Role of feline ANP32 proteins in regulating polymerase activity of influenza A virus

Recently,increasing natural infection cases and experimental animal challenge studies demonstrated domestic cats are susceptible to multiple subtypes influenza A virus(IAV)infections.Notably,some subtype IAV strains could circulate in domestic cats after cross-species transmission and even infected humans,posing a threat to public health.Host factors related to viral polymerase activity could determine host range of IAV and acidic nuclear phosphoprotein 32(ANP32)is the most important one among them.However,role of cat-derived ANP32 on viral polymerase activity and host range of IAV is still unknown.In the present study,a total of 10 feline ANP32(feANP32)splice variants(including 5 feANP32A,3 feANP32B,and 2 feANP32E)were obtained from domestic cats by RT-PCR.Sequence alignment results demonstrated amino acid deletions and/or insertions occurred among feANP32 variants,but all feANP32 proteins were primarily localized to cell nucleus.Minigenome replication systems for several representative IAV strains were established and the support ability of feANP32 on IAV polymerase activity was estimated.The results indicated that most feANP32A and feANP32B splice variants were able to support all the tested IAV strains,though the support activity of a single feANP32 protein on polymerase activity varied among different IAV strains.In addition,the role of feANP32 in supporting H3N2 canine influenza virus was determined by investigating viral replication in vitro.Collectively,our study systematically investigated the support activity of feANP32 on IAV,providing a clue for further exploring the mechanism of susceptibility of cats to IAV.

Isolation of a feline-derived feline panleukopenia virus with an A300P substitution in the VP2 protein and confrmation of its pathogenicity in dogs

Feline panleukopenia virus(FPV)is a single-stranded DNA virus that can infect cats and cause feline panleukopenia,which is a highly contagious and fatal disease in felines.The sequence of FPV is highly variable,and mutations in the amino acids of its capsid protein play crucial roles in altering viral virulence,immunogenicity,host selection,and other abilities.In this study,the epidemiology of FPV was studied using 746 gastrointestinal swab samples derived from cats that presented gastrointestinal symptoms specifcally,diarrhea or vomiting during the period spanning from 2018 to 2022.The overall prevalence of FPV-positive patients among these samples was determined to be 45.4%.Capsid(virion)protein 2(VP2)gene of each FPV-positive sample was sequenced and amplifed,yielding 65 VP2 sequences.Among them,six VP2 gene sequences were detected in the majority of the samples test positive for FPV,and these positive samples originated from a diverse range of geographical locations.These isolates were named FPV-6,FPV-10,FPV-15,FPV-251,FPV-271 and FPV-S2.Additionally,the substitution of Ala300Pro(A300P)in VP2 was detected for the frst time in feline-derived FPV(FPV-251).FPV-251 isolate,with this substitution in VP2 protein,exhibited stable proliferative capacity in Madin-Darby canine kidney(MDCK)cells and A72 cells.FPV-271 was selected as the FPV control isolate due to its single amino acid diference from VP2 protein of FPV-251 at position 300(FPV-271 has alanine,while FPV-251 has proline).After oral infection,both FPV-251 and FPV-271 isolates caused feline panleukopenia,which is characterized by clinical signs of enterocolitis.However,FPV-251 can infect dogs through the oral route and cause gastrointestinal(GI)symptoms with lesions in the intestine and mesenteric lymph nodes(MLNs)of infected dogs.This is the frst report on the presence of an A300P substitution in VP2 protein of feline-derived FPV.Additionally,FPV isolate with a substitution of A300P at VP2 protein demonstrated efcient replication capabilities in canine cell lines and the ability to infect dogs.

Nitric oxide activation of a potassium channel (BK_(Ca)) in feline lower esophageal sphincter

AIM:To assess the effect of nitric oxide (NO) on the large conductance potassium channel (BKCa) in isolated circular (CM) and sling (SM) muscle cells and muscle strips from the cat lower esophageal sphincter (LES) to determine its regulation of resting tone and relaxation.METHODS:Freshly enzymatically-digested and isolated circular smooth muscle cells were prepared from each LES region.To study outward K + currents,the perforated patch clamp technique was employed.To assess LES resting tone and relaxation,muscle strips were mounted in perfused organ baths.RESULTS:(1) Electrophysiological recordings from isolated cells:(a) CM was more depolarized than SM (-39.7 ± 0.8mV vs-48.1 ± 1.6 mV,P < 0.001),and maximal outward current was similar (27.1 ± 1.5 pA/pF vs 25.7 ± 2.0 pA/pF,P > 0.05);(b) The NO donor sodium nitroprusside (SNP) increased outward currents only in CM (25.9 ± 1.9 to 46.7 ± 4.2 pA/pF,P < 0.001) but not SM (23.2 ± 3.1 to 27.0 ± 3.4 pA/pF,P > 0.05);(c) SNP added in the presence of the BK Ca antagonist iberiotoxin (IbTX) produced no increase in the outward current in CM (17.0 ± 2.8 vs 13.7 ± 2.2,P > 0.05);and (d) L-NNA caused a small insignificant inhibition of outward K + currents in both muscles;and (2) Muscle strip studies:(a) Blockade of the nerves with tetrodotoxin (TTX),or BK Ca with IbTX had no significant effect on resting tone of either muscle;and (b) SNP reduced tone in both muscles,and was unaffected by the presence of TTX or IbTX.CONCLUSION:Exogenous NO activates BK Ca only in CM of the cat.However,as opposed to other species,exogenous NO-induced relaxation is predominantly by a non-BK Ca mechanism,and endogenous NO has minimal effect on resting tone.

Isolation and phylogenetic analysis of feline calicivirus strains from various region of China

Feline calicivirus(FCV)is an important feline pathogen mainly causing upper respiratory tract disease,conjunctivitis,and stomatitis,and it is classifed into genotype I and genotype II.To investigate the prevalence and molecular characteristics of FCV,this study collected 337 cat swab samples from animal hospitals in diferent regions of China from 2019 to 2021.The positive detection rate of FCV was 29.9%(101/337)by RT-PCR.Statistical analysis showed that FCV prevalence was signifcantly associated with living environment(p=0.0004),age(p=0.031)and clinical symptoms(p=0.00),but not with sex(p=0.092)and breed(p=0.171).The 26 strains of FCV were isolated using F81 cells.Phylogenetic analysis showed that 10 isolates belonged to genotype I,and 16 isolates belonged to genotype II.These 26 isolates were highly genetically diverse,of which HB7 isolate had three same virulence-related amino acid loci with VSD strains.Potential loci distinguishing diferent genotypes were identifed from 26 isolates,suggesting the genetic relationship between diferent genotypes.In addition,selection pressure analysis based on capsid protein of 26 isolates revealed that the protein is under diversifying selection.This study reveals the genetic diversity of FCV and provides a reference for the screening of vaccine candidate strains and the development of vaccines with better cross-protection efects.

Comparative evaluation of acute phase proteins by C-reactive protein(CRP)and serum amyloid A(SAA)in nonhuman primates and feline carnivores

The feasibility of a commercially available assay for C-reactive protein(CRP,CRP for humans:hCRP,and CRP for dogs:vCRP)and a trial reagent of serum amyloid A(SAA,vSAA for animals)were applied to the measurement of acute phase proteins in zoo animals,particularly in nonhuman primates and feline carnivores was evaluate.Results showed that hCRP and vSAA methods were applicable to measure CRP and SAA in Haplorhini.There was a highly signifcant correlation between both parameters with remarkably high correlation coefcient.A higher proportion of Bonnet macaques in Haplorhini,and the linear regression with good correlation between hCRP and vSAA levels were observed.Reference values in healthy Bonnet macaques were hCRP(46.86±30.97 nmol/L)and vSAA(9.06±1.95μg/mL).Although Ring-tailed lemur,which belonging to Strepsirrhini,showed low vSAA concentrations(reference values:1.08±0.47μg/mL),vSAA in patients was apparently elevated.The vCRP and vSAA methods were applicable to measurements of CRP and SAA in feline carnivores for highly signifcant correlation between both parameters.Theses two methods were also been deteded in lions,tigers and cheetahs.vSAA assays can be applied to measure SAA levels in other carnivores and herbivores.In conclusion,vSAA systems have potential utility as diagnostic tools for health screening and prediction in zoo animals.

Diagnosis,Treatment and Experience of A Case of Feline Lower Urinary Tract Syndrome

In February 2018,a 2-year-old American shorthair was treated conservatively for cystitis caused by crystalluria.However,as long as drug withdrawal was proceeding,the condition would be repeated,and the condition worsened since August.In order to diagnose and treat the sick cat,clinical examination,laboratory examination,X-ray,B-type ultrasound and other methods were used for diagnosis,and treatment was conducted according to the diagnosis results.The results showed that the cat was caused by ammoniomagnesium phosphate crystals blocking urethra,complicated with postrenal azotaemia and hyperkalemia.The relapse continued after conservative treatment,and the cat finally recovered by modifying urethra surgically.The results showed that the deficiency of drinking water,the change of urine pH,and high dietary magnesium and fiber content could promote the occurrence of the disease.For the problem of urethral obstruction that could not be solved by conservative treatment,operation was done to modify urethral stricture.

Expedited Management of Canine and Feline Vomiting and Diarrhea. Observational Study in 3952 Dogs and 2248 Cats Using Sucralfate-Like Potency-Enhanced Polyanionic Phyto-Saccharide—Elm Mucilage

A potency-enhanced polyanionic phyto-saccharide of elm mucilage (PEPPS) was prescribed by 197 small animal veterinarians in an open-labeled field trial. Clients provided informed consent to veterinarians to prescribe PEPPS to 3952 dogs and 2248 cats. A 2 day/4 dose response rate, determined by veterinarians’ consensus, provided clinical threshold for a significant clinical outcome. Data was collected through phone interviews conducted over a period of 3.5 years from June 2003 through December 2006. 82% of 1928 vomiting dogs and 77% of 1064 vomiting cats responded to PEPPS within 2 days or four doses. 93% of 2024 dogs and 79% of 1184 cats with diarrhea responded to PEPPS within 2 days or four doses. PEPPS appears useful for managing vomiting and diarrhea in dogs and cats. However, a randomized blinded placebo controlled trial is needed to quantify true clinical efficacy.

Case Report on Feline Polycythemia Vera

Polycythemia vera has been reported as a known condition in cats as early as 1966. This condition manifests as an increased mass in red blood cells and elevated hematocrit and is defined as an idiopathic chronic myeloproliferative disorder. The patient described in this paper presented with hyperemic gums and pinna and an acute onset of progressive ataxia and lethargy. Several possible underlying primary conditions such as cardiac disease and renal malignancy were excluded by running basic blood work and radiographic imaging. Initial blood work revealed a significantly elevated packed cell volume (88%). After diagnosis, treatment with phlebotomy and chemotherapy lead to a reduction in hematocrit and elimination of neurologic signs. This case study represents the diagnosis and successful management of this disease in a private practice setting. Polycythemia vera is relatively uncommon in dogs and cats, but should still be considered in cases of neurologic disorders, especially with the presence of bright red ears, paws, or gums.

Generalizations of the Feline and Texas Chainsaw Josephus Problems

We define and study the Extended Feline Josephus Game, a game in which n players, each with &#8467;lives, stand in a circle. The game proceeds by alternating between hitting k consecutive players—each of whom will consequently lose a life—and skipping s consecutive players. This cycle continues until every player except one loses all of their lives. Given the nonnegative integer parameters n, k, s and &#8467;, the goal of the game is to identify the surviving player. In this paper, we show how the defining parameters n, k, s, and &#8467;affect the survivor of games with specific constraints on those parameters and our main results provide new closed formulas to determine the survivor of these Extended Feline Josephus Games. Moreover, for cases where these formulas do not apply, we provide recursive formulas for reducing the initial game to other games with smaller parameter values. For the interested reader, we present a variety of directions for future work in this area, including an extension which considers players lying on a general graph, rather than on a circle.

Feline Hypertrophic Cardiomyopathy Associated with the p.A31P Mutation in cMyBP-C Is Caused by Production of Mutated cMyBP-C with Reduced Binding to Actin

Hypertrophic cardiomyopathy (HCM) is a myocardial disorder, with complications including heart failure, thromboemboli and sudden death. Human and feline HCM (fHCM) are clinically comparable, thus fHCM may serve as a spontaneous animal model. fHCM in Maine Coon (MC) cats is associated with the p.A31P mutation in the cMyBP-C protein. The mutation is located in the cMyBP-C C0-domain which is known to interact with actin. The presence and levels of the wild type and mutated protein in heart tissue from mutant and wild type MC cats were examined by SDS-PAGE and mass spectrometry (MS). Quantitative yeast-2-hybrid (Y2H) protein-protein interaction analysis was used to assess the effect of the mutation on C0C1/actin interaction. The NMR-based structure of the C0 domain was used to calculate the energetic consequence of replacing alanine with a proline residue. In the homozygous MC cat, the mutated cMyBP-C protein was present, and cMyBPC-C levels were not reduced compared to that of the wild type cat. However, the interaction of actin with mutant cMyBP-C C0C1 was reduced compared to that of wild type. This may be because the substitution of the alanine with proline in position 31 was energetically highly unfavorable and resulted in only one hydrogen bond within the anti-parallel beta-strand compared to two hydrogen-bonds for alanine, possibly destabilizing the structure of the actin-interacting domain. The p.A31P mutation is present in cardiac tissue and the most likely pathogenic mechanism is interference with contractility by reducing binding of the C0C1 domain of cMyBP-C to actin.

The Role of Relative Spinal Motion during Feline Galloping for Speed Performance

Felines use their spinal column to increase their running speed at rapid locomotion performance. However, its motion profile behavior during fast gait locomotion has little attention. The goal of this study is to examine the relative spinal motion profile during two different galloping gait speeds. To understand this dynamic behavior trend, a dynamic motion of the feline animal （Felis catus domestica） was measured and analyzed by motion capture devices. Based on the experiments at two different galloping gaits, we observed a significant increase in speed （from 3.2 m.s-1 to 4.33 m.s-1） during the relative motion profile synchronization between the spinal （range： 118.86~ to 168.00~） and pelvic segments （range： 46.35~ to 91.13~） during the hindlimb stance phase （time interval： 0.495 s to 0.600 s）. Based on this discovery, the relative angular speed profile was applied to understand the possibility that the role of the relative motion match during high speed locomotion generates bigger ground reaction force.

Optimal Center-of-Mass Pivot Effect of Spinal Motion in Feline Galloping

In this paper,we studied the acceleration behavior of a quadruped animal during a galloping motion.Because the development of many quadruped robotic systems has been focused on dynamic movements,it is obvious that guidance from the dynamic behavior of quadruped animals is needed for robotics engineers.To fulfill this demand,this paper deals with analysis of the galloping motions of a domestic cat,which is well known for its excellent acceleration performance among four-legged animals.Based on the planar motion capture environment,the movement data of a galloping feline was acquired and the dynamic motions were estimated using a spring-mass system.In particular,the effects of the position and angle of the center-of-mass of the cat,angular displacement of the spine,and angular velocity of the spine were analyzed and are discussed below.Through this process,it was possible to understand the dynamic movement characteristics of the cat,and to understand the relationships between,and the influences of,these parameters.From this analysis,we provide significant data applicable to the design of joint movements in quadruped robot systems.

Two Inhibitors Against the 3C-Like Proteases of Swine Coronavirus and Feline Coronavirus

Coronaviruses(CoVs)are important human and animal pathogens that cause respiratory and gastrointestinal diseases.Porcine epidemic diarrhoea(PED),characterized by severe diarrhoea and vomiting in pigs,is a highly lethal disease caused by porcine epidemic diarrhoea virus(PEDV)and causes substantial losses in the swine industry worldwide.However,currently available commercial drugs have not shown great therapeutic effects.In this study,a fluorescence resonance energy transfer(FRET)-based assay was applied to screen a library containing 1,590 compounds and identified two compounds,3-(aminocarbonyl)-1-phenylpyridinium and 2,3-dichloronaphthoquinone,that target the 3C-like protease(3CL^(pro))of PEDV.These compounds are of low molecular weight(MW)and greatly inhibited the activity of this enzyme(IC_(50) values were obtained in this study).Furthermore,these compounds exhibited antiviral capacity against another member of the CoV family,feline infectious peritonitis virus(FIPV).Here,the inhibitory effects of these compounds against CoVs on Vero cells and feline kidney cells were identified(with EC_(50) values)and cell viability assays were performed.The results of putative molecular docking models indicate that these compounds,labeled compound 1 and compound 2,contact the conserved active sites(Cys144,Glu165,Gln191)of 3CL^(pro) via hydrogen bonds.These findings provide insight into the antiviral activities of compounds 1 and 2 that may facilitate future research on anti-CoV drugs.

Role of Intracellular Distribution of Feline and Bovine SAMHD1 Proteins in Lentiviral Restriction

Human SAMHD1(h SAM)restricts lentiviruses at the reverse transcription step through its d NTP triphosphohydrolase(d NTPase)activity.Besides humans,several mammalian species such as cats and cows that carry their own lentiviruses also express SAMHD1.However,the intracellular distribution of feline and bovine SAMHD1(f SAM and b SAM)and its significance in their lentiviral restriction function is not known.Here,we demonstrated that f SAM and b SAM were both predominantly localized to the nucleus and nuclear localization signal(11KRPR14)-deleted f SAM and b SAM relocalized to the cytoplasm.Both cytoplasmic f SAM and b SAM retained the antiviral function against different lentiviruses and cytoplasmic f SAM could restrict Vpx-encoding SIV and HIV-2 more efficiently than its wild-type(WT)protein as cytoplasmic h SAM.Further investigation revealed that cytoplasmic f SAM was resistant to Vpx-induced degradation like cytoplasmic h SAM,while cytoplasmic b SAM was not,but they all demonstrated the same in vitro d NTPase activity and all could interact with Vpx as their WT proteins,indicating that cytoplasmic h SAM and f SAM can suppress more SIV and HIV-2 by being less sensitive to Vpx-mediated degradation.Our results suggested that f SAM-and b SAM-mediated lentiviral restriction does not require their nuclear localization and that f SAM shares more common features with h SAM.These findings may provide insights for the establishment of alternative animal models to study SAMHD1 in vivo.

Sequence variability analysis on major histocompatibility complex class Ⅱ DRB alleles in three felines

The variation of the exon 2 of the major histo-compatibility complex(MHC)class II gene DRB locus in three feline species were examined on clouded leopard(Neofelis nebulosa),leopard(Panthera pardus)and Amur tiger(Panthera tigris altaica).A pair of degenerated primers was used to amplify DRB locus covering almost the whole exon 2.Exon 2 encodes theβ1 domain which is the most vari-able fragments of the MHC class II molecule.Single-strand conformational polymorphism(SSCP)analysis was applied to detect different MHC class II DRB haplotypes.Fifteen recombinant plasmids for each individual were screened out,isolated,purified and sequenced finally.Totally eight distinct haplotypes of exon 2 were obtained in four individuals.With-in 237 bp nucleotide sequences from four samples,30 vari-able positions were found,and 21 putative peptide-binding positions were disclosed in 79 amino acid residues.The ratio of nonsynonymous substitutions(d_(N))was much higher than that of synonymous substitutions(d_(S)),which indicated that balancing selection probably maintain the variation of exon 2.MEGA neighbor joining(NJ)and PAUP maximum parsimo-ny(MP)methods were used to reconstruct phylogenetic trees among species,respectively.Results displayed a more close relationship between leopard and tiger;however,clouded leopard has a comparatively distant relationship form the other two.

Analysis of Genetic Variation of VP2 Gene in 6 FPV Strainsin China

In order to understand the variation of FPV strains in the Jinan area, Shandong Province, China, the VP2 gene of 6 FPV strains was sequenced, and the analysis of the genetic relationship, evolution and main functional site variation was carried out. It was found that FPV-XY2, FPV-XY3 and FPV-XY6 were the same strain with 100% homology, and also close to FPV-XY1, and the homology between FPV-XY4 and FPV-XY5 was close. The homology between the reference strain and the test strain was over 99.3%. According to the evolutionary analysis, the genetic relationship among FPV-XY1, FPV-XY2, FPV-XY3, FPV-XY6 was close, and the genetic relationship between FPV-XY4 and FPV-XY5 was close, and the result was similar to the homologous result. Compared with the VP2 amino acid sequence of the standard strain FPV-CU4, the VP2 protein of all the test strains changed from I to t on the 101st Amino acid, this may be the cause of immune failure in these 6 cases;the change of a to s in the 91st amino acid position of FPV-XY1, FPV-XY2, FPV-XY3, FPV-XY6 may be the cause of enhanced virulence of FPV. This study provides a reference for exploring the epidemic law of FP in the Jinan area, the standard of FP treatment plan and the research and development of FPV subunit vaccine.

The Comparison of Genetic Variation in the Envelope Protein Between Various Immunodeficiency Viruses and Equine Infectious Anemia Virus

The envelope protein （Env） of lentiviruses such as HIV, SIV, FIV and EIAV is larger than that of other retroviruses. The Chinese EIAV attenuated vaccine is based on Env and has helped to successfully control this virus, demonstrating that envelope is crucial for vaccine. We compared Env variation of the four kinds of lentiviruses. Phylogenetic analysis showed that the evolutionary relationship of Env between HIV and SIV was the closest and they appeared to descend from a common ancestor, and the relationship of HIV and EIAV was the furthest. EIAV had the shortest Env length and the least number of potential N-linked glyeosylation sites （PNGS） as well as glyeosylation density compared to various immunodefieiency viruses. However, HIV had the longest Env length and the most PNGS. Moreover, the alignment of HIV and SIV showed that PNGS were primarily distributed within extraeellular membrane protein gp120 rather than transmembrane gp41. It implies that the size difference among these viruses is associated with a lentivirus specific function and also the diversity of env. There arc low levels of modification of glycosylation sites of Env and selection of optimal protective epitopes might be useful for development of an effective vaccine against HIV/AIDS.

Molecular diagnosis in cat allergy

Domestic cats represent one of the most common sources of indoor allergens.All over the world,many households own cats,whose allergens are persistent and widespread.Cat allergy itself is frequent,and its symptoms vary from rhinoconjunctivitis to life-threatening asthma.In vitro diagnosis using precision medicine allergy immunoassays is important because natural cat dander extracts may differ in quality and quantity of some of the individual allergen components and other molecules.In the component-resolved diagnosis of cat allergy,singleplex and multiplex specific immunoglobulin(Ig)E assays include use of the cat-specific major allergen,secretoglobin Fel d 1(as a species-specific molecule),other allergen components(such as lipocalins Fel d 4,cross-reacting with other animal similar molecules,and Fel d 7,present in small quantities in natural extracts),and serum albumin Fel d 2(related to the cat-pork syndrome).IgA Fel d 5 and IgM Fel d 6 are not available as allergen components in the current commercial IgE immunoassays,but they may impair the in vitro diagnostic evaluation of cat allergy because galactose-α1,3-galactose is an IgE-binding epitope of these native feline allergens.The benefits of molecular-based cat allergy diagnosis are continually evaluated,as the role of recombinant allergen components already known is detailed and new other molecules of interest may be discovered in the future.

Experimental Researches on Carcinogenesis or Tumorigenicity of MDCK Canine Kidney Cell(CKC) Lines and Analysis of Their Chromosome Karyotypes

The chromosomal number variations & structural aberrations of the MDCK cell line, primary feline or canine kidney cell(FKC or CKC) and Hela cell line were investigated and their karyotypes of conventional chromosome bands were analyzed. The carcinogenesis or tumorigenicity testing of these cell lines in about 232 nude mice and for colony formation in soft agarose and for haemagglutination under different concentration of plant lectins of these cells were carried out. Under the prerequisite that the incidence of cancer or tumor in negative-control nude mice inoculated subcutaneously with primary feline or canine kidney cell cultures purified in vitro at passage 3 was 0 (0/22) and 0 (0/10), respectively. The incidence of the progressively-growing malignant tumor(MT) in positive-control nude mice inoculated subcutaneously with Hela cell cultures of KB, X, or NM20/X strain was 10/10, 25/25 and 5/51, respectively. The results showed that the incidence of tumor in nude mice with tetraploid YA strain of MDCK cell during 20 - 45 passages, with hy-podiploid JB strain of MDCK cell on passage 25, with di-and hypoploid JC strain of MDCK cell during 2-15 passages or with hypoploid M strain of MDCK cell during 9 - 27 passages was 28/58, 1/5, 4/18 and 0/31, respectively. The chromosomal analysis results showed that the ratio of difference in the rate of modal chromosome number between high(mcs+ n) and lowest (mcs) passages was not more than 5% - 15% and the structure aberrations was generally 0 - 3% . These results proved that the genetic characteristics of chromosomal number of cell lines determines their tumorigenicity, but it is species-specific. MDCK line has tumorigenicity no matter what its chromosome karyotype is, at least it has very low tumorigenicity even when its modal chromosome number is hypoploid. The repeatedly frozen, thawed and split controls of tumorigenicity-positive cell lines(X strain of Hela, M strain of BHK-21, JA strain of Vero, YA strain of MDCK) have much lower tumorigenicity or are even non-carcinogenesis, and the repeatedly frozen, thawed and split controls of very low tumorigenicity cell lines (M or JC strain of MDCK) are certainly non-carcinogenic and never have increased tumorigenicity. It is thus evident that MDCK cell of M, JB or JC strain can be approved as substrate for the preparation of attenuated viral vaccines, but MDCK cell of YA strain can not be approved as substrate for the preparation of attenuated viral vaccines. In summary, all strains of MDCK cell line have tunorigenicity, at least have low tumor igencity , never have non-cancinogenic MDCK, but very low tumorigenicity MDCK cell strains can certainly be used for the approval production of canine viral vaccines if the DNA content in viral cell cultures was remarkably decreased through conventional means in manufacturing process. Therefore, the master cell stock and working cell bank of MDCK line used for vaccine manufacture were established in China, which are free of infectious agents, and described with respect to cytogenetic characteristics and tumorigenicity.Tests showed that there were correlations among cell line chromosome number variations, anchorage independence in soft a-garose, haemagglutination under plant lectins, and tumor-forming ability in nude mice, thus all the in vitro tests are economic, simple and reliable means for monitoring the tumor-forming ability of MDCK line in nude mice.