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An attenuated infectious bronchitis virus (TW2575/98) vaccine strain was successfully developed after 75 serial passages in embryonated chicken eggs. However, the in ovo vaccination for disease control was not applied in practice because this vaccine strain is highly pathogenic to chicken embryos (CEs) causing early death, dwarfing and other harmful effects. We compared the differences in virus replication, pathological changes, and tissue tropism between the wild virus and attenuated vaccine strain in CEs inoculated with different viral titer levels, i.e. 0.1, 1 and 10 EID[Formula: see text]/egg. The wild virus caused dwarfing effect at high titer inoculation, whereas the attenuated vaccine strain caused the dwarfing effect only at a lower viral inoculation accompanied by the earlier infection establishment and embryonic death at high and medium titers. There were no significant differences in the pathological changes in CEs infected by both wild and attenuated strains. Detected by immunohistochemistry, the viral antigens of both strains could be found mainly at the epithelium of the chorioallantoic membrane, lung parabronchus, renal tubules and some in the spleen and heart serosa. These findings indicated that the early embryonic death and dwarfing is not related to the change in cell/tissue tropism of the vaccine strain, rather on the early infection establishment and viral load. We suggest that the vaccine strain inoculated titer could be adjusted to an optimal low level for in ovo vaccination to overcome the poor hatching rate for its higher virulence to chicken embryos.

The attenuated avian infectious bronchitis virus (IBV), derived from a wild strain (TW2575/98w) in chicken embryos after 75 passages, is designed as a commercial vaccine strain (TW2575/98vac) to control the disease in Taiwan. The differences in viral infectivity, replication efficiency, and genome sequences between TW2575/98w and TW2575/98vac were determined and compared. TW2575/98vac caused earlier death of chicken embryos and had higher viral replication efficiency. Thirty amino acid substitutions resulting from 44 mutated nucleotides in the viral genome were found in TW2575/98vac. All of the molecular variations lead to attenuation, found in TW2575/98, were not observed consistently in the other IBVs (TW2296/95, Ark/Ark-DPI/81, the Massachusetts strain, GA98/CWL0470/98, and CK/CH/LDL/97I) and vice versa. After further comparisons and evaluations from three aspects: (1) longitudinal analysis on the timing of variations appeared in specific homologous strain passages, (2) horizontal evaluations with the amino acid changes between wild and vaccine strains among the other 5 IBVs, and (3) inspection on alterations in the chemical characteristics of substituted amino acid residues in viral proteins, four amino acid substitutions [V342D in p87, S1493P and P2025S in HD1, as well as F2308Y in HD1(P41)] were selected as highly possible candidates for successful TW2575/98w attenuation. Our findings imply that molecular variations, which contribute to the successful attenuation of different IBVs, are diverse and not restricted to a fixed pattern or specific amino acid substitutions in viral proteins. In addition, four amino acid changes within the replicase gene-encoded proteins might be associated with TW2575/98 virus virulence.

Reverse transcription polymerase chain reaction (RT-PCR) was used routinely to detect the avian influenza virus (AIV) nucleoprotein (NP) gene. The purpose of the present study was to compare the correctness of a nested RT-PCR (nRT-PCR), one conventional RT-PCR with its outer primer (oRT-PCR) and the other conventional RT-PCR with its inner primer (iRT-PCR) to detect AIV NP gene. A total of 365 AI-free fecal swabs (73 pools), 7 tracheal swabs and anllantoic fluid from 25 chicken embryos were used to determine the analytic specificities of those tests. Compared with the iRT-PCR, the nRT-PCR was more sensitive for AIV detection. However, the specificities of nRT-PCR, oRT-PCR and iRT-PCR were 48.6% (35/72), 100% (67/67) and 91.3% (84/92), respectively. The amplifying band was sequenced and confirmed to be the AIV NP gene as the positive control. The specificity of this nRT-PCR is too low to be used for the AIV screening test.

Reticuloendotheliosis virus (REV) infects various animals including chickens, ducks, geese, pheasants, peafowl and other birds. REV causes tumor, runting disease syndrome and immunosuppression in infected birds. The purpose of this study was to develop a rapid, reliable and convenient method to detect anti-REV antibody. The REV envelope protein was expressed by baculovirus expression system and evaluated for the potential as an antigen in an enzyme linked-immunosorbent assay (ELISA). The env gene from a REV strain goose/3410/06 was cloned into bacmid vector and expressed in Sf9 insect cells. The expected recombinant envelope protein expressed in infected insect cells was demonstrated in western blot with size of 62 kDa, and purified for using as antigen in an ELISA. A total of 182 chicken serum samples were used to evaluate the suitability of the ELISA based on neutralization test as the gold standard. The results showed that the relative sensitivity and relative specificity of this ELISA were 88.5% and 97.7%, respectively. The present ELISA using baculovirus expression envelope protein can be used to detect anti-REV antibody in the field.

Immunohistochemistry features and cross-reactivity of commercially available antibodies are poorly understood in avian species. Here, we present a confirmative diagnosis of a rare systemically metastatic rhabdomyosarcoma in a seven-month-old male domestic chicken (Gallus domesticus). The animal had multiple grey to dark-red masses in the pectoral muscle, coelomic cavity, and multiple visceral organs. Microscopically, these masses were composed of spindloid neoplastic cells arranged into interwoven and patternless arrays with the presence of binucleated to multinucleated and karyomegalic neoplastic cells. A systemically metastatic rhabdomyosarcoma associated with avian leukosis viruses (ALV) infection was diagnosed by immunohistochemically demonstrating neoplastic cells dispersedly positive for desmin and p27 major capsid protein of ALVs, but diffusely negative for cytokeratin, vimentin, [Formula: see text]-smooth muscle actin, MyoD1, S-100, and neuron-specific enolase. These tumors and internal organs were further characterized positive for subgroups A, B, C, D, or E ALVs, but negative for subgroup J ALV, by polymerase chain reactions. The report suggests the potential of using IHC for a confirmative diagnosis of rhabdomyosarcoma and ALV infection in chickens. The role of endogenous and exogenous ALVs in the rhabdomyosarcoma is also discussed in the present report.

We determined the prevalence of influenza A virus in dogs in Taiwan and isolated A/canine/Taiwan/E01/2014. Molecular analysis indicated that this isolate was closely related to influenza A(H6N1) viruses circulating in Taiwan and harbored the E627K substitution in the polymerase basic 2 protein, which indicated its ability to replicate in mammalian species.

Tumor diseases occur frequently in chickens causing a great economic loss. Infected chickens’ pathological lesions are not pathognomonic. This study developed an accurate diagnosis for tumor diseases in chickens. Specific primers to reticuloendotheliosis virus (REV), avian leucosis virus subgroup A (ALV-A), avian leucosis virus subgroup J (ALV-J), and Marek’s disease virus (MDV) were combined into one tube with a single step multiplex reverse transcription polymerase chain reaction (mRT-PCR) performed to amplify the genes from each virus. A total of 117 sample pools containing blood and tissues were collected from chickens. Three of these pools (2.6%) showed REV positive, 22 (18.8%) ALV-J positive, 1 (0.8%) ALV-A positive, and 3 (2.6%) MDV positive. A total of 268 blood samples were used to compare the viral RNA detection from plasma using RT-PCR and provirus DNA from buffy coat using PCR. The result showed no difference from both tests. In conclusion, the present mRT-PCR could be used for tumor virus detections in chickens.

The H5 avian influenza virus subtype has huge impact on the poultry industry. Rapid diagnosis and accurate identification of the highly pathogenic avian influenza virus and low-pathogenicity avian influenza virus is essential, especially during H5 outbreaks and surveillance. To this end, a novel and rapid strategy for H5 virus molecular pathotyping is presented. The specific hemagglutinin gene of the H5 virus and the basic amino acid number of the motif at the hemagglutinin precursor protein cleavage site were detected using oligonucleotide microarray. Highly pathogenic and low-pathogenicity avian influenza viruses in Taiwan were differentiated using 13 microarray probes with the naked eye. The detection limit reached 3.4 viral RNA copies, 1000 times more sensitive than reverse transcription polymerase chain reaction. Thus, the oligonucleotide microarray would provide an alternative H5 pathogenicity determination using the naked eye for laboratories lacking facilities.

In this study, a multi-layer modulation technique was used in an SPR optical fiber sensor to enhance the sensitivity of the SPR optical fiber sensor by adjusting the SPR resonant wavelength. The sputtering process deposited 20 nm of TiO 2 , 11 nm of SiO 2 and 30 nm of gold film on the material surface to change the refractive index. Regardless of the different refractive index solutions (1.32 and 1.36), the sensitivities in wavelength interrogation of the SPR optical fiber with the single gold thin film and multi-layers modulation were 1.08×10 −5 RIUs and 1.74×10 −6 RIUs, respectively. The results showed the significant differences between the different refractive index solutions of 1.32 and 1.36 using the 850 nm light source to analyze the SPR optical fiber sensor in real-time. The sensitivities in intensity interrogation of the SPR optical fiber with the single gold thin film and multi-layers modulation were 1.08×10 −3 RIUs and 1.73×10 −4 RIUs, respectively, which indicated that the multi-layer modulation techniques could enhance the sensitivity of the SPR optical fiber sensor. The compact size of the multi-layer SPR fiber sensor had a wider detecting range of the refractive index and higher sensitivity, which had the potential for other applications in biological analysis with suitable wavelength.

Avian oncogenic viruses include the avian leukosis virus (ALV), reticuloendotheliosis virus (REV) and Marek's disease virus (MDV). Multiple oncogenic viral infections are frequently seen, with even Marek's disease vaccines reported to be contaminated with ALV and REV. The gross lesions caused by avian oncogenic viruses often overlap, making differentiation diagnosis based on histopathology difficult. The objective of this study is to develop a rapid approach to simultaneously differentiate, subgroup and pathotype the avian oncogenic viruses. The oligonucleotide microarray was employed in this study. Particular DNA sequences were recognized using specific hybridization between the DNA target and probe on the microarray, followed with colorimetric development through enzyme reaction. With 10 designed probes, ALV-A, ALV-E, ALV-J, REV, MDV pathogenic and vaccine strains were clearly discriminated on the microarray with the naked eyes. The detection limit was 27 copy numbers, which was 10-100 times lower than multiplex PCR. Of 102 field samples screened using the oligonucleotide microarray, 32 samples were positive for ALV-E, 17 samples were positive for ALV-J, 6 samples were positive for REV, 4 samples were positive for MDV, 7 samples were positive for both ALV-A and ALV-E, 5 samples were positive for ALV-A, ALV-E and ALV-J, one sample was positive for both ALV-J and MDV, and 3 samples were positive for both REV and MDV. The oligonucleotide microarray, an easy-to-use, high-specificity, high-sensitivity and extendable assay, presents a potent technique for rapid differential diagnosis of avian oncogenic viruses and the detection of multiple avian oncogenic viral infections under field conditions.

Neutralizing antibodies on the globular head of the hemagglutinin (HA) of avian influenza virus (AIV) are crucial for controlling this disease. However, most neutralizing antibodies lack cross reaction. This report describes the identification of a hemagglutinin epitope on the globular head near the receptor binding site of the H6N1 AIV. A monoclonal antibody named EB2 was prepared against the H6N1 AIV HA. Flow cytometry of AIV-infected chicken embryo fibroblast, DF-1 cells and specific-pathogen-free embryonated eggs were used to verify the neutralizing activity of this mAb. To narrow down the binding region, partially overlapping HA fragments and synthetic peptides were used to map the epitope by immune-blotting. The linear motif RYVRMGTESMN, located on the surface on the globular head of the HA protein, was identified as the epitope bound by EB2 mAb. Alignment of the EB2-defined epitope with other H6 AIVs showed that this epitope was conserved and specific to H6. We propose that this motif is a linear B-cell epitope of the HA protein and is near the receptor binding site. The identified epitope might be useful for clinical applications and as a tool for further study of the structure and function of the AIV HA protein.

Neoplasic diseases in chickens are frequently caused by avian leukosis virus (ALV), including subgroup A (ALV-A) and subgroup J (ALV-J) mostly. The objective of the present study was to develop a one-step, single-tube reverse transcription loop-mediated isothermal amplification (RT-LAMP) test for detecting ALV-A and ALV-J separately. Primers were designed from the conserved region of the pol and env genes of ALV-A and ALV-J for RT-LAMP tests. Other avian viruses were used to determine the specificity of these tests. The results showed that a ladder pattern on gel electrophoresis was observed for ALV-A and ALV-J specifically and no cross-reaction was observed with other avian viruses. Serial dilutions of ALV-A and ALV-J were used to evaluate the detection limits of these tests. The present RT-LAMP tests showed 10–100 times more sensitivity than the conventional reverse transcription polymerase chain reaction. Thus, these tests are rapid and accurate for the detection of ALV-A and ALV-J.

Protein A and protein G are extremely useful molecules for the immobilization of antibodies. However, there are limited comparative reports available to evaluate their immobilization performance for use as biosensors. In this study, a comparative analysis was made of approaches that use protein A and protein G for avian leukosis virus detection. The antibody-protein binding affinities were determined using surface plasmon resonance (SPR) analysis. The immobilization efficiency was obtained by calculating the number of the protein molecular binding sites. The positive influence of sensor response on antigen detection indicates that the amount of immobilized antibody plays a major role in the extent of immobilization. Moreover, the biosensors constructed using both proteins were found to be regenerative. The SPR results from this study suggest that the surfaces of protein G provide a better equilibrium constant and binding efficacy for immobilized antibodies, resulting in enhanced antigen detection.

An influenza pandemic poses a serious threat to humans and animals. Conventional treatments against influenza include two classes of pathogen-targeting antivirals: M2 ion channel blockers (such as amantadine) and neuraminidase inhibitors (such as oseltamivir). Examination of the mechanism of influenza viral infection has shown that endosomal acidification plays a major role in facilitating the fusion between viral and endosomal membranes. This pathway has led to investigations on vacuolar ATPase (v-ATPase) activity, whose role as a regulating factor on influenza virus replication has been verified in extensive genome-wide screenings. Blocking v-ATPase activity thus presents the opportunity to interfere with influenza viral infection by preventing the pH-dependent membrane fusion between endosomes and virions. This study aims to apply diphyllin, a natural compound shown to be as a novel v-ATPase inhibitor, as a potential antiviral for various influenza virus strains using cell-based assays. The results show that diphyllin alters cellular susceptibility to influenza viruses through the inhibition of endosomal acidification, thus interfering with downstream virus replication, including that of known drug-resistant strains. In addition, combinatorial treatment of the host-targeting diphyllin with pathogen-targeting therapeutics (oseltamivir and amantadine) demonstrates enhanced antiviral effects and cell protection in vitro.

The current reticuloendotheliosis virus (REV) antibody detection kit that uses enzyme-linked immunosorbent assay (ELISA) needs concentrated virus, which is difficult to obtain due to its poor propagation in cells. In addition, this kit detects only chicken antibody but not other species. To overcome these disadvantages, we cloned and expressed REV env gene to develop monoclonal antibodies (mAbs), which we used for antibody detection in ELISA. Three mAbs were prepared from mice. These three mAbs could recognize REVs from ducks and geese by immunodot assay. In addition, the epitopes that the three mAbs recognized were determined by using three different env protein fragments by western blotting. One mAb was used to develop a blocking ELISA (bELISA) coated with expressed env protein to detect anti-REV antibody in chicken serum. This assay had a 98.8% (79/80) agreement with a commercial ELISA kit. Another 146 chicken sera with known neutralization antibodies were used as positive controls to evaluate this bELISA. The sensitivity and specificity this bELISA were 88.9% (40/45) and 94.8% (91/96), respectively. Thus, this bELISA could be used for anti-REV antibody detection in birds.

To detect antibody on pen-side is a rapid way to know the avian influenza (AI) infectious status in a chicken flock. The purpose of this study was to develop an immunochromatographic strip (ICS) assay to detect the antibody against the AI virus (AIV) for field applications. The ICS was constructed by fixing an AIV strain A/chicken/Taiwan/2838V/2000 (H6N1) onto a nitrocellulose membrane as the antigen at the test line and goat anti-rabbit IgG antibody at the control line. The colloidal gold conjugated with rabbit anti-chicken IgG was used as the tracer. The present ICS was used to detect antibodies against avian influenza virus in 326 chicken serum samples from the field. Compared with HI, this ICS could detect antibodies against H5 and H6 AIVs. The hemagglutination inhibition (HI) test was used as the standard to evaluate the ICS accuracy. The results showed that the sensitivity and specificity of this ICS reached 95.2% (159/167) and 94.3% (150/159), respectively. The Kappa value of the HI and ICS was 0.896 (P0.001). In conclusion, this ICS could be used as a rapid test to detect antibodies against AIVs in the field.

An avian infectious bronchitis virus (IBV) strain 2575/98 was attenuated using serial chicken embryo passage to become a vaccine in Taiwan. The aim of this study was to investigate the replication ability, pathogenicity, and tissue tropism of the wild and vaccine strains in chicken embryos. The embryos were inoculated with different titers of wild and vaccine strains. Quantification of virus in allantoic fluid was evaluated using real time RT-PCR. The results showed that the vaccine strain replicated in higher titers than the wild strain, and caused embryo death so quickly that only a few dwarfisms occurred. The embryos inoculated with wild and vaccine strains had similar lesions that were confined primarily to the chorionallantoic membrane (CAM), liver, and kidneys. The immunohistochemical data showed that IBV was present predominantly in the lungs, kidneys, and CAM. Although both strains caused hepatic damage, very few virus antigens were detected in the hepatic tissue. The pathogenicity of the vaccine becomes higher in embryos although it is lower in chickens than its wild strain. The vaccine strain could be used as a possible new vaccine candidate for IBV control., Virus zaraznog bronhitisa peradi soj 2575/98 bio je oslabljen uzastopnim pasažama u pilećim zametcima da bi poslužio kao cijepni soj u Tajvanu. Cilj je ovog rada bio istražiti mogućnost umnožavanja, patogenost i tropizam terenskog i cijepnog soja u pilećim zametcima. Zametci su bili inokulirani cijepnim sojem različitog titra. Količina virusa u alantoisnoj tekućini bila je određena RT-PCR-om u stvarnom vremenu. Rezultati su pokazali da se cijepni soj umnažao u višem titru od divljeg soja i prouzročio uginuće zametaka tako brzo da se uspjelo razviti svega nekoliko kržljavih. U inokuliranih zametaka, bez obzira na divlji ili cijepni soj, razvile su se slične promjene pretežito na korioalantoisnoj opni, jetrima i bubrezima. Imunohistokemijski je dokazano da se virus zaraznog bronhitisa prvenstveno nalazio u plućima, bubrezima i korioalantoisnoj opni. Iako su oba soja prouzročila oštećenja jetara, neznatna količina virusnog antigena bila je dokazana u jetrenom tkivu. Patogenost cijepnog soja bila je u zametcima jača, a u pilićima slabija od divljeg soja. Cijepni soj bi se mogao rabiti kao mogući kandidat za proizvodnju novog cjepiva protiv zaraznog bronhitisa.

We have applied a reusable silicon nanowire field-effect transistor (SiNW-FET) as a biosensor to conduct ultrasensitive detection of H5N2 avian influenza virus (AIV) in very dilute so- lution. The reversible surface function- alization of SiNW-FET was made possi- ble using a disulfide linker. In the sur- face functionalization, 3-mercaptopro- pyltrimethoxysilane (MPTMS) was first modified on the SiNW-FET (referred to as MPTMS/SiNW-FET), with subse- quent dithiothreitol washing to reduce any possible disulfide bonding between the thiol groups of MPTMS. Subse- quently, receptor molecules could be immobilized on the MPTMS/SiNW- FET by the formation of a disulfide bond. The success of the reversible sur- face functionalization was verified with fluorescence examination and electrical measurements. A surface topograph of the SiNW-FET biosensor modified with a monoclonal antibody against H5N2 virus (referred to as mAbH5/ SiNW-FET) after detecting approxi- mately 10 17 m H5N2 AIVs was scanned by atomic force microscopy to demonstrate that the SiNW-FET is ca- pable of detecting very few H5N2 AIV particles.

The H6 subtype of avian influenza virus (AIV) infection occurs frequently in wild and domestic birds. AIV antigen detection is preferred for controlling AIV as birds are infected before they produce antibodies. The purpose of this study was to develop an early diagnostic method for AIV detection. Six monoclonal antibodies (mAbs) developed from a field H6N1 AIV strain were tested for their ability to bind to viruses. The two that showed the greatest binding ability to AIVs were used for antigen detection. An antigen-capture enzyme-linked immunosorbent assay (ELISA) to detect H6 AIVs was developed using these mAbs. One mAb was coated onto an ELISA plate as the capture antibody. The other mAb was used as the detector antibody after labeling with horseradish peroxidase. The antigen-capture ELISA detected H6N1 AIVs but not H5 AIVs, human H1N1, H3N2 influenza or other viruses. This antigen-capture ELISA could be used to specifically detect H6N1 AIV.

Innate immune response is important for viral clearance during influenza virus infection. Galectin-1, which belongs to S-type lectins, contains a conserved carbohydrate recognition domain that recognizes galactose-containing oligosaccharides. Since the envelope proteins of influenza virus are highly glycosylated, we studied the role of galectin-1 in influenza virus infection in vitro and in mice. We found that galectin-1 was upregulated in the lungs of mice during influenza virus infection. There was a positive correlation between galectin-1 levels and viral loads during the acute phase of viral infection. Cells treated with recombinant human galectin-1 generated lower viral yields after influenza virus infection. Galectin-1 could directly bind to the envelope glycoproteins of influenza A/WSN/33 virus and inhibit its hemagglutination activity and infectivity. It also bound to different subtypes of influenza A virus with micromolar dissociation constant ( K d ) values and protected cells against influenza virus-induced cell death. We used nanoparticle, surface plasmon resonance analysis and transmission electron microscopy to further demonstrate the direct binding of galectin-1 to influenza virus. More importantly, we show for the first time that intranasal treatment of galectin-1 could enhance survival of mice against lethal challenge with influenza virus by reducing viral load, inflammation, and apoptosis in the lung. Furthermore, galectin-1 knockout mice were more susceptible to influenza virus infection than wild-type mice. Collectively, our results indicate that galectin-1 has anti-influenza virus activity by binding to viral surface and inhibiting its infectivity. Thus, galectin-1 may be further explored as a novel therapeutic agent for influenza.

Infectious bronchitis virus (IBV) infection has been a major threat to the poultry industry worldwide. Current commercially available ELISA kits detect group-specific antibodies; however, to understand the status of field infection, a monoclonal antibody (mAb) blocking ELISA (b-ELISA) against local IBVs was developed. The selected mAb showed specificity to Taiwan IBV strains but had no cross-reactivity with the vaccine strain H120. Using the hemagglutination inhibition (HI) test as a standard, the cut-off value, sensitivity, and specificity of a b-ELISA using this mAb were evaluated in 390 field samples. The type-specificity of detection was validated using a panel of chicken hyperimmune sera. The results showed that the b-ELISA demonstrated high sensitivity (98.0%) and specificity (97.2%) of detection. The agreement between the results of the b-ELISA and the HI test was statistically significant (Kappa = 0.95), and there was no significant difference between these two methods (McNemar p = 0.72). The b-ELISA specifically detected Taiwan IBV serotypes but not three non-Taiwan IBV serotypes nor sera against other avian pathogens. This b-ELISA provides type-specific antibody detection of local IBV strains. It has the potential to serve as a rapid and reliable diagnostic method for IBV clinical infections in the field in Taiwan.

Feline infectious peritonitis (FIP) is a fatal disease in domestic and nondomestic felids caused by feline coronavirus (FCoV). Currently, no effective vaccine is available for the prevention of this disease. In searching for agents that may prove clinically effective against FCoV infection, 16 compounds were screened for their antiviral activity against a local FCoV strain in Felis catus whole fetus-4 cells. The results showed that Galanthus nivalis agglutinin (GNA) and nelfinavir effectively inhibited FCoV replication. When the amount of virus preinoculated into the test cells was increased to mimic the high viral load present in the target cells of FIP cats, GNA and nelfinavir by themselves lost their inhibitory effect. However, when the two agents were added together to FCoV-infected cells, a synergistic antiviral effect defined by complete blockage of viral replication was observed. These results suggest that the combined use of GNA and nelfinavir has therapeutic potential in the prophylaxis and treatment of cats with early-diagnosed FIP.

PB1-F2 is a viral protein that is encoded by the PB1 gene of influenza A virus by alternative translation. It varies in length and sequence context among different strains. The present study examines the functions of PB1-F2 proteins derived from various human and avian viruses. While H1N1 PB1-F2 was found to target mitochondria and enhance apoptosis, H5N1 PB1-F2, surprisingly, did not localize specifically to mitochondria and displayed no ability to enhance apoptosis. Introducing Leu into positions 69 (Q69L) and 75 (H75L) in the C terminus of H5N1 PB1-F2 drove 40.7% of the protein to localize to mitochondria compared with the level of mitochondrial localization of wild-type H5N1 PB1-F2, suggesting that a Leu-rich sequence in the C terminus is important for targeting of mitochondria. However, H5N1 PB1-F2 contributes to viral RNP activity, which is responsible for viral RNA replication. Lastly, although the swine-origin influenza virus (S-OIV) contained a truncated form of PB1-F2 (12 amino acids [aa]), potential mutation in the future may enable it to contain a full-length product. Therefore, the functions of this putative S-OIV PB1-F2 (87 aa) were also investigated. Although this PB1-F2 from the mutated S-OIV shares only 54% amino acid sequence identity with that of seasonal H1N1 virus, it also increased viral RNP activity. The plaque size and growth curve of the viruses with and without S-OIV PB1-F2 differed greatly. The PB1-F2 protein has various lengths, amino acid sequences, cellular localizations, and functions in different strains, which result in strain-specific pathogenicity. Such genetic and functional diversities make it flexible and adaptable in maintaining the optimal replication efficiency and virulence for various strains of influenza A virus.

Low pathogenic H6N1 avian influenza viruses (AIVs) have circulated in domestic chickens since 1972 in Taiwan. Detection of avian influenza (AI) antibody is a routine work for AI control in Taiwan. The currently available commercial enzyme-linked immunosorbent assays (ELISAs) are unable to differentiate antibody responses between different subtypes. To this end, a panel of monoclonal antibodies (mAbs) to A/chicken/Taiwan/2838V/00 (H6N1) was developed and implemented a blocking ELISA (bELISA) to detect the H6 antibody. These mAbs were confirmed specific to H6 AIVs. One monoclonal antibody was purified and labeled with horseradish peroxidase to set up a bELISA. The cut-off value was calculated to be 30% inhibition percentage from 138 H6-negative sera. The sensitivity and specificity of the bELISA were 100% and 97%, respectively. The bELISA detected seroconversion in H6-infected farms earlier than hemagglutination inhibition test. The results show that this bELISA could be used to detect H6 infection in the field.

Avian influenza virus (AIV) monitoring in migratory birds has been performed in Taiwan since 1998. From 1998 to 2007, 29,287 samples were collected from wild ducks, shorebirds, and other wild birds in the four wetlands around Taiwan and at two outside islets, Penghu and Kinmen. Virus isolation was performed for all collected samples by inoculating chicken embryos. The AIV in the allantoic fluid was identified using hemagglutination and reverse transcription PCR. The AIV prevalence from those samples was 0.81% (237/29,287). The peak prevalence reached 1.06% (186/17,493) from September to December, during which time migrating ducks came from the North. The prevalence from January to April was 0.51%. However, no virus was isolated from May to August. The partial HA genes of 28 H4 AIVs were sequenced and analyzed. The phylogenetic tree showed that a correlation existed between the isolation years and the evolutional distances. The pathogenicity of the isolated H5 and H7 AIVs was determined by intravenous pathogenicity index (IVPI) testing in specific-pathogen-free chickens and by HA cleavage sequencing. Using the IVPI test and the HA cleavage sequences, all of the H5 or H7 AIVs isolated were determined to be low pathogenicity AIVs.

Avian coronavirus infectious bronchitis virus (IBV) poses a major threat to the global poultry industry. New IBV geno- and serotypes are continually reported. However, information on IBV prevalence is not frequently addressed in these reports. This study reports on a viral surveillance program in Taiwan from 2005 to 2006 with sampling conducted in poultry slaughterhouses. The genetic features of the obtained field isolates were investigated using sequence analysis and SimPlot analysis. A 1-directional neutralization test was performed to examine the antigenic variations among the collected viruses. The selection pressures that may contribute to the evolution of Taiwan IBV during recent decades were assessed. The surveillance program revealed that 8 out of 47 flocks (17%) were IBV-infected, from which 13 IBV isolates were recovered. Based on the phylogenetic analysis of the S1 gene, 11 of 13 isolates (84.6%) clustered with Taiwan group I. One IBV isolate showed evidence of frequent recombination events with China-like IBV in the spike glycoprotein (S) gene. Another isolate demonstrated the incorporation of China-like and H120-like genome fragments within the S2 gene and the membrane protein (M) gene region, respectively. Some antigenic changes were found in the 1-directional neutralization test. However, no positive selection pressures were related to those variations in the S1 genes among Taiwan IBV. Based on our work, we suggest that sampling chickens in poultry slaughterhouses is an effective and valuable means of compiling viral prevalence data, particularly in situations where there is subclinical infection. Infectious bronchitis viruses from slaughtered chickens revealed intertypic genetic recombination and antigenic diversity.

The outcomes of feline coronavirus (FCoV) infection vary greatly from asymptomatic or mild enteric infection to fatal feline infectious peritonitis (FIP). On the basis of in vitro neutralization tests, FCoVs can be divided into two serotypes. To explore the correlation between different types of FCoV and FIP, clinical specimens collected from 363 naturally infected cats during 2003–2007 were analyzed. Amplification of a portion of the S gene from the FCoV was performed and a total of 222 cases were differentiated. Among them, 197 (88.7%) cats were type I-positive, 13 (5.9%) were type II-positive, and 12 (5.4%) were positive for both types. Irrespective of the predominance of type I FCoV infection in Taiwan, type II FCoV demonstrated a significantly higher correlation with FIP (p

Infectious bronchitis virus (IBV) infections in poultry cause great economic losses to the poultry industry worldwide. The emergence of viral variants complicates disease control. The IBV strains in Taiwan were clustered into two groups, Taiwan group I and Taiwan group II, based on the S1 gene. A variant was previously identified and showed a distinct S1 gene homology with other local strains. This study investigated the 3′ 7.3 kb genome of eight Taiwan strains isolated from 1992 to 2007. The genes of interest were directly sequenced. Sequence analyses were performed to detect any recombination event among IBVs. The results demonstrated that all of the examined viruses maintained the typical IBV genome organization as 5′-S-3a-3b-E-M-5a-5b-N-UTR-3′. In the phylogenetic analyses, various genes from one strain were clustered into separate groups. Moreover, frequent recombination events were identified in the Simplot analyses among the Taiwan and China CK/CH/LDL/97I-type strains. Putative crossover sites were located in the S1, S2, 3b, M genes and the intergenic region between the M and 5a genes. All of the recombinants showed chimeric IBV genome arrangements originated from Taiwan and China-like parental strains. Field IBVs in Taiwan undergo genetic recombination and evolution.

Molecular sexing of the diversified avian family Strigidae is difficult. Sex identification using the intron length difference between W and Z chromosomal CHD1 genes, as visualized by agarose gel electrophoreses, often produces ambiguous results. Here we describe a simple method for sexing a variety of Strigidae species using oligonucleotide microarrays, on which several sex-specific probes operated complementarily or in concert. The sex of 8 owl species was identified clearly on the microarrays through sequence recognition. This sequence-directed method can be easily applied to a wider range of Strigidae species.

Attenuated infectious bronchitis virus (IBV) vaccines are available but the relationship between sequence and virulence is not clear. In this study, the sequences of the 3' 7.3 kb of the genome, amplified using reverse transcription-polymerase chain reaction before and after attenuation, were compared to study the relationship between virulence and the sequences of three IBV strains. After attenuation, two to six amino acid substitutions were found in the spike 1 subunit, and two or three amino acid substitutions were found in the spike 2 subunit. None or one amino acid substitution was found in the small membrane protein, and one or three amino acid substitutions were found in the membrane protein. However, no amino acid substitution was found in the nucleocapsid (N) protein, indicating that the N protein might not be related to this attenuation. The 3' untranslated region after the N gene of one strain was partially deleted after attenuation, and might be correlated with virulence. This study is the first demonstration for IBV comparing sequence changes in the 3' 7.3 kb genome after attenuation. The aforementioned information on amino acid changes might be useful in future virulence studies.

Ferrets have demonstrated high susceptibility to the influenza virus. This study discusses a natural 2009 pandemic influenza A (H1N1) (A(H1N1)pdm09) virus infection in a pet ferret (Mustela putorius furo) identified in Taiwan in 2013. The ferret was in close contact with family members who had recently experienced an influenza-like illness (ILI). The ferret nasal swab showed positive results for influenza A virus using one-step RT-PCR. The virus was isolated and the phylogenetic analysis indicated that all of the eight segmented genes were closely related to the human A(H1N1)pdm09 virus linage isolated in Taiwan. This study may provide a perspective view on natural influenza A virus transmission from the local human population into pet ferrets.

A surface plasmon resonance (SPR) waveguide immunosensor fabricated by germanium-doped silicon dioxide was investigated in this study. The designed waveguide sensor consisted of a 10 microm SiO(2) substrate layer (n=1.469), a 10 microm Ge-SiO(2) channel guide (n=1.492) and a 50 nm gold film layer for immobilization of biomolecules and SPR signal detection. The resultant spectral signal was measured by a portable spectrophotometer, where the sensor was aligned by a custom-designed micro-positioner. The results of the glycerol calibration standards showed that the resonance wavelength shifted from 628 to 758 nm due to changes of refractive index from 1.36 to 1.418. Flow-through immunoassay on waveguide sensors also showed the interactions of protein A, monoclonal antibody (mAb ALV-J) and avian leucosis virus (ALVs) resulted in wavelength shifting of 4.17, 3.03 and 2.18 nm, respectively. The SPR dynamic interaction could also be demonstrated successfully in 4 min as the sensor was integrated with a lateral flow nitrocellulose strip. These results suggest that SPR detection could be carried out on designed waveguide sensor, and the integration of nitrocellulose strip for sample filtering and fluid carrier would facilitate applications in point-of-care portable system.

Due to variations in serotypes among different strains of avian infectious bronchitis viruses (IBV), vaccination of chicks with imported vaccines fails to protect them from IBV infections in Taiwan. Therefore, we develop attenuated vaccines from local strains in Taiwan. A Taiwan Group I (TW I) strain was passaged 74 times through specific pathogen-free (SPF) chicken embryonated eggs, and then tested in SPF chickens. The attenuated vaccine was not pathogenic in 1-week-old chicks, had a neutralization index (NI) of greater than 4.4 and efficacy of 90% when inoculated birds were challenged with a field IBV strain. Similar results were obtained for a vaccine made from a Taiwan Group II IBV strain. Additionally, the TW I attenuated vaccine strain had no reversion to virulence after five back passages in chicks. In conclusion, these attenuated vaccines have potential for controlling local Taiwanese IBV infections in chickens.

Sex determination of birds is important to ensure successful breeding strategies, especially for endangered species. Most birds are vulnerable to stress during handling, however, so obtaining a sufficient amount of genomic DNA (gDNA) while causing the least amount of harm is a critical issue. Avian gender can be determined based on different CHD1 gene intron sizes in W and Z sex chromosomes. We have compared various specimen sources and have found that the rachis segment of a feather is a good DNA source for determining sex. This indicates that plucking the whole feather is not necessary; a cut feather including the rachis is a superior method because it decreases stress on the examined birds and is accomplished easily. Zoo Biol. 0:1–5, 2006. © 2006 Wiley-Liss, Inc.

The disease caused by infectious bronchitis virus (IBV) produces great economic for the poultry industry. The purpose of this study is to investigate the molecular epidemiology of IBV in Taiwan. An old IBV strain isolated in 1964 and another 31 strains isolated from 1991 to 2003 were selected for N-terminal S1 gene analysis. Based on their phylogenetic tree, 13 strains were selected for sequencing the entire S1 and partial nucleocapsid (N) genes. The results indicated that Taiwanese IBV strains could be divided into two distinct lineages, Taiwan Group I and Taiwan Group II, with one Massachusetts strain and one Chinese strain. No recombination was found between H120 and the Taiwanese strains in the S1 gene. However, the S1 gene showed a noticeably higher divergence than the N gene. The phylogenetic trees constructed from the S1 and N genes indicate that intergenic recombination has occurred. Since most local strains are in Taiwanese clusters, developing vaccines from local strains is necessary for IBV control in Taiwan.

A mixture of viruses with variations in virulence is likely present within a low-pathogenicity avian influenza virus (LPAIV) population. An H6N1 AIV was isolated from a field case showing 3.8% weekly mortality and a 33% egg production drop in Taiwan. The pathologic lesions included proventricular hemorrhage and urate deposition in the kidneys and on visceral organs. From the field isolate, a done (2838N) that caused no lesions or death was obtained using limit dilution in chicken embryos and a done (2838V) that caused renal lesions and death was obtained using contact infection in chicks. Both clones were inoculated intranasally in 4-week-old specific pathogen free (SPF) chickens to test their virulence. Renal urate deposition was found in chickens inoculated with 2838V but not in chickens inoculated with 2838N. In situ hybridization, polymerase chain reaction, and virus isolation were used to confirm the spread of 2838V from the respiratory tract to the renal tissue. We found that contact infection in chickens is a good method to obtain a more virulent done from a heterogeneous virus population.

Avian Influenza A virus (AIV) subtype H5 is divided into American and Eurasian lineages, according to hemagglutinin gene sequences. Although methods for detecting H5 AIVs have been described, no H5 strain-specific detection method has been reported. The purpose of the present study was to develop an antigen-capture enzyme-linked immunosorbent assay (ACE) to detect and differentiate between the American and the Eurasian H5 AIVs. Monoclonal antibodies (mAbs) against the HA fragment of a Eurasian H5N2 AIV were used as the capture antibodies as well as the detector antibodies after labeling with horseradish peroxidase to develop an ACE. One mAb was selected for detecting the American as well as the Eurasian H5 AIVs. The other mAb was used for detecting only the Eurasian H5N2 but not the American H5N2 AIVs, H6N1 AIVs, or Newcastle disease virus. The ACEs developed would be useful for detection and differentiation of H5 AIVs from the Eurasian and the American H5 AIVs.

Subgroup J avian leukosis virus (ALV-J) causes serious economic losses in the commercial poultry industry. Measuring group-specific antigen (GSA) by enzyme-linked immunosorbent assay (ELISA) has been used to identify chickens infected with this virus. However, the inability of ELISA to discriminate the GSA from endogenous ALV (subgroup E ALV [ALV-E]) or ALV-J infection has limited its usage. The purpose of the present study was to develop a method to discriminate between uninfected flocks having ALV-E and ALV-J-infected flocks by ELISA. The GSA and anti-ALV-J antibody in the plasma samples from chickens at different ages in three grandparent farms were measured by ELISA. Infected flocks were confirmed by reverse transcription-polymerase chain reaction with different subgroup-specific primers and sequence analysis. The results indicated that the GSA of ALV-J-infected flocks increased, but that of the uninfected flocks decreased during young ages. The anti-ALV-J antibody of infected flocks was higher and increased earlier than that of uninfected flocks. Thus, measuring GSA in blood at the ages of 1 and 6 wk by ELISA is suitable to discriminate between ALV-J-infected flocks and uninfected flocks having ALV-E.

Sporadic outbreaks of Newcastle disease (ND) occurred in Taiwan during 1998-2000. In some cases, the disease occurred in broilers less than 2 wk old that originated in a broiler breeder farm, so spread of the ND virus (NDV) from the infected breeder farm to broiler ranches was suspected. The purpose of the present study was to examine the possibility of the transmission of NDV through eggs. Both clinical and experimental evidence were used to prove that this is possible. From epidemiological investigation, the possibility of transmission through eggs was suggested in two separate ND cases from a breeder farm and its progeny because two identical NDVs were isolated from both cases. In order to clarify the possibility of the transmission through eggs, one mean egg lethal dose (ELD50) of NDV was inoculated into the allantoic cavity of 155 9-to-11-day-old specific-pathogen-free (SPF) chicken embryos. Seventy-one hatching chicks from the inoculated embryos were raised for 14 days. The cloacal swabs from those chicks at the ages of 1, 4, and 7 days and the tissues after necropsy at the ages of 14 days were taken for virus isolation. The same NDV was reisolated from three hatching chicks. This experiment confirms that a few chicken embryos infected in ovo with a low titer of NDV can hatch and contain NDV after hatching, which results in NDV spreading through eggs.

Cleavage of the hemagglutinin (HA) precursor (HA0) by trypsin, which produces the active HA1 and HA2 complex, is a critical step for activating the avian influenza virus (AIV). However, other tryptic cleavage sites on HA might also cause HA degradation and affect the virulence. Otherwise, HA is modified by glycosylation in the host cell. The conjugated glycans on HA may hinder the antigenic epitopes, and thus prevent the virus from being recognized and attacked by the antibodies. In this study, we observed that glycosylation at the Asn-167 (N167) site on the HA1 of the H6N1 AIV strain A/chicken/Taiwan/2838V/00 (2838V) protected Arg-201 (R201) from tryptic cleavage. The 2838V HA protein became sensitive to tryptic cleavage, whereas the glycans at N167 were removed by N-glycosidase F (PNGase-F). Furthermore, the infectivity of 2838V decreased when pretreated with PNGase-F and trypsin. Our observations suggest that the inaccessibility of the R201 residue of HA1 for tryptic cleavage, which is sterically hindered by glycosylation at N167, is a crucial factor for determining the infectivity of the AIV.

A side-polished fiber optic surface plasmon resonance (SPR) sensor was fabricated to expose the core surface and then deposited with a 40 nm thin gold film for the near surface sensing of effective refractive index changes with surface concentration or thickness of captured avian influenza virus subtype H6. The detection surface of the SPR optical fiber sensor was prepared through the plasma modification method for binding a self-assembled monolayer of isopropanol chemically on the gold surface of the optical fiber. Subsequently, N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide/N-hydroxysuccinimide was activated to enable EB2-B3 monoclonal antibodies to capture A/chicken/Taiwan/2838V/00 (H6N1) through a flow injection system. The detection limit of the fabricated optical fiber sensor for A/chicken/Taiwan/2838V/00 was 5.14 × 10(5) EID50/0.1 mL, and the response time was 10 min on average. Moreover, the fiber optic sensor has the advantages of a compact size and low cost, thus rendering it suitable for online and remote sensing. The results indicated that the optical fiber sensor can be used for epidemiological surveillance and diagnosing of avian influenza subtype H6 rapidly.

Avian leukosis virus (ALV) infection in Taiwan Country chickens (TCCs) was investigated by using gene detection, virus isolation, and sequence analysis. The blood samples of 61 TCC flocks at market ages from a slaughter house were screened for exogenous ALVs using polymerase chain reaction to investigate the ALV infection status. The buffy coats from three breeder and four commercial chicken flocks were cocultured with DF-1 cells to isolate the virus. The full proviral DNA genomes of two ALV isolates were sequenced, analyzed, and compared with reference ALV strains. The gene detection results showed that 60 and 43 of the 61 flocks were infected with subgroup A of ALV (ALV-A) and subgroup J of ALV (ALV-J), respectively. Virus isolation results showed that five ALV-As and two ALV-Js were isolated from those seven TCC flocks. The full sequences of the isolates showed that isolate TW-3577 possessed a myeloblastosis-associated virus 1 gp85 coding region and an ALV-J 3'-untranslated region (3'UTR) and was similar to ordinary ALV-A. However, TW-3593 was unique. The 3'UTR of this isolate displayed high identity to endogenous counterpart sequence and its gp85 was different from all subgroups. This unique ALV is common in Taiwan.

The pathogenicity of an adenovirus isolated from pigeons (Pigeon adenovirus, PA) with inclusion body hepatitis in Taiwan was investigated in specific-pathogen-free (SPF) chicks and in racing pigeons. One-day-old SPF chicks were inoculated subcutaneously with 10(4) 50% tissue culture infective dose (TCID50, low dose) and 10(8) TCID50 (high dose) of the virus, respectively. The chicks began to die three days post inoculation (DPI) with high dose of the virus and the mortality reached 100%; the chicks began to die 6 DPI and the mortality reached 90% at 14 DPI with low dose. The adult pigeons seemed to be resistant to the PA. However, this virus decreased the production of antibody against Newcastle disease virus in pigeons. It is found that this PA belongs to genetic group D from the restriction patterns produced by BamH I and Hind III.

This study established a novel method of pre-screening peptides for monoclonal antibody (mAb) production. Whole virus particles were used as antigens to produce mAbs in the first stage. However, most mAbs obtained from this method were aimed toward hemagglutinin. For this reason, synthetic peptides were used as antigens for mAb production that aimed at the AIV proteins with low abundance or poor immunogenicity in the virus particle. The peptides that showed high immunogenicity were designed using bioinformatic tools for immunization. For high-throughput, a rabbit was used to screen the immunogenicities of the synthetic peptides. Those showed high immunity were used for mAb preparation in mice. Several new mAbs against PB2, PA, M1, M2, NS1 and NS2 proteins were successfully obtained in this study. Furthermore, the epitopes of M1 and NS1 mAbs were determined using competitive western blot assay and competitive ELISA. This study might simplify the mAb preparation and serves as the basis for developing mAb against poor immunogenic proteins.

Background The timely and accurate diagnosis of specific influenza virus strains is crucial to effective prophylaxis, vaccine preparation and early antiviral therapy. The detection of influenza A viruses is mainly accomplished using polymerase chain reaction (PCR) techniques or antibody-based assays. In conjugation with the immunoassay utilizing monoclonal antibody, mass spectrometry is an alternative to identify proteins derived from a target influenza virus. Taking advantage of the large surface area-to-volume ratio, antibody-conjugated magnetic nanoparticles can act as an effective probe to extract influenza virus for sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) and on-bead mass spectrometric analysis. Results Iron oxide magnetic nanoparticles (MNP) were functionalized with H5N2 viral antibodies targeting the hemagglutinin protein and capped with methoxy-terminated ethylene glycol to suppress nonspecific binding. The antibody-conjugated MNPs possessed a high specificity to H5N2 virus without cross-reactivity with recombinant H5N1 viruses. The unambiguous identification of the captured hemagglutinin on magnetic nanoparticles was realized by SDS-PAGE visualization and peptide sequence identification using liquid chromatography-tandem mass spectrometry (LC-MS/MS). Conclusions The assay combining efficient magnetic separation and MALDI-MS readout offers a rapid and sensitive method for virus screening. Direct on-MNP detection by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) provided high sensitivity (~103 EID50 per mL) and a timely diagnosis within one hour. The magnetic nanoparticles encapsulated with monoclonal antibodies could be used as a specific probe to distinguish different subtypes of influenza.