@misc{oai:az.repo.nii.ac.jp:00003175,
 author = {小山, 弘之 and Koyama, Hiroyuki},
 month = {2013-02-08, 2014-08-08, 2013-02-08},
 note = {Introduction
 The coexistence of turkey herpesvirus (HVT) particles and C-type particles in duck embryo fibroblast (DEF) and chick embryo fibroblast (CEF) cells infected with HT-1 strain (HVT) was proved by electron microscopy. To date, there have not been any reports, other than the author's of coexistence of C-type particles in electron microscopical observations of HVT. Since the observed particles were C-type, we believe that this virus morphologically belongs to the oncorna virus group. However, it is generally known that the avian leukosis-sarcoma virus group (ALSV) does not replicate in DEF cells. This suggested the possibility that the inoculum of HVT infected cells was contaminated by C-type particles other than ALSV.
 On the other hand, HVT is used world-wide as a live vaccine against Marek's disease and its protective effect is well recognized. This vaccine consists of HVT cultivated on DEF or CEF. It can not be neglected that there is a strong possibility of contamination of the vaccine by C-type particles during the HT-1 strain used by the author was passaged several times in chicken and duck cells following isolation. For these reasons, we undertook the isolation and viological characterization of these C-type virus particles.

Results
 The HT-1 strain, Which had been passaged 18 times in chick kidney cells, 17 times in duck kidney cells and 7 times in DEF after isolation from Japanese turkey, was kindly provided by Dr. Izawa (The Kitasato Institute). This material was further passaged 9 times in DEF by the author, coded as HT-1-(9) and used as the starting material for these studies. The HVT titer of the material was 10^6.0 TCID_50/ml.
 HT-1-(9) cell suspension was inoculated in DEF, CEF(C/O) and CEF(SPF) respectively for cultivation at 38°C. After 4 days of incubation, all of three types of cells showed giant cell formation and a strong CPE, while control cultures produced all perfect monolayers. The cells from each culture were harvested and observed by electron microscopy. All of the HT-1-(9) infected cell showed large numbers of HVT particles as well as some C-type particles. The size of the C-type particles was of the order of 80 nm. In contrast, no HVT or C-type particles were observed in either non-inoculated DEF or CEF(C/O). There were, however, a few C-type particles observed in CEF(SPF). The author's electron microscopic observations clearly demonstrated the coexistence of HVT and C-type particles in HT-1-(9) infected cells. Considering that ALSV generally cannot replicate in DEF, it appears that the C-type particles are unique and distinctly different from those of ALSV. In order to establish a biological assay for further studies on this virus, HT-1-(9) cell infected with C-type particles were inoculated into chicks to determine pathogenicity. Two-day-old chicks were infected intraperitoneally (ip) with O.2ml of 10-fold serial dilutions of HT-1-(9) infected cells. After 3 weeks, pathogenic effects were recognized at dilutions up to 10^-3, as evidenced by the pathological changes in the primary and secondary wings. When diluted HT-1-(9) infected cells were inoculated into 2 or 8-day-old chicks, there were no differences in the number of days until appearance of symptoms, and the number of birds affected. In order to determine the effect of inoculation site on the appearance of symptoms, HT-1-(9) infected cells were inoculated into 2 or 8-day-old chicks by the ip and subcutaneous (sc) routes respectively. Again, no differences due to the site of inoculation were observed. A characteristic wing aberration appeared among the chickens depending on the dilution: three weeks after inoculation, the barbs of the primary and secondary wings did not foliate normally but adhered tightly to the rachis. When observed after 3 to 5 weeks, the barbs on the top and bottom edges of the wing were foliating normally but in the middle, the barbs adhered tightly to the rachis. When these wings were observed as a whole, it appeared as if there were no barbs in the middle since they were adhering to the rachis. The author refers to this symptom with the Japanese expression ""nakanuke"" which means defoliation in the middle portion of wing. This type of aberration has not been reported elsewhere, being the author the first to describe it. Since our results show that HVT has no pathogenicity at all for chicken, an agent other than HVT, in particular the C-type particle observed by electron microscopy, is implicated in the induction of nakanuke.
 The nakanuke symptom in all cases appears symmetrically on the left and right wings of chicks. In the experiments, it was found that the observation was most favorably made at 3 to 4 weeks after inoculation and therefore all observation were made during this period.
 Since the earlier experiments suggested that the causative agent for nakanuke was already present in the starting material, i.e. HT-1-(9), the author investigated the possibility of passaging this agent in DEF or CEF infected with HT-1-(9). HVT titers and nakanuke effects were examined after the 1st passage, and it was found that the agent propagated in both DEF and CEF infected with HT-1-(9). Next, the author examined whether the agent is filtrable or not and how the titer of the co-infecting HVT affects the agent. Amplued suspensions of HT-1-(9) infected cells were thawed repidly in a water bath and pooled as starting material. This suspension had a HVT titer of 10^6.0 TCID_50/ml and produced nakanuke changes in chicks at 10^-3 dilution. The starting material was centrifuged and passed through a 0.45μ millipore filter. The HVT titer of the filtrate was relatively low, being 10^0.7 TCID_50/ml. However, upon inoculation into chicks, 14 out of 15 inoculated chicks showed nakanuke effects. These results indicate that the HVT content of the inoculation material is not directly related to the development of the nakanuke changes in inoculated chicks. Moreover, the agent is filtrable through a 0.45μ millipore filter.
 To isolate the agent from the HT-1-(9) infected cells, millipore filtrate mixed with an equal volume of anti-HVT serum was incubated at 37°C for 60 min. This step was performed in order to neutralize and exclude any HVT possibly remaining in the starting material. The incubated mixture was inoculated by ip injection into 2-day-old chicks. Within 22 days, 2 of 12 inoculated chicks showed nakanuke wing aberrations. This strongly indicates that the nakanuke-causing agent was present in the original inoculum. The above filtrate, neutralized with anti-HVT serum and inoculated into CEF (SPF) monolayers, showed no HVT-related CPE nor any other abnormal changes after 8 days of incubation. The cultured cells were then harvested and the cell suspension was inoculated onto freshly prepared CEF. On incubation, no abnormal changes were observable even after 2 passages. When these twice-passaged monolayers were harvested and similarly passaged three times on CEF, no observable CPE were produced. However, when the harvested cells were injected into 2-day-old chicks (ip), 5 of 16 injected chicks showed signs of nakanuke within 3 weeks after inoculation. These results indicate that the nakanuke-causing agent was still present in the cellular material from the third passage.
 It was possible to isolate the nakanuke-causing agent at this passage level. This isolation material was coded as nakanuke agent and was thereafter passaged in CEF(SPF) with the following results; (1) this agent replicated on CEF(SPF) without producing any CPE and was found intracellularly as well as in the culture medium; (2) electron microscopical observation of the infected CEF revealed numerous C-type particles (80 nm); (3) inoculation of the isolated agent into DEF resulted in replication with weak CPE, the observation of which served for the determination of titer; (4) the nakanuke symptom was transferred to healthy chicks by inoculation of the isolated agent as well as organ or feather follicle suspensions derived from affected chicks; (5) neutralizing antibody for this agent was present in nakanuke convalescent chicken serum.
 From the above, it was clear that the nakanuke-inducing agent isolated from HT-1-(9) consists of C-type particles which grow in CEF and DEF, induce nakanuke in HT-1-(9)-inoculated chicks, replicate in various chick organs and can be transferred from chick to chick.
 At present, reticuloendotheliosis virus group (REVs) are known to have C-type particles and to be able to replicate in both CEF and DEF. There has been no report, however, of the induction of nakanuke-like symptoms by these REVs. Nevertheless, the pathogenicity of REVs for chicks has been shown to range from extremely high to quite slight. Among the known viruses, the REVs are thought to be most closely related to the nakanuke agent isolated by the author. Therefore, the indirect fluorescent antibody (FA) test was performed using anti-agent chicken serum and anti-REV chicken serum. Both anti-REV and anti-agent sera gave positive reactions with both REV-infected and agent-infected cells. Only cells reacted with normal serum gave negative results. Staining by the FA assay gave the same results irrespective of the combination of anti-serum and virus, the cytoplasm staining heavily while the nucleus remained unstained. This indicates that the nakanuke agent and REV have common antigenic properties. However, in order to carry out more detailed experiments, it was necessary to use DEF for the following reasons. When CEF(SPF), CEF(C/O) and DEF cells were incubated for 4 days, perfect monolayers were produced. The cells from each culture were respectively harvested and observed by electron microscopy.  No C-type particles were observed in non-inoculated DEF or CEF(C/O). There were, however, a few C-type particles observed in CEF(SPF). Therefore, non-inoculated DEF, CEF(℅) and CEF(SPF) were further examined for the presence of spontaneous C-type particles after 3 subcultures. As a result, no C-type or other particles were observed in either DEF or CEF(C/O) but large numbers of C-type particles were found in CEF(SPF). The results indicate that in CEF(SPF), C-type particles which do not belong to subgroup A or B of ALSV are present as spontaneous contaminants.
 Because of this, and in order to localize the isolated agent virologically with further certainty, the author used DEF cells which are not spontaneously contaminated by C-type particles to isolate a C-type nakanuke-causing virus from the same material (HT-l-(9) infected cells) as in previous experiments but using DEF.
 The HT-1-(9) infected cells were freeze-thawed 3 times and the supernatant obtained after centrifugation (HVT titer:<10^1.0 TCID_50/ml) was used as the isolated nakanuke agent-containing material. An agent group was prepared by treating the isolated material with anti-HVT serum and a control group was prepared by treating the medium with anti-HVT serum. To ensure that the nakanuke agent did not spontaneously contaminate the anti-HVT serum, medium or DEF used for the control group, exactly the same passaging was performed as for the agent group. Equivalent amounts of anti-HVT serum were added to the undiluted isolated material and medium. These mixtures were incubated at 37°C for 60 min, inoculated in DEF and cultured at 37°C.
 The pathogenicity of the culture fluid passaged 9 times in chicks was investigated. The culture fluid (0.2ml of 10-fold serial dilutions) was inoculated sc into day-old chicks. After 21 days, the occurrence of nakanuke was investigated. The results indicate that nakanuke occurred in chicks receiving inoculated material diluted as much as 10^-4. In the control group inoculated with culture fluid, there was no nakanuke observed (0/10). While the agent group showed viral replication with CPE from the beginning, in the control group no abnormalities could be seen at any time. The content of agent virus in the DEF culture fluid was about 10^5.4 TCID_50/ml. The presence of HVT in nakanuke agent isolated in this way shown by means of the direct FA test using anti-Marek's disease virus chicken serum, and by electron mocroscopy. It was likely possible to isolate the nakanuke agent at this passage level.
 The cultured DEF cells of both agent and control groups were examined by electron microscopy. In the agent group, there were many C-type particles both inside and outside the cells with an 80 nm and an electron-dense core surrounded by spikes. In the control group, no HVT or C-type particles were found. Next, the C-type particles were concentrated from the culture fluid of the agent group for further purification and observation using a 10-30% sucrose gradient. The material was then observed by negative staining. The particles were found to have a diameter of 80-100 nm. The surfaces had many spikes with once spike in the center and six spikes arranged at constant intervals around it.
 From the conclusion that the nakanuke agent is a C-type particle, it would follow that the viral nucleic acid would be the RNA type. In order to determine the particle density and type, the following experiments were performed. The agent was cultured in DEF cells in the presence of 8μci/ml of ^3 H-uridine, and the culture fluid harvested after 7 days was analyzed by sucrose density gradient(10-60%) centrifugation. The results demonstrated that the C-type particle was an RNA virus with a density of 1.16g/cc.
 The isolated agent showed C-type particles similar to ALSV. However, it was proven that the agent is not contaminated with ALSV by experiment using the complement fixing antigen of Rous sarcoma virus (RSV). This made it clear that the nakanuke agent had been passaged as a single agent.
 In order to determine the presence of antigens common to REV-T strain and the nakanuke agent isolated from HT-1-(9) in CEF, the following experiments were carried out. Since the isolated agent showed C-type paricles, the correlation of nakanuke agent and REV antigenicity was investigated. The DEF inoculated with the agent group and REV both showed CPE while the CEF did negative. After 3 days, the coverslips were fixed and the presence of antigens was determined by means of indirect FA tests. There were cross-reactions for both the anti-agent serum and anti-REV serum on coverslips infected with the agent and REV. In addition, both FA test were positive. However, in the case of the RSV and control coverslips, there were no reactions with any of these sera and the results were negative. From this, it was evident that the isolated agent and REV have common antigenicity. Concomitantly, this also indirected that the viral material used was not contaminated with ALSV. Therefore, in vitro cross-neutralization test between nakanuke agent and REV-T strain were carreied out using duck serum against the isolated agent anti-REV duck serum, respectively. The virus-serum mixtures were inoculated into DEF cells, and inhibition of CPE and of virus antigen formation was determined by FA assay with the homologous and heterologous sera. The neutralization test demonstrated evident cross-reactivity.
 This led us to assume that the isolated agent is a virus with antigenicity very similar to that of the REV-T strain. In addition, neutralization tests were performed with REV-T strain and nakanuke agent isolated from HT-1-(9) in CEF demonstrated that the agent had been successfully isolated from both CEF and DEF.
 The REVs were first isolated in 1968 in U.S. from turkey with leukosis (T strain), 6 strain of this virus being isolated subsequently. These viruses do not have antigens in common with ALSV. They therefore from a single group. Among these viruses, the T and SNV (spleen necrosis virus) strains induce reticuloendotheliosis in chicks. The formation of tumors in the host is lethal. In contrast, the DIAV (duck infectious anemia virus), CSV (chick syncytial virus) as well as two other strains are weakly pathogenic. Nevertheless, none of these strains have been reported to induce the nakanuke symptoms described by the author.
 After its isolation, the REV-T strain used by the author was passaged in chick liver homogenate and proved highly pathogenic for chicks, the infected chicks developing enlarged livers and spleens and reticuloendotheliosis terminating in death within 2 weeks. The REV-T strain which originated from in vivo passaging was highly lethal for day-old chicks. The virus obtained by passaging this T-strain twice in DEF and containing 10^5.5 TCID_50/ml was used to make 10-fold serial dilutions which were then inoculated into day-old chicks. The inoculated chicks survived but when observed three weeks post inoculation, they showed wing changes identical with the nakanuke described by the author. The results indicated that nakanuke could be induced by inoculum dilutions up to 10^-4.
 The viral material passaged once or twice in DEF (10^5.4 TCID_50/ml) showed reduced lethality for chicks, death being delayed to 13-26 days. Among the chicks which died between 20-26 days, there were some which showed enlarged spleens and livers along with nekanuke symptoms. When liver tissue from these chicks was homogenized and passaged into healthy ones, the original lethality of the virus was readily restored.
 Therefore in vivo cross-neutralization tests were performed, the nati-agent and anti-REV sera being reacted with the isolated agent and the REV-T strain passaged twice in DEF. Investigations were carried out to determine if the nakanuke symptom was suppressed by these viruses.
 The results indicated both anti-agent end anti-REV sera suppressed the nakanuke symptoms caused by the isolated agent and REV and cross-neutralization was established. This proves that nakanuke is caused by the isolated agent and DEF-passaged REV-T strain.
 Nakanuke induced by inoculation of nakanuke agent or in vitro passaged REV-T strain in day-old chicks can be seen from 3 weeks until 5 weeks after inoculation. Between 5 and 6 weeks, the wings begin to show various aberrations depending on the severity of nakanuke. That is, there will be chicks which recover from nakanuke, chicks missing primary and secondary wing feathers or completely lacking body feathers which subsequently recover wing feather growth as well as chicks in which the nakanuke affected area ceases to develop resulting in anemia and eventually death. A comparison of the two viruses shows that REV-T strain is strongly lethal for chicks, 12 of the 25 chicks developing anemia followed by death. In contrast, the nakanuke agent caused death in 3 out 25 cases.
 Histopathological observation was carried out on the chicks inoculated with these viruses and affected severe atrophy of the thymus and Bursa of Fabricius (BF), some of them developed severe anemia dying. In the early stages of infection, there was enlargement of the spleen follicles, decreased numbers of lymphocytes from the thymus and BF, infiltration and multiplication of reticulum cells in the vicinity of the liver vascular system and interstitial tissue of the heart. At times, focal necrosis was observed in the liver. In these chicks, viral infection was generalized. However, these changes did not attain the level which occurred with tumorgenecity due to reticuloendotheliosis, nor were these any tumorgenic changes in chicks which died of severe anemia.

Discussion
 Current research on the REVs is drawing much attention because the REVs appear to be C-type viruses similar to ALSV and mammalian leukemia virus.
 In Japan, the author's research work was the first of this type to be reported. Subsequently, in 1974 several aberrations broke out among chicks inoculated with Marek's disease vaccine. It was found that the causative agent in that case induced the same king of symptoms as did the nakanuke agent isolated by the author and it was shown to belong to the REV group. Simultaneously, in vitro passaging of the REV-T strain was found to readily reduce its pathogenicity so as to produce nakanuke symptoms.
 It is not clear exactly at what point the nakanuke agent contaminated the HT-1-(9) strain employed by the author. Because, when two other strains isolated by Izawa et al and having different passage histories were used in similar experiments, they were found to be negative for nakanuke agent and contamination was ruled out. As a result, the agent is thought to have co-infected at some point during passage of the HT-1 strain virus via the cells employed for passaging, most probably DEF.
 REVs isolated in U.S. which produced clinical signs were derived from turkeys, ducks and chickens. In contrast, in Japan they have been isolated solely from HVT infected cells. Because of these reports, fertilized eggs chick embryos used for human as well as animal vaccines were required to be checked for the absence of REVs.
 The REVs, whose natural hosts are waterfowel, in some way infected domestic chickens, ducks and turkeys. However, the distribution of REVs in this country has yet to be clarified. The isolation of this virus by the author and from Mareck's disease vaccine are the only known reports of its occurrence in Japan. A furthermore detailed study of the mode and extent of infection by this virus is deemed necessary in future., 目的
　著者は七面鳥ヘルペスウイルス(herpesvirus of turkey, HVT)の発育過程について電子顕微鏡観察を行っていた際、HT-1株(HVT)感染アヒル胎児細胞(DEF)及びニワトリ胎児細胞(CEF)内においてHVT粒子と共にC-型粒子の存在することを発見した。この粒子は形態学的には80nmのC-型粒子として観察されることからoncorna virus groupに属するavian leukosis-sarcoma group(ALSV)のウイルスと類似しているが、対照細胞では観察されないことから接種材料に起因すると考えた。しかしDEFではALSVの増殖が不可能とされていることから推測すると、この粒子はALSV以外のC-型を示すウイルスであろうことが示唆される。
　現在までHVT感染においてこのようなC-型粒子が観察されたという報告は皆無である。
　一方、HVTはマレック病生ワクチンとして全世界でその防禦効果が認められDEF或はCEFでウイルスを増殖させた生ワクチンとして使用されており、著者の用いたHT-1株も分離以来、ニワトリ及びアヒル細胞で継代維持されて来たことを考えると、観察されたC-型粒子によるワクチンの汚染の可能性も考えられる。
　以上の理由から著者は今回観察されたC-型粒子のニワトリに対する病原性と、その粒子のウイルス学的位置付けを目的として実験を行った。

実験成績
1)　"nakanuke" agentの分離
　北里研究所、附属家畜衛生研究所、伊沢等によって我が国で最初に七面鳥から分離されたHVT(HT-1株)の内、ニワトリ腎臓、アヒル腎臓及びDEFで各々、18、17、7代通過したウイルスを入手後著者によって更にDEFで9代通過した材料をHT-1-(9)と付号して実験の出発材料とした。
　HT-1-(9)を感染させたDEF及びCEFにはHVT粒子の他にC-型粒子が電子顕微鏡によって観察された。そこでC-型粒子を含むと考えられるHT-1-(9)を幼若ヒナに接種し病原性を調べた。接種ヒナは3週後、正羽翼、副羽翼の中間層に特異な変化を示した。即ち、羽の中間部分の羽弁が正常に開かず、羽軸に密着するものである。このような症状は未報告であり著者らによって初めて観察され羽翼の"中抜け"("nakanuke")と呼ばれた。
　次にHT-1-(9)をDEF及びCEFで3継代しても同様な"nakanuke"を引き起すこと、HT-1-(9)から調製した0.45μ濾過材料及びその濾過材料に抗HVT血清を加えHVTを中和、不活化した材料の接種によっても"nakanuke"が引き起される事実から、出発材料中に"nakanuke"を誘発する因子が存在し、その因子はHVT以外の自己増殖力を持つ濾過性微生物であることが証明され、この因子を"nakanuke" agentと命名した。
　"nakanuke" agentはHT-1-(9)濾過材料を抗HVT血清処理し、初めにCEFを用いて分離し、二、三の実験を行った。しかしこれらの実験に使用したCEF自体がALSVのsubgrouo. A. B以外のC-型ウイルスによって垂直感染を受け汚染されていることを知った。この垂直感染したC-型粒子はヒナに"nakanuke"を誘発しないが、"nakanuke" agentと形態学的に類似しているため以後の実験が不明確になることが想像された。そこで、C-型粒子の垂直感染のないDEFを用いて再度出発材料からCEFで用いたと同様の方法で"nakanuke" agentの単離を行った。単離した"nakanuke" agentはDEF及びCEFで増殖し培養液中にagentを放出するが、前者のみに細胞変性(CPE)を示し、感染価の測定が可能であった。agentは感染細胞内で大きさ80nmのC-型粒子として観察され、HT-1-(9)で認めたものと類似していた。
2)　Agentの形状並びに生化学的性状
　"nakanuke" agentが養液中に放出されることを利用し、^3H-uridineの存在下で培養後、培養液からagentを濃縮し、sucrose gradientで遠心分画し測定した結果、粒子は比重1.16g/CCでアイソトープ活性の測定から粒子の核酸はRNA型であった。同様に培養液から濃縮精製した粒子をネガティブ染色により観察すると大きさ80～100nmで表面に多数のspikeを有するC-型粒子であった。
3)　Agentの病原性
　単離"nakanuke" agentはHT-1-(9)と同一の"nakanuke"をヒナに引き起した。これらヒナを長期観察すると、接種3週頃より正羽翼、副羽翼全体、或は一部に"nakanuke"を示し、それらは5～8週に至り"nakanuke"部分が羽の上端に移行し、ついには正常な羽で置換する例と、強い"nakanuke"により正羽及び全身の羽毛が脱落しそれらが後に正常の羽又は羽毛で置換し正常の発育をとげるもの、或は正羽全体に及ぶ"nakanuke"を示し、ヒナの発育は著しく抑制され、正常の羽に置換することなく、強い貧血を示し斃死する例、等が観察される。"nakanuke" agent接種ヒナの血液や多くの臓器からagentが再分離され、ヒナからヒナへの伝達が可能であることから、接種ヒナの体内におけるagentの増殖による衝撃の強さにより種々な"nakanuke"が観察されるものと考えられる。
4)　Agentのウイルス学的同定
　単離"nakanuke" agentがCEF及びDEFで増殖するC-型粒子であることから既知ウイルスでは七面鳥白血病ウイルス(reticuloendotheliosis virus group, REVs)が最も近縁と考へ、REVsの代表株であるT株を用いて抗原的関係を調べた。
　"nakanuke" agent、REV-T株及びALSVの代表株であるRous sarcoma virus(RSV)をDEF及びCEFに接種し、抗"nakanuke" agent及び抗REV-T株血清による間接螢光抗体法を行った結果、"nakanuke" agentとREV-T株間で交叉反応が認められた。又、抗"nakanuke" agent、抗REV-T株血清と両ウイルス間で交叉中和反応を行い、DEFに接種後、両ウイルスの抗原合成を螢光抗体法で測定した結果、完全に交叉中和が成立し、分離"nakanuke" agentがREVsに属することが同定された。この結果は農林省動物医薬品検査所、分与の抗REV-T株標準血清によっても確認された。
5)　REV-T株との病原性の比較
　REV-T株はニワトリヒナ、アヒルヒナ等に対しreticuloendotheliosisを引き起し接種後2週以内に宿主を高率に腫瘍死させることが知られている。著者はこのT株を入手後DEFで1～2代通過することによりその造腫瘍性の減弱と消失を確認するとともに、接種ヒナが著者らの観察した"nakanuke"を発症することを初めて発見した。この"nakanuke"は分離"nakanuke" agentによるものと同様の症状を示しその経過も類似していたが、T株はやゝ強い病原性を示した。
　次にDEF通過REV-T株が"nakanuke"を発症することを利用し、この"nakanuke"が抗"nakanuke" agent及び抗REV-T株血清によって阻止されるか否かを実験した。その結果、抗"nakanuke" agent血清は"nakanuke" agentとREV-T株による各々の"nakanuke"を阻止した。逆にREV-T株血清はREV-T株と"nakanuke" agentによる"nakanuke"を阻止し、交叉中和が成立した。それに対し、抗RSV血清、正常血清では両ウイルスによる"nakanuke"の発症は全く阻止出来なかった。
　ニワトリヒナ継代REV-T株で腫瘍死したヒナでは病理学的に多数のreticuloendothelial systemから端を発した細胞が観察されるが、それに対しDEF通過T株及び"nakanuke" agent接種ヒナではその細胞の出現は少く、腫瘍性の変化に至らないし、腫瘍死することもない。これらの接種ヒナが死亡する例では全て強い発育抑制と貧血を伴い斃死する。

考察
　REVsは米国において臨床症状を伴った七面鳥、アヒル、ニワトリから6株が分離され、これらの株は病原性の強いものから弱いものまで分布することが報告されている。しかし、これらの報告においては著者らの観察した"nakanuke"については未報告である。一方、我が国におけるREVsの研究は皆無であり、著者らの報告が最初となった。我が国では1974年春から秋にマレック病生ワクチン(HVTをDEF或はCEFで増殖させた感染細胞ワクチン)の接種事故が発生したが、この事故鶏は著者らの観察したと同様の"nakanuke"を示し、その後の研究でも事故例ワクチンから著者らと同様のREVsが分離されるに至った。同時にREV-T株をDEF及びCEFで通過することにより"nakanuke"を引き起すことが追試確認された。上記の如く我が国におけるREVsの分離は著者を初めとして全てHVT感染細胞から分離された。そして著者らの系を含めて分離ウイルスがどの時点で迷入して来たかについては全く不明である。米国におけるREVsは野性の水鳥を本来の宿主とし、それが何んらかの方法で、七面鳥、アヒル、ニワトリ等に伝染すると考えられていることから推測すると、著者らの使用したHT-1株の1亜系がたまたまREVsによって汚染されていたと考えられる。事実HT-1株の他の2つの亜系はREVsの迷入が著者らによって否定されている。これらのことからHT-1株の継代に使用した細胞、特にDEFからの迷入が考えられる。
　我が国におけるREVsの分布については全く不明の状態であるが、今後この種のウィルスによる野外での汚染が考えられ、鳥類の発育卵を用いる実験及びワクチンについての重要な問題となるであろう。我が国ではこれらの理由から発育卵を用いる動物用、人体用ワクチンは全てREVsの否定を行うことが実施されるに至った。

結論
　HT-1株(HVT)の1亜系に混在していたC-型ウイルスを初めて分離した。このウイルスは大きさ80～100nmのC-型を示すRNAウイルスで比重1.16g/CC、血清学的にはREV群に属するものであった。又、このウイルスはニワトリヒナに対し羽翼の特異な病変"中抜け"("nakanuke")を起した。更にREV-T株でもDEF細胞を通過することよりその造腫瘍性の減弱を来たし"nakanuke"を発症することを発見した。},
 title = {ニワトリヒナに特異な病変を起すC-型ウイルスに関する研究 : 特に七面鳥ヘルペスウイルス株に混在したウイルスの分離},
 year = {}
}