The genome of turkey herpesvirus

Here we present the first complete genomic sequence of Marek's disease virus serotype 3 (MDV3), also known as turkey herpesvirus (HVT). The 159,160-bp genome encodes an estimated 99 putative proteins and resembles alphaherpesviruses in genomic organization and gene content. HVT is very similar to MDV1 and MDV2 within the unique long (UL) and unique short (US) genomic regions, where homologous genes share a high degree of colinearity and their proteins share a high level of amino acid identity. Within the UL region, HVT contains 57 genes with homologues found in herpes simplex virus type 1 (HSV-1), six genes with homologues found only in MDV, and two genes (HVT068 and HVT070 genes) which are unique to HVT. The HVT US region is 2.2 kb shorter than that of MDV1 (Md5 strain) due to the absence of an MDV093 (SORF4) homologue and to differences at the UL/short repeat (RS) boundary. HVT lacks a homologue of MDV087, a protein encoded at the UL/RS boundary of MDV1 (Md5), and it contains two homologues of MDV096 (glycoprotein E) in the RS. HVT RS are 1,039 bp longer than those in MDV1, and with the exception of an ICP4 gene homologue, the gene content is different from that of MDV1. Six unique genes, including a homologue of the antiapoptotic gene Bcl-2, are found in the RS. This is the first reported Bcl-2 homologue in an alphaherpesvirus. HVT long repeats (RL) are 7,407 bp shorter than those in MDV1 and do not contain homologues of MDV1 genes with functions involving virulence, oncogenicity, and immune evasion. HVT lacks homologues of MDV1 oncoprotein MEQ, CxC chemokine, oncogenicity-associated phosphoprotein pp24, and conserved domains of phosphoprotein pp38. These significant genomic differences in and adjacent to RS and RL regions likely account for the differences in host range, virulence, and oncogenicity between nonpathogenic HVT and highly pathogenic MDV1.     

Marek's disease virus (MDV) ICP4, pp38, and meq genes are involved in the maintenance of transformation of MDCC-MSB1 MDV-transformed lymphoblastoid cells.

An antisense strategy has been used to identify genes important for the maintenance of transformation of MDCC-MSB1 (MSB1) Marek's disease virus-transformed lymphoblastoid cells. Oligodeoxynucleotides antisense to the predicted translation initiation regions of ICP4 and pp38 mRNAs inhibited proliferation of MSB1 cells but not MDCC-CU91 (CU91) reticuloendotheliosis virus-transformed cells. Control oligodeoxynucleotides having the same base composition but a different sequence did not inhibit MSB1 cell proliferation. In addition, ICP4 and pp38 antisense oligodeoxynucleotides resulted in 77- and 100-fold reductions in colony formation by MSB1 cells in soft agar, respectively. To extend and corroborate these results, a novel system based on efficiently regulated expression of eukaryotic genes by a chimeric mammalian transactivator, LAP267 (S. B. Baim, M. A. Labow, A. J. Levine, and T. Shenk, Proc. Natl. Acad. Sci. USA 88:5072-5076, 1991), was used. MSB1-derived stably transfected cell lines in which RNA antisense to Marek's disease virus ICP4, pp38, or meq could be induced by treatment of the cells with isopropyl-beta-D-thiogalactopyranoside (IPTG) were constructed. Control cell lines in which expression of ICP4 sense or pUC19 sequences could be induced by IPTG were also constructed. Induction of the cell lines indicated that ICP4 antisense RNA, but not ICP4 sense RNA or pUC19 RNA, inhibited proliferation of MSB1 cells. Induction of ICP4, meq, or pp38 antisense RNAs, but not ICP4 sense or pUC19 RNAs, had a dramatic effect on relative colony formation by MSB1 cells in soft agar. These results indicate that ICP4, pp38, and Meq are all involved in the maintenance of transformation of MSB1 cells.     

Homologies between herpesvirus of turkey and Marek's disease virus type-1 DNAs within two co-linearly arranged open reading frames, one encoding glycoprotein A

The genomes of two avian herpesviruses, Marek's disease virus type 1 (MDV1) and herpesvirus of turkey (HVT), share close homology only within certain DNA regions. One such homologous region of HVT DNA was cloned and sequenced. Two open reading frames (ORFs) were found in the long unique region, ORF1 encoding the glycoprotein A (gA), and ORF2 encoding a still unidentified protein. These two HVT-ORFs are located at almost the same positions as the homologous MDV1-ORFs. The nucleotide sequence homologies between HVT and MDV1 were 73% and 68% for ORF1 and ORF2, respectively. Both the 5'- and 3'-noncoding regions, however, are less conserved. The third letter within every codon of ORF1 and ORF2 showed a mismatch of greater than 50% between the two viruses. The amino acid (aa) sequence homologies between the corresponding putative viral proteins are 83% and 80% for ORF1 (gA) and ORF2, respectively. More than 90% homology was observed in the C-terminal region of ORF1 (gA). Furthermore, the deduced aa sequences for both of the ORFs in these two viruses showed considerable homology to two adjoining genes in herpes simplex virus type 1, the glycoprotein C and UL45 genes.     

Roles of avian herpesvirus microRNAs in infection, latency, and oncogenesis

MicroRNAs have been reported for the avian herpesviruses Marek's disease virus 1 (MDV1; oncogenic), Marek's disease virus 2 (MDV2; non-oncogenic), herpesvirus of turkeys (HVT), and infectious laryngotracheitis virus (ILTV). No obvious phylogenetic relationships exist among the avian herpesvirus microRNAs, but the general genomic locations of microRNA clusters are conserved, with microRNAs being located in the repeat regions of the genomes. In some cases, microRNAs are antisense to open reading frames. Among MDV1 field isolates with different virulence properties, microRNAs are highly conserved, and variations that have been observed lie in putative promoter regions. One cluster of MDV1 microRNAs lies upstream of the meq gene, and this cluster is more highly expressed in tumors caused by an extremely virulent MDV1 isolate compared to tumors caused by a less virulent isolate. Several of the avian herpesvirus microRNAs are orthologs of microRNAs in other species. For example, mdv1-miR-M4 shares a seed sequence with gga-miR-155 (also shared with Kaposi sarcoma herpesvirus (KSHV) kshv-miR-K12), mdv2-miR-M21 shares a seed with miR-29b, and hvt-miR-H14 shares a seed sequence with miR-221. Functional analyses of avian herpesvirus microRNAs include a variety of in vitro assays to demonstrate potential function as well as the use of mutants that can exploit the ability to assess phenotypes experimentally in the natural host. This article is part of a Special Issue entitled:MicroRNA's in viral gene regulation.     

Persistence of genomes of both herpesvirus of turkeys and Marek's disease virus in a chicken T-lymphoblastoid cell line

A cell line tentatively designated as MDCC-BO1(T), was established from spleen cells of an apparently healthy chicken inoculated with herpesvirus of turkey (HVT). BO1(T) cells were T lymphoblastoid cells and the more than 95% of them had Marek's disease (MD) tumor-associated surface antigen (MATSA). However, no viral internal antigens or membrane antigens could be demonstrated in them by immunofluorescence tests using chicken anti-HVT and -MD virus (MDV) sera. The virus could be rescued from BO1(T) cells by co-cultivation with chick embryo fibroblasts (CEF). The DNA of the rescued virus was characterized as HVT DNA by its sedimentation profile in a neutral glycerol gradient and its endonuclease Hind III cleavage-pattern. Ultrastructural studies on CEF infected with the rescued virus revealed the presence of HVT-like virions. However, DNA-DNA reassociation kinetics showed that the BO1(T) cells contained a few copies of NVT and also MDV genomes.     

The genome of a very virulent Marek's disease virus

Here we present the first complete genomic sequence, with analysis, of a very virulent strain of Marek's disease virus serotype 1 (MDV1), Md5. The genome is 177,874 bp and is predicted to encode 103 proteins. MDV1 is colinear with the prototypic alphaherpesvirus herpes simplex virus type 1 (HSV-1) within the unique long (UL) region, and it is most similar at the amino acid level to MDV2, herpesvirus of turkeys (HVT), and nonavian herpesviruses equine herpesviruses 1 and 4. MDV1 encodes 55 HSV-1 UL homologues together with 6 additional UL proteins that are absent in nonavian herpesviruses. The unique short (US) region is colinear with and has greater than 99% nucleotide identity to that of MDV1 strain GA; however, an extra nucleotide sequence at the Md5 US/short terminal repeat boundary results in a shorter US region and the presence of a second gene (encoding MDV097) similar to the SORF2 gene. MD5, like HVT, encodes an ICP4 homologue that contains a 900-amino-acid amino-terminal extension not found in other herpesviruses. Putative virulence and host range gene products include the oncoprotein MEQ, oncogenicity-associated phosphoproteins pp38 and pp24, a lipase homologue, a CxC chemokine, and unique proteins of unknown function MDV087 and MDV097 (SORF2 homologues) and MDV093 (SORF4). Consistent with its virulent phenotype, Md5 contains only two copies of the 132-bp repeat which has previously been associated with viral attenuation and loss of oncogenicity.     

Identification of a unique Marek's disease virus gene which encodes a 38-kilodalton phosphoprotein and is expressed in both lytically infected cells and latently infected lymphoblastoid tumor cells.

The identification of unique Marek's disease (MD) virus (MDV) antigens expressed not only in lytically infected cells but also in latently infected MD lymphoblastoid tumor cell lines is important in understanding the molecular mechanisms of latency and transformation by MDV, an oncogenic lymphotropic herpesvirus of chickens. Through cDNA and nucleotide sequence analysis, an open reading frame (designated the pp38 ORF) which encodes a predicted polypeptide of 290 amino acids was identified in BamHI-H. Demonstration that the pp38 ORF spans the junction of the MDV long unique and long internal repeat regions (MDV has an alphaherpesvirus genome structure) precludes the presence of the gene encoding the B-antigen complex (gp100, gp60, and gp49) in the same region of BamHI-H, where it was originally thought to exist. Duplication of the complete pp38 ORF was not observed in BamHI-D, but part of it (encoding 45 amino acids) was found in the long terminal repeat region of the fragment. By use of trpE-pp38 fusion proteins, antisera against pp38 were prepared. By immunoprecipitation and sodium dodecyl sulfate-polyacrylamide gel electrophoresis, a predominant virus-specific 38,000-dalton polypeptide (designated pp38) and a minor 24,000-dalton polypeptide (designated p24) were found. No precursor-product relationship was found between pp38 and p24 by pulse-chase analysis, and only pp38 was detected by Western blot (immunoblot) analysis with antiserum to pp38. pp38 was found to be phosphorylated and present in oncogenic serotype 1-but not nononcogenic serotype 3-infected cells. Expression of the gene encoding pp38 was relatively insensitive to phosphonoacetic acid inhibition, suggesting that pp38 may belong to one of the early classes of herpesvirus proteins. pp38 was also detected in the latently infected MSB-1 lymphoblastoid tumor cell line. The detection of antibody against pp38 in immune chicken sera indicates that pp38 is an immunogen in birds with MD. Most of the properties described here for a protein detected by methods based on finding the ORF first are identical to those of a 38-kDa phosphoprotein reported by others, suggesting that they are the same. Collectively, the data reported here provide (i) more definitive information on the complete ORF of another MDV gene and the protein that it encodes, (ii) clarification of the gene content within a specific region of the MDV genome, and (iii) the molecular means to conduct further studies to determine whether pp38 plays a role in MDV latency and transformation.     

The Leeuwenhoek Lecture, 1997. Marek's disease herpesvirus: oncogenesis and prevention.

There are a number of neoplasias for which a herpesvirus is an essential part of the aetiology. Of these, Marek''s disease is the most common and provides excellent opportunities for the study of a herpesvirus-induced tumour both experimentally and under natural conditions in the field. Marek''s disease is caused by an alpha herpesvirus; it differs from the other oncogenic herpesviruses which are gamma herpesviruses. It is a ubiquitous virus in poultry populations of the world and is highly cell-associated and contagious, yet only a proportion of infected fowl develop tumours. Evidence is presented to suggest that at least one of the reasons for a wide variation in the incidence of the disease is a temporal interplay between virulent viruses and viruses of low or no virulence. The viral genes associated with the oncogenicity of Marek''s disease virus (MDV) are discussed and it is concluded that it is likely that several genes are involved. Finally, a brief history of vaccination to control Marek''s disease is given and mode of action discussed. It is concluded that the mechanism of protection is mainly through an antiviral cell mediated immune response, resulting in a lowered challenge virus burden. Marek''s disease viruses over the past 40 years have been evolving greater oncogenicity, some of which are not adequately controlled by the vaccines that are currently available. It is suggested that for MDV to produce tumours, there is a need for the cytolytic infection phase and that infection must be with an MDV which possesses a functional gC, ICP4 for maintaining latency which allows the expression of at least the 1.8 kb family, pp38, meq, and possibly pp14 genes, for maintaining the tumour state and possibly initiating this state. Intervention in this process reduces the chance of tumour formation and incidence in a population which can occur through natural or man-mediated infection with non-pathogenic MDVs.     

Processing of glycoprotein gB related to neutralization of Marek's disease virus and herpesvirus of turkeys

The glycoprotein gB related to neutralization of Marek's disease virus (MDV) and herpesvirus of turkeys (HVT) is composed of several glycosylated polypeptides, which were immunoprecipitated with monoclonal antibodies and rabbit antiserum cross-reactive to MDV-gB and HVT-gB, and analyzed by SDS-polyacrylamide gel electrophoresis. The present pulse-chase experiments showed that the precursor forms of MDV- and HVT-gB were glycoproteins with molecular weights of 110K to 115K (gp115/110) and 115K (gp115), respectively. These precursor forms were processed to smaller gB's (gp63 and gp50 for MDV; gp62, gp52, and gp48 for HVT), at least in part by sialylation. The proteins synthesized in the presence of tunicamycin were two polypeptides of 88K and 83K in MDV-infected cells and a 90K polypeptide in HVT-infected cells, indicating the presence of unglycosylated precursor forms of MDV- and HVT-gB. Differences between virulent and avirulent MDV's and between HVT's with and without protective activity against Marek's disease were observed in the processed forms of MDV- and HVT-gB, especially at the processing step of sialylation.     

Evaluation of DNA homology of Marek's disease virus, herpesvirus of turkeys and Epstein-Barr virus under varied stringent hybridization conditions

Marek's disease virus (MDV) showed only 0.3-0.6% homology in DNA sequence with herpesvirus of turkeys (HVT) at Tm-24.4 degrees C in spite of its antigenic similarity to the latter. Southern blot hybridization under stringent conditions showed that the homology between MDV and HVT is located in the restricted portion of these viral genomes. At Tm-49.6 degrees C, which permits the detection of homology with one base mismatch in three between the MDV and HVT DNAs, sequences with weak homology were found to be distributed over most of these viral genomes. No homology was detected between Epstein-Barr virus and either MDV or HVT DNA.     

Monoclonal antibodies with specificity for three different serotypes of Marek's disease viruses in chickens

A total of 50 antibody-secreting hybridoma cells against Marek's disease virus (MDV) and turkey herpesvirus (HVT) have been produced. Eleven hybridomas were used for serotyping a panel of 15 pathogenic and nonpathogenic strains of MDV and HVT, representing three serotypes. The antibodies from the culture medium have fluorescence antibody (FA) titers of up to 100 and those from mouse ascitic fluid have titers ranging from 10(4) to 10(6). Monoclonal antibody T81 is type-common, i.e., it reacts at equal titer with all MDV and HVT tested. Of the remaining 10 antibodies, eight react only with pathogenic and attenuated strains of MDV (presumably serotype 1), one reacts only with nonpathogenic MDV (presumably) serotype 2), and one reacts only with strains of HVT (presumably serotype 3). Two hybridomas belong to IgG2a and IgG2b subclasses, respectively, and the remaining nine belong to IgG1 subclass. None of the antibodies specific for MDV strains reacted with homologous viruses in serum neutralization (SN), agar gel precipitin (AGP), or membrane immunofluorescence tests. Antibody L78, which is specific for HVT, was reactive with its homologous virus in the SN test; antibody from the culture medium showed an SN titer of 10 and that from mouse ascites a titer of 10,000. None of the antibodies specific for MDV or HVT reacted with other avian or mammalian herpesviruses, avian leukosis viruses (ALV), reticuloendotheliosis viruses (REV), or Marek's disease tumor-associated surface antigen (MATSA) expressed in a lymphoblastoid cell line, MDCC-MSB-1.     

表达强毒pp38决定簇的马立克病病毒疫苗毒CVI988点突变株的构建和特性

用鸡马立克病病毒（ＭＤＶ）强毒ＧＡ株的３８ｋＤ磷蛋白（ｐｐ３８）基因克隆ＤＮＡ转染Ｉ型弱毒疫苗ＣＡＩ９８８／Ｒｉｓｐｅｎｓ株ＭＤＶ感染的鸡胚成纤维细胞,再用能识别Ｉ型强毒ｐｐ３８的单克隆抗体Ｈ１９做免疫荧光试验,筛选到能在ｐｐ３８基因上表达强毒株特异性抗原决定簇的定向点突变弱毒株ＣＶＩ／ｒｐｐ３８。用＾３５Ｓ－蛋氨酸标记的细胞裂解物做免疫沉淀反应表明,单抗Ｈ１９不能识别天然ＣＶＩ９８８株ＭＤＶ中的     

Inhibitory effects of nitric oxide and gamma interferon on in vitro and in vivo replication of Marek's disease virus

The replication of Marek's disease herpesvirus (MDV) and herpesvirus of turkeys (HVT) in chicken embryo fibroblast (CEF) cultures was inhibited by the addition of S-nitroso-N-acetylpenicillamine, a nitric oxide (NO)-generating compound, in a dose-dependent manner. Treatment of CEF culture, prepared from 11-day-old embryos, with recombinant chicken gamma interferon (rChIFN-gamma) and lipopolysaccharide (LPS) resulted in production of NO which was suppressed by the addition of N(G)-monomethyl L-arginine (NMMA), an inhibitor of inducible NO synthase (iNOS). Incubation of CEF cultures for 72 h prior to treatment with rChIFN-gamma plus LPS was required for optimal NO production. Significant differences in NO production were observed in CEF derived from MDV-resistant N2a (major histocompatibility complex [MHC], B(21)B(21)) and MDV-susceptible S(13) (MHC, B(13)B(13)) and P2a (MHC, B(19)B(19)) chickens. N2a-derived CEF produced NO earlier and at higher levels than CEF from the other two lines. The lowest production of NO was detected in P2a-derived CEF. NO production in chicken splenocyte cultures followed a similar pattern, with the highest levels of NO produced in cultures from N2a chickens and the lowest levels produced in cultures from P2a chickens. Replication of MDV and HVT was significantly inhibited in CEF cultures treated with rChIFN-gamma plus LPS and producing NO. The addition of NMMA to CEF treated with rChIFN-gamma plus LPS reduced the inhibition. MDV infection of chickens treated with S-methylisothiourea, an inhibitor of iNOS, resulted in increased virus load compared to nontreated chickens. These results suggest that NO may play an important role in control of MDV replication in vivo.     

马立克氏病病毒pp38基因上游的一个双向启动子研究

马立克氏病病毒（MDV）pp38基因上游是病毒基因组DNA复制原点。在其两侧均含有启动子TATAbox、CAATbox等特征性的保守基元,推测是一个天然的双向启动子。为了在体外验证其双向启动活性,本研究以MDVpp38为报告基因,并将其ORF插入到pUC18中,构建了pUCpp38质粒。将包含该启动子完整区域的789bp序列分别以正反两个方向克隆进pUCpp38质粒中pp38报告基因的上游,获得的重组质粒pProfpp38和pProrpp38。将所获得的重组质粒分别转染鸡胚成纤维细胞（CEF）,通过间接免疫荧光试验检测pp38基因的表达以验证该启动子的双向启动活性。结果表明,马立克氏病病毒复制原点区的启动子无论以何种方向插入pUCpp38质粒中,在转染细胞24h内能检测到pp38基因的表达,48h后能获得高效和持续的表达。逐渐缩小该启动子的范围,最终在320bp时,仍能检测到两个方向较强的启动活性。     

马立克氏病毒单克隆抗体的研究

获得了4株分泌马立克氏病毒(MDV)特异性单克隆抗体(McAb)的杂交瘤细胞:4BS10对MDV所有毒株呈阳性反应;4CN8 对MDV血清1,3型毒株发生反应;2BN90和4CN24只对MDV血清1型毒株有阳性反应。3个McAb属IgG1,1个为IgG2b,均不中和MDV,免疫扩散试验也无沉淀线。对禽白血病毒(ALV)无交叉反应。 以2BN90和辣根过氧化物酶、异硫氰酸荧光素的结合物进行直接酶联免疫吸附试验和直接荧光抗体试验,均获得成功。抗体滴度前者为1/51,200,后者为1/640。对ALV无交叉反应。     

利用细菌人工染色体技术构建整合F基因的重组MDV疫苗株*

目的:预防马立克氏病病毒(MDV)和新城疫病毒(NDV)混合感染鸡引起的疾病,构建表达NDV F蛋白的MDV疫苗株CVI988 BAC重组载体,并包装成重组病毒,为疫苗免疫提供更多的重组疫苗选择。方法:首先利用PCR扩增带有卡那霉素(Kanamycin,Kana)抗性基因片段的F基因,采用同源重组的方法将其整合到CVI988 BAC上,进一步诱导I-SceI表达敲除Kana基因而获得重组质粒CVI988 BAC-F。通过磷酸钙法转染鸡胚成纤维细胞获得重组病毒。结果:Western blot和间接免疫荧光实验证实重组病毒能够表达F蛋白。病毒生长曲线和蚀斑大小测定结果表明,F基因的插入不影响病毒的体外增殖。结论:利用BAC技术成功构建了整合F基因的重组MDV病毒CVI988 BAC-F,为MDV重组疫苗研发,防控NDV与MDV共感染奠定了基础。     

Identification of the neoplastically transformed cells in Marek's disease herpesvirus-induced lymphomas: recognition by the monoclonal antibody AV37

Understanding the interactions between herpesviruses and their host cells and also the interactions between neoplastically transformed cells and the host immune system is fundamental to understanding the mechanisms of herpesvirus oncology. However, this has been difficult as no animal models of herpesvirus-induced oncogenesis in the natural host exist in which neoplastically transformed cells are also definitively identified and may be studied in vivo. Marek's disease (MD) herpesvirus (MDV) of poultry, although a recognized natural oncogenic virus causing T-cell lymphomas, is no exception. In this work, we identify for the first time the neoplastically transformed cells in MD as the CD4(+) major histocompatibility complex (MHC) class I(hi), MHC class II(hi), interleukin-2 receptor alpha-chain-positive, CD28(lo/-), phosphoprotein 38-negative (pp38(-)), glycoprotein B-negative (gB(-)), alphabeta T-cell-receptor-positive (TCR(+)) cells which uniquely overexpress a novel host-encoded extracellular antigen that is also expressed by MDV-transformed cell lines and recognized by the monoclonal antibody (MAb) AV37. Normal uninfected leukocytes and MD lymphoma cells were isolated directly ex vivo and examined by flow cytometry with MAb recognizing AV37, known leukocyte antigens, and MDV antigens pp38 and gB. CD28 mRNA was examined by PCR. Cell cycle distribution and in vitro survival were compared for each lymphoma cell population. We demonstrate for the first time that the antigen recognized by AV37 is expressed at very low levels by small minorities of uninfected leukocytes, whereas particular MD lymphoma cells uniquely express extremely high levels of the AV37 antigen; the AV37(hi) MD lymphoma cells fulfill the accepted criteria for neoplastic transformation in vivo (protection from cell death despite hyperproliferation, presence in all MD lymphomas, and not supportive of MDV production); the lymphoma environment is essential for AV37(+) MD lymphoma cell survival; pp38 is an antigen expressed during MDV-productive infection and is not expressed by neoplastically transformed cells in vivo; AV37(+) MD lymphoma cells have the putative immune evasion mechanism of CD28 down-regulation; AV37(hi) peripheral blood leukocytes appear early after MDV infection in both MD-resistant and -susceptible chickens; and analysis of TCR variable beta chain gene family expression suggests that MD lymphomas have polyclonal origins. Identification of the neoplastically transformed cells in MD facilitates a detailed understanding of MD pathogenesis and also improves the utility of MD as a general model for herpesvirus oncology.     

火鸡疱疹病毒在鸡胚成纤维细胞上有效代次的测定及其免疫原性恢复的研究

本试验证明,火鸡疱疹病毒(HVT)FC126株在鸡胚成纤维细胞(CEF)传55代的病毒,仍有预防马立克氏病(MD)的效力,至第70代效力明显下降。若将HVT CEF第35代病毒复归火鸡连续传4代,能明显恢复其免疫原性,用C细胞传代则有效代次可达到75代。因而,此方法能有效地解决免疫原性下降的种毒的更新问题。本试验还为该种毒和疫苗的有效代次的使用范围提供了可靠依据。