Establishment and application of a quantitative real-time PCR detection system for Brucella S2 vaccine strain / 中华地方病学杂志

Objective:To establish a quantitative real-time PCR detection system for Brucella S2 vaccine strain. Methods:Based on the differences in the entire genome sequence between Brucella S2 vaccine strain and other reference strains of Brucella, primers and probes were designed to establish a quantitative real-time PCR detection system for Brucella S2 vaccine strain. The DNA of 22 reference strains of Brucella and 8 non- Brucella control strains were obtained from the National Institute for Infectious Disease Control and Prevention of the Chinese Center for Disease Control and Prevention. At the same time, environmental samples were obtained from the brucellosis vaccine manufacturers, and bacterial DNA from environmental samples was extracted using a blood/tissue genomic DNA extraction kit. The obtained DNA was pre-amplified by conventional PCR, and then subjected to quantitative real-time PCR secondary amplification (nested fluorescence quantitative PCR) using the amplified PCR product as a template. The specific fluorescence curve and corresponding number of cycles (Ct value) were observed, and the sensitivity was tested. Results:The quantitative real-time PCR detection system established did not detect specific fluorescence curves (without Ct values) for 21 reference strains of Brucella and 8 non- Brucella control strains, except for S2 vaccine strains. The established detection system had a minimum detection limit of 4.34 fg (genomic DNA) for detecting the DNA of Brucella S2 vaccine strain; DNA of Brucella S2 vaccine strain was detected in 3 of the 14 environmental samples collected. Conclusion:The quantitative real-time PCR detection system established can detect Brucella S2 vaccine strain in samples, with good sensitivity and specificity.

Etiological analysis of clinical cases of live attenuated varicella vaccine / 中国生物制品学杂志

@#Objective To analyze the etiology of clinical cases of live attenuated varicella vaccine.Methods 64 samples of varicella vesicle fluid from 49 patients clinically diagnosed as varicella cases in phase Ⅲ clinical trial of live attenuated varicella vaccine in enterprises(the test site was Henan Province) were collected,extracted for DNA,and distinguished for wild-type strains and vaccine strains by PCR and restriction fragment length polymorphism(PCR-RFLP).Genotype was analyzed using the genotyping method recommended at the international(varicella-zoster virus,VZV) nomenclature meeting2008.Results 64 vesicle fluids were all wild-type strains(Pst Ⅰ~+Sma Ⅰ~-BssH Ⅱ~-Nae Ⅰ~-),and no vaccine-related cases occurred.All 49 isolates belonged to Clade 2.Additionally,compared with Clade 2,a synonymous mutation(T→C) in SNP18 082 was detected in all 49 isolates,and a mutation(C→A) in SNP790 was detected in one isolate.Conclusion In the clinical trial of live attenuated varicella vaccine in Henan Province,all the clinical cases were caused by infection of wild-type strain which belonged to Clade 2 genetic branch.

Research progress in antigenic evolution of influenza A (H3N2) virus / 中华微生物学和免疫学杂志

Influenza viruses are common pathogens causing respiratory infections in humans. Among the four seasonal influenza viruses, influenza A virus H3N2 has become the leading cause of seasonal influenza illness and death, posing a great threat to public health and the economy. Since it first emerged and caused a pandemic in 1968, H3N2 has been circulating repeatedly in human beings and continually evades host immune attack by antigenic drift, resulting in a decrease in vaccine efficacy. In this paper, the antigenic evolution of influenza A virus H3N2, the impact of antigenic evolution on the selection of vaccine strains and some models for predicting the evolution of influenza viruses were analyzed and reviewed, which paved the road for understanding the antigenic evolution of influenza virus and vaccine development.

Two cases of measles after vaccination with measles mumps and rubella combined live attenuated vaccine / 上海预防医学

Measles is an acute respiratory infectious disease caused by the measles virus. It is highly infectious and easy to occur in children. It causes many serious complications such as tracheitis， otitis media and pneumonia. Since the promotion of the measles vaccine in China， the measles epidemic has been effectively controlled. From June 1， 2020， the immunization procedure of measles-containing vaccine for children in Shanghai has been adjusted to one dose of measles， mumps and rubella combined live attenuated vaccine （MMR） at the age of 8 months， 18 months and 6 years. There is generally no local reaction after the injection of the MMR vaccine. A few individuals may have transient fever and scattered rash， which generally fade away by themselves. However， because it is a live vaccine， it may cause vaccine related diseases in extremely rare cases. This paper reports two cases of measles after vaccination with the MMR vaccine.

Synergistic Effect of NF-κB Signaling Pathway Inhibitor and Oncolytic Measles Virus Vaccine Strain against Lung Cancer and Underlying Mechanisms / 中国肺癌杂志

BACKGROUND@#Lung cancer is one of the leading causes of cancer-related morbidity and mortality. Oncolytic virotherapy is an emerging therapeutic modality that utilizes replication-competent viruses to destroy cancers. As a powerful tool to kill tumor cells with excellent safety profile, attenuated measles virus of the Edmonston strain (MV-Edm) has been widely applied in the development of tumor therapy and preclinical trials. The aim of this study was to investigate the synergistic effect of nuclear factor kappa B (NF-κB) signaling pathway inhibitor and oncolytic measles virus vaccine against lung cancer and the involved mechanisms.@*METHODS@#Using Western blot to detect MV-Edm infection of A549 and H1299 were infected by MV-Edm alone or used the NF-κB pathway inhibitor PS1145/cell autophagy related siRNA, expression level of p-IκBα, IκBα, PARP and BAX were determined by western blot. Using flow cytometry to analysis the rate of apoptosis, and using MTT [3-(4,5)-dimethylthiahiazo(-z-y1)-3,5-di-phenytetrazoliumromide] method to detect the cell survival rate.@*RESULTS@#Inhibition of cell autophagy could obviously inhibit the MV-Edm infection induced the NF-κB pathway activation in A549 and H1299. In MV-Edm infected A549 and H1299, p-IκBα level increased and IκBα level decreased over infection time, compared with control group. Inhibition of the NF-κB pathway by PS1145 could promote the apoptosis of MV-Edm infected A549 and H1299 and amplify the tumor killing effect.@*CONCLUSIONS@#The combination of NF-κB signaling pathway inhibitor pS1145 and oncolytic measles virus vaccine strains can promote the apoptosis of human lung cancer cells A549 and H1299 and enhance their oncolytic effect.

Pathogenic potential of Brucella ovis field isolates with different genotypic profile and protection provided by the vaccine strain B. ovis ΔabcBA against B. ovis field isolates in mice / Vacina viva atenuada Brucella ovis Δ abcBA encapsulada protege camundongos frente a desafios de B. ovis isoladas de campo

Brucella ovis causes economic and reproductive losses in sheep herds. The goal of this study was to characterize infection with B. ovis field isolates in a murine model, and to evaluate protection induced by the candidate vaccine strain B. ovis ΔabcBA in mice challenged with these field isolates. B. ovis field strains were able to colonize and cause lesions in the liver and spleen of infected mice. After an initial screening, two strains were selected for further characterization (B. ovis 94 AV and B. ovis 266 L). Both strains had in vitro growth kinetics that was similar to that of the reference strain B. ovis ATCC 25840. Vaccination with B. ovis ΔabcBA encapsulated with 1% alginate was protective against the challenge with field strains, with the following protection indexes: 0.751, 1.736, and 2.746, for mice challenged with B. ovis ATCC25840, B. ovis 94 AV, and B. ovis 266 L, respectively. In conclusion, these results demonstrated that B. ovis field strains were capable of infecting and inducing lesions in experimentally infected mice. The attenuated vaccine strain B. ovis ΔabcBA induced protection in mice challenged with different B. ovis field isolates, resulting in higher protection indexes against more pathogenic strains.(AU)

Brucella ovis é responsável por perdas econômicas e reprodutivas em rebanhos ovinos. O objetivo deste trabalho foi caracterizar a infecção com as cepas isoladas de campo de B. ovis em modelo murino e avaliar a eficiência vacinal da mutante B. ovis ΔabcAB para proteção contra desafio com as cepas isoladas de campo. Foram utilizadas sete cepas isoladas de campo foram capazes de colonizar e provocar lesões no fígado e no baço de camundongos após sete dias pós-infecção. Após triagem, duas cepas foram selecionadas para a melhor caracterização (B. ovis 94 AV and B. ovis 266L). Ambas apresentaram crescimento em placa de cultivo semelhante ao da cepa de referência B. ovis ATCC 25840. A vacinação com a cepa de Brucella ovis ΔabcBA encapsulada com alginato a 1% foi capaz de proteger camundongos desafiados com as cepas isoladas de campo, com os seguintes índices de proteção: 0,751, 1,736 e 2,746, para camundongos desafiados com B. ovis ATCC 25840, B. ovis 94 AV e B. ovis 266 L, respectivamente. Estes resultados demonstraram que as cepas isoladas de campo de B. ovis são capazes de infectar e induzir lesão em camundongos experimentalmente infectados. O uso da cepa mutante atenuada B. ovis ΔabcBA para vacinação de fêmeas C57BL/6 desafiados com diferentes cepas de B. ovis induziu proteção nos camundongos desafiados com diferentes cepas de B. ovis. Deste modo, mostrando-se eficiente na proteção das cepas de campo de B. ovis.(AU)

Genetic characterization of varicella zoster virus in Jilin province in 2017, China / 中华实验和临床病毒学杂志

Objective@#To clarify the genotype of varicella-zoster virus (VZV) in Jilin province in 2017, and to discriminate between vaccine strain and wild-type strain.@*Methods@#Vesicle fluid and throat swab samples were collected from 10 individuals with suspected VZV in Jilin province from January to March of 2017. Real-time fluorescent quantitative PCR was used to detect viral nucleic acid. Specific regions of ORF22, ORF38 and ORF62 of VZV were amplified by PCR. Viral genotype was determined by five SNPs of ORF 22 and vaccine strain or wild-type strain was distinguished by four SNPs of ORF 38 and ORF 62. The results were analyzed with MEGA5 and BioEdit software, using the VZV reference strain sequences from GenBank.@*Results@#VZV-positive strains were detected in 10 samples, all belonged to Clade 2. There was a synonymous mutation (C→T) in position 38 048 of JL17-7 strain. The nucleotide homology of ORF22 showed that all 10 samples were on the same branch with the Clade 2 referenced strains. Compared with Clade 2 referenced strains, the homology of nucleotide and amino acid for all 10 samples were 99.5%-100% and 99.3%-100%, respectively. The four specific SNPs of ORF38 and ORF62 in 10 samples were A-T-T-T, which were consistent with wild-type strain.@*Conclusions@#This study reveals that the VZV strains circulating in Jilin province in 2017 were all wild-type strains belonging to Clade 2.

Safety of yellow fever vaccine strain (Tiantan strain) in China and WHO vaccine strain 17D-213 / 中华微生物学和免疫学杂志

Objective@#To study the intracerebral pathogenicity of the yellow fever vaccine strains of Tiantan strain used in China and WHO vaccine strain 17D-213 in mice.@*Methods@#Mice of different ages and strains were intracerebrally injected with same amount of Tiantan strain and 17D-213 strain. The death and survival of mice were observed and recorded. The LD50/ml and half survival time of the two vaccine strains were compared and analyzed.@*Results@#There was no difference in LD50/ml between the Tiantan strain and 17D-213 strain when used through intracerebral injection in one-day-old suckling mice, 7-9 g mice or 12-14 g mice. Moreover, no significant difference in survival trend was found in 7-9 g mice or 12-14 g mice injected with the two vaccine strains. However, the two strains had statistically different influences on the survival trend of one-day-old suckling mice. The half survival time of the Tiantan strain was 11 d, while that of the WHO vaccine strain 17D-213 was 6 d. Excepting in NIH mice, no significant differences in LD50/ml were detected between the same amount of two strains in BALB/c, KM, ICR or C57 mice.@*Conclusions@#The yellow fever Tiantan vaccine strain and WHO vaccine strain 17D-213 have no significant difference in the intracerebral pathogenicity in mice of different ages and strains with good safety.

Safety of yellow fever vaccine strain (Tiantan strain) in China and WHO vaccine strain 17D-213 / 中华微生物学和免疫学杂志

Objective To study the intracerebral pathogenicity of the yellow fever vaccine strains of Tiantan strain used in China and WHO vaccine strain 17D-213 in mice. Methods Mice of different ages and strains were intracerebrally injected with same amount of Tiantan strain and 17D-213 strain. The death and survival of mice were observed and recorded. The LD50/ml and half survival time of the two vaccine strains were compared and analyzed. Results There was no difference in LD50/ml between the Tiantan strain and 17D-213 strain when used through intracerebral injection in one-day-old suckling mice, 7-9 g mice or 12-14 g mice. Moreover, no significant difference in survival trend was found in 7-9 g mice or 12-14 g mice injected with the two vaccine strains. However, the two strains had statistically different influences on the survival trend of one-day-old suckling mice. The half survival time of the Tiantan strain was 11 d, while that of the WHO vaccine strain 17D-213 was 6 d. Excepting in NIH mice, no significant differences in LD50/ml were detected between the same amount of two strains in BALB/c, KM, ICR or C57 mice. Con-clusions The yellow fever Tiantan vaccine strain and WHO vaccine strain 17D-213 have no significant difference in the intracerebral pathogenicity in mice of different ages and strains with good safety.

Successive subculture and genetic stability of hepatitis A virus vaccine strain H2 / 中华实验和临床病毒学杂志

Objective@#To investigate the genetic stability of virus seed H2M20K7 (K7) of live attenuated Hepatitis A virus H2 strain (HAV, H2 strain) for production of hepatitis A (Live) vaccine, lyophilized after continuous passages.@*Methods@#The virus seed K7 of H2 strain was proliferated and passaged in KMB17 cells in cell factories. Viruses of different passages were harvested after continuous passages. Virus RNA was extracted and the complete genomes of different virus passages (K7, K10, K11, K13, K15, K18) were sequenced by using next-generation deep sequencing. The mutation rates of different passages were compared. The infectivity titers of different virus passages of H2 strain were tested by ELISA.@*Results@#The mutation rates of complete genomes of different passages were low after continuous passages of master virus seed. The structure of gene was stable and non-synonymous mutation rate was lower than 0.57%. The mutation rate of 5 ’non-coding regions was lower than 0.1%. There was no significant mutation in VP1/2 A and 2C virulence site. The infectious titers of H2 strains of different passages were within 7.76-8.50 lgCCID50/ml. No statistically significant difference was found in this study.@*Conclusions@#The gene structure of the master virus seed, working seed and different passages of H2M20K7 after subculture was stable and the mutation rate was low. No significant mutation was found in 5’non-coding regions, and the critical virulence sites such as VP1/2 A, 2B and 2C showed attenuated characteristics with low mutation rate. Virulence of the virus did not changed. The H2 strain maintained stable viral infectivity and genetic stability and comply with the requirements as virus seed for vaccine manufacturing.

Evolution of influenza A(H3N2) virus hemagglutinin (HA1) gene in Shangluo, China, 2014-2015 / 中国人兽共患病学报

Comparative analysis of the variations in HA 1 gene of the influenza A (H3N2) virus and the vaccine recommended were conducted in Shangluo city of China,during the surveillance year of 2014-2015.In this study,we collected the samples of H3N2 subtype strain from the Shanglou City of China during the surveillance period of 2014-2015.The strain was cultured in MDCK cells,HA gene fragment was amplified by RT-PCR and the nucleotide sequence was determined.Sequence alignment was performed using the clustax2.1 software.The phylogenetic tree was constructed by Mega6.0 software and was analyzed by Neighboring-joining method.Results showed that the homology of isolated strain during 2014-2015 was 97.2 ％-99.9％ and homology with the recommended vaccine strain A/Texas/50/2012 was 97.3％-98.5％.The amino acid sequence of the HA 1 gene of the isolated strain was compared with that of the vaccine strain.The major antigenic determinants of the isolates in 2014,having mutations were section B,Y159F,S198P,while the major antigenic determinants of isolates in 2015,having amino acid mutations were A zone G142R,B region S159F,S198P.These results indicated that the key antigenic determinant of influenza H3N2 subtype strain in Shangluo City has changed in 2014-2015 and A/Texas/50/2012 vaccine component is no more effective.Hence,there is an urgent need to update the influenza H3N2 subtype vaccine components and in future we should be deeply concerned about the evolution ofinfluenza H3N2 gene trends.

Evolución Genética del Virus Influenza A(H1N1) pdm09 en Paraguay 2009-2016

RESUMEN Una cepa triple reasortante del virus Influenza A emergió en al año 2009 dando origen a una pandemia que alcanzó a Paraguay en junio del mismo año. Con el fin de investigar la evolución genética del virus influenza A (H1N1) pdm09 en Paraguay fueron analizadas las secuencias nucleotídicas del Gen de la Hemaglutinina de 20 cepas de Influenza A(H1N1)pdm09, aisladas en el Centro Nacional de Influenza de Paraguay entre los años 2009 y 2016, y secuenciadas en el Centro Colaborador de OPS/OMS en Atlanta USA. El análisis filogenético muestra la circulación de al menos 5 grupos genéticos bien diferenciados de Influenza A(H1N1)pdm09 en Paraguay desde el 2009. Solamente los virus aislados en el 2016 pertenecen al sub Grupo genético 6B.1 en el cual se encuentra la actual cepa vacunal A/Michigan/45/2015 recomendada para el hemisferio Sur desde el año 2017. Los virus circulantes en años anteriores pertenecen a grupos antigénicamente indistinguibles de la cepa vacunal previa A/California/7/2009. No se encontraron diferencias resaltantes en las secuencias de los virus, relacionadas a severidad clínica ni a distribución geográfica. Los resultados de este estudio reafirman la necesidad de una vigilancia virológica sistemática para orientar el establecimiento de estrategias adecuadas de prevención y control de la influenza.

ABSTRACT A triple reassortant strain of Influenza A virus emerged in 2009, leading to a pandemic that reached Paraguay by June the same year. In order to investigate the genetic evolution of influenza A (H1N1)pdm09 virus in Paraguay, we analized the nucleotide sequences of the Hemagglutinin gene of 20 Influenza A (H1N1)pdm09 strains, isolated at the Paraguayan National Influenza Centre between 2009 and 2016, and sequenced at the PAHO/WHO Collaborating Center in Atlanta, USA. Phylogenetic analysis shows the circulation of at least 5 well-differentiated genetic groups of Influenza A (H1N1) pdm09 in Paraguay since 2009. Only the viruses isolated in 2016 belong to genetic subgroup 6B.1, the same as the current vaccine strain A/Michigan/45/2015, recommended for the Southern hemisphere since 2017. The viruses circulated previous years belong to groups antigenically indistinguishable from the previous vaccine strain A/California/7/2009. No significant differences were found in sequences of the viruses, related to clinical severity or geographic distribution. The results of this study reaffirm the need for systematic virological surveillance to guide the establishment of adequate strategies for the prevention and control of influenza.

Serotypes of duck Escherichia coli and strain selection for vaccine / 中国人兽共患病学报

In order to obtain the serotype distribution of E.coli from duck and to screen the vaccine bacterial strains,the serotype identifications and biological characteristics of E.coli were analyzed in recent years from Shandong,Hebei and other areas of commercial duck field;selections of vaccine strains were detected by the virulence and immunogenicity.Totally 44 isolated bacterial strains of E.coli from duck were identified to a total of six serotypes:O78,O93,O76,O2,O92 and O32.The O78 serotype was the dominant serotype,accounting for 56.8％ (25/44);O93 serotype for 15.9％ (7/44) according to bacterial Oantigen typing.The strain SD (O78 serotype) was confirmed to have strong virulence and good immunogenicity.The O78,O93 and O76 are the dominant serotypes of duck E.coli in the study areas.The SD strain could be used as the candidate for the next development of inactivated vaccine.

Cross-neutralization between three mumps viruses & mapping of haemagglutinin-neuraminidase (HN) epitopes.

Background & objectives: The reports from the countries where mumps vaccine is given as routine immunization suggest differences in mumps virus neutralizing antibody titres when tested with vaccine and wild type viruses. Such reports are unavailable from countries like India where mumps vaccine is not included in routine immunization. We, therefore, undertook this study to understand the cross-neutralization activity of Indian mumps viruses. Methods: By using commercial mumps IgG enzyme immunoassay (EIA) and a rapid focus reduction neutralization test (FRNT), a panel of serum samples was tested. The panel consisted of 14 acute and 14 convalescent serum samples collected during a mumps outbreak and 18 archived serum samples. Two wild types (genotypes C and G) and Leningrad-Zagreb vaccine strain (genotype N) were used for the challenge experiments and FRNT titres were determined and further compared. The HN protein sequence of three mumps viruses was analyzed for the presence of key epitopes. Results: All serum samples effectively neutralized mumps virus wild types and a vaccine strain. However, significantly lower FRNT titres were noted to wild types than to vaccine strain (P<0.05). The comparison between EIA and FRNT results revealed 95.6 per cent agreement. No amino acid changes were seen in the epitopes in the Indian wild type strains. All potential N-linked glycosylation sites were observed in Indian strains. Interpretation & conclusions: Good cross-neutralization activity was observed for three mumps virus strains, however, higher level of FRNT titres was detected for mumps virus vaccine strain compared to Indian wild type isolates.

Study on the differences of glycoprotein gene between rabies virus epidemic strains from Guizhou province and vaccine strains / 中华微生物学和免疫学杂志

Objective To study the differences of glycoprotein gene (G gene) between rabies virus epidemic strains of Guizhou province in recent years and vaccine strains,and to provide scientific basis for the development of rabies vaccine and establishment of effective control and prevention measures.Methods RT-PCR assay was used to amplify the G gene of rabies positive brain tissues samples of human and dog derived from Guizhou province in recent years.The amplification products were sequenced and comparatively analyzed with that of vaccine strains by using bioinformatics software.Results Eight full-length G gene sequences were obtained by RT-PCR amplification,sequencing and splicing.The homogeny of G gene between 8 epidemic strains of Guizhou province and 9 vaccine strains were 82.0％-94.1％ and 87.6％-97.5％ on nucleotide and deduced amino acid level,respectively,and the highest homogeny were found with the human vaccine strain CTN (87.0％-94.1％ for nucleotide and 93.7.％-97.5％ for amino acid) among the 6 human rabies vaccine strains,while highest homogeny were found with strain Flury (83.9％-84.6％ for nucleotide and 91.1％-93.0％ for amino acid) among the three animal vaccine strains.Besides,among the 8 epidemic strains from Guizhou province,strain GZ09 collected in the year of 2005 was of the highest homogeny with human rabies vaccine strain CTN and animal rabies vaccine strain Flury,while strain GZ30 collected in the year of 2010 was of lowest homogeny with human rabies vaccine strain CTN and animal rabies vaccine strain Flury.Moreover,phylogenetic analysis based on the G gene indicated that the relationship of 8 epidemic strains derived from Guizhou,the 9 vaccine strains and genotype 1 Lyssavirus were clustered to a same branch.Vaccine strain CTN among the 9 vaccine strains was closest to the 8 epidemic strains,and the other 8 vaccine strains were relatively more distant from the epidemic strains of Guizhou province.In addition,phylogenetic analysis indicated that among the 8 epidemic strains from Guizhou province,strain GZ09 collectcd in the year of 2005 was of closest evolutionary relationship to CTN,while the other 7 epidemic strains were relatively more distant from CTN.Conclusion This study confirmed on the G gene level that rabies virus strains circulated in Guizhou province in recent years and the vaccine strains used in China belonged to rabies virus genotype 1,and the virus strains circulated in Guizhou province in recent years is of smallest difference with the human vaccine strain CTN and animal vaccine strain Flury.Besides,as time goes on,the difference between the epidemic strain and the vaccine strains becomes more and more obvious.The results of this study will provide scientific basis for the development of rabies vaccine and establishment of effective control and prevention measures.

Molecular Identification of the Vaccine Strain from the Inactivated Rabies Vaccine

Since 1994, several different inactivated rabies vaccines have been used to immunize domestic animals such as dogs, cats, and cattle in South Korea. The Korean Veterinary Authority has conducted safety and efficacy testes of inactivated vaccines using laboratory animals. In this study, we applied a molecular method to investigate the genetic characterization of the rabies virus (RABV) genes in six commercial inactivated rabies vaccines, and determined the efficiency of two extraction reagents (i.e., sodium citrate or isopropyl myristate) to separate the vaccine antigens from the antigen/adjuvant complexes. Six partial nucleocapsid (N: 181 bp) and five partial glycoprotein (G: 306 bp) genes were successfully amplified with specific primer sets, which demonstrated that sodium citrate is more efficient than isopropyl myristate in extracting viral RNA from inactivated gel vaccines. In addition, we identified the viral strain of the vaccine by analyzing the nucleotide sequences of the N and the G genes. The nucleotide similarity of the partial N and G genes ranged from 97.1 to 99.4% and from 91.8 to 100% among rabies vaccine strains, respectively, indicating that each manufacturer used different rabies virus strains to produce their vaccines. The molecular method used in this study could also be used to identify viral strains in other inactivated vaccines.

Rotavirus A genotype G1P[8]: a novel method to distinguish wild-type strains from the Rotarix® vaccine strain

Rotaviruses are important enteric pathogens for humans and animals. Group A rotaviruses (RV-A) are the most common agents of severe gastroenteritis in infants and young children and vaccination is the most effective method to reduce RV-A-associated diseases. G1P[8], the most prevalent RV-A genotype worldwide, is included in the RV-A vaccine Rotarix®. The discrimination between wild-type G1P[8] and vaccine G1P[8] strains is an important topic in the study of RV-A epidemiology to manage outbreaks and to define control measures for vaccinated children. In this study, we developed a novel method to segregate the wild-type and vaccine strains using restriction endonucleases. The dsRNA from the Rotarix® vaccine was sequenced and the NSP3 gene was selected as the target gene. The vaccine strain has a restriction pattern that is different than that of wild-type RV-A G1P[8] isolates after digestion with the restriction endonuclease BspHI. This pattern could be used as a marker for the differentiation of wild-type G1P[8] strains from the vaccine strain.

Molecular identification of the vaccine strain from the inactivated oil emulsion H9N2 low pathogenic avian influenza vaccine

In order to control the H9N2 subtype low pathogenic avian influenza (LPAI), an inactivated vaccine has been used in Korea since 2007. The Korean veterinary authority permitted the use of a single H9N2 LPAI vaccine strain to simplify the evolution of the circulating virus due to the immune pressure caused by the vaccine use. It is therefore important to determine the suitability of the vaccine strain in the final inactivated oil emulsion LPAI vaccine. In this study, we applied molecular rather than biological methods to verify the suitability of the vaccine strain used in commercial vaccines and successfully identified the strain by comparing the nucleotide sequences of the hemagglutinin and neuraminidase genes with that of the permitted Korean LPAI vaccine strain. It is thought that the method used in this study might be successfully applied to other viral genes of the LPAI vaccine strain and perhaps to other veterinary oil emulsion vaccines.

Cross-reactivity of Vaccine and Fields Strains of Bovine Coronaviruses in Korea

Bovine coronavirus (BCoV) causes severe diarrhea in newborn calves, and is associated with winter dysentery in adult cattle and respiratory infections in calves and feedlot cattle. Although the Korean BCoV vaccine strain, BC94, was isolated in 1995, there has still been no report of a molecular characterization of the vaccine strain. To characterize the vaccine strain, relationships between BC94 and field strains were investigated, based on sequence analysis and cross-immunity. We determined the complete sequences of the HE, N, and S genes from BC94 and four NVRQS isolates (SUN5, A3, 0501, 0502). Due to its major role in antigenicity, the spike proteins of the BCoVs were analyzed. BC94 showed distinctive genetic divergence from field isolates collected from 2002 to 2005. BC94, SUN5, and A3 had no virulence-specific sequence and there was a single amino acid change, from asparagine to lysine at residue 175, in the polymorphic region. Strains 0501 and 0502 had virulence-specific sequences at all seven sites. Although the recently isolated Korean BCoVs and BC94 were genetically different, the cleavage site of spike genes at 763~768 (KRRSRR) and the antigenic domain II of the spike protein, amino acid position 528, were conserved in all NVRQS isolates. The antigenic relatedness of KCD9, representative of recent Korean BCoVs, was compared with the Korean vaccine strain BC94. KCD9 showed cross-reactivity against BC94 by virus neutralization (VN) test. These results suggest that BC94 is antigenically closely related to field isolates and is still effective as a vaccine strain.

Cross-reactivity of Vaccine and Fields Strains of Bovine Coronaviruses in Korea

Bovine coronavirus (BCoV) causes severe diarrhea in newborn calves, and is associated with winter dysentery in adult cattle and respiratory infections in calves and feedlot cattle. Although the Korean BCoV vaccine strain, BC94, was isolated in 1995, there has still been no report of a molecular characterization of the vaccine strain. To characterize the vaccine strain, relationships between BC94 and field strains were investigated, based on sequence analysis and cross-immunity. We determined the complete sequences of the HE, N, and S genes from BC94 and four NVRQS isolates (SUN5, A3, 0501, 0502). Due to its major role in antigenicity, the spike proteins of the BCoVs were analyzed. BC94 showed distinctive genetic divergence from field isolates collected from 2002 to 2005. BC94, SUN5, and A3 had no virulence-specific sequence and there was a single amino acid change, from asparagine to lysine at residue 175, in the polymorphic region. Strains 0501 and 0502 had virulence-specific sequences at all seven sites. Although the recently isolated Korean BCoVs and BC94 were genetically different, the cleavage site of spike genes at 763~768 (KRRSRR) and the antigenic domain II of the spike protein, amino acid position 528, were conserved in all NVRQS isolates. The antigenic relatedness of KCD9, representative of recent Korean BCoVs, was compared with the Korean vaccine strain BC94. KCD9 showed cross-reactivity against BC94 by virus neutralization (VN) test. These results suggest that BC94 is antigenically closely related to field isolates and is still effective as a vaccine strain.