Primary Hamster Kidney Cells Research Articles

Genetic and neuroattenuation phenotypic characteristics and their stabilities of SA14-14-2 vaccine seed virus

Japanese encephalitis (JE) live attenuated vaccine SA14-14-2 is the most widely used JE vaccine in the world. Large-scale clinical trials have demonstrated satisfactory safety and efficacy profiles. The establishment of genetic and attenuated neurovirulence characteristics and their stabilities of SA14-14-2 virus are important in relation to vaccine safety in humans. Therefore, several researchers have studied and analyzed the full-length gene sequences of the SA14-14-2 virus strain. However, sequencing results have shown a significant difference. Here, we further studied the full-length sequence of three class seed virus banks of the vaccine as well as two vaccine viruses with different passages in primary hamster kidney cells, and compared them with our original stored SA14 parent virus (low passage in mouse brain). The full-length gene sequence determined in this study indicates there were 57 nucleotide and 25 amino acid substitutions of the SA14-14-2 strain compared to its parental SA14 virus strain. The full-length sequences of the three class seed bank viruses and the vaccine virus PHKC8 were completely identical among them, but the working seed virus passaged in primary hamster kidney cells for 17 generations (PHKC17) had a single nucleotide change at the 5′ NCR. Both KM and ICR mice tested by intracerebral (i.c.) or subcutaneous (s.c.) routes with the three class seed viruses and vaccine viruses with ≥5.7 lgpfu/mL remained healthy, but all the mice inoculated with the SA14 parental virus strain died as early as day 5 post-inoculation. The present study provided new information on the full-length gene sequence and attenuated neurovirulence of SA14-14-2. They can be used as a reference sequence for vaccine quality control and surveillance of neurovirulence reversion following vaccination. Moreover, the present results further demonstrated the high genetic and phenotypic stabilities of the SA14-14-2 virus, suggesting the neurovirulence reversion of the vaccine strain will be highly unlikely.

A novel dengue virus serotype 1 vaccine candidate based on Japanese encephalitis virus vaccine strain SA14-14-2 as the backbone.

To develop a potential dengue vaccine candidate, a full-length cDNA clone of a novel chimeric virus was constructed using recombinant DNA technology, with Japanese encephalitis virus (JEV) vaccine strain SA14-14-2 as the backbone, with its premembrane (prM) and envelope (E) genes substituted by their counterparts from dengue virus type 1 (DENV1). The chimeric virus (JEV/DENV1) was successfully recovered from primary hamster kidney (PHK) cells by transfection with the in vitro transcription products of JEV/DENV1 cDNA and was identified by complete genome sequencing and immunofluorescent staining. No neuroinvasiveness of this chimeric virus was observed in mice inoculated by the subcutaneous route (s.c.) or by the intraperitoneal route (i.p.), while some neurovirulence was displayed in mice that were inoculated directly by the intracerebral route (i.c.). The chimeric virus was able to stimulate high-titer production of antibodies against DENV1 and provided protection against lethal challenge with neuroadapted dengue virus in mice. These results suggest that the chimeric virus is a promising dengue vaccine candidate.

Comparing the immunogenicity and safety of 3 Japanese encephalitis vaccines in Asia-Pacific area: A systematic review and meta-analysis

Japanese encephalitis virus (JEV), a leading cause of Japanese encephalitis (JE) in children and adults, is a major public health problem in Asian countries. This study reports a meta-analysis of the immunogenicity and safety of vaccines used to protect infants or children from JE. Three types of JE vaccine were examined, namely, Japanese encephalitis live-attenuated vaccine (JEV-L), Japanese encephalitis inactivated vaccine (Vero cell) (JEV-I(Vero)), and Japanese encephalitis inactivated vaccine (primary hamster kidney cell) (JEV-I(PHK)). These vaccines are used to induce fundamental immunity against JE; however, few studies have compared their immunogenicity and safety in infants and young children less than 2 years of age. Data were obtained by searching 5 databases: Web of Science, PubMed, China National Knowledge Infrastructure, the China Wanfang database, and the Cochrane database. Fifteen articles were identified and scored using the Jadad score for inclusion in the meta-analysis. Random effect models were used to calculate the pooled seroconversion rate and adverse reaction rate when tests for heterogeneity were significant. The results showed that the pooled seroconversion rate for JEV-I(PHK) (62.23%) was lower than that for JEV-I(Vero) (86.49%) and JEV-L (83.52%), and that the pooled adverse reaction rate for JEV-L (18.09%) was higher than that for JEV-I(PHK) (10.08%) and JEV-I(Vero) (12.49%). The pooled relative risk was then calculated to compare the seroconversion and adverse reaction rates. The results showed that JEV-I(Vero) and JEV-L were more suitable than JEV-I(PHK) for inducing fundamental immunity to JE in infants and children less than 2 years of age.

Genetic and phenotypic properties of vero cell-adapted Japanese encephalitis virus SA14-14-2 vaccine strain variants and a recombinant clone, which demonstrates attenuation and immunogenicity in mice.

The live-attenuated Japanese encephalitis virus (JEV) SA14-14-2 vaccine, produced in primary hamster kidney cells, is safe and effective. Past attempts to adapt this virus to replicate in cells that are more favorable for vaccine production resulted in mutations that significantly reduced immunogenicity. In this study, 10 genetically distinct Vero cell-adapted JEV SA14-14-2 variants were isolated and a recombinant wild-type JEV clone, modified to contain the JEV SA14-14-2 polyprotein amino acid sequence, was recovered in Vero cells. A single capsid protein mutation (S66L) was important for Vero cell-adaptation. Mutations were also identified that modulated virus sensitivity to type I interferon-stimulation in Vero cells. A subset of JEV SA14-14-2 variants and the recombinant clone were evaluated in vivo and exhibited levels of attenuation that varied significantly in suckling mice, but were avirulent and highly immunogenic in weanling mice and are promising candidates for the development of a second-generation, recombinant vaccine.

Construction and preliminary investigation of a novel dengue serotype 4 chimeric virus using Japanese encephalitis vaccine strain SA14-14-2 as the backbone

For the purpose of developing a novel dengue vaccine candidate, recombinant plasmids were constructed which contained the full length cDNA clone of Japanese encephalitis (JE) vaccine strain SA14-14-2 with its premembrane (PreM) and envelope (E) genes replaced by the counterparts of dengue virus type 4 (DENV4). By transfecting the in vitro transcription products of the recombinant plasmids into BHK-21 cells, a chimeric virus JEV/DENV4 was successfully recovered. The chimeric virus was identified by complete genome sequencing, Western blot and immunofluorescent staining. Growth characteristics revealed it was well adapted to primary hamster kidney (PHK) cells. Its genetic stability was investigated and only one unintentional mutation in 5′-untranslated region (5′-UTR) was found after 20 passages in PHK cells. Neurotropism, neurovirulence and immunogenicity of the chimeric virus were tested in mice. Besides, the influence of JE vaccine pre-immunization on the neutralizing antibody level induced by the chimeric virus was illuminated. To our knowledge, this is the first chimeric virus incorporating the JE vaccine stain SA14-14-2 and DENV4. It is probably a potential candidate to compose a tetravalent dengue chimeric vaccine.

Isolation and Molecular Characterization of Foot and Mouth Disease SAT2 Virus during Outbreak 2012 in Egypt

During April and May 2012, six outbreaks of FMD type SAT 2 were reported in Egyptian governorates: EL- Gharbiya (Kafr Qeretna, Al – Mahala Alkobra, Ebshaway Almalak, Qutoor), local cattle, buffaloes and dairy farms in Kafr El-Shaykh (Alsalmya, Fouah), AL - Minya (Village 3, Samalout, Mansafees, Abo Querkas), Dumyat (Ezbet 20, Kafr saad), ALexandria (Iraq Village, Al Aamerya), EL minufya (Monshaat Gerges, Ashmoun, Shatanoof, Ezbet Algalayla, Berket Alsabaa, Mahalet Sabak),Luxor (Al Gorna) and Al- Sharqiya (Al -Telleen, Menya Al kamh) Governorates. Suspected FMD virus samples (22 Tongue epithelium (TE), 12 vesicular fluid samples (VF)), in addition to, 10 cell culture grown virus of tongue epithelium origin, 6 cell culture grown virus of vesicular fluid origin. The assays used in this study involved CFT and Indirect ELISA typing kits for typing of FMDV virus samples; 3ABC-FMD for differentiation between vaccinated and infected animals in any revealed protective immune response in the examined sera from Egypt 2012 outbreak. PCR as an advanced confirmatory technique was done before submitting FMDV suspected clinical samples for Reference Laboratory of Foot and Mouth Disease in Pirbright, United Kingdom. Samples were isolated on primary bovine kidney and Baby Hamster kidney cell cultures, where all samples gave cytopathic effect. Also, all virus samples inoculated in swiss albino suckling baby mice showed pathognomonic paralysis effect of the virus in hind limbs of mice. Serum samples collected from suspected infected cattle were examined using FMD-3ABC. All positive samples to 3ABC antibodies indicated that the animals were infected by FMDV. FMD antibody detection revealed infection of 62%, 100%, 61%, 37.5%, 45.5% , 72.7% and 66.6% of sera collected from Al-Gharbiya, Kafr El-Shaykh, Dumyat, Alexandria, Al-Monufya, Luxor and Al-Sharqiya respectively. The obtained results of 3ABC antibodies were subsequently confirmed by Indirect ELISA Kit, which revealed that the causative agent of the outbreak was FMDV SAT2. The prefinal confirmatory technique was the Reverse transcription polymerase chain reaction (RT-PCR), which was done to detect clinically suspicious samples using target primers. Also, DNA sequencing of the current FMDV strain was performed and revealed that there was no significant divergence among the isolated strains as the divergence was 0.3 to 2.2% among them, and also there was no significant divergence between the isolated strains and FMDV SAT2/EGY/5/2012 and SAT2/EGY/15/2012 The closest percentage of identity of the isolated strains in comparison with reference strains was 89.4% with FMDV SAT2/LIB/2003. For official confirmation, the isolated viruses were sent to the Reference Laboratory for Foot and Mouth Disease in Pirbright, United Kingdom. Isolation of the causative agent is the first step for diagnosis of the disease. Finally, the PCR product was sequenced and analyzed using the gene bank data to confirm that the new out

The Japanese Encephalitis Vaccine: Worldwide and Korean Status

Japanese encephalitis is the leading cause of viral encephalitis in Asia, where it accounts for up to 50,000 cases. Approximately 20% of affected patients die, and 30-50% of survivors have significant neurological sequelae. Inactivated mouse-brain derived Japanese encephalitis vaccines has been effectively implemented to control the disease effectively in Korea and several other Asian countries. However, the vaccine is expensive and difficult to produce, requires multiple doses, and has been associated with hypersensitivity reactions and rare adverse neurologicale events. The live-attenuated SA14-14-2 vaccine derived from primary hamster kidney (PHK) cells was developed in China and has been used there since 1988. Outside China, it has been licensed and used in Korea and several other Asian countries. This vaccine is effective and inexpensive. However, the lack of precedence for using a PHK cell substrate in a live-attenuated vaccine is a special issue of concern. The WHO working group has recommended additional safety studies in selected high-risk groups, as well as ongoing post-marketing studies to ensure long-term safety. Recently, a new inactivated vaccine and live-attenuated chimeric vaccine have been developed from vero cells. With this background, this article summarized the current status of Japanese encephalitis vaccination worldwide and in Korea. (Korean J Pediatr Infect Dis 2008;15:108-114)

Empirically Derived Live-Attenuated Vaccines Against Dengue and Japanese Encephalitis

Abstract Although studies have been intensively conducted for over 50 years, there is still no commercial vaccine available against dengue disease. The lack of a suitable animal disease model, together with the lack of in vitro or in vivo markers of attenuation have been detrimental to the development of a tetravalent live-attenuated dengue vaccine. Historically, development of monovalent vaccine has substantiated the difficulty in maintaining a balance between a sufficiently attenuated vaccine and immunogenicity. Associating the four serotypes into a single dose (tetravalent vaccine) established the complex interactions among the four dengue serotypes. Here we review the major milestones in the long history of the dengue vaccine development, which recently led to phase 1 clinical studies in children in Thailand. Data from clinical studies with two dengue tetravalent live attenuated vaccines are examined and discussed. In contrast to dengue, vaccines against Japanese encephalitis (JE) virus played a major role in controlling JE disease in Asia. A lived-attenuated vaccine (SA14-14-2) was licensed in China in 1988. More than 100 million doses have been distributed there since. The vaccine appears safe, inducing >95% seroconversion after two doses. However, the vaccine is in need of improvement in terms of quality; there is a need to change the cell substrate (primary hamster kidney cells). This will be a significant step toward an international acceptance of this vaccine, which can play a major role in controlling the disease, particularly if the vaccine is integrated into the routine childhood immunization schedule.

Immunogenicity of wild-type and vaccine strains of Japanese encephalitis virus and the effect of haplotype restriction on murine immune responses

The Chinese live attenuated Japanese encephalitis (JE) virus vaccine clone SA14-14-2 produced in primary hamster kidney (PHK) cells has been adapted to primary dog kidney cells (PDK) for use as a live attenuated human vaccine. In this study we have compared the immunogenicity in mice of SA14-14-2 (PDK) and SA14-14-2 (PHK); also included was the wild-type parent to the vaccine clones, SA14, and another wild-type JE virus strain Nakayama (original). It was found that Balb/c (H-2 d) mice given a single dose of 10 3 or 10 6 p.f.u. of live SA14-14-2 (PHK) virus elicited a superior neutralizing (N) antibody response as compared to the same dosages of live SA14-14-2 (PDK) virus. However, if the vaccine clones were inactivated and administered in a two-dose regime the N antibody response elicited was similar for the two viruses. This observation may be explained by differences in the replication efficiency in vivo of the respective vaccine clones. The humoral immune response to all the virus strains in this study elicited by different inbred mouse strains each carrying a discrete haplotype (Balb/c (H-2 d), C3H (H-2 k), C57BL/6 (H-2 b)) were also assessed using haemagglutination inhibition (HAI) and N assays. Viruses were shown to elicit patterns of high and low N-antibody response depending on the major histocompatibility complex (MHC) make-up of the mouse strains. However, the patterns did not necessarily coincide when HAI and N titre reactivity patterns were compared for the same virus strain.

Human Rabies and Its Prevention: An Overview

Human rabies is underreported, but it clearly is still a frequent medical problem that is virtually confined to tropical countries. Rabies encephalitis remains essentially incurable, and most patients die at home, their terrible symptoms unpalliated by sedatives or analgesics. Recent attempts to cure rabies in well-equipped intensive care units have failed, a situation reemphasizing the importance of preventive measures. The great advances that have been made in understanding the rabies virus and the associated improvements in rabies vaccines have had little or no impact in the tropical endemic zone. Most patients who have been exposed to rabies are still given nervous tissue vaccines for postexposure prophylaxis. An urgent priority is the development of a regimen using tissue culture vaccine that is sufficiently economical to replace nervous tissue vaccine. This has been achieved in China with primary hamster kidney cell vaccine.

Use of Serum and Vaccine in Combination for Prophylaxis Following Exposure to Rabies

Persons seriously wounded by rabid animals in China were administered postexposure rabies treatment with serum and vaccine. Some of these exposures were similar to those described in an attack by a rabid wolf in Iran in 1954, in which some persons developed rabies. Postexposure treatment consisted of the administration of primary hamster kidney cell rabies vaccine and purified equine antirabies serum. Persons with severe wounds from the bites of rabid animals survived without sequelae when the serum and vaccine were administered together within 3 days of the bite, and persons with superficial exposures survived after treatment with vaccine alone. Serum neutralizing antibodies were present 1 year after the patients had received a full course of treatment. Some persons with severe wounds who had not received antiserum and vaccine died of rabies.

Study of the Protective Effect of the Primary Hamster Kidney Cell Rabies Vaccine

Cette etude confirme l'efficacite du vaccin antirabique prepare sur cellules de rein de hamster

Biochemical characterization of a foot-and-mouth disease virus strain attenuated for cattle. Brief report.

Wild-type, virulent (A-24 Cruzeiro subtype) foot-and-mouth disease virus (FMDV), a related attenuated strain and revertants of the attenuated strain were examined by titration on primary bovine kidney (PBK) and baby hamster kidney (BHK-21) cells, as well as, by infection of unweaned mice. Wild type virus grew equally well in all three systems, whereas the attenuated strain had a titer 2-3 log lower in PBK cells than in the other 2 assays. Within 9 successive passages in BHK-21 cells the attenuated strain gave rise to revertants that had regained the growth properties of wild-type virus in PBK cells. After cloning of the attenuated strain by plaque isolations, the same revertant phenotype was obtained within 9 successive passages. Oligonucleotide mapping indicated that the attenuated strain differed from the wild-type and revertants by at least one additional oligonucleotide. Differences in poly(C) length were not found among any of the three strains of FMDV. These results correlate attenuation and virulence with point mutation(s) and not with deletions. Possible reversions in nature with this attenuated strain may be anticipated.

The primary hamster kidney cell rabies vaccine: adaptation of viral strain, production of vaccine, and pre- and postexposure treatment.

The hamster kidney cell rabies vaccine was investigated as a substitute for classical nervous tissue rabies vaccine. The Beijing strain of fixed rabies virus was adapted to primary hamster kidney cells (PHKCs), and four types of rabies vaccine (plain, adjuvant, concentrated, and concentrated adjuvant vaccines) were developed for human use. The potencies of the vaccines met the requirements of the World Health Organization, and these vaccines elicited rather satisfactory antibody responses in volunteers. The postexposure use of vaccine was evaluated in 301 individuals, 97 of whom had been bitten by proven rabid animals. None of the individuals contracted rabies during the observation period. After several years of field trials with both pre- and postexposure vaccines, the evidence indicates that the PHKC rabies vaccines are effective and safe for human use.

Chemically-induced temperature sensitive mutants of dengue virus type 2. I. Isolation and partial characterization.

Temperature sensitive (ts) mutants of dengue virus type 2 (DEN-2, TH-36 isolate) were induced by replication in primary hamster kidney cells treated with 5-azacytidine. Seven ts mutants were obtained from 138 clones isolated by an immunofluorescent cloning technique. Of these 7 ts mutants, 5 were sufficiently stable to permit partial characterization. Complementation was detected at very low but statistically significant levels between some ts mutants at 40 degrees C. Viral double-stranded RNA production was evaluated in LLC-MK2 cells at 30 degrees and 40 degrees C by micro-quantitative complement fixation. The results of complementation tests and RNA production tests indicated that the 4 of 5 stable ts mutants constitute 3 separate complementation groups (2 RNA+ and 1 RNA-groups), while a fifth ts mutant was RNA- but non-complementable. The data presented here indicate that a genetic system can be developed without employing traditional plaque or cytopathology methods. Further, the 5 DEN-2 ts mutants are believed to represent the only set of complementation-positive flavivirus mutants so far isolated.

Malignant transformation of hamster kidney cells by BK virus

Primary hamster kidney cells were transformed by BK virus, a new human papovavirus. Transformed (HKBK) cell produced BK virus T antigen and induced tumors in hamsters that developed antibodies to BK virus T antigen. BK virus was rescued from HKBK cells by Sendai virus-assisted fusion with permissive cells. One out of six cell lines derived from HKBK cell-induced tumors showed the same characteristics as HKBK cells.

Susceptibility of Guinea Pig Cell Cultures to Infection with Cell-Bound and Cell-Free Marek's Disease Virus (JM Strain)

The etiologic agent of Marek's disease (Type II leukosis) has been identified as a highly cell-associated herpes-type virus (7) which manifests its presence in susceptible avian cell cultures by producing characteristic degenerative changes (5,7). The JM strain of Marek's disease virus (MDV) was recently found to infect primary hamster kidney cell cultures (1,2), in contrast to previous findings from which it was concluded that only cell cultures of avian origin were susceptible (4,6). To further investigate the susceptibility of mammalian cells to infection by JM virus (9), primary guinea pig kidney cell cultures were prepared and infected with cell-free and cell-bound JM virus.

Wild birds as eastern (EEE) and western (WEE) equine encephalitis sentinels.

Six species of wild birds were studied for their suitability as sentinels to detect the transmission of EEE and WEE viruses in the Pocomoke Cypress Swamp, Maryland. Blood specimens from birds were tested for virus and for neutralizing antibodies in tubes of primary hamster kidney cell culture. Virus isolations and serological data from bobwhite quail, white-throated sparrows, red-winged blackbirds and English sparrows indicated that transmission of WEE virus began in late July, 1968, and preceded the onset of EEE virus transmission by several weeks and that transmission of both viruses continued into November. Of the species tested, bobwhite quail and white-throated sparrows survived best. Selection and use of wild birds as sentinels are discussed.

Sensitive Procedure for Detecting Residual Viable Virus in Inactivated Rabies Vaccine

A procedure for testing inactivated rabies vaccines of tissue culture origin for residual viable virus is reported in which the vaccine to be tested is passed in primary hamster kidney cell culture (PHK) before mouse inoculation. In preliminary experiments, titrations of rabies virus in which each dilution was passed in PHK before inoculating mice yielded titers 100 to 10,000 times higher than the titers obtained for the same virus by direct mouse inoculation. This rabies virus amplification procedure was evaluated by testing 18 lots of inactivated rabies vaccine of tissue culture origin. No viable virus was found in these vaccine lots when tested by direct intracerebral inoculation of mice. Eight of these 18 lots were found to contain viable virus, however, when tested by passage in PHK cell culture. The significance of low levels of viable virus in rabies vaccines is discussed. It is recommended that the amplification procedure described in this report be used in the safety testing of rabies vaccines of tissue culture origin and that it be evaluated for use in testing other rabies vaccines of low tissue content.

Behavior of bovine ephemeral fever virus in laboratory animals and cell cultures.

A Japanese strain (Yamaguchi) of bovine ephemeral fever virus, propagated in BHK2I-WI2 cells of hamster kidney origin, was used. Adult rats inoculated intracardially or intracerebrally remained healthy, but produced neutralizing antibody, suggesting actual viral growth in the animals. However, no clinical and serological evidence for infection with the virus was obtained in adult sheep, goats, swine and rabbits inoculated intravenously. One-day-old guinea pigs inoculated intracerebrally and adult guinea pigs inoculated intracardially remained healthy and produced no neutralizing antibody. The virus multiplied not only in BHK2I-WI2 cell culture but also in cultures of stable line hamster cells (HmLu-1 and HmT), primary hamster kidney and embryo cells, long-term cultured bovine embryo cells (BEK1), primary calf kidney and embryo cells, and primary suckling rat kidney and embryo cells. However, no evidence for cytopathogenesis and viral growth was obtained in primary cultures of bovine leukocytes, bovine bone marrow. thymus, thyroid and adrenal cells, guinea pig, mouse and chick kidney cells, and mouse and chick embryo cells, and in culture of African green monkey kidney cells (Vero). The virus multiplied best and induced marked cytopathogenesis in BHK21-WI2 cells; lower temperatures such as 34 C or 30 C appeared preferable to obtain constantly better viral yields, although the virus grew more slowly. Similar effects of the incubation temperatures were also obvious in the other susceptible cell cultures, in which the cytopathogenesis was less marked than in BHK21-WI2 cells or absent, roughly coinciding with decreasing grades of viral growth.