Construction and characterization of H5N1-recombinant fowlpox viruses co-expressing host cytokines

Possessing a large double stranded DNA genome up to 300 kb, fowlpox virus (FWPV) has been developed to express avian influenza virus (AIV) antigens since the late 1980s. A more advanced approach would be to coexpress host cytokines from such recombinants. This thesis describes the strategy to construct H5N1-recombinant FWPV (rFWPV) coexpressing chicken Interleukin 12 (IL-12) or Interleukin 15 (IL-15), and discusses the immunogenicity of the recombinants following inoculation into specific-pathogen-free (SPF) chickens. Previously cloned and sequenced cDNAs encoding full-length H5 and N1 of influenza strain A/Chicken/Malaysia/5858/2004 genes were amplified by PCR and inserted into plasmid pEFL29, under the control of a copy of the vaccinia virus p7.5 early/late promoter. The expression cassettes were recombined into the genome of the FP9 strain of FWPV at the fpv002 locus. Recombinant viruses were produced by transfection of the plasmid into chicken embryo fibroblasts (CEFs) after infection with FP9, and isolated by six fold plaque purification on CEFs using X-Gal selection. Chicken IL-12 or IL-15 genes, under control of a synthetic/hybrid poxvirus promoter, were inserted into a ‘transient dominant selection’ recombination plasmid, pPC1.X. The cytokine expression cassettes were then recombined, at the fpPC1 (fpv030) locus, into rFWPV already carrying AIV genes. Following three rounds of passage in CEFs in the presence of mycophenolic acid (MPA), recombinant viruses carrying the gpt gene were isolated. These unstable recombinants were plaque-purified in the absence of MPA until they lost the gpt gene spontaneously, verified by their failure to replicate in the presence of MPA. Recombinant proteins were successfully detected using western blotting and indirect immunofluorescence assay (IFAT). Parental and rFWPV (105 PFU) were inoculated subcutaneously into one-day-old SPF chickens. Sera from chickens immunized with rFWPV/H5 and rFWPV/H5/IL-15 demonstrated viral neutralizing activities, based on the haemagglutation inhibition (HI) test, in which reached a peak at Week 3. A competitive enzyme-linked immunosorbent (ELISA) assay detected N1-specific antibodies induced by rFWPV/N1 and rFWPV/N1/IL-12 at Weeks 4 and 5. Non-specific cellular immune responses were assessed by flow cytometric analysis to enumerate CD4+ and CD8+ T-lymphocytes in peripheral blood. Results of Experiment 2 showed chickens vaccinated with rFWPV/H5, rFWPV/H5/IL-15, rFWPV/N1 and rFWPV/N1/IL-12 demonstrated a higher increase in CD8+ than CD4+ T cell population, relative to control and chickens vaccinated with parental FWPV. Weekly weighing showed that chickens vaccinated with rFWPV/H5/IL-15 had the highest body weight compared to other groups, while the rFWPV/N1/IL-12 group showed the significantly lowest body weight. In summary, this study showed diverse immunogenicity of H5N1-rFWPV coexpressing IL-12 or IL-15. It also demonstrated a weight sparing effect of co-expressing IL-15 in rFWPV vaccines. The results provide the basis for future homologous challenge studies, using live H5N1 virus to evaluate the protective efficacy of the rFWPV vaccines

Light and life

Since the 10th century B.C., light has been appreciated as a source of energy. Psychologically, a powerful ray of light can induce calmness and excitement in humans. We have been told in the holy books of Islam, Judaism, and Christianity, how the Queen of Sheba (Balqis) had lived with hundreds of windows that allowed beams of sunlight in every morning before she prostrated in front of them. It is probably through this ancient practice that the physician Niels Finsen was inspired to use light to treat diseases and developed the first artificial light source for this purpose back in the 1900s

Innovation impulse

A perfect blend of design and innovation could serve as catalysts for future change. Designers offer knowledge and competencies to enable innovators envisage new ideas, with the ultimate aim of finding better and long-lasting solutions, by pushing boundaries and changing the norm. Inordinate power of good design will lead to invention of products or services with greater value and encouraging demand

Culture stimuly

People manifest beliefs, beliefs represent culture. Culture of certain comunities, or even from one generation to the next, defines the originality or character of people of the world. Accumulation of knowledge, experience, language, attitudes, religion, values, hierarchies and material objects distinguishes one group of people from another. Influencing stimulus can be many, including observation of natural ecological and environmental factors, particularly from species of animal and plant, or even the advancement of technology by time

Malady in the sky

Birds migrating across national and intercontinental borders can play a role in the spread of infectious diseases. Their capability to travel over long distances and through a diversity of habitats exposes them to a wide range of microorganisms such as bacteria, virus, parasite or drug-resistant organisms. Similar to wild birds, these migratory birds are hosts or natural reservoirs for many zoonotic diseases. Without showing any clinical signs, they act as a silent disease carrier by possessing pathogens that can threaten the well-being of other animals and humans, hence, raising concern on public confidence in health care, food safety, and security

The ambiguous mystery

Zika virus is not new. The first case of human infection was detected in Uganda and Tanzania in 1952. This flavivirus was associated with diseases in 1964. The virus is spread mainly by the bite of an infected mosquito, and may also be transmitted sexually

Avian polyomavirus: a recent update

Avian polyomavirus disease is among the most common viral diseases of domesticated exotic birds as such in psittacine families. Caused by avian polyomavirus (APV) which possess a circular, double-stranded DNA which encodes for major structural virus protein 1 (VP1) and minor structural proteins VP2, VP3 and VP4, the disease is also known as Budgerigar fledgling disease polyomavirus (BFPyV), Papovavirus, and Psittacine polyomavirus. Infections from APV may lead to cutaneous haemorrhage, abdominal distension, feather abnormalities and even death. The APV virus has a broad avian host range and is known to cause acute chronic disease in several psittacine birds such as parrot, cockatoo, macaw, and budgerigar. The current status of APV epidemiology globally has not been fully recorded. Only the studies of the virus and disease caused within several countries are used as references, and few were done together with detection of beak and feather disease virus. Despite the common occurrence of APV among bird breeders in Malaysia, a very limited study has been done to evaluate the prevalence status of APV in Malaysia. In this review, we wish to disseminate knowledge, particularly to pet owners and bird breeders, on APV characterisations, its updated occurrence worldwide and prevention strategies. This information may be useful to trigger in depth study on the epidemiology of disease and better management practises among breeders

Prevalence of avian polyomavirus in psittacine birds in the Klang Valley

Avian polyomavirus (APV) primarily affects young birds and can cause mortality in a wide range of psittacine birds. This is the first study to detect the presence of APV in Malaysia. A total of 85 faecal samples were collected from symptom-free psittacines species from four different breeders in the Klang Valley. Upon genomic DNA extraction, the presence of APV was analysed by PCR using primers APVfull-AF (5'- ACAATGCCTAACGGAACGCC-3') and APVfull-AR (5'-CACCGAAGCGGCGATACTATA-3'). Positive results of 3 kbp PCR amplicon were detected in six out of the 30 samples (20%), which were from yellow-collared macaws, blue-headed parrots, red-crowned macaws, sulphur-crested cockatoos, blue-throated macaws, and Pesquet's parrots. As a conclusion, the prevalence of APV was clearly indicated

Stingless bee rearing and colony splitting

Stingless bee is a diverse group of highly eusocial bees (meliponines) comprising the Meliponini subfamily of the Apidae family. Throughout the tropics, various kinds of stingless bee have been reared by beekeepers for their products (honey, propolis and bee bread) and pollination services for fruits and vegetables. This meliponiculture in logs or hives is considered safe due to the bee’s inability to sting, with the latter being more preferable for monitoring and managing the colonies and harvesting the products. The source of stingless bee colonies is depending on efficient hunting of feral colonies, which might affect the ecosystem. Following to the growing trend of bee rearing in Malaysia, mainly due to the smart branding strategy of the government through research institutes and universities, e.g. Malaysian Agricultural Research and Development Institute (MARDI), Universiti Malaysia Terengganu and Universiti Sains Malaysia, it is crucial to increase the number of honey-producing hives by colony splitting. The basis of colony splitting includes provision oviposition (POP), swarming and antennation. Colony splitting is necessary due to deforestation, to expand income generation and for entomological study purposes. A more advanced approach in colony splitting is to prepare and rear the virgin queen bee in vitro, resulting in more queens per one single colony. With the advancement in stingless bee rearing techniques, people may apply the colony splitting strategy to maximise stingless bee products and enhance crop production

The bouncy beam

The nervous system consists of nerve cells or neurons, which can transmit information in the form of electrical signals to our sense organs. Neurons generally are not a good electrical conductor; however, they have elaborate mechanisms for generating electrical signals by eliminating negative resting potentials