Detection of porcine epidemic diarrhea virus-neutralizing antibody using high-throughput imaging cytometry.

Porcine epidemic diarrhea virus (PEDV) is an emerging porcine coronavirus that causes a tremendous economic burden on the swine industry. The assessment of PEDV-neutralizing antibody levels provides a valuable tool to assess and predict herd immunity. We evaluated the performance of a PEDV imaging cytometry-based high-throughput neutralization test (HTNT) and compared the HTNT to a fluorescent focus neutralization (FFN) assay using serum samples from pigs of known PEDV infection status (n = 159). Estimates of diagnostic sensitivity and specificity for HTNT and FFN assays derived from receiver-operator characteristic (ROC) curve analyses showed that both PEDV FFN and HTNT provided excellent diagnostic performance. However, in the laboratory, imaging cytometry provided an objective and semi-automated approach that removed human subjectivity from the testing process and reduced the read-time of a 96-well plate to < 4 min. In addition, imaging cytometry facilitated the rapid collection and long-term storage of test images and data for further evaluation or client consultation. For PEDV and other pathogens, imaging cytometry could provide distinct advantages over classic virus neutralization or FFN assays for the detection and quantitation of neutralizing antibody.

EFFECTS OF LONG-TERM ROUX-EN-Y GASTRIC BYPASS ON BODY WEIGHT AND CLINICAL METABOLIC COMORBIDITIES IN BARIATRIC SURGERY SERVICE OF A UNIVERSITY HOSPITAL.

Background: Due to the high failure rate observed in the clinical treatment of morbid obesity an increase in bariatric surgery indications, as an alternative for the control of obesity and comorbidities, is noticeable. Aim: To evaluate the performance of type 2 diabetes mellitus, high blood pressure and dyslipidemia in patients submitted to Roux-en-Y gastric bypass in late follow-up. Methods: Retrospective analysis of 59 patients included in the bariatric surgery program. Anthropometric (height and body weight) and laboratory (LDLc, HDLc, VLDLc, triglyceride -TG - and glucose) data were collected on pre- and postoperative stages, through medical records. Results: Among the patients, 86% were female aged 43±11, of whom 52% had attended high school. The average postoperative time was 7±3 years. During the postoperative period, there were decreases of weight and body mass index, respectively (133±06 kg vs 91±04 kg p<0.05 e 49±74 kg/m2 vs 33±79 kg/m2, p<0.05). In comparison to the preoperative stage, lower concentrations of glucose (101.00±26.99 vs 89,11±15.19, p=0.014), total cholesterol rates (179.00±37,95 vs 167.48±28,50, p=0.016), LDLc (104.30±33.12 vs 91.46±24.58, p=0.016), VLDLc (25.40±11,12 vs 15.68±7.40, p<0.01), and TG (143.35±86.35 vs 82.45±37.39, p<0.01) and higher concentrations of HDLc (43.53±8.23 vs 57.90±15.60, p<0.01) were identified in the postoperative stage. 40% of hypertensive patients were still undergoing high blood pressure treatment during the postoperative stage. There was remission of type 2 diabetes mellitus and dyslipidemia on 81% and 94% of the cases, respectively. Conclusion: Roux-en-Y gastric bypass has proven itself to be an effective long term procedure, promoting weight loss, remission of DM2 and dyslipidemia.

Racional: Em função do alto grau de falência que se observa no tratamento clínico da obesidade mórbida, observa-se um aumento da procura pela cirurgia bariátrica como alternativa para o controle da obesidade e comorbidades. Objetivo: Avaliar a evolução do diabete melito tipo 2, da hipertensão arterial sistêmica e da dislipidemia em pacientes submetidos à gastroplastia redutora em Y-de-Roux no período de pós-operatório tardio. Métodos: Análise retrospectiva de 59 pacientes inseridos em programa de cirurgia bariátrica. Foram coletados dados antropométricos (altura e peso corporal) e laboratoriais (LDLc, HDLc, VLDLc, triglicerídeo -TG - e glicose) nos períodos pré e pós-operatório por meio de prontuários médicos. Resultados: Entre os pacientes, 86% eram mulheres com idade de 43±11 anos e 52% tinham cursado o ensino médio. O tempo médio de pós-operatório foi de 7±3 anos. Houve redução no peso e no índice de massa corporal no pós-operatório, respectivamente (133±06 kg vs 91±04 kg p<0,05 e 49±74 kg/m2 vs 33±79 kg/m2, p<0,05). Observou-se concentrações inferiores no pós-operatório, comparado com o pré-operatório, da glicose (101,00±26,99 vs 89,11±15,19, p=0,014), colesterol total (179,00±37,95 vs 167,48±28,50, p=0,016), LDLc (104,30±33,12 vs 91,46±24,58, p=0,016), VLDLc (25,40±11,12 vs 15,68±7,40, p<0,01), e TG (143,35±86,35 vs 82,45±37,39, p<0,01) e maiores de HDLc (43,53±8,23 vs 57,90±15,60, p<0,01 ). No pós-operatório 40% dos pacientes hipertensos ainda estavam em tratamento para hipertensão arterial sistêmica. Houve remissão do diabete melito tipo 2 e da dislipidemia em 81% e 94% dos casos, respectivamente. Conclusão: A gastroplastia redutora em Y-de-Roux mostrou ser procedimento eficaz em longo prazo, com resultados persistentes na perda de peso, remissão do DM2 e da dislipidemia.

Does Circulating Antibody Play a Role in the Protection of Piglets against Porcine Epidemic Diarrhea Virus?

The contribution of circulating antibody to the protection of naïve piglets against porcine epidemic diarrhea virus (PEDV) was evaluated using a passive antibody transfer model. Piglets (n = 62) derived from 6 sows were assigned to one of 6 different treatments using a randomized block design which provided for allocation of all treatments to all sows' litters. Each treatment was designed to achieve a different level of circulating anti-PEDV antibody via intraperitoneally administration of concentrated serum antibody. Piglets were orally inoculated with PEDV (USA/IN/2013/19338E, 1 x 103 TCID50 per piglet) 24 hours later and then monitored for 14 days. Piglets remained with their dam throughout the experiment. Sow milk samples, piglet fecal samples, and data on piglet clinical signs, body weight, and body temperature were collected daily. Fecal samples were tested by PEDV real-time reverse transcriptase PCR. Serum, colostrum, and milk were tested for PEDV IgG, IgA, and virus-neutralizing antibody. The data were evaluated for the effects of systemic PEDV antibody levels on growth, body temperature, fecal shedding, survival, and antibody response. The analysis showed that circulating antibody partially ameliorated the effect of PEDV infection. Specifically, antibody-positive groups returned to normal body temperature faster and demonstrated a higher rate of survivability than piglets without PEDV antibody. When combined with previous literature on PEDV, it can be concluded that both systemic antibodies and maternal secretory IgA in milk contribute to the protection of the neonatal pig against PEDV infections. Overall, the results of this experiment suggested that passively administered circulating antibodies contributed to the protection of neonatal piglets against PEDV infection.

EFFECTS OF LONG-TERM ROUX-EN-Y GASTRIC BYPASS ON BODY WEIGHT AND CLINICAL METABOLIC COMORBIDITIES IN BARIATRIC SURGERY SERVICE OF A UNIVERSITY HOSPITAL / EFEITOS NO LONGO PRAZO DA GASTROPLASTIA REDUTORA EM Y-DE-ROUX SOBRE O PESO CORPORAL E COMORBIDADES CLÍNICO METABÓLICAS EM SERVIÇO DE CIRURGIA BARIÁTRICA DE UM HOSPITAL UNIVERSITÁRIO

ABSTRACT Background: Due to the high failure rate observed in the clinical treatment of morbid obesity an increase in bariatric surgery indications, as an alternative for the control of obesity and comorbidities, is noticeable. Aim: To evaluate the performance of type 2 diabetes mellitus, high blood pressure and dyslipidemia in patients submitted to Roux-en-Y gastric bypass in late follow-up. Methods: Retrospective analysis of 59 patients included in the bariatric surgery program. Anthropometric (height and body weight) and laboratory (LDLc, HDLc, VLDLc, triglyceride -TG - and glucose) data were collected on pre- and postoperative stages, through medical records. Results: Among the patients, 86% were female aged 43±11, of whom 52% had attended high school. The average postoperative time was 7±3 years. During the postoperative period, there were decreases of weight and body mass index, respectively (133±06 kg vs 91±04 kg p<0.05 e 49±74 kg/m2 vs 33±79 kg/m2, p<0.05). In comparison to the preoperative stage, lower concentrations of glucose (101.00±26.99 vs 89,11±15.19, p=0.014), total cholesterol rates (179.00±37,95 vs 167.48±28,50, p=0.016), LDLc (104.30±33.12 vs 91.46±24.58, p=0.016), VLDLc (25.40±11,12 vs 15.68±7.40, p<0.01), and TG (143.35±86.35 vs 82.45±37.39, p<0.01) and higher concentrations of HDLc (43.53±8.23 vs 57.90±15.60, p<0.01) were identified in the postoperative stage. 40% of hypertensive patients were still undergoing high blood pressure treatment during the postoperative stage. There was remission of type 2 diabetes mellitus and dyslipidemia on 81% and 94% of the cases, respectively. Conclusion: Roux-en-Y gastric bypass has proven itself to be an effective long term procedure, promoting weight loss, remission of DM2 and dyslipidemia.

RESUMO Racional: Em função do alto grau de falência que se observa no tratamento clínico da obesidade mórbida, observa-se um aumento da procura pela cirurgia bariátrica como alternativa para o controle da obesidade e comorbidades. Objetivo: Avaliar a evolução do diabete melito tipo 2, da hipertensão arterial sistêmica e da dislipidemia em pacientes submetidos à gastroplastia redutora em Y-de-Roux no período de pós-operatório tardio. Métodos: Análise retrospectiva de 59 pacientes inseridos em programa de cirurgia bariátrica. Foram coletados dados antropométricos (altura e peso corporal) e laboratoriais (LDLc, HDLc, VLDLc, triglicerídeo -TG - e glicose) nos períodos pré e pós-operatório por meio de prontuários médicos. Resultados: Entre os pacientes, 86% eram mulheres com idade de 43±11 anos e 52% tinham cursado o ensino médio. O tempo médio de pós-operatório foi de 7±3 anos. Houve redução no peso e no índice de massa corporal no pós-operatório, respectivamente (133±06 kg vs 91±04 kg p<0,05 e 49±74 kg/m2 vs 33±79 kg/m2, p<0,05). Observou-se concentrações inferiores no pós-operatório, comparado com o pré-operatório, da glicose (101,00±26,99 vs 89,11±15,19, p=0,014), colesterol total (179,00±37,95 vs 167,48±28,50, p=0,016), LDLc (104,30±33,12 vs 91,46±24,58, p=0,016), VLDLc (25,40±11,12 vs 15,68±7,40, p<0,01), e TG (143,35±86,35 vs 82,45±37,39, p<0,01) e maiores de HDLc (43,53±8,23 vs 57,90±15,60, p<0,01 ). No pós-operatório 40% dos pacientes hipertensos ainda estavam em tratamento para hipertensão arterial sistêmica. Houve remissão do diabete melito tipo 2 e da dislipidemia em 81% e 94% dos casos, respectivamente. Conclusão: A gastroplastia redutora em Y-de-Roux mostrou ser procedimento eficaz em longo prazo, com resultados persistentes na perda de peso, remissão do DM2 e da dislipidemia.

Nutritional profile of patients in a multidisciplinary treatment program for severe obesity and preoperative bariatric surgery.

BACKGROUND: Along with the augmentation in obesity rates in recent years, the demand for bariatric surgery has startlingly increased. Nutritional counseling in the preoperative period is very important because it contributes to higher success rate in the post-operative period. AIM: To assess the nutritional status of patients in a multidisciplinary program for the treatment of severe obesity and pre-operatively for bariatric surgery, characterizing the consumption of healthy nutrients. METHODS: A retrospective analysis of 30 patients was conducted. Personal information, anthropometric data and dietary assessment by 24-hour food record were collected. The analysis of energy intake was performed in Dietpro 5.1 Professional(r) program. The statistical treatment of the caloric intake was performed by an ANOVA test with Bonferroni's post hoc and for anthropometric data the paired t test was used. RESULTS: From the total, 73% of the patients were women and 27% male, mean age was 48.4+12.9 and 49.8+8.1, respectively. A lower weight in the 5th appointment was observed when compared with the 1st one. There was a reduction in caloric intake in the 2nd, 3rd, 4th and 5th appointments when compared with the 1st. It was observed that in the 5th appointment more than 50% of the patients underwent six meals a day. There was an increase in the consumption of fruit along the appointments and 72.2% of patients consumed 1-2 servings of fruit a day. Vegetables intake was high in all appointments in the pre-operative period and, although low, the whole grain products consumption has increased during the pre-operative period achieving 30% of the study population. CONCLUSION: There was a decrease in body weight, a trend in the decrease of the body mass index and waist circumference and quantitative and qualitative improvement of food consumption.

Nutritional profile of patients in a multidisciplinary treatment program for severe obesity and preoperative bariatric surgery / Perfil nutricional de pacientes em programa multidisciplinar de tratamento da obesidade grave e em pré-operatório de cirurgia bariátrica

BACKGROUND: Along with the augmentation in obesity rates in recent years, the demand for bariatric surgery has startlingly increased. Nutritional counseling in the preoperative period is very important because it contributes to higher success rate in the post-operative period. AIM: To assess the nutritional status of patients in a multidisciplinary program for the treatment of severe obesity and pre-operatively for bariatric surgery, characterizing the consumption of healthy nutrients. METHODS: A retrospective analysis of 30 patients was conducted. Personal information, anthropometric data and dietary assessment by 24-hour food record were collected. The analysis of energy intake was performed in Dietpro 5.1 Professional(r) program. The statistical treatment of the caloric intake was performed by an ANOVA test with Bonferroni´s post hoc and for anthropometric data the paired t test was used. RESULTS: From the total, 73% of the patients were women and 27% male, mean age was 48.4+12.9 and 49.8+8.1, respectively. A lower weight in the 5th appointment was observed when compared with the 1st one. There was a reduction in caloric intake in the 2nd, 3rd, 4th and 5th appointments when compared with the 1st. It was observed that in the 5th appointment more than 50% of the patients underwent six meals a day. There was an increase in the consumption of fruit along the appointments and 72.2% of patients consumed 1-2 servings of fruit a day. Vegetables intake was high in all appointments in the pre-operative period and, although low, the whole grain products consumption has increased during the pre-operative period achieving 30% of the study population. CONCLUSION: There was a decrease in body weight, a trend in the decrease of the body mass index and waist circumference and quantitative and qualitative improvement of food consumption. .

RACIONAL: Com o avanço da obesidade nos últimos anos, a procura pela cirurgia bariátrica tem aumentado de forma alarmante. O aconselhamento nutricional no período pré-operatório é de grande importância, pois contribui para maior taxa de sucesso no pós-operatório. OBJETIVO: Avaliar o perfil nutricional dos pacientes inseridos em um programa multidisciplinar para o tratamento da obesidade grave e em pré-operatório de cirurgia bariátrica, caracterizando o consumo de nutrientes saudáveis. MÉTODOS: Foi realizada análise retrospectiva de 30 pacientes sendo coletadas informações pessoais, antropométricas e dietéticas por meio de recordatórios de 24 horas. A análise da ingestão energética foi realizada no programa Dietpro 5.1 Profissional(r). O tratamento estatístico do consumo calórico foi realizado pelo teste ANOVA com post hoc por Bonferroni e para os dados antropométricos foi utilizado o teste t pareado. RESULTADOS: Do total dos pacientes 73% eram mulheres e 27% homens, com idade de 48,4+12,9 e 49,8+8,1, respectivamente. Foi observado menor peso na 5ª consulta quando comparado com a 1ª. Houve diminuição no consumo calórico nas 2ª, 3ª, 4ª e 5ª consultas quando comparadas com a 1ª. Observou-se que na 5ª consulta mais de 50% dos pacientes realizavam seis refeições ao dia. Houve aumento no consumo de frutas ao longo das consultas chegando a 72,2% dos pacientes que consumiam de uma a duas porções de frutas ao dia. A ingestão de verduras e legumes era elevada em todas as consultas no período pré-operatório e o consumo de produtos integrais, apesar de baixo, aumentou ao longo do período pré-operatório, alcançando 30% da população ao final do estudo. CONCLUSÃO: Houve ...

Comparison of genome diversity of Brucella spp. field isolates using Universal Bio-signature Detection Array and whole genome sequencing reveals limitations of current diagnostic methods.

The detection and identification of bio-threat agents and the study of host-pathogen interactions require a high-resolution detection platform capable of discerning closely related species. Diverse analysis methods are used to identify pathogens, specifically Brucella species or biovars. In this study, we compared four diagnostic approaches including serology-based biochemical test, PCR assay, microarray analysis using a Universal Bio-signature Detection Array (UBDA) and whole genome "deep" sequencing for Brucella organisms including a number of field isolates. We found that although there was frequent agreement among the different tests, some tests gave compound/contradictory results that were a consequence of species diversity due to mixed infections or minor contaminants as measured by UBDA and validated from whole genome sequence. By comparing these analysis techniques, we demonstrate that standard diagnostics used in the field are limited in their ability to identify genomic DNA contaminants in field isolates while UBDA and sequencing analysis are highly sensitive in tracing genomic differences among the isolates.

Effect of age on the pathogenesis and innate immune responses in Pekin ducks infected with different H5N1 highly pathogenic avian influenza viruses.

The pathogenicity of H5N1 highly pathogenic avian influenza (HPAI) viruses in domestic ducks varies between different viruses and is affected by the age of the ducks, with younger ducks presenting a more severe disease. In order to better understand the pathobiology of H5N1 HPAI in ducks including the role of host responses, 2 and 5-week-old Pekin ducks were infected with three different H5N1 HPAI viruses. Virus-induced pathology ranged from no clinical signs to severe disease and mortality, with the 2-week-old ducks being more severely affected by the more virulent viruses. However, these more virulent viruses induced higher body temperatures in the 5-week-old ducks than in the 2-week-old ducks indicating possible differences in innate immune responses. To analyze the ducks host responses to H5N1 HPAI virus infection, expression of innate immune-related genes was measured in the spleens and lungs of infected ducks at the peak of virus infection. IFN-α, RIG-I, and IL-6 RNA levels were increased in spleens regardless of the virus given and the age of the ducks, however differences were observed in the levels of up-regulation of IFN-α and RIG-I between the 2 and the 5-week-old ducks with the more virulent virus. Differences in IL-2 gene expression were also observed. In the lungs, the levels of expression of innate immune-related genes were lower than in the spleen, with mostly up-regulation of RIG-I and IL-6 and down-regulation of IFN-α and IL-2; no significant difference in expression was found between the 2 and the 5-week-old ducks. The differences observed in the innate immune responses to infection with H5N1 HPAI viruses could explain in part the differences in pathogenicity found between the 2 and 5-week-old ducks, however earlier time points after infection and additional innate immune-related genes should be examined.

H7 avian influenza virus vaccines protect chickens against challenge with antigenically diverse isolates.

Vaccination has been a critical tool in the control of some avian influenza viruses (AIV) and has been used routinely in Pakistan to help control sporadic outbreaks of highly pathogenic (HP) H7 AIV since 1995. During that time, several AIV isolates were utilized as inactivated vaccines with varying degrees of success. In order to evaluate which H7 AIV strains may serve as optimal vaccines for diverse H7 AIVs from Pakistan we conducted vaccination-challenge studies with five H7 vaccines against challenge with two HPAIVs: A/chicken/Murree/NARC-1/1995 H7N3 and A/chicken/Karachi/SPVC-4/2004 H7N3. To further characterize the isolates antigenic cartography was used to visually demonstrate the antigenic relationships among the isolates. All vaccines provided similar protection against mortality, morbidity and shedding of challenge virus from the respiratory tract. However, some minor (not statistically significant) differences were observed and correlated with antibody levels induced by the vaccine prior to challenge.

Efficacy of commercial vaccines in protecting chickens and ducks against H5N1 highly pathogenic avian influenza viruses from Vietnam.

Highly pathogenic (HP) H5N1 avian influenza (AI) viruses continue to circulate in Asia and have spread to other regions of the world. Though attempts at eradication of the viruses during various outbreaks have been successful for short periods of time, new strains of H5N1 viruses continue to emerge and have become endemic in parts of Asia and Africa. Vaccination has been employed in Vietnam as part of AI control programs. Domestic ducks, which make up a large part of poultry in Vietnam, have been recognized as one of the primary factors in the spread of AI in this country. As a result, ducks have been included in the vaccination programs. Despite the effort to control AI in Vietnam, eradication of the disease has not been possible, due in part to the emergence and spread of new viruses. Here, we tested the abilities of avian influenza oil emulsion vaccines of different genetic origins to protect against disease and viral shedding in both 2-wk-old white leghorn chickens and 1-wk-old Pekin ducks. Seventy-five to 100% of vaccinated chickens were protected from mortality, but viral shedding occurred for at least 4 days post challenge. All but one vaccinated duck were protected from mortality; however, all groups shed virus up through at least 5 days postchallenge, depending on the vaccine and challenge virus used. Differences in levels of hemagglutination inhibition (HI) antibody titers induced by the vaccines were observed in both chickens and ducks. Although the vaccines tested were effective in protecting against disease and mortality, updated and more efficacious vaccines are likely needed to maintain optimal protection.

The effects of NS gene exchange on the pathogenicity of H5N1 HPAI viruses in ducks.

Until 2002, H5N1 highly pathogenic avian influenza (HPAI) viruses caused only mild respiratory infections in ducks. Since then, new viruses have emerged that cause systemic disease and high mortality in ducks and other waterfowl. Studies on HPAI virus pathogenicity in ducks have been limited, and there is no clear explanation of why the pathogenicity of some H5N1 HPAI viruses has increased. The nonstructural protein 1 (NS1 protein) is known to suppress immune responses in influenza virus-infected hosts affecting virus pathogenesis. In order to determine if the NS1 protein contributes to increased virulence in ducks, single-gene reassortant viruses were generated. Exchanging the NS genes from A/Ck/HK/220/97 (a virus that produces mild disease in ducks) and A/Dk/VN/201/05 (a very virulent virus for ducks) in the rEgret/02 background (a recombinant virus derived from A/Egret/HK/757.2/02, a highly pathogenic virus in ducks) resulted in decreased mean death times compared to infection with the rEgret/02 virus in ducks, but the change was not statistically significant. Infection with the reassortant viruses affected the expression of immune-related genes in spleens and lungs when compared to controls, but when compared among them, the expression of the duck genes was similar. Furthermore, virus titers in spleen, lung, and brain as well as antigen distribution in various tissues were similar in ducks infected with the reassortant viruses. All together these data show that, under these experimental conditions, exchanging the NS gene had minimal effect on the virus pathogenicity, and it suggests that other viral genes, or combination of genes, are most likely contributing to the increased virulence of H5N1 HPAI viruses in ducks.

Sequence and phylogenetic analysis of H7N3 avian influenza viruses isolated from poultry in Pakistan 1995-2004.

BACKGROUND: Avian influenza virus (AIV) infections have caused heavy economic losses to the poultry industry in Pakistan as well as numerous other regions worldwide. The first introduction of H7N3 AIV to Pakistan occurred during 1995, since then H7N3, H9N2 and H5N1 AIVs have each been sporadically isolated. This report evaluates the genetic origin of the H7N3 viruses from Pakistan collected 1995-2004 and how they disseminated within the country. To accomplish this we produced whole genome sequences for 6 H7N3 viruses and data for the HA and NA genes of an additional 7 isolates. All available sequence from H7N3 AIV from Pakistan was included in the analysis. RESULTS: Phylogenetic analysis revealed that there were two introductions of H7 into Pakistan and one N3 introduction. Only one of the H7 introductions appears to have become established in poultry in Pakistan, while the other was isolated from two separate outbreaks 6 years apart. The data also shows that reassortment has occurred between H7N3 and H9N2 viruses in the field, likely during co-infection of poultry. Also, with the exception of these few reassortant isolates, all 8 genes in the predominant H7N3 virus lineage have evolved to be phylogenetically distinct. CONCLUSIONS: Although rigorous control measures have been implemented in commercial poultry in Pakistan, AIV is sporadically transmitted to poultry and among the different poultry industry compartments (broilers, broiler breeders, table egg layers). Since there is one primary H7 lineage which persists and that has reassorted with the H9N2 AIV in poultry, it suggests that there is a reservoir with some link commercial poultry. On a general level, this offers insight into the molecular ecology of AIV in poultry where the virus has persisted despite vaccination and biosecurity. This data also illustrates the importance of sustained surveillance for AIVs in poultry.

Cell surface display of highly pathogenic avian influenza virus hemagglutinin on the surface of Pichia pastoris cells using alpha-agglutinin for production of oral vaccines.

Yeast is an ideal organism to express viral antigens because yeast glycosylate proteins more similarly to mammals than bacteria. Expression of proteins in yeast is relatively fast and inexpensive. In addition to the convenience of production, for purposes of vaccination, yeast has been shown to have natural adjuvant activity making the expressed proteins more immunogenic when administered along with yeast cell wall components. Development of genetic systems to display foreign proteins on the surface of yeast via fusion to glycosylphosphatidylinositol-anchored (GPI) proteins has further simplified the purification of recombinant proteins by not requiring harsh treatments for cellular lysis or protein purification. We have expressed the hemagglutinin protein from a highly pathogenic avian influenza (HPAI) virus [A/Egret/HK/757.2/02], subtype H5N1, on the surface of the yeast strain Pichia pastoris, as an anchored C-terminal fusion with the Saccharomyces cerevisiae GPI-anchored cell wall protein, alpha-agglutinin. Surface expression of the hemagglutinin fusion protein was demonstrated by immunofluorescence microscopy. Functionally, the fusion protein retained hemagglutinin agglutinating activity, and oral vaccination with the yeast resulted in production of virus neutralizing antibodies. This study represents the first steps in the generation of a yeast-based vaccine for protection against highly pathogenic strains of avian influenza.

A single substitution in amino acid 184 of the NP protein alters the replication and pathogenicity of H5N1 avian influenza viruses in chickens.

Changes in the NP gene of H5N1 highly pathogenic avian influenza (HPAI) viruses have previously been shown to affect viral replication, alter host gene expression levels and affect mean death times in infected chickens. Five amino acids at positions 22, 184, 400, 406, and 423 were different between the two recombinant viruses studied. In this study, we individually mutated the five amino acids that differed and determined that the difference in virus pathogenicity after NP gene exchange was a result of an alanine to lysine change at position 184 of the NP protein. Infection with viruses containing a lysine at NP 184 induced earlier mortality in chickens, increased virus titers and nitric oxide levels in tissues, and resulted in up-regulated host immune genes, such as alpha-interferon (IFN-alpha), gamma-interferon (IFN-gamma), orthomyxovirus resistance gene 1 (Mx1), and inducible nitric oxide synthase (iNOS). This study underlines the importance of the NP in avian influenza virus replication and pathogenicity.

Domestic pigs have low susceptibility to H5N1 highly pathogenic avian influenza viruses.

Genetic reassortment of H5N1 highly pathogenic avian influenza viruses (HPAI) with currently circulating human influenza A strains is one possibility that could lead to efficient human-to-human transmissibility. Domestic pigs which are susceptible to infection with both human and avian influenza A viruses are one of the natural hosts where such reassortment events could occur. Virological, histological and serological features of H5N1 virus infection in pigs were characterized in this study. Two- to three-week-old domestic piglets were intranasally inoculated with 10(6) EID(50) of A/Vietnam/1203/04 (VN/04), A/chicken/Indonesia/7/03 (Ck/Indo/03), A/Whooper swan/Mongolia/244/05 (WS/Mong/05), and A/Muscovy duck/Vietnam/ 209/05 (MDk/VN/05) viruses. Swine H3N2 and H1N1 viruses were studied as a positive control for swine influenza virus infection. The pathogenicity of the H5N1 HPAI viruses was also characterized in mouse and ferret animal models. Intranasal inoculation of pigs with H5N1 viruses or consumption of infected chicken meat did not result in severe disease. Mild weight loss was seen in pigs inoculated with WS/Mong/05, Ck/Indo/03 H5N1 and H1N1 swine influenza viruses. WS/Mong/05, Ck/Indo/03 and VN/04 viruses were detected in nasal swabs of inoculated pigs mainly on days 1 and 3. Titers of H5N1 viruses in nasal swabs were remarkably lower compared with those of swine influenza viruses. Replication of all four H5N1 viruses in pigs was restricted to the respiratory tract, mainly to the lungs. Titers of H5N1 viruses in the lungs were lower than those of swine viruses. WS/Mong/05 virus was isolated from trachea and tonsils, and MDk/VN/05 virus was isolated from nasal turbinate of infected pigs. Histological examination revealed mild to moderate bronchiolitis and multifocal alveolitis in the lungs of pigs infected with H5N1 viruses, while infection with swine influenza viruses resulted in severe tracheobronchitis and bronchointerstitial pneumonia. Pigs had low susceptibility to infection with H5N1 HPAI viruses. Inoculation of pigs with H5N1 viruses resulted in asymptomatic to mild symptomatic infection restricted to the respiratory tract and tonsils in contrast to mouse and ferrets animal models, where some of the viruses studied were highly pathogenic and replicated systemically.

Differential host gene expression in cells infected with highly pathogenic H5N1 avian influenza viruses.

In order to understand the molecular mechanisms by which different strains of avian influenza viruses overcome host response in birds, we used a complete chicken genome microarray to compare early gene expression levels in chicken embryo fibroblasts (CEF) infected with two avian influenza viruses (AIV), A/CK/Hong Kong/220/97 and A/Egret/Hong Kong/757.2/02, with different replication characteristics. Gene ontology revealed that the genes with altered expression are involved in many vital functional classes including protein metabolism, translation, transcription, host defense/immune response, ubiquitination and the cell cycle. Among the immune-related genes, MEK2, MHC class I, PDCD10 and Bcl-3 were selected for further expression analysis at 24 hpi using semi-quantitive RT-PCR. Infection of CEF with A/Egret/Hong Kong/757.2/02 resulted in a marked repression of MEK2 and MHC class I gene expression levels. Infection of CEF with A/CK/Hong Kong/220/97 induced an increase of MEK2 and a decrease in PDCD10 and Bcl-3 expression levels. The expression levels of alpha interferon (IFN-alpha), myxovirus resistance 1 (Mx1) and interleukin-8 (IL-8) were also analyzed at 24 hpi, showing higher expression levels of all of these genes after infection with A/CK/Hong Kong/220/97 compared to A/Egret/Hong Kong/757.2/02. In addition, comparison of the NS1 sequences of the viruses revealed amino acid differences that may explain in part the differences in IFN-alpha expression observed. Microarray gene expression analysis has proven to be a useful tool on providing important insights into how different AIVs affect host gene expression and how AIVs may use different strategies to evade host response and replicate in host cells.

NP, PB1, and PB2 viral genes contribute to altered replication of H5N1 avian influenza viruses in chickens.

The virulence determinants for highly pathogenic avian influenza viruses (AIVs) are considered multigenic, although the best characterized virulence factor is the hemagglutinin (HA) cleavage site. The capability of influenza viruses to reassort gene segments is one potential way for new viruses to emerge with different virulence characteristics. To evaluate the role of other gene segments in virulence, we used reverse genetics to generate two H5N1 recombinant viruses with differing pathogenicity in chickens. Single-gene reassortants were used to determine which viral genes contribute to the altered virulence. Exchange of the PB1, PB2, and NP genes impacted replication of the reassortant viruses while also affecting the expression of specific host genes. Disruption of the parental virus' functional polymerase complexes by exchanging PB1 or PB2 genes decreased viral replication in tissues and consequently the pathogenicity of the viruses. In contrast, exchanging the NP gene greatly increased viral replication and expanded tissue tropism, thus resulting in decreased mean death times. Infection with the NP reassortant virus also resulted in the upregulation of gamma interferon and inducible nitric oxide synthase gene expression. In addition to the impact of PB1, PB2, and NP on viral replication, the HA, NS, and M genes also contributed to the pathogenesis of the reassortant viruses. While the pathogenesis of AIVs in chickens is clearly dependent on the interaction of multiple gene products, we have shown that single-gene reassortment events are sufficient to alter the virulence of AIVs in chickens.

Rabies virus-induced apoptosis involves caspase-dependent and caspase-independent pathways.

Previously, it has been shown that the laboratory attenuated rabies virus CVS-B2C, but not the wild-type virus SHBRV, induces apoptosis in mice and the induction of apoptosis is mediated by viral glycoprotein. Induction of apoptosis by CVS-B2C limits the spread of the virus in the CNS. In the present study, we characterized the pathways by which CVS-B2C induces apoptosis. BSR cells were infected with CVS-B2C or SHBRV and harvested at different time points for detection of apoptosis by immunofluorescence and flow cytometry. Apoptosis was detected only in cells infected with CVS-B2C, but not SHBRV. Caspase activity and expression of several apoptotic proteins were analyzed by fluorometric assay and Western blotting. Activation of caspase-8 and -3, but not of caspase-9, was observed in CVS-B2C-infected cells. In addition, the level of expression of Apaf-1 did not change. Furthermore, PARP was cleaved confirming activation of downstream caspases. All these data suggest that CVS-B2C infection activates the extrinsic, but not the intrinsic, apoptotic pathway. In addition, AIF, a caspase-independent apoptotic protein was up-regulated and translocated from the cytoplasm to the nucleus post-infection, suggesting that apoptosis induced by CVS-B2C also involves the activation of a caspase-independent pathway.

Glycoprotein-mediated induction of apoptosis limits the spread of attenuated rabies viruses in the central nervous system of mice.

Induction of apoptosis by rabies virus (RV) has been reported to be associated with the expression of the glycoprotein (G), but inversely correlated with pathogenicity. To further delineate the association between the expression of the G and the induction of apoptosis, recombinant RVs with replacement of only the G gene were used to infect mice by the intracerebral route. Recombinant viruses expressing the G from attenuated viruses expressed higher level of the G and induced more apoptosis in mice than recombinant RV expressing the G from wild-type (wt) or pathogenic RV, demonstrating that it is the G gene that determines the level of G expression and, consequently, the induction of apoptosis. Likewise, recombinant viruses expressing the G from wt or pathogenic RV are more pathogenic in mice than those expressing G from attenuated RV, confirming the inverse correlation between RV pathogenicity and the induction of apoptosis. To investigate the mechanism by which induction of apoptosis attenuates viral pathogenicity, mice were infected with wt or attenuated RV by the intramuscular route. It was found that low doses of attenuated RV induced apoptosis in the spinal cord and failed to spread to the brain or produce neurological disease. On the other hand, apoptosis was not observed in the spinal cord of mice infected with the same doses of wt RV and the virus spread to various parts of the brain and induced fatal neurologic disease. These results suggest that glycoprotein-mediated induction of apoptosis limits the spread of attenuated rabies viruses in the central nervous system (CNS) of mice.

Attenuated rabies virus activates, while pathogenic rabies virus evades, the host innate immune responses in the central nervous system.

Rabies virus (RV) induces encephalomyelitis in humans and animals. However, the pathogenic mechanism of rabies is not fully understood. To investigate the host responses to RV infection, we examined and compared the pathology, particularly the inflammatory responses, and the gene expression profiles in the brains of mice infected with wild-type (wt) virus silver-haired bat RV (SHBRV) or laboratory-adapted virus B2C, using a mouse genomic array (Affymetrix). Extensive inflammatory responses were observed in animals infected with the attenuated RV, but little or no inflammatory responses were found in mice infected with wt RV. Furthermore, attenuated RV induced the expression of the genes involved in the innate immune and antiviral responses, especially those related to the alpha/beta interferon (IFN-alpha/beta) signaling pathways and inflammatory chemokines. For the IFN-alpha/beta signaling pathways, many of the interferon regulatory genes, such as the signal transduction activation transducers and interferon regulatory factors, as well as the effector genes, for example, 2'-5'-oligoadenylate synthetase and myxovirus proteins, are highly induced in mice infected with attenuated RV. However, many of these genes were not up-regulated in mice infected with wt SHBRV. The data obtained by microarray analysis were confirmed by real-time PCR. Together, these data suggest that attenuated RV activates, while pathogenic RV evades, the host innate immune and antiviral responses.