A prospective CSFV-PCV2 bivalent vaccine effectively protects against classical swine fever virus and porcine circovirus type 2 dual challenge and prevents horizontal transmission.

Classical swine fever virus (CSFV) infection leading to CSF outbreaks is among the most devastating swine diseases in the pig industry. Porcine circovirus type 2 (PCV2) infection, resulting in porcine circovirus-associated disease (PCVAD), is also a highly contagious disease affecting pig health worldwide. To prevent and control disease occurrence, multiple-vaccine immunization is necessary in contaminated areas or countries. In this study, a novel CSFV-PCV2 bivalent vaccine was constructed and demonstrated to be capable of eliciting humoral and cellular immune responses against CSFV and PCV2, respectively. Moreover, a CSFV-PCV2 dual-challenge trial was conducted on specific-pathogen-free (SPF) pigs to evaluate vaccine efficacy. All of the vaccinated pigs survived and showed no clinical signs of infection throughout the experimental period. In contrast, placebo-vaccinated pigs exhibited severe clinical signs of infection and steeply increased viremia levels of CSFV and PCV2 after virus challenge. Additionally, neither clinical signs nor viral detections were noted in the sentinel pigs when cohabitated with vaccinated-challenged pigs at three days post-inoculation of CSFV, indicating that the CSFV-PCV2 bivalent vaccine completely prevents horizontal transmission of CSFV. Furthermore, conventional pigs were utilized to evaluate the application of the CSFV-PCV2 bivalent vaccine in field farms. An adequate CSFV antibody response and a significant decrease in PCV2 viral load in the peripheral lymph nodes were observed in immunized conventional pigs, suggesting its potential for clinical application. Overall, this study demonstrated that the CSFV-PCV2 bivalent vaccine effectively elicited protective immune responses and the ability to prevent horizontal transmission, which could be a prospective strategy for controlling both CSF and PCVAD in commercial herds.

Cryptococcal granulomas of basal ganglia due to Cryptococcus neoformans in a cat: a case report and literature review.

A 3-year-old spayed female domestic short-haired cat presented with a head turning to the left, circling to the right, seizures, and opisthotonos for approximately one month. Neurological examination revealed a deficit in the postural reaction of the left limbs and visual abnormalities. Forensic computed tomography revealed a hyperattenuating round mass of 1.3 cm diameter with a hypoattenuating center in the right hemisphere. Histopathology showed multifocal granuloma lesions with the major mass mostly affecting the right basal ganglia. Cryptococcus neoformans variety grubii molecular type VNI/ST31 was isolated from the cryptococcal granulomas. This report highlights the epidemiological distribution and differential diagnosis of a feline central nervous system cryptococcosis caused by C. neoformans that occurred in an Asian country.

Characterization and Immunogenicity of Influenza H7N9 Vaccine Antigens Produced Using a Serum-Free Suspension MDCK Cell-Based Platform.

Human infections with avian-origin H7N9 influenza A viruses were first reported in China, and an approximately 38% human mortality rate was described across six waves from February 2013 to September 2018. Vaccination is one of the most cost-effective ways to reduce morbidity and mortality during influenza epidemics and pandemics. Egg-based platforms for the production of influenza vaccines are labor-intensive and unable to meet the surging demand during pandemics. Therefore, cell culture-based technology is becoming the alternative strategy for producing influenza vaccines. The current influenza H7N9 vaccine virus (NIBRG-268), a reassortant virus from A/Anhui/1/2013 (H7N9) and egg-adapted A/PR/8/34 (H1N1) viruses, could grow efficiently in embryonated eggs but not mammalian cells. Moreover, a freezing-dry formulation of influenza H7N9 vaccines with long-term stability will be desirable for pandemic preparedness, as the occurrence of influenza H7N9 pandemics is not predictable. In this study, we adapted a serum-free anchorage-independent suspension Madin-Darby Canine Kidney (MDCK) cell line for producing influenza H7N9 vaccines and compared the biochemical characteristics and immunogenicity of three influenza H7N9 vaccine antigens produced using the suspension MDCK cell-based platform without freeze-drying (S-WO-H7N9), the suspension MDCK cell-based platform with freeze-drying (S-W-H7N9) or the egg-based platform with freeze-drying (E-W-H7N9). We demonstrated these three vaccine antigens have comparable biochemical characteristics. In addition, these three vaccine antigens induced robust and comparable neutralizing antibody (NT; geometric mean between 1016 and 4064) and hemagglutinin-inhibition antibody (HI; geometric mean between 640 and 1613) titers in mice. In conclusion, the serum-free suspension MDCK cell-derived freeze-dried influenza H7N9 vaccine is highly immunogenic in mice, and clinical development is warranted.

The feasibility of field collected pig oronasal secretions as specimens for the virologic surveillance of Japanese encephalitis virus.

Virologic surveillance of Japanese encephalitis virus (JEV) relies on collecting pig blood specimens and adult mosquitoes in the past. Viral RNAs extracted from pig blood specimens suffer from low detecting positivity by reverse transcription PCR (RT-PCR). The oronasal transmission of the virus has been demonstrated in experimentally infected pigs. This observation suggested oronasal specimens could be useful source in the virus surveillance. However, the role of this unusual route of transmission remains unproven in the operational pig farm. In this study, we explore the feasibility of using pig oronasal secretions collected by chewing ropes to improve the positivity of detection in commercial pig farms. The multiplex genotype-specific RT-PCR was used in this study to determine and compare the positivity of detecting JEV viral RNAs in pig's oronasal secretions and blood specimens, and the primary mosquito vector. Oronasal specimens had the overall positive rate of 6.0% (95% CI 1.3%-16.6%) (3/50) to 10.0% (95% CI 2.1%-26.5%) (3/30) for JEV during transmission period despite the negative results of all blood-derived specimens (n = 2442). Interestingly, pig oronasal secretions and female Culex tritaeniorhynchus mosquito samples collected from the same pig farm showed similar viral RNA positive rates, 10.0% (95% CI 2.1%-26.5%) (3/30) and 8.9% (95% CI 2.5%-21.2%) (4/45), respectively (p> 0.05). Pig oronasal secretion-based surveillance revealed the seasonality of viral activity and identified closely related genotype I virus derived from the mosquito isolates. This finding indicates oronasal secretion-based RT-PCR assay can be a non-invasive, alternative method of implementing JEV surveillance in the epidemic area prior to the circulation of virus-positive mosquitoes.

Evaluation of classical swine fever E2 (CSF-E2) subunit vaccine efficacy in the prevention of virus transmission and impact of maternal derived antibody interference in field farm applications.

BACKGROUND: Classical swine fever (CSF) is one of the most devastating pig diseases that affect the swine industry worldwide. Besides stamping out policy for eradication, immunization with vaccines of live attenuated CSF or the CSF-E2 subunit is an efficacious measure of disease control. However, after decades of efforts, it is still hard to eliminate CSF from endemically affected regions and reemerging areas. Most of previous studies demonstrated the efficacy of different CSF vaccines in laboratories under high containment conditions, which may not represent the practical performance in field farms. The inadequate vaccine efficacy induced by unrestrained factors may lead to chronic or persistent CSF infection in animals that develop a major source for virus shedding among pig populations. In this study, a vaccination-challenge-cohabitation trial on specific-pathogen-free (SPF) pigs and long-term monitoring of conventional sows and their offspring were used to evaluate the efficacy and the impact of maternally derived antibody (MDA) interference on CSF vaccines in farm applications. RESULTS: The trials demonstrated higher neutralizing antibody (NA) titers with no clinical symptoms and significant pathological changes in the CSF-E2 subunit vaccine immunized group after CSFV challenge. Additionally, none of the sentinel pigs were infected during cohabitation indicating that the CSF-E2 subunit vaccine could provoke adequately acquired immunity to prevent horizontal transmission. In field farm applications, sows immunized with CSF-E2 subunit vaccine revealed an average of higher and consistent antibody level with significant reduction of CSF viral RNA detection via saliva monitoring in contrast to those of live attenuated CSF vaccine immunized sows possessing diverse antibody titer distributions and higher viral loads. Furthermore, early application of the CSF-E2 subunit vaccine in 3-week-old piglets illustrated no MDA interference on primary immunization and could elicit consistent and long-lasting adequate antibody response suggesting the flexibility of CSF-E2 subunit vaccine on vaccination program determination. CONCLUSIONS: The CSF-E2 subunit vaccine demonstrated significant efficacy and no MDA interference for immunization in both pregnant sows and piglets. These advantages provide a novel approach to avoid possible virus shedding in sow population and MDA interference in piglets for control of CSF in field farm applications.

Ultrasonographic appearance of pseudo-placentational endometrial hyperplasia in a dog.

A 5-year-old, clinically normal intact female Miniature Schnauzer was presented for demonstrative ultrasonography in a seminar. She had two pregnancies in the past and had a natural mating 2 months previously. Ultrasonography revealed a segmental and circumferential mural thickening of the right uterine horn. The endometrium was markedly thickened with multiple organized hyperechoic linear striations, perpendicular to the mucosal surface. Histology revealed focal endometrial hyperplasia resembling the maternal tissue of the normal canine placenta. A diagnosis of spontaneous pseudo-placentational endometrial hyperplasia (PEH) was made. This study described a unique ultrasonographic appearance of PEH, which may facilitate the diagnosis of PEH.

Autochthonous Lactic Acid Bacteria Isolated From Dairy Cow Feces Exhibiting Promising Probiotic Properties and in vitro Antibacterial Activity Against Foodborne Pathogens in Cattle.

Bovine enteric bacterial pathogens are a major cause of health decline in agricultural cattle populations. The identification of host-derived microbiota with probiotic characteristics is key for the development of treatments utilizing pathogen displacement and recolonization by commensal flora. In this study, intestinal microbiota in fecal samples from four Holstein dairy cows were analyzed using 16S ribosomal RNA gene next-generation sequencing, leading to the identification of three Lactobacillus isolates (Lactobacillus gasseri, Lactobacillus reuteri, and Lactobacillus salivarius). By taking advantage of the preferential growth in acidified culture media, bacterial characteristics examination, and restriction fragment length polymorphism analysis of 16S rRNA genes, the three lactic acid bacteria (LAB) strains were successfully isolated. The three LAB isolates possess the prerequisite growth tolerances for probiotic functionality, as well as exhibit effective antimicrobial potency against enteric bacterial pathogens of cattle, including Escherichia coli O157:H7, Mycobacterium avium subspecies paratuberculosis, and Salmonella species (Salmonella enteritidis, Salmonella typhimurium, and Salmonella Dublin). Moreover, the LAB isolates showed significant adhesion to cattle intestine, implying greater survivability potential due to their species specificity when administered in the same host species. The LAB isolates were sensitive to most antibiotics with notable resistances of L. gasseri to streptomycin and L. salivarius to kanamycin. Genes attributed to specific antibiotic resistances demonstrated a low risk of lateral transfer in a conjugation study. Our in vitro results demonstrate the promising probiotic characteristics of these newly identified Lactobacillus strains and their considerable potential to serve as probiotics feed supplements for cows.

Efficacy of fungal immunomodulatory protein to promote swine immune responses against porcine reproductive and respiratory syndrome virus infection.

Fungal immunomodulatory protein (FIP) is one of the bioactive compounds of edible mushrooms, which has been shown to trigger type 1 T helper (Th1) pathway activation in research with mice. This study was designated to assess immunomodulatory effects of recombinant FIP-Flammulina velutipes (rFIP-fve) on swine and the protective efficacy against PRRSV infection. In the in vitro evaluations, rFIP-fve significantly triggered up-regulation of IL-2 and IFN-γ mRNA in porcine PBMCs and stimulated natural killer cytotoxicity. Porcine pulmonary alveolar macrophages (PAMs) treated with rFIP-fve showed prolonged life times, up-regulation of both MHC I and II molecules and enhanced abilities to present antigen. In the in vivo trial, two doses of 2 mg rFIP-fve significantly reduced drops in the CD4/CD8 ratio after PRRSV challenge, and the cytokine mRNA profile of PBMC revealed a tendency of IFN-γ up-regulation and a decrease in IL-10 in the rFIP-treated group. Moreover, administration of rFIP-fve also decreased the PRRSV viremia with 1 log10 in titer (p = 0.07) and alleviated the severity of clinical signs after PRRSV challenge. Conclusively, these results illustrate the in vitro and in vivo immunological changes of rFIP-fve administered to pigs and reveal its potential to be used as an immunomodulatory therapeutic against PRRSV infection.

Evaluation of a type 2 modified live porcine reproductive and respiratory syndrome vaccine against heterologous challenge of a lineage 3 highly virulent isolate in pigs.

Porcine reproductive and respiratory syndrome (PRRS) is one of the most common diseases in the global swine industry. PRRSV is characterized by rapid mutation rates and extensive genetic divergences. It is divided into two genotypes, which are composed of several distinct sub-lineages. The purpose of the present study was to evaluate the cross-protective efficacy of Fostera PRRS MLV, an attenuated lineage 8 strain, against the heterologous challenge of a lineage 3 isolate. Eighteen pigs were randomly divided into mock, MLV and unvaccinated (UnV) groups. The pigs in the MLV group were administered Fostera PRRS vaccine at 3 weeks of age and both the MLV and UnV groups were inoculated with a virulent PRRSV isolate at 7 weeks. Clinically, the MLV group showed a shorter duration and a lower magnitude of respiratory distress than the UnV group. The average days of fever in the MLV group was 3.0 ± 0.5, which was significantly lower than the 6.2 ± 0.5 days of the UnV group (P < 0.001). The average daily weight gains of the mock, MLV and UnV groups were 781 ± 31, 550 ± 44 and 405 ± 26 g/day, respectively, during the post-challenge phase. The pathological examinations revealed that the severity of interstitial pneumonia in the MLV group was milder compared to the UnV group. Furthermore, PRRSV viremia titers in the MLV pigs were consistently lower (101-101.5 genomic copies) than those of the UnV pigs from 4 to 14 DPC. In conclusion, vaccination with Fostera PRRS MLV confers partial cross-protection against heterologous challenge of a virulent lineage 3 PRRSV isolate.

Development and characterization of standard reagents for cell-based prepandemic influenza vaccine products.

Outbreaks of infection by novel avian influenza virus strains in humans cause public health issues worldwide, and the development of vaccines against such novel strains is the most effective method for the prevention of these virus outbreaks. All types of vaccines must be tested for potency before use; thus, quantitative potency assays are needed for influenza vaccines. The single radial immunodiffusion (SRID) assay is considered the gold standard for quantification of influenza virus antigens, and the SRID reference reagents are essential for the determination of vaccine potency. However, it remains debatable whether reference reagents derived from egg-based vaccine platforms can be used to precisely quantify non-egg-derived vaccines; thus, influenza vaccine production using cell-based platforms has attracted increasing attention. To evaluate the utility of reference reagents derived from a cell-based influenza vaccine platform, we prepared cell-based reference reagents from MDCK cell-grown viruses and compared them with egg-derived reference reagents. A primary liquid standard (PLS) was purified from cell-derived candidate influenza vaccine viruses, and hemagglutinin (HA) antigen content was determined by a densitometric method. The produced PLS could be stored at 4°C for more than 10 months. We also established a simple HA protein purification method for goat antiserum preparation, and the performance of the resulting antiserum was compared to that of standard reagents obtained using different production platforms. The results of this study indicate that these reference reagents can be used for both cell-based and egg-based production platforms and that the differences between these two types of platforms are negligible.

Updated phylogenetic analysis of the spike gene and identification of a novel recombinant porcine epidemic diarrhoea virus strain in Taiwan.

New variants of porcine epidemic diarrhoea virus (PEDV) causing a highly contagious intestinal disease, porcine epidemic diarrhoea virus (PED), have resulted in high mortality in suckling pigs across several countries since 2013. After 2015, the prevalence of the genogroup 2b (G2b) PEDVs decreased in a cyclical pattern with endemic seasonal outbreaks occasionally seen. To better understand the genetic diversity of PEDVs recently circulating in Taiwan, full-length spike (S) genes of 31 PEDV strains from 28 pig farms collected during 2016-2018 were sequenced. While the majority of S gene sequences (from 27/28 farms) were closely related to the previous G2b PEDV strains, increased genetic diversities leading to several nonsynonymous mutations scattering in the neutralizing epitopes of the S gene were detected in PEDVs recently circulating in Taiwan. Furthermore, novel recombinant variants, the PEDV TW/Yunlin550/2018 strains exhibiting recombinant events between a previously isolated Taiwan PEDV G2b strain and a wild-type PEDV G1a strain, were identified and further classified into a new genogroup, G1c. These results provide updated information about the genetic diversity of currently circulating PEDVs in the field and could help to develop more suitable strategies for controlling this disease.

A comparably high virulence strain of porcine reproductive and respiratory syndrome virus isolated in Taiwan.

Porcine reproductive and respiratory syndrome virus (PRRSV) has been endemic in Taiwan since 1991. This study aimed to present a highly virulent PRRSV in Taiwan based on farm data collection and both in vitro and in vivo evaluations in virus challenge studies. This virulent PRRSV strain was first noticed on Farm TSYM due to continuously high nursery mortality rate and severe PRRSV-associated pneumonia. In phylogenetic surveillance, the PRRSV TSYM-strain remained in the predominant position for years, even with several other PRRSV strain invasions. In laboratory challenge trials, the TSYM-strain led to prolonged pyrexia, growth retardation, high mortality rates and high viremia titer that similar to the highly pathogenic PRRSV. The TSYM-strain isolate also triggered early interleukin-10 up-regulation and significantly higher infection rates under in vitro experiments. This study provides information of a comparably virulent strain in Taiwan and its appearance in both farm and laboratory levels.

Development of American-Lineage Influenza H5N2 Reassortant Vaccine Viruses for Pandemic Preparedness.

Novel low-pathogenic avian influenza (LPAI) H5N2 viruses hit poultry farms in Taiwan in 2003, and evolved into highly pathogenic avian influenza (HPAI) viruses in 2010. These viruses are reassortant viruses containing HA and NA genes from American-lineage H5N2 and six internal genes from local H6N1 viruses. According to a serological survey, the Taiwan H5N2 viruses can cause asymptomatic infections in poultry workers. Therefore, a development of influenza H5N2 vaccines is desirable for pandemic preparation. In this study, we employed reverse genetics to generate a vaccine virus having HA and NA genes from A/Chicken/CY/A2628/2012 (E7, LPAI) and six internal genes from a Vero cell-adapted high-growth H5N1 vaccine virus (Vero-15). The reassortant H5N2 vaccine virus, E7-V15, presented high-growth efficiency in Vero cells (512 HAU, 107.6 TCID50/mL), and passed all tests for qualification of candidate vaccine viruses. In ferret immunization, two doses of inactivated whole virus antigens (3 µg of HA protein) adjuvanted with alum could induce robust antibody response (HI titre 113.14). In conclusion, we have established reverse genetics to generate a qualified reassortant H5N2 vaccine virus for further development.

Improving immunogenicity of influenza virus H7N9 recombinant hemagglutinin for vaccine development.

Human infections of novel avian influenza A virus (H7N9) emerged in early 2013 and caused about 40% case-fatality through 2017. Therefore, development of influenza H7N9 vaccines is critical for pandemic preparedness. Currently, there are three means of production of commercial influenza vaccines: egg-based, mammalian cell-based, and insect cell-based platforms. The insect cell-based platform has the advantage of high speed in producing recombinant protein. In this study, we evaluate the stability and immunogenicity of two different influenza H7 HA expression constructs generated using the baculovirus system, including membrane-based full-length HA (mH7) and secreted ectodomain-based H7 (sH7). The mH7 construct could form an oligomer-rosette structure and had a high hemagglutinin (HA) titer 8192. In contrast to mH7, the sH7 construct could not form an oligomer-rosette structure and did not have HA titer before cross-linking with anti-His antibody. Thermal stability tests showed that the sH7 and mH7 constructs were unstable at 43 °C and 52 °C, respectively. In a mice immunization study, the mH7 construct but not the sH7 construct could induce robust HI and neutralizing antibody titers. In conclusion, further development of the mH7 vaccine candidate is desirable.

Development of a high-growth enterovirus 71 vaccine candidate inducing cross-reactive neutralizing antibody responses.

Although Enterovirus 71 (EV71) has only one serotype based on serum neutralization tests using hyperimmune animal antisera, three major genogroups (A, B and C) including eleven genotypes (A, B1-B2, and C1-C5) can be well classified based on phylogenetic analysis. Since 1997, large-scale EV71 epidemics occurred cyclically with different genotypes in the Asia-Pacific region. Therefore, development of EV71 vaccines is a national priority in several Asian countries. Currently, five vaccine candidates have been evaluated in clinical trials in China (three C4 candidates), Singapore (one B2 candidate), and Taiwan (one B4 candidate). Overall, the peak viral titers of these 5 vaccine candidates could only reach about 107 TCID50/mL. Moreover, genotypes of these 5 candidates are different from the current predominant genotype B5 in Taiwan and South-Eastern Asia. We adapted a high-growth EV71 genotype B5 (HG-B5) virus after multiple passages and plaque selections in Vero cells and the HG-B5 virus could reach high titers (>108 TCID50/mL) in a microcarrier-based cell culture system. The viral particles were further purified and formulated with alum adjuvant. After two doses of intramuscular immunization in rabbits, the HG-B5 vaccine candidate could induce cross-reactive neutralizing antibodies against the three major EV71 genogroups. In conclusion, a high-growth EV71 virus was successfully adapted in Vero cells and could induce broad spectrum neutralizing antibody titers against three (A, B5, and C4) genotypes in rabbits.

Ultrasonographic features of insulinoma in six ferrets.

Insulinoma is a functional, insulin-secreting tumor, arising from the beta islet cells of the pancreas. It is one of the most common neoplasms in ferrets and has been associated with clinical signs of hypoglycemia, such as ptyalism, pawing at the mouth, seizures, lethargy, and coma. The ultrasonographic features of insulinoma in ferrets have not been previously reported. The purpose of this retrospective case series study was to describe the ultrasonographic features of confirmed insulinoma in a group of ferrets. Inclusion criteria were abdominal ultrasound examination and histological confirmed insulinoma by surgical biopsy. Six ferrets met the inclusion criteria, all of which had multiple hypoglycemic episodes. Ultrasonographic images were reviewed and the characteristics of the pancreatic nodules were recorded. Twenty-eight pancreatic nodules were observed in the six ferrets and were primarily hypoechoic (89.3%, 25/28) and homogenous (46.4%, 13/28) with a smooth margin (78.6%, 22/28). The distribution of the pancreatic nodules was 46.4% in the left lobe, 50% in the right lobe, and 3.6% in the body of the pancreas. The sizes of the pancreatic nodules varied from 1.5 × 1.5 to 4.1 × 5.6 mm. All of the pancreatic nodules removed from surgery were histopathologically confirmed as insulinoma. The findings indicated that insulinoma in ferrets could be detected through ultrasonography, which may facilitate diagnosis and preoperative surgical planning.

Assessment of pathogenicity and antigenicity of American lineage influenza H5N2 viruses in Taiwan.

During December 2003 and March 2004, large scale epidemics of low-pathogenic avian influenza (LPAI) H5N2 occurred in poultry farms in central and southern Taiwan. Based on genomic analysis, these H5N2 viruses contain HA and NA genes of American-lineage H5N2 viruses and six internal genes from avian influenza A/H6N1 viruses endemic in poultry in Taiwan. After disappearing for several years, these novel influenza H5N2 viruses caused outbreaks in poultry farms again in 2008, 2010 and 2012, and have evolved into high pathogenic AI (HPAI) since 2010. Moreover, asymptomatic infections of influenza H5N2 were detected serologically in poultry workers in 2012. Therefore, we evaluated antigenicity and pathogenicity of the novel H5N2 viruses in ferrets. We found that no significant antigenic difference was detected among the novel H5N2 viruses isolated from 2003 to 2014 and the novel H5N2 viruses could cause mild infections in ferrets. Monitoring zoonotic transmission of the novel H5N2 viruses is necessary.

Evaluation of MDCK cell-derived influenza H7N9 vaccine candidates in ferrets.

Avian-origin influenza A (H7N9) viruses emerged as human pathogens in China in early 2013 and have killed >100 persons. Influenza vaccines are mainly manufactured using egg-based technology which could not meet the surging demand during influenza pandemics. In this study, we evaluated cell-based influenza H7N9 vaccines in ferrets. An egg-derived influenza H7N9 reassortant vaccine virus was adapted in MDCK cells. Influenza H7N9 whole virus vaccine antigen was manufactured using a microcarrier-based culture system. Immunogenicity and protection of the vaccine candidates with three different formulations (300 µg aluminum hydroxide, 1.5 µg HA, and 1.5 µg HA plus 300 µg aluminum hydroxide) were evaluated in ferrets. In ferrets receiving two doses of vaccination, geometric mean titers of hemagglutination (HA) inhibition and neutralizing antibodies were <10 and <40 for the control group (adjuvant only), 17 and 80 for the unadjuvanted (HA only) group, and 190 and 640 for the adjuvanted group (HA plus adjuvant), respectively. After challenge with wild-type influenza H7N9 viruses, virus titers in respiratory tracts of the adjuvanted group were significantly lower than that in the control, and unadjuvanted groups. MDCK cell-derived influenza H7N9 whole virus vaccine candidate is immunogenic and protective in ferrets and clinical development is highly warranted.

Reemergence of enterovirus 71 epidemic in northern Taiwan, 2012.

BACKGROUND: Enterovirus 71 (EV71) belongs to picornavirus family and could be classified phylogenetically into three major genogroups (A, B and C) including 11 genotypes (A, B1-B5 and C1-C5). Since 1997, EV71 has caused large-scale of epidemics with neurological complications in Asian children. In Taiwan, nationwide EV71 epidemics with different predominant genotypes have occurred cyclically since 1998. A nationwide EV71 epidemic occurred again in 2012. We conducted genetic and antigenic characterizations of the 2012 epidemic. METHODS: Chang Gung Memorial Hospital (CGMH) is a medical center in northern Taiwan. In CGMH, specimens were collected from pediatric inpatients with suspected enterovirus infections for virus isolation. Enterovirus isolates were serotyped and genotyped and sera from EV71 inpatients were collected for measuring neutralizing antibody titers. RESULTS: There were 10, 16 and 99 EV71 inpatients identified in 2010, 2011 and 2012, respectively. There were 82 EV71 isolates genotyped, which identified 17 genotype C4a viruses and 65 genotype B5 viruses. The genotype B5 viruses were not detected until November 2011 and caused epidemics in 2012. Interestingly, the B5-2011 viruses were genetically distinguishable from the B5 viruses causing the 2008 epidemic and are likely introduced from China or Southeastern Asia. Based on antigenic analysis, minor antigenic variations were detected among the B5-2008, B5-2011, C4a-2008 and C4a-2012 viruses but these viruses antigenically differed from genotype A. CONCLUSIONS: Genotype B5 and C4a viruses antigenically differ from genotype A viruses which have disappeared globally for 30 years but have been detected in China since 2008. Enterovirus surveillance should monitor genetic and antigenic variations of EV71.

Monitoring antigenic variations of enterovirus 71: implications for virus surveillance and vaccine development.

Enterovirus 71 (EV71) causes life-threatening epidemics in Asia and can be phylogenetically classified into three major genogroups (A ∼ C) including 11 genotypes (A, B1 ∼ B5, and C1 ∼ C5). Recently, EV71 epidemics occurred cyclically in Taiwan with different genotypes. In recent years, human studies using post-infection sera obtained from children have detected antigenic variations among different EV71 strains. Therefore, surveillance of enterovirus 71 should include phylogenetic and antigenic analysis. Due to limitation of sera available from children with EV71 primary infection, suitable animal models should be developed to generate a panel of antisera for monitoring EV71 antigenic variations. Twelve reference strains representing the 11 EV71 genotypes were grown in rhabdomyosarcoma cells. Infectious EV71 particles were purified and collected to immunize rabbits. The rabbit antisera were then employed to measure neutralizing antibody titers against the 12 reference strains and 5 recent strains. Rabbits immunized with genogroup B and C viruses consistently have a lower neutralizing antibody titers against genogroup A (≧ 8-fold difference) and antigenic variations between genogroup B and C viruses can be detected but did not have a clear pattern, which are consistent with previous human studies. Comparison between human and rabbit neutralizing antibody profiles, the results showed that ≧ 8-fold difference in rabbit cross-reactive antibody ratios could be used to screen EV71 isolates for identifying potential antigenic variants. In conclusion, a rabbit model was developed to monitor antigenic variations of EV71, which are critical to select vaccine strains and predict epidemics.