Lymphoproliferative Disease Virus in Wild Turkeys (Meleagris gallopavo) from Manitoba and Quebec, Canada.

This study describes the first recognized clinical case of lymphoproliferative disease virus (LPDV) in Canada and extends the range of LPDV in Canada through its detection in Manitoba and Quebec. We assessed the prevalence of LPDV in eastern wild turkeys (Meleagris gallopavo silvestris) with the use of whole, clotted blood from live birds in Manitoba (n = 65) and tissue samples collected postmortem in Quebec (n = 4). We tested for LPDV proviral DNA through PCR amplification and sequencing of a portion of the gag (p31) gene. Samples were also tested for reticuloendotheliosis virus (REV) by PCR. Twenty-four birds (34.8%) were positive for LPDV, including all diagnostic cases. One bird (1.4%) from Quebec had gross and microscopic lesions consistent with LPDV. Two turkeys (2.9%) were REV positive, one (1.4%) of which was co-infected with LPDV. Phylogenetic analysis of LPDV strains from Quebec and Manitoba grouped with previously sequenced samples from Ontario and publicly available sequences from a North American lineage. This study contributes valuable information toward ongoing surveillance and monitoring of LPDV in North America.

Virus de la enfermedad linfoproliferativa en pavos silvestres (Meleagris gallopavo) de Manitoba y Quebec, en Canadá. Este estudio describe el primer caso clínico reconocido del virus de la enfermedad linfoproliferativa (LPDV) en Canadá y extiende el rango de detección de este virus a través de su detección en Manitoba y Quebec. Se evaluó la prevalencia del virus de la enfermedad linfoproliferativa en pavos silvestres (Meleagris gallopavo silvestris) de la parte oriental, mediante el uso de sangre coagulada de aves vivas en Manitoba (n = 65) y de muestras de tejidos recolectadas postmortem en Quebec (n = 4). Se analizó el ADN proviral del virus de la enfermedad linfoproliferativa del pavo a través de la amplificación por PCR y la secuenciación de una parte del gene gag (p31). Las muestras también se analizaron para detectar el virus de la reticuloendoteliosis (REV) mediante PCR. Veinticuatro aves (34.8%) resultaron positivas para la presencia del virus de la enfermedad linfoproliferativa, incluyendo todos los casos diagnósticos. Un ave (1.4%) de Quebec tenía lesiones macroscópicas y microscópicas compatibles con este virus. Dos pavos (2.9%) fueron positivos a la presencia del virus de la reticuloendoteliosis, uno (1.4%) de los cuales se co-infectó con el virus de la enfermedad linfoproliferativa. El análisis filogenético de cepas del virus de la enfermedad linfoproliferativa de Quebec y Manitoba agrupó a estos virus con muestras previamente secuenciadas de Ontario y secuencias disponibles públicamente de un linaje de América del Norte. Este estudio aporta información valiosa para la vigilancia y el monitoreo continuos del virus de la enfermedad linfoproliferativa en América del Norte.

DETECTION OF LYMPHOPROLIFERATIVE DISEASE VIRUS IN CANADA IN A SURVEY FOR VIRUSES IN ONTARIO WILD TURKEYS ( MELEAGRIS GALLOPAVO).

The successful reintroduction of Wild Turkeys ( Meleagris gallopavo) to Ontario, Canada, has led to established populations in southern portions of the province and currently allows for biannual hunting seasons. These populations geographically overlap Domestic Turkey farms, an important sector of the provincial agri-food industry. Potential pathogen transmission between Wild Turkeys and Domestic Turkeys ( Meleagris gallopavo) is a concern, because they are susceptible to infection with many of the same pathogens and have direct and indirect contact in outdoor or open farm settings and contaminated environmental substrates. However, data concerning potential poultry pathogens in Wild Turkeys in Canada are scarce. Thus, we assessed the prevalence and geographic distribution of geographically relevant viruses in Ontario Wild Turkeys. Oropharyngeal and cloacal swabs were tested for avian influenza viruses (AIV) by real-time reverse transcriptase (RT)-PCR ( n=207), pooled tissues for lymphoproliferative disease virus (LPDV; n=183) and reticuloendotheliosis virus ( n=119) by PCR, and gross skin lesions by real-time RT-PCR for avian poxvirus ( n=8). We sequenced a fragment of the gag polyprotein (p31) gene of LPDV on a subset ( n=10) of LPDV-positive samples for phylogenetic analysis and tested additional upland game bird species ( n=39) and domestic fowl for LPDV ( n=17). To the best of our knowledge, we document the first detection of LPDV in Wild Turkeys in Canada, with a prevalence of 65% (119/183). Phylogenetic analysis revealed that LPDV sequences from Ontario were genetically similar to other North American strains and did not group into separate clades. Reticuloendotheliosis virus was detected in 4% (5/119) of LPDV-positive Wild Turkeys. Grossly evident skin lesions from five Wild Turkeys tested positive for poxvirus, and all turkeys tested negative for AIV. This study provides evidence of LPDV circulation in Canada and provides a baseline for comparison with future Wild Turkey pathogen surveillance and monitoring in Ontario and elsewhere.

Avian oncogenesis induced by lymphoproliferative disease virus: a neglected or emerging retroviral pathogen?

Lymphoproliferative disease virus (LPDV) is an exogenous oncogenic retrovirus that induces lymphoid tumors in some galliform species of birds. Historically, outbreaks of LPDV have been reported from Europe and Israel. Although the virus has previously never been detected in North America, herein we describe the widespread distribution, genetic diversity, pathogenesis, and evolution of LPDV in the United States. Characterization of the provirus genome of the index LPDV case from North America demonstrated an 88% nucleotide identity to the Israeli prototype strain. Although phylogenetic analysis indicated that the majority of viruses fell into a single North American lineage, a small subset of viruses from South Carolina were most closely related to the Israeli prototype. These results suggest that LPDV was transferred between continents to initiate outbreaks of disease. However, the direction (New World to Old World or vice versa), mechanism, and time frame of the transcontinental spread currently remain unknown.

No evidence of infectious retroviruses in measles virus vaccines produced in chicken embryo cell cultures.

All vaccines that are prepared in chicken embryo fibroblasts (CEFs) contain a low level of particle-associated reverse transcriptase (RT) activity, which is produced from the avian cell substrate. The RNAs present in the particles have sequence homology to viral DNAs belonging to the ancient endogenous avian virus (EAV) family or to the avian sarcoma-leukosis virus (ALV)-related subgroup E endogenous virus loci. Although no replication-competent retrovirus has been associated with the RT activity produced from CEFs, there have been some theoretical safety concerns regarding potential consequences of integration of EAV and ALV sequences in human DNA, which may result from nonproductive infection with replication-defective particles or infection with EAV and ALV pseudotypes bearing measles virus envelopes. To address these possibilities, we have analyzed EAV and ALV particles in a measles virus vaccine equivalent (MVVE) preparation, obtained from a U.S. manufacturer, for integration and for replication in human peripheral blood mononuclear cells (PBMCs). The results show the absence of EAV and ALV integrants in DNA prepared from MVVE-inoculated human cells by direct DNA PCR and Alu PCR assays and no propagation of retrovirus in 18-day cultures of MVVE-inoculated human PBMCs by a highly sensitive PCR-based RT assay. These results provide further confidence regarding the safety of chicken RT activity in live viral vaccines and support the continued use of chick-cell-derived vaccines in humans.

A PCR-based test for the presence of endogenous virus gene evA in chickens.

An endogenous virus, denoted evA, is present at high frequency in all brown egg layer lines. Using inverse polymerase chain reaction (PCR) based on the viral LTR regions, products were obtained containing cellular sequences 5' and 3' to the viral insertion point. PCR of chicken genomic DNA was carried out, using primers chosen from the 5' and 3' cellular sequences and a primer chosen from either the U3 or U5 portions of the viral LTR. Amplification of DNA from birds that did not carry evA with the primer triplets always gave a single 364 bp reaction product, interpreted as representing the flank-to-flank amplification product. Amplification of DNA from known homozygous or heterozygous evA carriers, with the same primer triplets, always gave both the expected junction product and 364 bp product. Therefore, these primer sequences can be used to distinguish evA carriers from non-carriers but cannot distinguish between homozygous and heterozygous evA carriers.

Poultry oncogenic retroviruses and humans.

Viruses of the avian leukosis/sarcoma group (ALSV) and reticuloendotheliosis viruses (REV) are highly prevalent in chickens and turkeys and naturally cause tumors in them. Commercial chickens are positive for antibodies, and a proportion actually carry infectious virus. Virus may be present in chicken products and in eggs, thus human exposure is virtually universal. The viruses show little potential for producing infectious viral particles in mammalian cells; nevertheless, they have the capacity to infect and transform mammalian cells (including human cells) in vitro, and to induce tumors in a variety of mammals, including primates. Most, but not all, of the serological studies in humans have been negative. Given the known behavior of these viruses in mammals, this was not unexpected. Moreover, there were methodological problems with most of the studies. There is some epidemiological evidence associating putative poultry exposure with cancer in humans. However, this has not been rigorously investigated. This paper is a comprehensive review of the extent of the carcinogenic potential these viruses show for humans. It is concluded, virological evidence indicates, that these viruses could conceivably have a carcinogenic potential for humans, but if so, at a level much less than in chickens. Whether this is insignificant, or translates to a real risk, is not known at the moment. Therefore, there is a need for definitive studies to completely rule out this possibility.

An overview of retrovirus replication and classification.

This introductory chapter has presented an overview of how retroviruses replicate and how they are classified within the family Retroviridae. The genomic structure of retroviruses, so reminiscent of bacterial transposons and other similar genetic elements, and reverse transcriptase, which leads to the reverse flow of genetic information from RNA to DNA, are responsible for many of the properties of these viruses which make them both fascinating and important as causes of cancer and other diseases. The requirement for integration shared by most retroviruses leads directly to most of the phenomena resulting from their interaction with target cells. Certainly latency, at the level of the organism, is one such property relevant to how we think of vaccines and therapeutic reagents. The ability of retroviruses to acquire oncogenes from cellular DNA has greatly facilitated our understanding of the genetics of neoplasia. Additionally, the use of retroviral vectors to introduce new genes into genetically defective animals is a consequence of the genetic organization of retroviruses. Classification of viruses at the species level is difficult for several reasons. In particular, viruses do not sexually reproduce in any conventional sense, and it is difficult to identify a population of virions which make up a genetically distinct pool. Thus, the definition of individual species is often controversial and is not necessarily aided by the criteria used to define larger phylogenetic groups. In the latter case, retroviruses have distinctive morphological and biochemical features which allow their classification at the family, subfamily, genus, and subgenus levels. Additional classification occurs by accounting for factors such as host range, cross neutralization, ability to compete in interspecies radioimmunoassays, and genetic homology detected by hybridization under conditions of relaxed stringency. Direct comparison of nucleotide sequences offers the hope that mathematical criteria will be developed that can define the level of differences characteristic of individual species, genuses, and subfamilies.

Avian erythroblastosis virus isolated from chick erythroblastosis induced by lymphatic leukemia virus subgroup A.

A new strain of avian erythroblastosis virus (AEV), designated "AEV-H," was established by serial passages of a field isolate of avian lymphatic leukemia virus subgroup A [LLV(A)] in chicks from a White Leghorn flock of line 151 chicks. One stock of AEV-H contained 10(4) focus-forming units/ml virus and 10(9) tissue culture infective dose/ml LLV(A). All of the chicks that received ip inoculations of 0.2 ml AEV-H developed erythroblastosis complicated by fibrosarcoma 16-24 days (approximately equal to 17.7 days) after inoculation. Pathologic changes of erythroblastosis were macroscopically observed, mainly in such visceral organs as the liver, spleen, and bone marrow. In addition, microscopic changes were observed in the lung, kidney, ovary, and heart. Pathologic changes of fibrosarcoma were so conspicuous that they were recognizable by the naked eye in the pancreas and in the serous membrane of the intestine.

Some biological properties of two new avian sarcoma viruses.

The new avian retroviruses UR1 and UR2 were isolated from spontaneous tumors of chickens by cocultivation of tumor material with susceptible chicken embryo fibroblasts. In vitro, UR1 induced formation of small foci of round and fusiform cells. On the other hand, cells infected by UR2 assumed an extremely elongated morphology. In vivo, both viruses induced fibrosarcomas and myxosarcomas with short latencies. Infectivity assays with and without mitomycin C showed that both viruses were defective for replication, but transformed nonproducing cell clones were obtained only with UR1. UR1-infected transformed nonproducing clones did not release particles detectable by reverse transcriptase assays, and fusion of transformed nonproducing cells with quail cells chronically infected with Rous sarcoma virus (a Bryan strain) failed to rescue infectious virus. This suggested that UR1 does not code for functional envelope glycoproteins. In this regard, UR1 appeared to be similar to Fujinami, PRCII, and Y73 viruses. The helper viruses of partially purified stocks of UR1 and UR2 appeared to belong to subgroup A, but these helper viruses were distinguishable from each other, as shown by host range experiments and neutralization tests. Hybridization studies with DNA complementary to the src gene of Rous sarcoma virus and RNAs extracted from both UR1 and UR2 showed no homology between the genomes of the new isolates and the transforming gene of Rous sarcoma virus.

Normal and dystrophic embryonic chicken pectoralis muscle cultures: III. Viral infection of normal cell cultures.

Twelve-day normal and dystrophic chick embryo breast muscle cells were cultured for up to 14 days, using normal embryo extract in the culture medium. Fluorodeoxyuridine was added on day 3 to suppress fibroblast overgrowth. Three of 6 experiments with normal cells were severely infected with avian leucosis/sarcoma (ALS) virus particles. The findings appear to lend support to the suggestion that the ALS virus is a mitochondriophage. Infected cultures demonstrated a depressed rate of total protein synthesis as reflected by incorporation of [3H]leucine. Extractable protein and total protein content were also depressed within several days after ALS particles were identified. These findings reinforce caution in acceptance of the common assumption that viral infections are an unavoidable and metabolically benign component of muscle cell cultures.

["Pharaoh" line culture of Japanese quail cells as a leukosis-free system for virus reproduction]. / Kul'tura kletok iaponskikh perepelov linii faraon kak bezleikoznaia sistema dlia reproduktsii virusov.

Contamination of Japanese quail, strain Pharaoh, cell culture with oncogenous and infectious avian viruses was studied. The susceptibility of the embryonal cell cultures of the Japanese quail, strain Pharaoh, to measles, parotitis and fixed rabies viruses was also determined. It was found that the sera of pubertal quails had no antibody to Rous sarcoma virus (RSV), strains Brian, RSV (RAV-1), Schmidt-Ruppin, Carr-Zilber, as well as to Marek's disease and Newcastle disease viruses. No reverse transcriptase activity was detected in the embryonal alantoic fluid of this avian species. The quails were less susceptible, as compared to the chicken, to Schmidt-Ruppin and Carr-Zilber strains of RSV. Measles, parotitis and fixed rabies viruses reproduced actively in the Japanese quail, Pharaoh strain, embryonal cell cultures. It is suggested that the embryonal cell cultures of this avian species can be used as a leukemia-free substrate for experimental studies and manufacturing of viral vaccines.

Low incidence of lymphoid tumors in chickens continuously producing endogenous virus.

A flock of 258 male and 243 female chickens of a cross of Regional Poultry Research Laboratory lines 15B and 7(2) were kept in a filtered-air positive-pressure house and observed for tumors from 100 to more than 729 days of age. These birds produced high titers of a subgroup E endogenous virus from the middle of the embryonic incubation period through the end of the experiment. No neoplasms were observed in the males. The females had two neoplasms indistinguishable from lymphoid leukosis and three other neoplasms not involving lymphoid cells. No evidence was found of infection with exogenous lymphoid leukosis viruses, Marek's disease virus, reticuloendotheliosis virus, or adenovirus (isolated on the isolation farm). Inoculation of another sample of this cross with a lymphoid leukosis virus of subgroup A resulted in 88% mortality with neoplasms (mostly lymphoid leukosis) by 167 days of age. The conclusion is that high levels of spontaneously produced endogenous virus do not induce high levels of neoplasms in chickens susceptible to lymphoid leukosis.

Isolation of a sarcoma virus from a spontaneous chicken tumor.

A spontaneous tumor was obtained from a hen of White-Leghorn stock raised in a local farm in Yamaguchi City, in December 1973. The tumor had characteristics of fibro- or myxofibro-sarcoma and could be maintained by transplantation in chickens of similar flocks. This tumor was proved to produce sarcoma virus at 33rd passage and the transforming virus was designated as Y73 sarcoma virus. Biological studies suggested that the virus recovered from the tumor belonged to avian leukosis-sarcoma complex having subgroup A specificity. The possibility of defectiveness of this virus will be discussed.

Expression of Marek's disease virus in producer and non-producer lymphoblastoid cell lines.

Continuous in vitro propagation of a producer Marek's disease (MD) lymphoblastoid cell line reduced the number of cells producing virus antigens and the inducibility of these antigens to 5-iododeoxyuridine (IUDR) but did not change the oncogenicity of the cells for chickens. In contrast, propagation of a non-producer MD cell line did not change the non-productivity of the cell line, but decreased the transplantability of the cells. The MD lines were free of replicating avian leukosis sarcoma viruses (LSV) and were susceptible to infection with LSV of subgroup A.