif, is involved in virus release. We also showed that the PSAP motif interacts with the host protein tumor suppressor gene 101 (TSG101) and that altering any proline within the PSAP motif disrupts this interaction. However, we showed that the ORF2 protein expressed in LMH cells is efficiently released from the cells in the absence of ORF3 and that coexpression of ORF2 and ORF3 did not act synergistically in this release, suggesting that another factor(s) such as ORF1 or viral genomic RNA may be necessary for proper particle release.

Mutational analysis of the hypervariable region of hepatitis e virus reveals its involvement in the efficiency of viral RNA replication.

The RNA genome of the hepatitis E virus (HEV) contains a hypervariable region (HVR) in ORF1 that tolerates small deletions with respect to infectivity. To further investigate the role of the HVR in HEV replication, we constructed a panel of mutants with overlapping deletions in the N-terminal, central, and C-terminal regions of the HVR by using a genotype 1 human HEV luciferase replicon and analyzed the effects of deletions on viral RNA replication in Huh7 cells. We found that the replication levels of the HVR deletion mutants were markedly reduced in Huh7 cells, suggesting a role of the HVR in viral replication efficiency. To further verify the results, we constructed HVR deletion mutants by using a genetically divergent, nonmammalian avian HEV, and similar effects on viral replication efficiency were observed when the avian HEV mutants were tested in LMH cells. Furthermore, the impact of complete HVR deletion on virus infectivity was tested in chickens, using an avian HEV mutant with a complete HVR deletion. Although the deletion mutant was still replication competent in LMH cells, the complete HVR deletion resulted in a loss of avian HEV infectivity in chickens. Since the HVR exhibits extensive variations in sequence and length among different HEV genotypes, we further examined the interchangeability of HVRs and demonstrated that HVR sequences are functionally exchangeable between HEV genotypes with regard to viral replication and infectivity in vitro, although genotype-specific HVR differences in replication efficiency were observed. The results showed that although the HVR tolerates small deletions with regard to infectivity, it may interact with viral and host factors to modulate the efficiency of HEV replication.

Construction of an infectious cDNA clone of avian hepatitis E virus (avian HEV) recovered from a clinically healthy chicken in the United States and characterization of its pathogenicity in specific-pathogen-free chickens.

A genetically distinct strain of avian hepatitis E virus (avian HEV-VA strain) was isolated from a healthy chicken in Virginia, and thus it is important to characterize and compare its pathogenicity with the prototype strain (avian HEV-prototype) isolated from a diseased chicken. Here we first constructed an infectious clone of the avian HEV-VA strain. Capped RNA transcripts from the avian HEV-VA clone were replication-competent after transfection of LMH chicken liver cells. Chickens inoculated intrahepatically with RNA transcripts of avian HEV-VA clone developed active infection as evidenced by fecal virus shedding, viremia, and seroconversion. To characterize the pathogenicity, RNA transcripts of both avian HEV-VA and avian HEV-prototype clones were intrahepatically inoculated into the livers of chickens. Avian HEV RNA was detected in feces, serum and bile samples from 10/10 avian HEV-VA-inoculated and 9/9 avian HEV-prototype-inoculated chickens although seroconversion occurred only in some chickens during the experimental period. The histopathological lesion scores were lower for avian HEV-VA group than avian HEV-prototype group in the liver at 3 and 5 weeks post-inoculation (wpi) and in the spleen at 3 wpi, although the differences were not statistically significant. The liver/body weight ratio, indicative of liver enlargement, of both avian HEV-VA and avian HEV-prototype groups were significantly higher than that of the control group at 5 wpi. Overall, the avian HEV-VA strain still induces histological liver lesions even though it was isolated from a healthy chicken. The results also showed that intrahepatic inoculation of chickens with RNA transcripts of avian HEV infectious clone may serve as an alternative for live virus in animal pathogenicity studies.

Complete genomic sequence of bacteriophage felix o1.

Bacteriophage O1 is a Myoviridae A1 group member used historically for identifying Salmonella. Sequencing revealed a single, linear, 86,155-base-pair genome with 39% average G+C content, 131 open reading frames, and 22 tRNAs. Closest protein homologs occur in Erwinia amylovora phage φEa21-4 and Escherichia coli phage wV8. Proteomic analysis indentified structural proteins: Gp23, Gp36 (major tail protein), Gp49, Gp53, Gp54, Gp55, Gp57, Gp58 (major capsid protein), Gp59, Gp63, Gp64, Gp67, Gp68, Gp69, Gp73, Gp74 and Gp77 (tail fiber). Based on phage-host codon differences, 7 tRNAs could affect translation rate during infection. Introns, holin-lysin cassettes, bacterial toxin homologs and host RNA polymerase-modifying genes were absent.

Persistent infection of turkeys with an avirulent strain of turkey hemorrhagic enteritis virus.

The Virginia avirulent strain (VAS) of turkey hemorrhagic enteritis virus (THEV), which is commonly used in live vaccines for commercial turkeys, was studied to determine characteristics of infection. It has been observed that turkeys infected with the VAS maintain protective antibody levels in excess of 20 wk postvaccination. It is theorized that this immune response is modulated by either a persistent or latent infection. A series of studies have been undertaken to determine changes in virus location and serology over time. A trial was also conducted to evaluate the effect of corticosteroid administration on viral recrudescence, and an attempt was made to isolate live virus from tissues of birds 10 wk postinfection (pi). Antibody titers were determined by enzyme-linked immunosorbent assay, and PCR was used to detect viral DNA. Histopathology was performed on formalin-fixed paraffinized tissues. Viral DNA was detected in various tissues through 15 wk pi in the presence of high antibody titers. Viral DNA was detected at 3-5 days pi in the spleens of susceptible turkeys inoculated with tissues collected from infected birds at 10 wk pi. It is unknown whether the viral DNA is associated with live virus or rather is the result of persistent maintenance of the viral genome within lymphoid/macrophage target cells. Future studies will test for viral RNA in order to confirm the presence of replicating THEV. Regardless of the actual status of the THEV DNA detected at 10-15 wk pi, it is clear that THEV does not cause a simple acute infection. The characteristics of THEV infection are identical to the nonlytic persistent infections seen in human adenoviruses, and therefore THEV may serve as a model for the study of virus-cell interactions mediating persistence.

Comparative pathogenesis in specific-pathogen-free chickens of two strains of avian hepatitis E virus recovered from a chicken with Hepatitis-Splenomegaly syndrome and from a clinically healthy chicken.

Avian hepatitis E virus (avian HEV) is the primary causative agent of Hepatitis-Splenomegaly (HS) syndrome in chickens. Recently, a genetically unique strain of avian HEV, designated avian HEV-VA, was recovered from healthy chickens in Virginia. The objective of this study was to experimentally compare the pathogenicity of the prototype strain recovered from a chicken with HS syndrome and the avian HEV-VA strain in specific-pathogen-free chickens. An infectious stock of the avian HEV-VA strain was first generated and its infectivity titer determined in chickens. For the comparative pathogenesis study, 54 chickens of 6-week-old were assigned to 3 groups of 18 chickens each. The group 1 chickens were each intravenously inoculated with 5x10(2.5) 50% chicken infectious dose of the prototype strain. The group 2 received the same dose of the avian HEV-VA strain, and the group 3 served as negative controls. Six chickens from each group were necropsied at 2, 3 and 4 weeks post-inoculation (wpi). Most chickens in both inoculated groups seroconverted by 3wpi, and the mean anti-avian HEV antibody titers were higher for the prototype strain group than the avian HEV-VA strain group. There was no significant difference in the patterns of viremia and fecal virus shedding. Blood analyte profiles did not differ between treatment groups except for serum creatine phosphokinase levels which were higher for prototype avian HEV group than avian HEV-VA group. The hepatic lesion score was higher for the prototype strain group than the other two groups. The results indicated that the avian HEV-VA strain is only slightly attenuated compared to the prototype strain, suggesting that the full spectrum of HS syndrome is likely associated with other co-factors.

Comparison of 12 turkey hemorrhagic enteritis virus isolates allows prediction of genetic factors affecting virulence.

Turkey hemorrhagic enteritis virus (THEV) is a member of the genus Siadenovirus and causes disease in turkey poults characterized by splenomegaly, bloody diarrhoea and death. The mechanism responsible for intestinal lesion formation and mortality is not known, although there is strong evidence that it is immune-mediated. All strains of THEV are serologically indistinguishable, although there are naturally occurring avirulent strains of THEV that replicate efficiently in turkeys without the intestinal haemorrhage or mortality associated with more virulent strains. The purpose of this study was to determine which viral genes are involved in virulence. The full-length genome of an avirulent vaccine strain was sequenced and compared with the genome of a virulent field isolate from Israel that was sequenced in 1998. Comparison of the two 26.3 kb genomes revealed 49 nucleotide differences resulting in 14 putative amino acid changes within viral proteins. Sequencing of the regions surrounding the 14 missense mutations revealed variations in ORF1, E3 and the fiber (fib) knob domain in five additional strains with varying degrees of virulence. Complete sequences of these genes were determined in a total of 11 different strains of THEV. All strains had at least one missense mutation in ORF1, and all but two of the strains had one missense mutation in E3. At least one missense mutation was found in the fiber knob domain in six out of seven virulent strains. Sequence variation of ORF1, E3 and fib in strains of THEV with different phenotypes strongly indicates that these genes are the key factors affecting virulence.

Deletions of the hypervariable region (HVR) in open reading frame 1 of hepatitis E virus do not abolish virus infectivity: evidence for attenuation of HVR deletion mutants in vivo.

Hepatitis E virus (HEV) is an important human pathogen, although little is known about its biology and replication. Comparative sequence analysis revealed a hypervariable region (HVR) with extensive sequence variations in open reading frame 1 of HEV. To elucidate the role of the HVR in HEV replication, we first constructed two HVR deletion mutants, hHVRd1 and hHVRd2, with in-frame deletion of amino acids (aa) 711 to 777 and 747 to 761 in the HVR of a genotype 1 human HEV replicon. Evidence of HEV replication was detected in Huh7 cells transfected with RNA transcripts from mutant hHVRd2, as evidenced by expression of enhanced green fluorescent protein. To confirm the in vitro results, we constructed three avian HEV mutants with various HVR deletions: mutants aHVRd1, with deletion of aa 557 to 585 (Delta557-585); aHVRd2 (Delta612-641); and aHVRd3 (Delta557-641). Chickens intrahepatically inoculated with capped RNA transcripts from mutants aHVRd1 and aHVRd2 developed active viral infection, as evidenced by seroconversion, viremia, and fecal virus shedding, although mutant aHVRd3, with complete HVR deletion, was apparently attenuated in chickens. To further verify the results, we constructed four additional HVR deletion mutants using the genotype 3 swine HEV as the backbone. Mutants sHVRd2 (Delta722-781), sHVRd3 (Delta735-765), and sHVRd4 (Delta712-765) were shown to tolerate deletions and were infectious in pigs intrahepatically inoculated with capped RNA transcripts from the mutants, whereas mutant sHVRd1 (Delta712-790), with a nearly complete HVR deletion, exhibited an attenuation phenotype in infected pigs. The data from these studies indicate that deletions in HVR do not abolish HEV infectivity in vitro or in vivo, although evidence for attenuation was observed for HEV mutants with a larger or nearly complete HVR deletion.