South China Biological Medicine (China)

H9N2 subtype avian influenza virus (AIV) was the highly contagious pathogen which has caused severe losses in the poultry industry throughout China in recent years. Using current epidemic viruses as vaccine was an effective way to prevent infection of H9N2 subtype AIV. In this study, a total of 23 H9N2 subtype AIV strains were isolated in 200 samples from 13 provinces of China during 2012-2016. The sequencing and phylogenetic analysis of the hemagglutinin gene sequence of the isolation strains showed that 22 isolation strains were clustered to h9.4.2.5 lineage, while only 1 belonged to h9.4.2.6. The data of cross-HI, neutralization and cross-immune protection shown that the A/chicken/Hunan/HN/2015 (HN) and A/chicken/Shandong/SD/2014 (SD) strains as vaccine could effectively protect present viruses infection compared with other strains. These results indicated that current epidemic viruses were mainly belong to h9.4.2.5 lineage and HN and SD strains as candidate vaccine strains were potentiality for the protection of present H9N2 subtype AIV infection.

First identified as belonging to the H5N1 subtype in Guangdong province, China, in 1996, the Gs/GD lineage H5 avian influenza viruses have continuously evolved and spread since.1Wille M Barr IG Resurgence of avian influenza virus.Science. 2022; 376: 459-460Crossref PubMed Scopus (7) Google Scholar, 2Dai M Yan N Huang Y Zhao L Liao M Survivability of highly pathogenic avian influenza virus on raw chicken meat in different environmental conditions.Lancet Microbe. 2022; 3: e92Summary Full Text Full Text PDF PubMed Scopus (2) Google Scholar H5 subtype viruses evolved into multiple distinct subclades, among which 2.3.4.4 has become dominant in China,3Yamaji R Saad MD Davis CT et al.Pandemic potential of highly pathogenic avian influenza clade 2.3.4.4 A(H5) viruses.Rev Med Virol. 2020; 30e2099Crossref PubMed Scopus (34) Google Scholar and H5N6 viruses of this subclade led to an apparent increase in human infections in 2021 and 2022. During this period, 33 cases (of a total of 76 recorded cases infected with subclade 2.3.4.4 H5N6 viruses) have been documented, resulting in 11 deaths and posing an alarming threat to public health.1Wille M Barr IG Resurgence of avian influenza virus.Science. 2022; 376: 459-460Crossref PubMed Scopus (7) Google Scholar Almost all confirmed cases were in individuals who had reported exposure to poultry. To address concerns regarding the sharp increase in the number of human infections, we explored the evolutionary dynamics and conducted a risk assessment of H5N6 viruses from birds in live poultry markets during 2021.In 2021, 5883 cloacal and tracheal swab samples from chickens, ducks, and geese were obtained from live poultry markets in China and analysed for influenza viruses (appendix pp 9–13). We determined the full-length genomes of 19 H5N6 viruses from chickens, ducks, and geese and found that the genomes of these viruses were more complex than those of H5N6 viruses circulating in China previously (appendix pp 2–3). Of particular concern is that the genomes of all H5N6 viruses were continuously reassorted with those of 2.3.4.4b H5N8 viruses of wild bird-origin, and were genetically closely related to the H5N6 virus that infected humans in 2021 (appendix pp 2–3, 14–28). We found that these 2.3.4.4b H5N6 viruses were novel reassortants. The HA and M genes of all isolates were derived from 2.3.4.4b H5N8 viruses of wild-bird origin, whereas other internal genes of all isolates exhibited several separate clusters. On the basis of phylogenetic trees, we identified five distinct genotypes of H5N6 viruses (G1–G5; appendix pp 3, 28), and found that both the G1 and G3 genotype H5N6 viruses were 100% lethal to mice (appendix pp 3–4, 28–29). The high genetic diversity and the virulence in mammals of H5N6 viruses in 2021 pose an increasing threat to public health. During the 2020–21 influenza season, novel H5N8 viruses repeatedly entered Europe, Russia, South Korea, and China, causing numerous outbreaks in wild birds and poultry.4Zhang J Li X Wang X et al.Genomic evolution, transmission dynamics, and pathogenicity of avian influenza A (H5N8) viruses emerging in China, 2020.Virus Evol. 2021; 7veab046Crossref Scopus (11) Google Scholar Notably, these H5N8 viruses infected humans in Russia,5Pyankova OG Susloparov IM Moiseeva AA et al.Isolation of clade 2.3.4.4b A(H5N8), a highly pathogenic avian influenza virus, from a worker during an outbreak on a poultry farm, Russia, December 2020.Euro Surveill. 2021; 262100439Crossref PubMed Scopus (34) Google Scholar highlighting the increasing threat to humans posed by the co-circulation of H5N6 and H5N8 viruses.Wild waterfowl are regarded as natural reservoirs that contribute to the global spread of avian influenza viruses through long-distance migration.1Wille M Barr IG Resurgence of avian influenza virus.Science. 2022; 376: 459-460Crossref PubMed Scopus (7) Google Scholar, 4Zhang J Li X Wang X et al.Genomic evolution, transmission dynamics, and pathogenicity of avian influenza A (H5N8) viruses emerging in China, 2020.Virus Evol. 2021; 7veab046Crossref Scopus (11) Google Scholar Frequent contact with wild birds remains the most probable cause of viral introduction into domestic poultry. Countries in the Middle East, Europe, and Asia have a particularly important role in the global dissemination of influenza viruses.4Zhang J Li X Wang X et al.Genomic evolution, transmission dynamics, and pathogenicity of avian influenza A (H5N8) viruses emerging in China, 2020.Virus Evol. 2021; 7veab046Crossref Scopus (11) Google Scholar The co-circulation of H5N6 and H5N8 viruses in migratory birds accelerated the evolution of novel variants. Nowadays, clade 2.3.4.4b H5 influenza viruses are widely prevalent in China. However, based on our assessment, the circulating 2.3.4.4b H5N6 viruses are antigenically distinct from the strains in the commercial vaccine in China (appendix pp 3–4, 29), suggesting that these novel viruses will continue to circulate in poultry if the commercial vaccine is not updated. The ongoing 2021–22 wave of avian influenza, which involves not only H5N6 but also the current outbreak of H5N1 viruses, is unprecedented in terms of its rapid spread and high frequency of outbreaks in birds, and poses a continuous threat to public health. First identified as belonging to the H5N1 subtype in Guangdong province, China, in 1996, the Gs/GD lineage H5 avian influenza viruses have continuously evolved and spread since.1Wille M Barr IG Resurgence of avian influenza virus.Science. 2022; 376: 459-460Crossref PubMed Scopus (7) Google Scholar, 2Dai M Yan N Huang Y Zhao L Liao M Survivability of highly pathogenic avian influenza virus on raw chicken meat in different environmental conditions.Lancet Microbe. 2022; 3: e92Summary Full Text Full Text PDF PubMed Scopus (2) Google Scholar H5 subtype viruses evolved into multiple distinct subclades, among which 2.3.4.4 has become dominant in China,3Yamaji R Saad MD Davis CT et al.Pandemic potential of highly pathogenic avian influenza clade 2.3.4.4 A(H5) viruses.Rev Med Virol. 2020; 30e2099Crossref PubMed Scopus (34) Google Scholar and H5N6 viruses of this subclade led to an apparent increase in human infections in 2021 and 2022. During this period, 33 cases (of a total of 76 recorded cases infected with subclade 2.3.4.4 H5N6 viruses) have been documented, resulting in 11 deaths and posing an alarming threat to public health.1Wille M Barr IG Resurgence of avian influenza virus.Science. 2022; 376: 459-460Crossref PubMed Scopus (7) Google Scholar Almost all confirmed cases were in individuals who had reported exposure to poultry. To address concerns regarding the sharp increase in the number of human infections, we explored the evolutionary dynamics and conducted a risk assessment of H5N6 viruses from birds in live poultry markets during 2021. In 2021, 5883 cloacal and tracheal swab samples from chickens, ducks, and geese were obtained from live poultry markets in China and analysed for influenza viruses (appendix pp 9–13). We determined the full-length genomes of 19 H5N6 viruses from chickens, ducks, and geese and found that the genomes of these viruses were more complex than those of H5N6 viruses circulating in China previously (appendix pp 2–3). Of particular concern is that the genomes of all H5N6 viruses were continuously reassorted with those of 2.3.4.4b H5N8 viruses of wild bird-origin, and were genetically closely related to the H5N6 virus that infected humans in 2021 (appendix pp 2–3, 14–28). We found that these 2.3.4.4b H5N6 viruses were novel reassortants. The HA and M genes of all isolates were derived from 2.3.4.4b H5N8 viruses of wild-bird origin, whereas other internal genes of all isolates exhibited several separate clusters. On the basis of phylogenetic trees, we identified five distinct genotypes of H5N6 viruses (G1–G5; appendix pp 3, 28), and found that both the G1 and G3 genotype H5N6 viruses were 100% lethal to mice (appendix pp 3–4, 28–29). The high genetic diversity and the virulence in mammals of H5N6 viruses in 2021 pose an increasing threat to public health. During the 2020–21 influenza season, novel H5N8 viruses repeatedly entered Europe, Russia, South Korea, and China, causing numerous outbreaks in wild birds and poultry.4Zhang J Li X Wang X et al.Genomic evolution, transmission dynamics, and pathogenicity of avian influenza A (H5N8) viruses emerging in China, 2020.Virus Evol. 2021; 7veab046Crossref Scopus (11) Google Scholar Notably, these H5N8 viruses infected humans in Russia,5Pyankova OG Susloparov IM Moiseeva AA et al.Isolation of clade 2.3.4.4b A(H5N8), a highly pathogenic avian influenza virus, from a worker during an outbreak on a poultry farm, Russia, December 2020.Euro Surveill. 2021; 262100439Crossref PubMed Scopus (34) Google Scholar highlighting the increasing threat to humans posed by the co-circulation of H5N6 and H5N8 viruses. Wild waterfowl are regarded as natural reservoirs that contribute to the global spread of avian influenza viruses through long-distance migration.1Wille M Barr IG Resurgence of avian influenza virus.Science. 2022; 376: 459-460Crossref PubMed Scopus (7) Google Scholar, 4Zhang J Li X Wang X et al.Genomic evolution, transmission dynamics, and pathogenicity of avian influenza A (H5N8) viruses emerging in China, 2020.Virus Evol. 2021; 7veab046Crossref Scopus (11) Google Scholar Frequent contact with wild birds remains the most probable cause of viral introduction into domestic poultry. Countries in the Middle East, Europe, and Asia have a particularly important role in the global dissemination of influenza viruses.4Zhang J Li X Wang X et al.Genomic evolution, transmission dynamics, and pathogenicity of avian influenza A (H5N8) viruses emerging in China, 2020.Virus Evol. 2021; 7veab046Crossref Scopus (11) Google Scholar The co-circulation of H5N6 and H5N8 viruses in migratory birds accelerated the evolution of novel variants. Nowadays, clade 2.3.4.4b H5 influenza viruses are widely prevalent in China. However, based on our assessment, the circulating 2.3.4.4b H5N6 viruses are antigenically distinct from the strains in the commercial vaccine in China (appendix pp 3–4, 29), suggesting that these novel viruses will continue to circulate in poultry if the commercial vaccine is not updated. The ongoing 2021–22 wave of avian influenza, which involves not only H5N6 but also the current outbreak of H5N1 viruses, is unprecedented in terms of its rapid spread and high frequency of outbreaks in birds, and poses a continuous threat to public health. We declare no competing interests. We acknowledge the authors and the originating and submitting laboratories of the sequences from GISAID's EpiFlu Database on which this research is based. All submitters of data can be contacted directly through the GISAID website (https://www.gisaid.org). This work was supported by the Guangdong Major Project of Basic and Applied Basic Research (2020B0301030007), the National Natural Science Foundation of China (31830097 and 31672586), the Guangdong Province Universities and Colleges Pearl River Scholar Funded Scheme (2018, WQ), and the Young Scholars of Yangtze River Scholar Professor Program (2019, WQ). JZ, HY, ML, and WQ planned and conceptualised the laboratory work and wrote the Correspondence. HY and YL isolated the virus from samples and sequenced the genomes. JZ, HY, and YL conducted the animal experiments and antigenic analysis. JZ conducted bioinformatics analysis and data interpretation. ML and WQ provided the funding. JZ and HY contributed equally. Supplementary Material Download .pdf (1.79 MB) Help with pdf files Supplementary appendix Download .pdf (1.79 MB) Help with pdf files Supplementary appendix Survivability of highly pathogenic avian influenza virus on raw chicken meat in different environmental conditionsHighly pathogenic avian influenza (HPAI) viruses, such as H7N9 and H5N8, can cause huge economic losses in the poultry industry, and they can pose a serious risk to public health.1,2 In Japan, HPAI viruses have been repeatedly detected on raw poultry, reportedly transported by international flight passengers from China and Vietnam.1 Here, we report the survivability of HPAI viruses on raw chicken meat in different environmental conditions. Full-Text PDF Open Access

Multiple recent outbreaks of highly pathogenic H5N8 viruses originating in aquatic birds frequently occurred in most European countries, Russia, South Korea, and Japan during the winter of 2020-21, and one zoonotic event of poultry workers infected with novel H5N8 viruses were reported in Russia. Strikingly, these novel H5N8 viruses had emerged and been co-circulating in wild birds and poultry in multiple provinces of China during 2020-21. In China, the population of aquatic birds has risen significantly in the past twenty years, and China is regarded as the largest reservoir for influenza viruses carried in aquatic birds across the globe. Hence, the co-circulation of these novel H5N8 viruses poses an alarming threat to not only poultry industry but also human health. In this study, we sequenced full-length genomes of these H5N8 viruses circulating in China. Phylogenetic analysis demonstrated that poultry-origin H5N8 viruses in China fell within wild birds-origin clade 2.3.4.4b H5N8 viruses from Europe during 2020-21, and notably, were genetically closely related to human-infecting H5N8 viruses in Russia. Moreover, they possessed several molecular markers associated with mammalian adaption. Bayesian coalescent analysis showed that these H5N8 viruses might have introduced into China during June-September 2020, suggesting that these H5N8 viruses might have introduced via wild bird migration or poultry trade. Besides, we also found that the effective population size of clade 2.3.4.4b H5N8 viruses dramatically increased during the winter season of 2020/21, as is consistent with previous increase of genetic diversity during the winter seasons of 2013/14 and 2016/17, which indicated that the wild bird migration accelerates the genetic diversity of these H5N8 viruses during the winter season of 2020/21. Notably, these novel H5N8 viruses were lethal to chickens and mice, highly transmissible to ducks, and were antigenically distinct from 2.3.4.4h H5 viruses circulating in China, posing considerable threats to public health. Our findings offer novel insights into the evolution and risk assessment of H5N8 viruses during the winter season of 2020-21.

S ince emerging in China in 2013, influenza A(H7N9) viruses have continued to circulate in mainland China, sporadically causing human infection (1-3). As of February 2020, a total of 1,568 laboratory-confirmed human cases and 616 related deaths had been reported, for a fatality rate of 40% (http://www.fao.org/ag/againfo/programmes/ en/empres/H7N9/situation_update.html). In mid-2016, a highly pathogenic avian influenza (HPAI) virus of subtype H7N9 emerged, and the number of cases in humans began to rise sharply during a fifth wave (4,5). Animal studies indicated that these HPAI H7N9 viruses are highly virulent in chickens and have gained transmissibility among ferrets (5-7). Also, the cocirculation of HPAI (H7N9) viruses caused high genetic diversity and host adaption (8), posing public health concerns.

The H5N1 subtype highly pathogenic avian influenza virus (HPAIV) reveals high variability and threatens poultry production and public health. To prevent the spread of H5N1 HPAIV, we developed an H5N1 virus-like particle (VLP) vaccine based on the insect cell-baculovirus expression system. Single immunization of the H5N1 VLP vaccines induced high levels of HI antibody titres and provided effective protection against homologous virus challenge comparable to the commercial inactivated vaccine. Meanwhile, we assessed the relative efficacy of different adjuvants by carrying out a head-to-head comparison of the adjuvants ISA 201 and ISA 71 and evaluated whether the two adjuvants could induce broadly protective immunity. The ISA 71 adjuvanted vaccine induced significantly higher levels of Th1 and Th2 immune responses and provided superior cross-protection against antigenically divergent H5N1 virus challenge than the ISA 201 adjuvanted vaccine. Importantly, increasing the vaccine dose could further enhance the cross-protective efficacy of H5N1 VLP vaccine and confer completely sterilizing protection against antigenically divergent H5N1 virus challenge, which was mediated by neutralizing antibodies. Our results suggest that the H5N1 VLP vaccine can provide broad-spectrum protection against divergent H5N1 influenza viruses as determined by adjuvant and vaccine dose.

Highly pathogenic influenza A(H5N8) viruses had caused several outbreaks among wild bird and poultry populations across the globe, and strikingly, caused human infection, posing serious public health concerns. In this study, we conducted influenza surveillance in China during 2021 to monitor the evolution of influenza viruses in poultry. A total of 35 influenza viruses were obtained in chickens, ducks, and geese, of which 30 H5N8 viruses, 3 H5N1 viruses, and 2 H5N6 viruses. Phylogenetic analysis suggested all of H5N1, H5N6, and H5N8 isolates were derived from clade 2.3.4.4b H5N8 viruses during 2020/21 season, and notably, the internal genes of H5N1 and H5N6 viruses shared different genetic heterogeneity with H5N8 viruses and had been reassorted with wild bird-origin H5N1 viruses from Europe. By contrast, almost all H5N8 viruses exhibited only one phylogenic cluster with wild bird-origin H5N8 viruses in China and Korea, indicating that H5N8 viruses in China were more stable. Besides, we found that Korea is the main output geographic location in the spread of these H5N8 viruses to northern and eastern China, and especially, the co-circulation of H5N8 viruses occurred within China, with central China acted as a seeding population during the H5N8 epidemic. The statistical support was strong for viral migration from wild birds to chickens and ducks, indicating that 2.3.4.4b poultry-origin H5N8 viruses during 2020-2021 were originated from wild birds. Our findings provide novel insights into evolution and transmission dynamics of H5 subtype influenza viruses among poultry after novel H5N8 viruses invaded China for nearly one year.

Infectious bronchitis virus (IBV) is distributed worldwide and causes significant losses in the poultry industry. In recent decades, lineages GI-19 and GI-7 have become the most prevalent IBV strains in China. However, the molecular evolution and phylodynamics of the lineage GI-7 IBV strains remain largely unknown. In this study, we identified 19 IBV strains from clinical samples from January 2021 to June 2022 in China, including 12 strains of GI-19, 3 strains of GI-7, and 1 strain each of GI-1, GI-9, GI-13, and GI-28. These results indicated that lineages GI-19 and GI-7 IBVs are still the most prevalent IBVs in China. Here, we investigated the evolution and transmission dynamics of lineage GI-7 IBVs. Our results revealed that the Taiwan province might be the origin of lineage GI-7 IBVs and that South China plays an important role in the spread of IBV. Furthermore, we found low codon usage bias of the S1 gene in lineage GI-7 IBVs. This allowed IBV to replicate in the host during evolution as a result of reduced competition, mainly driven by natural selection and mutational pressure, where the role of natural selection is more prominent. Collectively, our results reveal the genetic diversity and evolutionary dynamics of lineage GI-7 IBVs, which could assist in the prevention and control of viral infection.

The direct competitive enzyme-linked immunosorbent assay (dcELISA) kit was developed for detecting tetrodotoxin (TTX). The working conditions of the dcELISA kit including the anti-TTX mAb coating concentration, coating method, enzyme-labeled antigen concentration, the antigen diluents, reaction time and temperature were all optimized. The result showed that mAb coating concentration was 3.72 μg/ml, it was coated at the condition of minimal power treatment of microwave oven for 3 min. The enzyme-labeled antigen concentration was 4.08 μg/ml. The competitive reaction was under the condition of room temperature 25 °C for 30 min. The half maximal inhibitory concentration (IC 50 ) of the standard curve was 20.4 ng/ml, detection limit was 1.1 ng/ml, linear range 3.3～137 ng/ml, the intra-assay CV and inter-assay CV were 6.25% and 7.34% respectively. And recovery rate of TTX ranged from 65.0% to 93.2% with the CV of 9.41～12.77%. This method is convenient, sensitive and time-saving, hope this dcELISA kit can bring benefits and reference for TTX detection.

The study was aimed to develop a loop-mediated isothermal amplification (LAMP) method which amplifies DNA with high specificity and rapidity for the detection of Shigella dysenteriae . A set of four primers was designed for recognizing six distinct sequences on the target ipaH of S. dysenteriae . By the method, the target DNA was amplified within 1h under isothermal condition at 65 °C. The sensitivities of the LAMP for detecting pure culture and genomic DNA were 1.04 CFU/ml and 1.06 fg/μl, while the sensitivities of PCR method were 1.04×10 2 CFU/ml and 1.06 pg/μl. Furthermore, the LAMP assay was examined for its ability to detect S. dysenteriae in artificially contaminated lettuce sample, the detection limits of this LAMP assay and the PCR method were 4.60 CFU/g and 4.60×10 2 CFU/g, respectively.

Rabies virus (RABV) is a highly pathogenic virus that causes a fatal disease in humans and other mammals, but the mechanism of its evolution, spread, and spillover remains unknown. In this study, we analyzed the codon usage pattern of 2,018 RABV full-length genome sequences from 79 countries collected between 1931 and 2021 to provide an insight into its molecular evolution and unravel its unknown host-adapted pattern. We found that RABV exhibited a weak codon usage bias, with a preference for the codons ending in A (28.10 ± 0.01) or U (26.43 ± 0.02). Moreover, natural selection plays a major role in shaping the codon usage bias of the RABV. Notably, nearly half of the 18 codons in the virus were best matched to the hosts' most abundant isoacceptor tRNAs, which might account for the wide range of RABV hosts. Furthermore, significant differences were observed in the codon usage patterns of RABV for different host species, suggesting that codon usage bias may be influenced by host-specific factors. In conclusion, our study reveals codon usage patterns of RABV that may help in the development of control strategies and effective vaccines and therapies against this deadly virus.

Dead chickens,ducks and geese suspected of fowl cholera were collected in 2007~2013 from Fujian and its neighbor provinces and subject to isolation of Pasteurella multocida.A total of 95 Pasteurella multocida isolates were obtained,among which 74 isolates were isolated from ducks,17 from chickens and 4 from geese.These bacterial strains were then determined for species and capsular types in PCR and for heat-stable somatic antigens in agar diffusion test.All isolates belonged to capsular type A and 64 isolates to heat-stable somatic antigens 1.These results have indicated that the main serotype of Pasteurella multocida isolates from avian(excluding turkey) in China has been remained A:1 since 1980's.

Vibrio parahaemololyticus is the main cause of foodborne disease,and it is also a common pathogen detected from seafood. Vibrio parashaemoloyticus would cause enormous loss when fish and shrimp and so on are infected by it. In this study,laying hens were divided into two groups,and were immunized with two different doses of prepared antigen. The low dose group of hens was immunized with 5×107 CFU/ml,and the high dose group of hens was immunized with 5×108 CFU/ml Vibrio parahaemololyticus. ELISA was established to detect the antibody activity. The results demonstrated that the highest titer of IgY against Vibrio parahaemololyticus in the high and low dose group could reach 1:57600 and 1:28800,respectively. The titer of IgY produced by the low dose group could maintain for 250 days,but the titer of IgY produced by the high dose group could maintain for more than 300 days.

The objectives of the present study were to establish an indirect ELISA for the detection of Vibrio parahaemololyticus,and analyse the specificity,sensitivity and reliability of the method.Water dilution method,ethanol precipitation and DEAE-Sepharose FF chromatography were used to extract and purify the anti-V.parahaemololyticus IgY.Then the purified anti-V.parahaemololyticus IgY was used as the first antibody and rabbit anti-chicken IgY-HRP was used as the second antibody.The suitable concentration of anti-V.parahaemololyticus IgY and rabbit anti-chicken IgY-HRP were decided,and the indirect ELISA for the detection of V.parahaemololyticus was established.The suitable mass concentration of the first antibody was 15 μg/mL,and the dilution of the second antibody was 1∶10 000.The detection limits of this method were 1.0×105 cfu/mL,and the specificity of the method was also determined,V.parahaemololyticus can be detected,while other bacteria strains can not be detected.The established indirect ELISA is stable,highly specific and sensitive,and it can detect V.parahaemololyticus quickly.

Objective To compare the effect of various types of newborn bovine sera on culture in vitro of BHK-21 cells.Methods BHK-21 cells were cultured with six kinds of newborn bovine sera,manufactured by various manufacturers or of different batches manufactured by the same manufacturer,respectively,and observed for morphology,increased fold in quantity,and survival time after subculture.Results The cells cultured with serum No.6 grew well,of which the increased fold in quantity was high and survival time was long as compared with those cultured with other newborn bovine sera.The culture efficacy of cells with serum No.3 was only second to that with serum No.6.However,no monolayer was formed in the cells on day 4 after culture with sera No.2,4 and 5,and little growth was observed in the cells cultured with serum No.1.Conclusion Significant differences were observed in culture efficacies of BHK-21 cells with newborn bovine sera manufactured by various manufacturers or of different batches manufactured by the same manufacturer.