Dissertations / Theses on the topic 'Influenza viruses. Influenza'

En el primer estudi de la present tesi, es van estudiar brots de malaltia respiratòria compatible amb virus de la influença de tipus A (IAV) així com granges que no mostraven simptomatologia clínica. Per a l'estudi dels brots, es van recollir mostres de hisops nasals d'animals amb signes respiratoris i febre (≥40 ° C), mentre que a les granges sense simptomatologia clínica, es van recollir hisops nasals de garrins de maternitat, transició i porcs d'engreix (20 per grup). Es va estudiar un total de 211 brots i 19 granges aparentment subclíniques. La presència i llinatge es van determinar per RT-qPCR, i es va fer l'aïllament de mostres seleccionades usant cèl·lules MDCK. Els aïllats van ser seqüenciats (genoma complet) mitjançant la tecnologia Illumina Miseq. Es va confirmar la presència de IAV en 145 casos de brots (68.7%), i en 15 granges aparentment subclíniques (78.9%). Els llinatges majorment detectats van ser H1avN2hu (33.6%), H1avN1av (24.3%) i H1huN2hu (18.7%). Es va obtenir un total de 60 aïllats, i els seus genomes van ser completament seqüenciats. Els genotips majoritàriament detectats van ser el tipus D i l'A, que es corresponen als llinatges H1avN2hu i H1avN1av, respectivament. Es van detectar un total de 14 genotips diferents, dels quals, 7 d'ells no havien estat prèviament reportados.En el segon estudi de la present tesi, es va estudiar la dinàmica de transmissió de IAV en les transicions d'una granja endèmica abans i després de l'aplicació de diferents esquemes de vacunació a les truges. Es van realitzar un total de tres estudis longitudinals: abans de la vacunació, després de la vacunació amb una vacuna comercial polivalent inactivada H1N1-H1N2-H3N2 i després de la vacunació amb una vacuna comercial monovalent pandèmica H1N1. Es van recollir mostres setmanals de hisops nasals dels garrins des de les 3-9 setmanes de vida, i mostres de sang a les 3, 6 i 9 setmanes de vida. En el primer longitudinal abans de la vacunació, es va avaluar la circulació vírica basal en 50 garrins de 4 lots consecutius. En el segon longitudinal, es va realitzar vacunació en llençol de truges usant la vacuna comercial polivalent (grup control) i la meitat d'aquestes van ser revacunades 3 setmanes abans de el part (grup tractament). Es va seleccionar un grup aleatori de 10 truges de cada grup i es va fer el seguiment setmanal de 5 garrins per truja. L'estudi va ser repetit en 4 lots consecutius. En el tercer estudi longitudinal, el procediment va ser el mateix que en l'anterior, però fent servir la vacuna inactivada pandèmica H1N1. Hisops nasals van ser examinats per RT-qPCR i els sèrums van ser analitzats utilitzant un ELISA comercial (CIVTEST ©-Suis Influenza). En el segon longitudinal després de l'aplicació de la primera vacuna, l'inici de la infecció es va retardar en dues setmanes, però no es van observar diferències significatives entre els dos grups; i en el tercer, l'inici de la infecció es va moure cap a l'esquerra en tots els grups, sense diferències significatives entre ells. En els tres estudis, es van detectar animals que excretaron virus en dos o fins a en tres mostrejos consecutius, així com alguns casos de re-infeccions. El llinatge present a la granja durant els dos primers estudis longitudinals es correspon a un H1avN1av. No obstant això, durant el tercer estudi, es va detectar circulant en tots els grups d'animals 1 H3huN2hu que portava un nou llinatge H3 humà derivat d'un virus de la grip estacional humana.
En el primer estudio de la presente tesis, se estudiaron brotes de enfermedad respiratoria compatible con virus de la influenza de tipo A (IAV) así como granjas que no mostraban sintomatología clínica. Para el estudio de los brotes, se recogieron muestras de hisopos nasales de animales con signos respiratorios y fiebre (≥40°C), mientras que en las granjas sin sintomatología clínica, se recogieron hisopos nasales de lechones de maternidad, transición y cerdos de engorde (20 por grupo). Se estudió un total de 211 brotes y 19 granjas aparentemente subclínicas. La presencia y linaje se determinaron por RT-qPCR, y se hizo el aislamiento de muestras seleccionadas usando células MDCK. Los aislados fueron secuenciados (genoma completo) mediante la tecnología Illumina Miseq. Se confirmó la presencia de IAV en 145 casos de brotes (68.7%), y en 15 granjas aparentemente subclínicas (78.9%). Los linajes mayormente detectados fueron H1avN2hu (33.6%), H1avN1av (24.3%) y H1huN2hu (18.7%). Se obtuvo un total de 60 aislados, y sus genomas fueron completamente secuenciados. Los genotipos mayoritariamente detectados fueron el tipo D y el A, que se corresponden a los linajes H1avN2hu y H1avN1av, respectivamente. Se detectaron un total de 14 genotipos diferentes, de los cuales, 7 de ellos no habían sido previamente reportados.En el segundo estudio de la presente tesis, se estudió la dinámica de transmisión de IAV en las transiciones de una granja endémica antes y después de la aplicación de diferentes esquemas de vacunación en las cerdas. Se realizaron un total de tres estudios longitudinales: antes de la vacunación, después de la vacunación con una vacuna comercial polivalente inactivada H1N1-H1N2-H3N2 y después de la vacunación con una vacuna comercial monovalente pandémica H1N1. Se recogieron muestras semanales de hisopos nasales de los lechones desde las 3-9 semanas de vida, y muestras de sangre a las 3, 6 y 9 semanas de vida. En el primer longitudinal antes de la vacunación, se evaluó la circulación vírica basal en 50 lechones de 4 lotes consecutivos. En el segundo longitudinal, se realizó vacunación en sábana de cerdas usando la vacuna comercial polivalente (grupo control) y la mitad de estas fueron revacunadas 3 semanas antes del parto (grupo tratamiento). Se seleccionó un grupo aleatorio de 10 cerdas de cada grupo y se hizo el seguimiento semanal de 5 lechones por cerda. El estudio fue repetido en 4 lotes consecutivos. En el tercer estudio longitudinal, el procedimiento fue el mismo que en el anterior, pero usando la vacuna inactivada pandémica H1N1. Hisopos nasales fueron examinados por RT-qPCR y los sueros fueron analizados usando un ELISA comercial (Civtest-Suis Influenza). En el segundo longitudinal después de la aplicación de la primera vacuna, el inicio de la infección se retrasó en dos semanas, pero no se observaron diferencias significativas entre ambos grupos; y en el tercero, el inicio de la infección se movió hacia la izquierda en todos los grupos, sin diferencias significativas entre ellos. En los tres estudios, se detectaron animales que excretaron virus en dos o hasta en tres muestreos consecutivos, así como algunos casos de re-infecciones. El linaje presente en la granja durante los dos primeros estudios longitudinales se corresponde a un H1avN1av. Sin embargo, durante el tercer estudio, se detectó circulando en todos los grupos de animales un H3huN2hu que llevaba un nuevo linaje de H3 humano derivado de un virus de la gripe estacional humana.
In the first study of the present thesis, we investigated outbreaks of respiratory disease (n=211) compatible with influenza A virus (IAV) as well as farms without overt respiratory disease (n=19) for the presence of IAV. In the outbreak investigations, nasal swabs were taken from animals with respiratory signs and fever (≥40°C) while in the farms with no evident respiratory disease, nasal swabs were randomly taken from suckling piglets, weaners and fatteners (20 animals per phase). Presence of IAV and lineage determination were assessed by RT-qPCR and isolation was attempted in selected samples using MDCK cells. Isolates were sequenced (full genome) by using Illumina Miseq technology. IAV participation was confirmed in 145 (68.7%) of the outbreaks, and in 15 (78.9%) of the farms without overt disease. The most commonly detected lineages were H1avN2hu (33.6%), H1avN1av (24.3%) and H1huN2hu (18.7%). Sixty IAV isolates were obtained and the genomes were fully sequenced. Genotypes D and A, H1avN2hu and H1avN1av, respectively, were predominant but up to 14 genotypes were identified, of which seven had not been previously reported. Four isolates containing a new H3hu lineage derived from a human seasonal virus were detected, and isolates containing genes from the pandemic virus represented a 31.7 % of the total. In the second study of the present thesis, the transmission dynamics of IAV in the nurseries from an endemic farm were assessed before and after the application of different vaccination schemes for sows. Three follow-up periods were examined: before vaccination, after vaccination with a commercial inactivated polyvalent H1N1-H1N2-H3N2 and after vaccination with a monovalent pandemic H1N1. Nasal swabs of piglets were taken weekly from 3-9 weeks of age and blood samples were taken at three, six and nine weeks of age. In the first follow-up before vaccination, the basal IAV circulation was assessed by sampling 50 piglets in 4 batches. In the second longitudinal study, sows were blanket vaccinated with the polyvalent vaccine (control group) and half of them received an extra dose 3 weeks pre-farrowing (treatment group). A random cohort of 10 sows in each group was selected and 5 piglets per sow were weekly followed. The trial was replicated in 4 consecutive batches. In the third follow-up period, the procedure was the same as in the second, but using a pandemic H1N1 inactivated vaccine. Nasal swabs were examined by RT-qPCR and serum samples were analysed using a commercial ELISA (Civtest-Suis Influenza). Incidences and beta values per week and pen were calculated after the RT-qPCR results. Before applying any vaccination scheme, the patterns of incidence were diverse in the examined pens but often viral circulation was detected as early as 4 weeks of age. At three weeks of age, most of the analysed animals were positive with high S/P ratios. In the second follow-up period after the application of the first vaccination scheme, the onset of infection was delayed by two weeks but there were no other significant differences between both groups, and in the third, the onset of infection shifted to the left for all groups, without significant differences among them. In all of the three studies, animals that shed virus in two and even three consecutive sampling times were detected, as well as some cases of re-infection. Interestingly, an H1avN1av virus was initially detected in the farm, but during the third study, a H3huN2hu was found circulating in the batches, carrying a new H3 human-like derived from human seasonal virus.
Universitat Autònoma de Barcelona. Programa de Doctorat en Medicina i Sanitat Animals

A number of different cell cultures were examined for their susceptibility to the influenza virus A/swine/Weybridge/86(H1N1) and A/swine/Weybridge/87(H3N2). PK1 (porcine kidney) was found to be the most susceptible to the viruses, and MDCK (canine kidney), the best cell line for primary isolation. A method of infectivity assay by immunoperoxidase in microplate cultures of MDCK cells was developed which was simple enough for routine use and practically as sensitive as the egg infectivity test. The potential risks of accidental importation of influenza infection in pig was assessed by determining the survival time of the porcine influenza virus H1N1 in pig tissues. It was found that the virus may keep its infectivity in frozen (-20°C) pig tissues for up to 15 days. The interspecies transmission of porcine influenza viruses was studied using turkeys infected with porcine influenza isolates. Although both A/swine/Weybridge/86 and A/swine/Weybridge/87 were transmitted from infected turkeys to pigs, only A/swine/Weybridge/86(H1N1) infected turkeys presented clinical signs of disease. More than 50% of the pigs presented the virus in the nostrils and/or faeces, at some time during the experiment, and all seroconverted. Transmission from these pigs to newly introduced turkeys was not observed, nor was seroconversion detected. Influenza epidemiology in Brazil was investigated by serological studies using pig sera collected in different areas of that country, using human, porcine and avian isolates of influenza viruses. Highest antibody titres were found against A/Leningrad/86(H3N2) (19%) and A/Port Chalmers/73(H3N2) (17%), but not against specific porcine isolates. Only serological evidence was found to suggest that reassortant influenza viruses occur in English pig herds. However, interspecies transmission of influenza viruses between man and pigs, and the maintenance of human strains in English pig herds was demonstrated by the isolation of two H3N2 influenza viruses very similar to A/Port Chalmers/73, present in the human population in the 1970s.

Human populations are constantly exposed to emerging pathogens such as influenza A viruses that result from cross-species transmissions. Generally these sporadic events are evolutionary dead-ends, but occasionally, viruses establish themselves in a new host that offers a novel genomic context to which the virus must adjust to avoid attenuation. However, the dynamics of this process are unknown. Here we present a novel method to characterize the time it takes to G+C composition at third codon positions (GC3 content) of influenza viruses to adjust to that of a new host. We compare the inferred dynamics in two subtypes, H1N1 and H3N2, based on complete genomes of viruses circulating in humans, swine and birds between 1900-2009. Our results suggest that both subtypes have the same fast-adjusting genes, which are not necessarily those with the highest absolute rates of evolution, but those with the most relaxed selective pressures. Our analyses reveal that NA and NS2 genes adjust the fastest to a new host and that selective pressures of H3N2 viruses are relaxed faster than for H1N1. The asymmetric nature of these processes suggests that viruses with the greatest adjustment potential to humans are coming from both birds and swine for H3N2, but only from birds for H1N1.

The influenza A virus non-structural (NS1) protein is multifunctional, and during virus-infection NS1 interacts with several factors in order to manipulate host-cell processes. This study reports that NS1 binds directly to p85β, a regulatory subunit of phosphoinositide 3-kinase (PI3K), but not to the related p85α. Expression of NS1 was sufficient to activate PI3K and cause the phosphorylation of a downstream mediator of PI3K signalling, Akt. However, in virus-infected MDCK cells, the kinetics of Akt phosphorylation did not correlate with NS1 expression, and suggested that negative regulation of this signalling pathway occurs subsequent to ~8h post-infection. Mapping studies showed that the NS1:p85β interaction is primarily mediated by the NS1 C-terminal domain and the p85β inter-SH2 (Src homology 2) domain. Additionally, the highly conserved tyrosine at residue 89 (Y89) of NS1 was found to be important for binding and activating PI3K in a phosphorylation-independent manner. The inter-SH2 domain of p85β is a coiled-coil structure that acts as a scaffold for the p110 catalytic subunit of PI3K. As NS1 does not displace p110 from the inter-SH2 domain, a model is proposed whereby NS1 forms an active heterotrimeric complex with PI3K, and disrupts the ability of p85β to control p110 function. Biological studies revealed that a mutant influenza A virus (Udorn/72) expressing NS1 with phenylalanine substituted for tyrosine-89 (Y89F) exhibited a small-plaque phenotype, and grew more slowly in MDCK cells than wild-type virus. Unexpectedly, another mutant influenza A virus strain (WSN/33) expressing NS1-Y89F was not attenuated in MDCK cells, yet appeared to be less pathogenic than wild-type in vivo. Overall, these data indicate a role for NS1-mediated PI3K activation in efficient influenza A virus replication. The potential application of this work to the design of novel anti-influenza drugs and vaccine production is discussed.

Respiratory infection is a major disease burden worldwide. Statistical reports revealed it is one of the main causes of mortality and morbidity especially in young children. Influenza infection is one of the predominant cause associate with respiratory infection. Traditionally, studies have been emphasized on the detection of influenza A and B virus owing to their significance in clinical and economic impact. Attention of influenza C virus is rarely recognized due to its difficulty in isolation. However, recently, increasing reports have been illustrated the co-circulation of influenza C virus globally. Serological studies also suggested majority of people worldwide acquired influenza C virus infection in their early childhood or adolescent stage, yet information regarding influenza C virus is still inadequate. Epidemiological and clinical impact of influenza C virus in pediatric patients in Hong Kong was examined by the approach of real-time PCR. From November 2007 to April 2011, a total of 1, 037 specimens were obtained from pediatric patients exhibited apparent respiratory tract illness in Hong Kong. Eleven strains of influenza C virus were detected by real-time PCR approach. All patients with influenza C virus infection were below 5 years of age with the youngest age of 11 months. The ratio of infection in male to female was approximately one to one. High grade fever appeared to be the most frequent clinical manifestations (10/11) of influenza C virus infection. Upper respiratory tract infection was also occasionally observed. The clinical presentation of influenza C virus was similar to its influenza counterpart. Phylogenetic analysis of influenza C virus was examined in 6/11 of the isolates to determine the lineages of co-circulating influenza C viruses in Hong Kong. Nucleotide sequencing was performed with primer targeting the hemagglutinin-esterase (HE) gene. Result revealed that most of the detected influenza C virus associate with the C/Sao Paulo/378/82 related lineage. Results from this study revealed the positive rate of influenza C was comparable to influenza B and resultant respiratory symptoms could be severe in pediatric patients It is suggested to consider the inclusion of influenza C virus detection in routine diagnostic panel and real-time PCR could be a desirable detection platform account for its sensitivity and rapidity.
published_or_final_version
Microbiology
Master
Master of Medical Sciences

Doctor of Philosophy
Department of Diagnostic Medicine/Pathobiology
Wenjun Ma
Influenza A virus (IAV) is an enveloped, segmented, negative-sense RNA virus that infects avian species and mammals. Its segmented feature enables antigenic shift which can generate novel IAVs that pose a threat to animal and public health due to lack of immunity to these viruses. Pigs have been considered the “mixing vessels” of influenza A viruses to generate novel reassortant viruses that may threaten animal and public health. Therefore, it is necessary to understand the pathogenicity and transmissibility of newly emerged reassortant viruses in swine. Adding to this complexity is the newly identified bat influenza A-like viruses which have roused interest in understanding the evolutionary history and pandemic potential of bat influenza. At least 10 different genotypes of novel reassortant H3N2 IAVs with gene(s) from 2009 pandemic H1N1 [A(H1N1)pdm09] have been identified in pigs in the United States. To date, only three genotypes of these viruses have been evaluated in animal models leaving the pathogenicity and transmissibility of the other seven genotype viruses unknown. We showed that reassortant viruses with genes from A(H1N1)pdm09 are pathogenic and transmissible in pigs. Further studies showed that avian-like glycine at position 228 of the HA receptor binding site is responsible for inefficient transmission of the reassortant H3N2 IAV with five A(H1N1)pdm09 genes. Studying the recently discovered IAV-like sequences from bats has been hindered by the lack of live virus isolation or culturing. Using synthetic genomics, we successfully rescued modified bat influenza viruses that had the HA and NA coding regions replaced with two classical IAVs. Additional studies were performed with truncations on NS1 protein and substitution of a putative virulence mutation in bat influenza PB2. Virus reassortment experiments demonstrated that bat influenza has limited genetic and protein compatibility with other influenza viruses; however, it readily reassorts with another divergent bat influenza virus. Taken together, our results provide insights into the pathogenicity and transmissibility of novel reassortant H3N2 IAVs in pigs. It also indicates that the bat influenza viruses recently identified are viable viruses that pose little pandemic threat to humans. Moreover, they provide new insights into the evolution and basic biology of influenza viruses.

Background: The seriousness of human influenza virus infection, in combination with the transmissibility of the virus, determines the impact that the virus will have in a population. However, the uncertainty surrounding the seriousness and changes in seriousness hindered the calibration of the early public health response. Methods: I applied statistical models to population-based mortality data and hospitalizations among patients with laboratory-confirmed H1N1pdm09. I estimated the disease burden, retrospectively and prospectively determined seriousness of influenza virus infections including the risk of death on a per-infection basis (IFR) and on a per-hospitalization (HFR) of H1N1pdm09, and investigated changes in seriousness. I used serologic surveillance data to estimate the cumulative incidence of infection in a population, and used it as the denominator of the IFR. I also conducted systematic reviews and meta-analysis to summarize published estimates of the risk of death among cases (CFR) and HFR of the pandemic influenza H1N1pdm09 virus. Results: I estimated that the first wave of H1N1pdm09 was associated with approximately 232 (95% confidence interval: 136, 328) excess deaths in all ages in Hong Kong, which was around 4 times the observed number of laboratory-confirmed deaths of H1N1pdm09. The point estimates for the IFR and HFR increased substantially with age. I included 77 estimates for the CFR from 50 published studies; whereas I included 187 estimates for the HFR from 184 published studies. The CFR was widely used to assess seriousness but the variation of a ‘case’ varied considerably in the literature. Variability in published estimates of the HFR was much less than variability in the CFR. Conclusions: Early in the next pandemic, estimation of the HFR, IFR or symptomatic CFR may provide a timely picture of the seriousness of infection, particularly if presented in comparison between two influenza virus infections in the same setting. Ongoing monitoring of mortality and influenza activity could permit identification of changes in seriousness of influenza virus infections.
published_or_final_version
Public Health
Doctoral
Doctor of Philosophy

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Biological Engineering, 2012.
Cataloged from PDF version of thesis.
Includes bibliographical references.
Human adaptation of avian influenza viruses pose an enormous public health challenge as the human population is predominantly naive to avian influenza antigens. As such, constant surveillance is needed to monitor the circulating avian strains. Of particular importance are strains belonging to H5N1, H7N7, H7N2 and H9N2 subtypes that continue to circulate in birds worldwide and have on occasions caused infections in humans. A key step in influenza human adaptation is the accumulation of substitutions/mutations in the viral coat glycoprotein, hemagglutinin (HA), that changes HA's binding specificity and affinity towards glycan receptors in the upper respiratory epithelia (referred to as human receptors). Unlike for the H1, H2, H3 and more recently H5 HA a correlation between the quantitative binding of HA to human receptors and respiratory droplet transmissibility has not been established for H9 and H7 subtypes. This thesis is a systematic investigation of determinants that mediate changes in HA-glycan receptor binding specificity, with focus on the molecular environments within and surrounding the glycan receptor binding site (RBS) of avian HAs, particularly the H9 and H7 subtypes. The glycan receptor binding properties of HA were studied using a combination of biochemical and molecular biology approaches including dose dependent glycan binding, human tissue staining and structural modeling. Using these complementary analyses, it is shown that molecular interactions between amino acids in and proximal to the RBS, including interactions between the RBS and the glycan receptor converge to provide high affinity binding of avian HA to human receptors. For the H9 HA [alpha]2-->6 glycan receptor-binding affinity of a mutant carrying Thr-189-->Ala amino acid change correlated with the respiratory droplet transmission in ferrets conferred by this change. Further, it was demonstrated for the first time that two specific mutations; Gln226-->Leu and Gly228-->Ser in glycan receptor-binding site of H7 HA substantially increase its binding affinity to human receptors. These approaches and findings contribute to a framework for monitoring the evolution of HA and the development of general rules that govern human adaption applicable to strains beyond ones currently under study.
by Karunya Srinivasan.
Ph.D.

Master of Science
Department of Diagnostic Medicine/Pathobiology
Wenjun Ma
With introduction of the 2009 pandemic H1N1 virus (pH1N1) into swine herds, reassortment between the pH1N1 and endemic swine influenza viruses (SIVs) has been reported worldwide. Recently, reassortant H3N2 and H1N2 variant SIVs that contain the M gene from pH1N1 virus and the remaining seven genes from North American triple-reassortant (TR) SIVs have emerged. These variant viruses have caused more than 300 cases of human infections and one death in the USA, creating a major public health concern. To date, the pathogenicity and transmissibility of H1N2 variant viruses in pigs has not been investigated. Through passive surveillance, we have isolated two genotypes of reassortant H1N2 viruses with pH1N1 genes from diseased pigs in Kansas. Full genome sequence and phylogenetic analysis showed that one is a swine H1N2 variant virus (swH1N2v) with the M gene from pH1N1; the other is a reassortant H1N2 virus (2+6 rH1N2) with six internal genes from pH1N1 and the two surface genes from endemic North American TR H1N2 SIVs. Furthermore, we determined the pathogenicity and transmissibility of the swH1N2v, a human H1N2 variant (huH1N2v), and the 2+6 rH1N2 in pigs using an endemic TR H1N2 SIV (eH1N2) isolated in 2011 as a control. All four viruses were able to infect pigs and replicate in the lungs. Both H1N2 variant viruses caused more severe lung lesions in infected pigs when compared to the eH1N2 and 2+6 rH1N2 viruses. Although all four viruses are transmissible in pigs and were detected in the lungs of contact animals, the swH1N2v shed more efficiently than the other three viruses in the respective sentinel animals. The huH1N2v displayed delayed and inefficient nasal shedding in sentinel animals. Taken together, the human and swine H1N2 variant viruses are more pathogenic and the swH1N2v more transmissible in pigs and could pose a threat to public and animal health.

Influenza B virus contributes to a significant portion of influenza disease burden in men. It is structurally similar and replicates in the same manner as the influenza A virus, leading to a comparable clinical presentation between the two viral species. Since 1977, influenza B has caused seasonal epidemics around the world together with A/H1N1 and A/H3N2 subtypes, and has a strong affinity to affect children of school age and young adults. In the 1980s, two antigenically distinct lineages of influenza B virus emerged, one being the B/Yamagata lineage and the other known as B/Victoria lineage. The most significant antigenic difference between the two is located in the HA1 domain of the viral hemagglutinin. Host immunity is not shared between the two viral lineages. Therefore, the global prevalence of the two influenza B lineages is closely monitored by the World Health Organization in order to decide which viral lineage to include in the annual trivalent influenza vaccines. Surprisingly, the current methods used in influenza B viral surveillance and lineage discrimination have not seen much technical advancement in nearly 25 years since the emergence of two viral lineages. The current study presents a novel, asymmetric real-time PCR assay which is able to determine the viral lineage in addition to detecting the presence of influenza B virus in clinical specimens. Asymmetric PCR is performed by deliberately limiting the amount of primers in one side of a PCR reaction. This significantly affects the replication efficiency and sensitivity of the PCR reaction, but at the same time facilitates target sequence detection by hybridization probes, due to an increased number of single stranded products in the reaction. Nevertheless, the use of asymmetric PCR has been avoided in the past. The recent introduction of linear-after-the-exponential (LATE) PCR refines the method by adjusting melting temperature of PCR primers so that TmLimiting – TmExcess ≥ 0°C. The modification is shown to raise the efficiency of asymmetric PCR to those of symmetric PCR, as well as allowing more relaxed criteria for PCR primer and probe design. In the current asymmetric assay, pan-influenza B primers and probes targeting Victoria and Yamagata linage specific regions of the influenza B HA were evaluated against a similar symmetric influenza B assay published by the World Health Organization. HA plasmid standards and 155 clinical specimens were tested by both assays, in which the two had intra-assay CV% of less than 5%. Albeit the efficiency and sensitivity of WHO published assay was slightly higher, LATE-PCR based assay performed influenza B detection and genotyping simultaneously with the use of hydrolysis probes. The overall sensitivity/ specificity of the genotyping assay are 96.81%/100% while the WHO recommended assay is at 98.94%/100% for influenza B detection. The LATE-PCR based genotyping assay also successfully genotyped 89 out of 94 clinical specimens. In conclusion, the influenza B genotyping assay evaluated in this study performed favorably and could serve as an alternative to cumbersome viral culture methods to aid in high-throughput global influenza surveillance.
published_or_final_version
Microbiology
Master
Master of Medical Sciences

Avian H5N1 viruses have perpetuated in poultry and caused sporadic human transmission since 1997. Vaccine candidates for the potential pandemic caused by H5N1 viruses have been continuously updated by World Health Organization. Multiple genetic lineages of H5N1 viruses which co-circulate and rapidly evolve in different regions, together with periodic population replacement of newly emerged genetic and antigenic variants in the field, pose great challenge for H5N1 vaccine candidate selection. The complexity of avian H5N1 viruses evolution raises an important issue for studying antigenic properties and also for projecting antigenic trend of this virus since the model established for the seasonal influenza viruses may not apply to H5N1 viruses which they are still in the animal phase. In contrast, the 2009 pandemic H1N1 viruses have established as another seasonal influenza viruses in humans. How will this swine originated viruses evolve genetically and antigenically in humans? For the first in human history, we are able to track the changes of pandemic viruses from the very beginning when they transmitted to human. This study focuses on antigenic and receptor binding properties of avian H5N1 viruses from 1997 to 2010 and 2009 pandemic H1N1 viruses from 2009 to 2011. It is found that avian H5N1 viruses continue to display highly diverse antigenic profile. The newly emerged H5N1 virus variants of clade 2.3.4 in 2008 and clade 2.3.2 in 2010 exhibit distinct antigenic properties as compared to the genetically similar viruses that were characterized previously. Receptor binding analysis showed H5N1 viruses still exhibit binding preference for avian type receptor. However, analysis of escape mutants selected from H5N1 viruses exposed to H5 monoclonal antibodies in cell based assay indicates that mutations in the conserved sites may cause switch of receptor binding specificity to human type or dual specificity for both human and avian. Based on antigenic and receptor binding analyses, it is found that the 2009 pandemic H1N1 viruses isolated from 2009 to 2011 are relatively stable. Most of the antigenic variants to monoclonal antibodies are transient and not able to become prevalent. It remains to be investigated if more significant antigenic variants may emerge in the coming seasons when population immunity prevails this virus. In conclusion, this study showed that clade 2.3 avian H5N1 viruses become increasingly antigenic distinct as compared to clade 2.1 and 2.2 viruses. Antigenic variation in antigenic sites may change receptor binding specificity in avian H5N1 viruses. The 2009 pandemic H1N1 viruses remain stable up to date but continue monitoring in coming seasons is necessary.
published_or_final_version
Microbiology
Master
Master of Philosophy

Cold-adapted (ca) live attenuated influenza vaccines (LAIVs) have been introduced as alternatives to existing inactivated influenza vaccines. The influenza A components of the FDA-approved ca LAIVs (Flumist®; Medimmune) have common internal genes derived from the donor strain A/Ann Arbor/6/60 ca and surface genes derived from current wild-type (wt) epidemic strains. The aim of this thesis was to investigate determinants of immunogenicity for reassortants of A/Ann Arbor/6/60 ca, using a range of immunological assays, including recently developed MHC tetramer techniques. From the study, the extent of viral replication in the respiratory tract of mice, the primary site of inoculation, was a key factor in determining ca vaccine immunogenicity. Replication was shown to be influenced by both viral surface Ags and the host MHC. The H3 ca reassortants CR6, CR18, CR29 and CR6-35* exhibited greater replication efficiency (as determined by their PFU:HAU ratios) than the H1 ca reassortants CR35 and CR6-35. The H3 ca reassortant CR6 caused a 3.79% loss in body weight but no losses were observed for the H1 ca reassortant CR35 and the ca H2N2 donor strain A/Ann Arbor/6/60 ca. Higher HI responses were detected after 3 weeks in groups infected with the H3 ca reassortant CR6 (GMT 80) than with the H1 reassortant CR35 (GMT 10) and the H2 ca donor strain A/Ann Arbor/6/60 ca (GMT 13). Recently developed techniques were used to evaluate specific T-cell response to ca LAIVs. Fluorescent-labelled tetramer is the key reagent for use in tetramer-based flow cytometry assays. The NP366-374 peptide of influenza A viruses comprises an immunodominant epitope that is highly conserved between subtypes. Tetramers developed for A/PR/8/34 (H1N1) were able to detect NP-specific cytotoxic T lymphocytes (CTLs) induced by A/Ann Arbor /6/60 ca (H2N2). An attempt to prepare the A/Ann Arbor/6/60 ca-specific-NP-tetramer is described. H-2Db monomers were successfully refolded with the peptide, but only 20% were able to form tetramers through biotin-streptavidin linkage, resulting in a poor capacity to stain. By contrast, an IFN-γ ICC assay developed in parallel demonstrated that peptide NP366-374 was able to restimulate A/Ann Arbor/6/60 NP ca-specific CTLs and secrete IFN-γ when tested in vitro. Specific-B and T cell responses induced in the lungs in response to infection by ca reassortants exhibited great variability that was determined by the growth characteristics of different viruses. Type I (CTL) responses were induced by low yielding ca reassortants, such as CR35 (H1N1). Viruses with enhanced growth characteristics, such as CR6 (H3N2), produced higher Type II (HA-specific Ab) responses. In addition, host factors, such as MHC type, were found to play an important role in responses to the same viruses. Susceptible mouse strains, such as C57BL/6, showed higher CTL but lower serum Ab responses than more resistant strains, such as BALB/c. Throughout this PhD project, a fine balance between the humoral and CMI, local and systemic immune responses induced by ca LAIVs was demonstrated. The need to assess local immune responses, in addition to serum antibody levels, for the evaluation of vaccine efficacy was an important conclusion of the thesis.

Influenza A viruses cause seasonal epidemics and pandemics in the human population, resulting in significant morbidity and mortality. Influenza can be controlled by vaccination and antiviral therapy. However, antigenic drift, reducing vaccine effectiveness, and the development of antiviral resistance can result in reduced efficacy of the control measures. Drugs that target host cell processes, such as glycosylation, may be employed to complement drugs that target the virus, and iminosugar compounds which inhibit α-glucosidases have been reported to show antiviral activity against some viruses. Here, I have examined the effect of two iminosugars on human influenza A viruses. I have shown that two α-glucosidase inhibitors, N-butyl deoxynojirimycin (NB-DNJ) and N-nonyl deoxynojirimycin (NN-DNJ), show antiviral activity in cell culture against three human influenza A viruses: a recently circulating seasonal H3N2 virus strain, A/Brisbane/10/2007, an older H3N2 strain, A/Udorn/307/72, and a representative of the currently circulating pandemic H1N1 virus, A/Lviv/N6/2009. Of the two, NN-DNJ was the more potent drug. The virus target and mode of action of NN-DNJ has been examined. The effect of the drug was most marked after infection. Consistent with its action as an α-glucosidase inhibitor, NN-DNJ treatment resulted in altered glycan processing, as shown by a reduction in electrophoretic mobility of both influenza virus glycoproteins, haemagglutinin (HA) and neuraminidase (NA). NN-DNJ treatment was found to reduce cell surface expression of H3 HA. The level of sialidase activity of NA was reduced in the infected cell, however addition of exogenous sialidase to cells did not complement NN-DNJ mediated inhibition of virus replication. Using reassortant viruses, the drug susceptibility profile correlated with the HA. Reverse genetics was used to determine the effect of altering the glycosylation status of the HA; engineered viruses carrying modified sites seemed slightly more sensitive to the inhibitor than the parent virus. These results show that NN-DNJ inhibits influenza A virus replication in a strain-specific manner which is dependent on the HA.

Influenza A viruses are important pathogens of humans, other mammals and birds. Swine are considered to be the ‘mixing vessel’ for influenza viruses because of their susceptibility to infection with not only swine influenza viruses but also human and avian influenza viruses. After infection of pigs with different influenza viruses, reassortment events between genomic RNA segments and point mutations can take place which can result in novel influenza virus strains capable of causing human pandemics. To combat infections, vaccination is available in many countries for humans, but not typically used in pigs. However, anti-influenza drugs have been used to treat livestock, and mutations conferring drug resistance occur in circulating strains. The mechanisms responsible for the emergence and spread of drug resistant mutations against amantadine and oseltamivir have been studied previously but often gave conflicting results. Therefore, this PhD thesis focused on resolving the mechanisms responsible for this rapid drug resistance spread. In chapter one I examine the extent of reassortment events in swine influenza A viruses by analysing within subtype reassortment and extrapolating the results for the between subtype reassortment. Reassortment is one of the mechanisms that can be responsible for mutations, conferring resistance to drugs, to spread between strains, and thus spread in the host population. The findings of this chapter show that the genomic segments most prone to reassortment code for a polymerase (PB1) and both glycoproteins, within all three subtypes studied. Since particular mutations in the matrix protein (MP) segment cause resistance to amantadine, my study focused on MP compared to other segments and revealed moderate level of reassortment. MP reassorts well with polymerases, both within and between subtype, while nonstructural (NS) is least likely to reassort. Chapter two of this thesis aimed at resolving the origin and spread of the most common drug resistance conferring mutation in swine influenza viruses which causes amantadine resistance. I show first that this mutation occurred in swine influenza viruses and was therefore not transmitted from the recently ancestral avian influenza strains, and second that the prevalence of resistance in swine influenza viruses is due to functional linkage of mutations at other sites and not by direct drug pressure. In chapter three I examine the mechanisms responsible for the rapid rise and spread of oseltamivir resistance in human influenza H1N1 viruses which arose in the absence of drug use. The primary mutation lies in the neuraminidase glycoprotein but because of the close functional interaction I focus on changes in haemagglutinin that occurred in association with resistance. The results showed several mutations in haemagglutinin were associated with resistance suggesting selection acting on haemagglutinin in order to balance the activity of both glycoproteins. Overall these results show the importance of functional linkage between segments as a mechanism for the occurrence of drug resistance conferring mutations, and reassortment as a means of spreading these mutations into newly emerging strains.

PB2 ist ein essentieller Bestandteil der trimeren RNA abhängigen RNA Polymerase von Influenzaviren und ist bekannt für seine Schlüsselrolle in der Bestimmung des Viruswirtsspektrums. Diese Arbeit diente der Identifizierung neuer Interaktionspartner von PB2 eines saisonalen und eines hochpathogenen Influenzavirus Stammes im Kontext infizierter humaner alveolar Epithelzellen (A549) unter Einsatz massenspektrometrischer Analysen. Die anschließende Untersuchung ausgewählter zellulärer Interaktoren hatte zum Ziel, deren Einfluss auf den Replikationszyklus der Influenzaviren zu bestimmen, sowie Unterschiede in ihrer Relevanz für das saisonale und das hochpathogene Virus aufzuzeigen. Die Erzeugung und Nutzung von Influenzaviren die einen Strep-tag an ihrem PB2 Protein tragen ermöglichte eine Anreicherung von PB2 und seiner Interaktionspartner. Die anschließende massenspektrometrische Analyse identifizierte 22 potentielle PB2 Interaktionspartner. Eine Auswahl an 13 Proteinen wurde tiefer gehend analysiert und eine Komplexbildung mit PB2 konnte für 9 Proteine bestätigt werden. Darüber hinaus zeigten 11 Proteine einen Polymerase stimulierenden bzw. hemmenden Effekt. Das Polymerase stimulierende Protein HSPA8 wurde zur weiteren Untersuchung ausgewählt. Während ein Einfluss von HSPA8 auf den hochpathogenen Influenzastamm nicht abschließend geklärt werden konnte, wurde seine Bedeutung für den Vermehrungszyklus des saisonalen Stammes aufgezeigt. Die Überexpression von HSPA8 führte zu einer Steigerung der Polymerase-Aktivität, wohingegen die Erniedrigung des HSPA8 Spiegels in einer Verringerung der viralen Replikation und der Polymerase-Aktivität resultierte. Interessanterweise führte die Erniedrigung des HSPA8 Spiegels auch zu stark verminderter PB2-Expression, jedoch nur im Falle des saisonalen Influenzastammes. Dieser Befund deutet auf eine Rolle von HSPA8 als PB2-Chaperon, notwendig für Proteinstabilität von saisonalen aber nicht hochpathogenen Influenzaviren, hin.
PB2 is an essential component of the influenza virus trimeric RNA dependent RNA polymerase and is known to play a key role in virus host range determination. Here, a combined affinity-purification/mass spectrometric approach was performed to identify novel interaction partners of PB2 of seasonal and highly pathogenic viral strains in infected human alveolar epithelial cells (A549). The subsequent analysis of selected cellular interaction partners aimed to determine the influence of these proteins on the replication cycle, as well as to determine differences in their relevance for the seasonal and the highly pathogenic influenza virus strain. Generation and use of recombinant influenza viruses carrying a Strep-tag at their PB2 protein allowed for enrichment of PB2 and its interaction partners. The subsequent mass spectrometric analysis identified 22 potential PB2 interaction partners. A selection of 13 proteins was further analyzed, and co-precipitation with PB2 was confirmed for 9 proteins. Moreover, an inhibitory or stimulatory effect on polymerase activity was observed for 11 proteins. The polymerase stimulating protein HSPA8 was selected for further investigation. While the influence of HSPA8 on the highly pathogenic strain remained unclear, its importance for seasonal influenza virus life cycle was demonstrated. Overexpression of HSPA8 resulted in increased polymerase activity while HSP8 knock down resulted in reduction of viral replication and viral polymerase activity. Intriguingly, the knock down of HSPA8 led to a strong decrease of PB2 protein expression. However, this was only observed for seasonal PB2. These results indicate a role of HSPA8 as a PB2 chaperone, necessary for protein stability of seasonal but not highly pathogenic influenza virus.

Resuspended floor dust constitutes up to sixty percent of the total particulate matter in indoor air. This fraction may also include virus-laden particles that settle on the floor after being emitted by an infected individual. This research focuses on predicting the concentration of influenza A viruses in resuspended dust, generated by people walking in a room, at various heights above the floor. Using a sonic anemometer, we measured the velocity field from floor to ceiling at 10-cm intervals to estimate the magnitude of turbulence generated by walking. The resulting eddy diffusion coefficients varied between 0.06 m2 s-1 and 0.20 m2 s-1 and were maximal at ~0.75-1 m above the floor, approximately the height of the swinging hand. We used these coefficients in an atmospheric transport model to predict virus concentrations as a function of the carrier particle size and height in the room. Results indicate that the concentration of resuspended viruses at 1 m above the floor is about seven times the concentration at 2 m. Thus, shorter people may be exposed to higher concentrations of pathogens in resuspended dust indoors. This study illuminates the possibility that particle resuspension could be a mode of disease transmission. It also emphasizes the importance of considering resuspension of particulate matter when designing ventilation systems and flooring in hospitals and residences.
Master of Science

Influenza infections continue to be a global health concern that causing both seasonal epidemics and unpredictable pandemics. Hemagglutinin (HA) and Neuraminidase (NA) are the two major surface glycoproteins of influenza viruses, which are important for their host cell sialic acid (Sia) receptor binding and cleaving activities. Although numerous methods have been developed to study the HA and NA interactions with sialic acid, x-ray crystallography remained the only method to provide detailed information at atomic resolution. The aim of this study is to develop and evaluate a novel strategy for the investigation of influenza virus-receptor interactions, which is able to provide information about an interaction down to atomic resolution. Influenza virus-like particles (VLPs) containing HA and NA separately were developed and it was reported here for the first time that sole expression of NA in mammalian cell led to VLP formation. Characterization of these VLPs demonstrated that they are non-infectious, but morphologically and biochemically mimic the native viruses. Therefore the VLPs can be regarded as an ideal research model to study the HA-Sia interaction without the interference of NA, or vice versa. Saturation transfer difference (STD) NMR spectroscopy is a state-of-the-art technology to determine how a binding-ligand interacts with its target protein. Modification of STD-NMR methodology was performed to adapt the technique to influenza VLP system. HA-Sia interaction was investigated in great detail and group epitope mapping of the interacting ligands was performed by analyzing the STD-NMR spectra. The data obtained are in a good agreement with the well established crystallography technique, reflecting the reliability of the STD-NMR technology. Regarding the NA-Sia interaction, my data demonstrated that substrate-hydrolysis specificity of NA is dependent on the binding of NA to those ligands. In addition, using competition experiments with NA inhibitor, a secondary sialic acid binding site was detected. It is the first direct experimental evidence that confirms avian, seasonal human and human pandemic swine-origin influenza virus N1 neuraminidases exhibit a distinct secondary binding site. In conclusion, here I presented a novel interdisciplinary strategy using VLP and NMR technology to study the interaction of influenza virus with its receptor. This method is unique in its ability to provide detailed information on the HA and NA interactions with sialic acid leading to group epitope mapping of the binding ligands, which will help us not only to understand the virus tropism but also to define new therapeutic targets.
published_or_final_version
Microbiology
Doctoral
Doctor of Philosophy

Thesis (Ph. D.)--University of Kentucky, 2004.
Title from document title page (viewed Oct. 11, 2004). Document formatted into pages; contains ix, 114 p. : ill. Includes abstract and vita. Includes bibliographical references (p. 90-113).