NS2B/NS3 mutations enhance the infectivity of genotype I Japanese encephalitis virus in amplifying hosts.

Genotype I (GI) virus has replaced genotype III (GIII) virus as the dominant Japanese encephalitis virus (JEV) in the epidemic area of Asia. The mechanism underlying the genotype replacement remains unclear. Therefore, we focused our current study on investigating the roles of mosquito vector and amplifying host(s) in JEV genotype replacement by comparing the replication ability of GI and GIII viruses. GI and GIII viruses had similar infection rates and replicated to similar viral titers after blood meal feedings in Culex tritaeniorhynchus. However, GI virus yielded a higher viral titer in amplifying host-derived cells, especially at an elevated temperature, and produced an earlier and higher viremia in experimentally inoculated pigs, ducklings, and young chickens. Subsequently we identified the amplification advantage of viral genetic determinants from GI viruses by utilizing chimeric and recombinant JEVs (rJEVs). Compared to the recombinant GIII virus (rGIII virus), we observed that both the recombinant GI virus and the chimeric rJEVs encoding GI virus-derived NS1-3 genes supported higher replication ability in amplifying hosts. The replication advantage of the chimeric rJEVs was lost after introduction of a single substitution from a GIII viral mutation (NS2B-L99V, NS3-S78A, or NS3-D177E). In addition, the gain-of-function assay further elucidated that rGIII virus encoding GI virus NS2B-V99L/NS3-A78S/E177E substitutions re-gained the enhanced replication ability. Thus, we conclude that the replication advantage of GI virus in pigs and poultry is the result of three critical NS2B/NS3 substitutions. This may lead to more efficient transmission of GI virus than GIII virus in the amplifying host-mosquito cycle.

Loss of the Capsule Increases the Adherence Activity but Decreases the Virulence of Avibacterium paragallinarum.

Avibacterium paragallinarum is the causative agent of infectious coryza, an important respiratory disease of chickens. The capsule is an important virulence determinant of many pathogenic bacteria, but the function of the capsule in Av. paragallinarum is not well defined. In this study, acapsular mutants of Av. paragallinarum were constructed by inactivation of the hctA gene using the TargeTron gene knockout system. The acapsular mutants were found to have greater hemagglutination activity than did the wild-type strain. Further, acapsular mutants exhibited an increased ability to adhere to DF-1 cells and to form biofilms on abiotic surfaces. Virulence assays showed that acapsular mutants were less virulent than the wild-type strain. Taken together, these results indicated that loss of capsule increases hemagglutination and adhesion activities but decreases the virulence of Av. paragallinarum. These results could be valuable to further elucidate the function of the capsule and the mechanism of pathogenicity of Av. paragallinarum.

Identification and genomic characterization of feline calicivirus from a leopard cat (Prionailurus bengalensis) in Taiwan.

The leopard cat (Prionailurus bengalensis) is an endangered wildlife that is protected under Taiwan's regulations. The body of a road-killed leopard cat was found to contain sequences of feline calicivirus (FCV), designated W109-1443. Analysis of the complete genomic sequence revealed that it shared approximately 81% similarity with a Chinese strain of FCV found in a domestic cat. Phylogenetic analysis of the VP1 gene indicated that the W109-1443 isolate belonged to genogroup II. Recombination analysis revealed that the W109-1443 isolate may have resulted from recombination between two FCV strains. Given the potential impact of FCV on the health and survival of wild felids, further investigation is necessary to assess its pathogenicity in the leopard cat population.

The conservation and diversity of the exons encoding the glycine and arginine rich domain of the fibrillarin gene in vertebrates, with special focus on reptiles and birds.

The nucleolar rRNA 2'-O-methyltransferase fibrillarin (FBL) contains a highly conserved methyltransferase domain at the C-terminus and a diverse glycine arginine-rich (GAR) domain at the N-terminus in eukaryotes. We found that a nine-exon configuration of fbl and exon 2-3 encoded GAR domain are conserved and specific in vertebrates. All internal exons except exon 2 and 3 are of the same lengths in different vertebrate lineages. The lengths of exon 2 and 3 vary in different vertebrate species but the ones with longer exon 2 usually have shorter exon 3 complementarily, limiting lengths of the GAR domain within a certain range. In tetrapods except for reptiles, exon 2 appears to be longer than exon 3. We specifically analyzed different lineages of reptiles for their GAR sequences and exon lengths. The lengths of exon 2 in reptiles are around 80-130-nt shorter and the lengths of exon 3 in reptiles are around 50-90 nt longer than those in other tetrapods, all in the GAR-coding regions. An FSPR sequence is present at the beginning of the GAR domain encoded by exon 2 in all vertebrates, and a specific FXSP/G element (X can be K, R, Q, N, and H) exist in the middle of GAR with phenylalanine as the 3rd exon 3-encoded amino acid residue starting from jawfish. Snakes, turtles, and songbirds contain shorter exon 2 compared with lizards, indicating continuous deletions in exon 2 and insertions/duplications in exon 3 in these lineages. Specifically, we confirmed the presence the fbl gene in chicken and validated the RNA expression. Our analyses of the GAR-encoding exons of fbl in vertebrates and reptiles should provide the basis for further evolutionary analyses of more GAR domain encoding proteins.

NS2 is a key determinant of compatibility in reassortant avian influenza virus with heterologous H7N9-derived NS segment.

Influenza A viruses are common pathogens with high prevalence worldwide and potential for pandemic spread. While influenza A infections typically elicit robust cellular innate immune responses, the non-structural protein 1 (NS1) antagonizes host anti-viral responses and is critical for efficient virus replication and virulence. The avian influenza virus (AIV) H7N9 initially emerged in China in 2013 and has since crossed the avian-human barrier, causing severe disease in humans. To investigate the influence of the H7N9 NS gene (NS079) on viral replication and innate immune response, we generated several recombinant AIVs bearing various NS079 segments on the backbone of H6N1 (strain 0702). Intriguingly, the recombinant virus bearing the heterologous NS079 gene was highly attenuated compared with virus carrying the homologous NS gene (NS0702). Furthermore, we generated a NS079-0702R virus that expresses a chimeric NS gene in which part of the NS079 effector domain was replaced with the sequence from NS0702. The NS079-0702R virus exhibited significantly enhanced viral yield, approximately 100-fold more than virus bearing NS079. The high infection rate of NS079-0702R virus was reflected by strong induction of IFN and Mx expression in human A549 cells. Intriguingly, our in vitro comparative analysis suggested that the increased NS079-0702R infection capacity was independent of the ability of NS1 to interact with cellular partners, such as PKR and CPSF30. Since partial substitution of the effector domain from NS0702 altered the coding sequence of NS2, we further generated another recombinant virus with NS2 derived from H7N9. Surprisingly, the virus with H7N9-derived NS2 exhibited growth characteristics similar to NS079. Our data demonstrate that swapping NS2 components changes infection efficiency, suggesting a key role for NS2 as a determinant of viral compatibility upon reassortment. These findings warrant further investigation into the precise mechanisms by which NS2 contributes to viral replication and host immunity.1.

Epidemiology of Severe Fever with Thrombocytopenia Syndrome in Dogs and Cats in Taiwan.

Severe Fever with Thrombocytopenia Syndrome (SFTS), caused by the SFTS Virus (SFTSV), is a global health threat. SFTSV in Taiwan has only been reported in ruminants and wild animals. Thus, we aimed to investigate the infection statuses of dogs and cats, the animals with closer human interactions. Overall, the SFTSV RNA prevalence was 23% (170/735), with dogs showing a 25.9% (111/429) prevalence and cats at 19.3% (59/306) prevalence. Noticeably, the prevalence in stray animals (39.8% 77/193) was significantly higher than in domesticated ones (17.2%, 93/542). Among the four categories analyzed, the highest SFTSV prevalence was found in the stray dogs at 53.9% (120/193), significantly higher than the 24.2% prevalence noted in stray cats. In contrast, domesticated animals exhibited similar prevalence rates, with 17.1% for dogs and 17.2% for cats. It is noteworthy that in the domesticated animal groups, a significantly elevated prevalence (45%, 9/20) was observed among cats exhibiting thrombocytopenia compared to those platelet counts in the reference range (4.8%, 1/21). The high infection rate in stray animals, especially stray dogs, indicated that exposure to various outdoor environments influences the prevalence of infections. Given the higher human interaction with dogs and cats, there is a need for proactive measures to reduce the risk associated with the infection of SFTSV in both animals and humans.

The First Nationwide Surveillance of Severe Fever with Thrombocytopenia Syndrome in Ruminants and Wildlife in Taiwan.

Since the first discovery of severe fever with thrombocytopenia syndrome virus (SFTSV) in China in 2009, SFTSV has rapidly spread through other Asian countries, including Japan, Korea, Vietnam and Pakistan, in chronological order. Taiwan reported its first discovery of SFTSV in sheep and humans in 2020. However, the prevalence of SFTSV in domestic and wildlife animals and the geographic distribution of the virus within the island remain unknown. A total of 1324 animal samples, including 803 domestic ruminants, 521 wildlife animals and 47 tick pools, were collected from March 2021 to December 2022 from 12 counties and one terrestrial island. The viral RNA was detected by a one-step real-time reverse transcription polymerase chain reaction (RT-PCR). Overall, 29.9% (240/803) of ruminants showed positive SFTSV RNA. Sheep had the highest viral RNA prevalence of 60% (30/50), followed by beef cattle at 28.4% (44/155), goats at 28.3% (47/166), and dairy cows at 27.5% (119/432). The bovine as a total of dairy cow and beef cattle was 27.8% (163/587). The viral RNA prevalence in ticks (predominantly Rhipicephalus microplus) was similar to those of ruminants at 27.7% (13/47), but wild animals exhibited a much lower prevalence at 1.3% (7/521). Geographically the distribution of positivity was quite even, being 33%, 29.1%, 27.5% and 37.5% for northern, central, southern and eastern Taiwan, respectively. Statistically, the positive rate of beef cattle in the central region (55.6%) and dairy cattle in the eastern region (40.6%) were significantly higher than the other regions; and the prevalence in Autumn (September-November) was significantly higher than in the other seasons (p < 0.001). The nationwide study herein revealed for the first time the wide distribution and high prevalence of SFTSV in both domestic animals and ticks in Taiwan. Considering the high mortality rate in humans, surveillance of other animal species, particularly those in close contact with humans, and instigation of protective measures for farmers, veterinarians, and especially older populations visiting or living near farms or rural areas should be prioritized.

A variant NS1 protein from H5N2 avian influenza virus suppresses PKR activation and promotes replication and virulence in mammals.

Highly pathogenic avian influenza viruses (HPAIVs) frequently receive global attention as threats to public health. The NS1 protein is a key virulence factor known to impair host antiviral responses. The study herein revealed HPAIV H5N2 NS gene encoded additional protein; a truncated NS1 variant, designated NS3, produced by alternative splicing of the NS transcript. To examine the function of NS3 during infection, we generated recombinant viruses expressing either full-length NS1 (RG-AIV-T375G) or NS3 (RG-AIV-NS3). Interestingly, RG-AIV-NS3 virus produced higher titres than RG-AIV-T375G in multiple mammalian cell lines. However, RG-AIV-T375G exhibited a replication advantage over RG-AIV-NS3 in chicken DF-1 cells, indicating that host cell identity dictates the effect of NS3 on viral replication. In mice and mammalian cells, RG-AIV-NS3 infection elicited higher level of cytokines, including IFN-ß, MX and TNF-α, potentially due to its higher replication activity. Based on mini-genome assay, NS3 had pronounced effects on viral replication machinery. Surprisingly, NS3 retained an interaction with PKR and suppressed PKR activation despite its lack of amino-acid residues 126-167. The poor replication ability of RG-AIV-T375G was partially restored in cells deficient in PKR suggesting that full-length NS1 may be insufficient to suppress PKR function. Notably, virulence of the full-length NS1-expressing RG-AIV-T375G virus was highly attenuated in mice when compared to RG-AIV-NS3. In summary, our study reveals the existence and function of a previously unidentified H5N2 viral protein, NS3. We found that NS3 is functionally distinct from NS1 protein, as it enhances viral replication and pathogenicity in mammalian systems, potentially via suppression of PKR activity.

Molecular Characterization and Pathogenicity of the Novel Recombinant Muscovy Duck Parvovirus Isolated from Geese.

Goose parvovirus (GPV) and Muscovy duck parvovirus (MDPV) are the main agents associated with waterfowl parvovirus infections that caused great economic losses in the waterfowl industry. In 2020, a recombinant waterfowl parvovirus, 20-0910G, was isolated in a goose flock in Taiwan that experienced high morbidity and mortality. The whole genome of 20-0910G was sequenced to investigate the genomic characteristics of this isolate. Recombination analysis revealed that, like Chinese rMDPVs, 20-0910G had a classical MDPV genomic backbone and underwent two recombination events with classical GPVs at the P9 promoter and partial VP3 gene regions. Phylogenetic analysis of the genomic sequence found that this goose-origin parvovirus was highly similar to the circulating recombinant MDPVs (rMDPVs) isolated from duck flocks in China. The results of experimental challenge tests showed that 20-0910G caused 100% mortality in goose embryos and in 1-day-old goslings by 11 and 12 days post-inoculation, respectively. Taken together, the results indicated that this goose-origin rMDPV was closely related to the duck-origin rMDPVs and was highly pathogenic to young geese.

Effects of Coronavirus Persistence on the Genome Structure and Subsequent Gene Expression, Pathogenicity and Adaptation Capability.

Coronaviruses are able to establish persistence. However, how coronaviruses react to persistence and whether the selected viruses have altered their characteristics remain unclear. In this study, we found that the persistent infection of bovine coronavirus (BCoV), which is in the same genus as SARS-COV-2, led to alterations of genome structure, attenuation of gene expression, and the synthesis of subgenomic mRNA (sgmRNA) with a previously unidentified pattern. Subsequent analyses revealed that the altered genome structures were associated with the attenuation of gene expression. In addition, the genome structure at the 5' terminus and the cellular environment during the persistence were responsible for the sgmRNA synthesis, solving the previously unanswered question regarding the selection of transcription regulatory sequence for synthesis of BCoV sgmRNA 12.7. Although the BCoV variants (BCoV-p95) selected under the persistence replicated efficiently in cells without persistent infection, its pathogenicity was still lower than that of wild-type (wt) BCoV. Furthermore, in comparison with wt BCoV, the variant BCoV-p95 was not able to efficiently adapt to the challenges of alternative environments, suggesting wt BCoV is genetically robust. We anticipate that the findings derived from this fundamental research can contribute to the disease control and treatments against coronavirus infection including SARS-CoV-2.

The Impacts of Antivirals on the Coronavirus Genome Structure and Subsequent Pathogenicity, Virus Fitness and Antiviral Design.

With the global threat of SARS-CoV-2, much effort has been focused on treatment and disease control. However, how coronaviruses react to the treatments and whether the surviving viruses have altered their characteristics are also unanswered questions with medical importance. To this end, bovine coronavirus (BCoV), which is in the same genus as SARS-CoV-2, was used as a test model and the findings were as follows. With the treatment of antiviral remdesivir, the selected BCoV variant with an altered genome structure developed resistance, but its pathogenicity was not increased in comparison to that of wild type (wt) BCoV. Under the selection pressure of innate immunity, the genome structure was also altered; however, neither resistance developed nor pathogenicity increased for the selected BCoV variant. Furthermore, both selected BCoV variants showed a better efficiency in adapting to alternative host cells than wt BCoV. In addition, the previously unidentified feature that the spike protein was a common target for mutations under different antiviral treatments might pose a problem for vaccine development because spike protein is a common target for antibody and vaccine designs. The findings derived from this fundamental research may contribute to the disease control and treatments against coronaviruses, including SARS-CoV-2.

The Impacts of Reassortant Avian Influenza H5N2 Virus NS1 Proteins on Viral Compatibility and Regulation of Immune Responses.

Avian influenza virus (AIV) can cause severe diseases in poultry worldwide. H6N1 AIV was the dominant enzootic subtype in 1985 in the chicken farms of Taiwan until the initial outbreak of a low pathogenic avian influenza (LPAI) H5N2 virus in 2003; thereafter, this and other LPAIs have been sporadically detected. In 2015, the outbreak of three novel H5Nx viruses of highly pathogenic avian influenza (HPAI) emerged and devastated Taiwanese chicken and waterfowl industries. The mechanism of variation in pathogenicity among these viruses is unclear; but, in light of the many biological functions of viral non-structural protein 1 (NS1), including interferon (IFN) antagonist and host range determinant, we hypothesized that NS genetic diversity contributes to AIV pathogenesis. To determine the impact of NS1 variants on viral infection dynamics, we established a reverse genetics system with the genetic backbone of the enzootic Taiwanese H6N1 for generation of reassortant AIVs carrying exogenous NS segments of three different Taiwanese H5N2 strains. We observed distinct cellular distributions of NS1 among the reassortant viruses. Moreover, exchange of the NS segment significantly influenced growth kinetics and induction of cytokines [IFN-α, IFN-ß, and tumor necrosis factor alpha (TNF-α)] in an NS1- and host-specific manner. The impact of NS1 variants on viral replication appears related to their synergic effects on viral RNA-dependent RNA polymerase activity and IFN response. With these approaches, we revealed that NS1 is a key factor responsible for the diverse characteristics of AIVs in Taiwan.

The first discovery of severe fever with thrombocytopenia syndrome virus in Taiwan.

Severe fever with thrombocytopenia syndrome (SFTS), an emerging tick-borne zoonosis, has been rapidly spread in many Asian counties since 2010, which raises the great concern in East Asia. Nevertheless, the infection status of SFTS in Taiwan remains unclear. To investigate the existence of SFTSV in Taiwan, a total of 151 serum samples collected from 31 sheep, 63 bovine and 57 dogs were enrolled this study. Furthermore, 360 adult female Rhipicephalus microplus were also included. One-step RT-nested PCR and IgG ELISA were conducted to test SFTSV specific RNA and antibodies, respectively. The result provided the first evidence of the existence of SFTSV RNA and antibodies in ruminants and ticks in Taiwan.

Prevalence and Genotyping of Chlamydia psittaci from Domestic Waterfowl, Companion Birds, and Wild Birds in Taiwan.

Chlamydia psittaci, the causative agent of avian chlamydiosis, an important zoonotic disease, infects a wide range of birds. Infected birds, whether symptomatic or asymptomatic, intermittently shed the agent through respiratory and intestinal routes. Therefore, it is essential to investigate the epizootiology of C. psittaci in poultry, pet birds, and wild birds. In this study, cloacal or fecal swabs collected from domestic waterfowl, psittacine birds, Columbidae, and wild birds were used to determine the prevalence of C. psittaci in Taiwan between 2014 and 2017. The C. psittaci infection rate was as high as 34.2% among domestic waterfowl farms. The waterfowl isolates clustered into two groups based on ompA phylogeny: one group (G1-like) clustered with the Polish G1 strains; the other group (waterfowl-TW) clustered near, but independently from, the classical ABE genotype cluster. Separately, 3.1% of parrot samples tested positive for C. psittaci belonging to genotype A. C. psittaci isolates of genotype B were detected in 10.1% of racing pigeons and other Columbidae. Wild bird samples from a wildlife refuge had a 2.2% prevalence rate; among these, two atypical C. psittaci genotypes were detected in samples from a Malayan night heron (Gorsachius melanolophus) and a Taiwan barbet (Megalaima nuchalis). Taken together, our results revealed that the risk of C. psittaci transmission from domestic waterfowl and Columbidae birds to humans could be underestimated, given the high prevalence rates in these birds. Furthermore, the free-range rearing system of waterfowl in Taiwan may promote C. psittaci transmission between poultry and wild birds. Pet birds and racing pigeons, which are in close contact with people, are also possible sources for cross-species transmission. Further studies are necessary to elucidate the virulence, biological and genetic characteristics, and modes of transmission of Taiwanese C. psittaci isolates to facilitate the prevention and control of C. psittaci infection.

Preparation of Chicken Anemia Virus (CAV) Virus-Like Particles and Chicken Interleukin-12 for Vaccine Development Using a Baculovirus Expression System.

Chicken infectious anemia (CIA) is a poultry disease that causes huge economic losses in the poultry industry worldwide. Commercially available CIA vaccines are derived from wild-type chicken anemia viruses (CAVs) by serial passage in cells or chicken embryos. However, these vaccinal viruses are not completely attenuated; therefore, they can be transmitted vertically and horizontally, and may induce clinical symptoms in young birds. In this study, we sought to eliminate these issues by developing a subunit vaccine exploiting the CAV structural proteins, engineering recombinant baculovirus-infected Spodoptera frugiperda (Sf9) cells that contained both the viral protein 1 (VP1) and VP2 of CAV. Moreover, we produced single-chain chicken interleukin-12 (chIL-12) in the same system, to serve as an adjuvant. The recombinant VP1 was recognized by chicken anti-CAV polyclonal antibodies in Western blotting and immunofluorescence assays, and the bioactivity of the recombinant chIL-12 was confirmed by stimulating interferon-γ (IFN-γ) secretion in chicken splenocytes. Furthermore, the ability of the recombinant VP1 to generate self-assembling virus-like particles (VLPs) was confirmed by transmission electron microscopy. Specific pathogen-free (SPF) chickens inoculated with VLPs and co-administered the recombinant chIL-12 induced high CAV-specific antibodies and cell-mediated immunity. Taken together, the VLPs produced by the baculovirus expression system have the potential to be a safe and effective CIA vaccine. Finally, we demonstrated the utility of recombinant chIL-12 as an adjuvant for poultry vaccine development.

The haemagglutinin of Avibacterium paragallinarum is a trimeric autotransporter adhesin that confers haemagglutination, cell adherence and biofilm formation activities.

The haemagglutinin (HA) protein plays a key role in the immunogenicity and pathogenicity of Avibacterium paragallinarum. A 210-kDa protein (HMTp210) was previously reported to be the HA of Av. paragallinarum, but the biological function of HMTp210 is not well defined. In this study, mutant strains that lacked HMTp210 were constructed using the TargeTron(®) gene knockout system. Haemagglutination and haemagglutination-inhibition (HI) assays showed that the HMTp210-deficient mutants exhibited no HA activity and failed to elicit HI antibodies in immunized chickens. Additionally, HMTp210-deficient mutants exhibited reduced ability to adhere to HeLa cells and to form biofilms on abiotic surfaces. Virulence assays showed that HMTp210-deficient mutants are less virulent than their isogenic wild-type strains. HMTp210 bears significant similarity to proteins of the trimeric autotransporter adhesin (TAA) family, and recombinant HMTp210 expressed in E. coli formed a trimeric structure. Taken together, these results indicated that HMTp210 is a trimeric autotransporter adhesin that confers haemagglutination, cell adherence and biofilm formation activities. These results should prove valuable to further elucidate the biological function of HA and the mechanism of pathogenicity of Av. paragallinarum.

Infectious laryngotracheitis virus in chickens.

Infectious laryngotracheitis (ILT) is an important respiratory disease of chickens and annually causes significant economic losses in the poultry industry world-wide. ILT virus (ILTV) belongs to alphaherpesvirinae and the Gallid herpesvirus 1 species. The transmission of ILTV is via respiratory and ocular routes. Clinical and post-mortem signs of ILT can be separated into two forms according to its virulence. The characteristic of the severe form is bloody mucus in the trachea with high mortality. The mild form causes nasal discharge, conjunctivitis, and reduced weight gain and egg production. Conventional polymerase chain reaction (PCR), nested PCR, real-time PCR, and loop-mediated isothermal amplification were developed to detect ILTV samples from natural or experimentally infected birds. The PCR combined with restriction fragment length polymorphism (RFLP) can separate ILTVs into several genetic groups. These groups can separate vaccine from wild type field viruses. Vaccination is a common method to prevent ILT. However, field isolates and vaccine viruses can establish latent infected carriers. According to PCR-RFLP results, virulent field ILTVs can be derived from modified-live vaccines. Therefore, modified-live vaccine reversion provides a source for ILT outbreaks on chicken farms. Two recently licensed commercial recombinant ILT vaccines are also in use. Other recombinant and gene-deficient vaccine candidates are in the developmental stages. They offer additional hope for the control of this disease. However, in ILT endemic regions, improved biosecurity and management practices are critical for improved ILT control.

Comparison of a TaqMan real-time polymerase chain reaction assay with a loop-mediated isothermal amplification assay for detection of Gallid herpesvirus 1.

A TaqMan real-time polymerase chain reaction (PCR) and loop-mediated isothermal amplification (LAMP) assay were developed to detect Gallid herpesvirus 1 (GaHV-1, formerly Infectious laryngotracheitis virus). The standard curve of real-time PCR was established, and the sensitivity reached 10 copies/µl. In the current study, the conversion between viral titer and GaHV-1 genomic copy number was constructed. Six primers for LAMP assay amplified target gene at 65°C within 45 min, and the detection limit was 60 copies/µl. The 6 primers were highly specific, sensitive, and reproducible for detection of GaHV-1. Although the sensitivity of LAMP was lower than that of real-time PCR, LAMP was faster, less expensive, and did not require a thermocycler. The LAMP assay would be a viable alternative assay in diagnostic laboratories that do not employ real-time PCR technology.

Development of TaqMan real-time RT-PCR for detection of avian reoviruses.

Avian reoviruses (ARVs) are an important cause of economic losses in commercial poultry. A TaqMan real-time RT-PCR assay for detecting of ARVs was developed. The primer-probe set was from the conserved region of ARV S4 genome segment. Real-time RT-PCR detected ARV strains including CO8 and ss412 strains, which belonged to different serological subgroups, and the test had no cross-reaction with other avian viruses. The detection limit of this assay was 5 ARV genome copies per 5 µl and was 150 times more sensitive than traditional RT-PCR. Statistical analyses indicated excellent reproducibility. For ARV strain 2408, a titer of 50% embryo infection dose and 50% tissue culture infectious dose equivalent to 3.9 ± 0.8, and 2.9 ± 0.3 ARV genome copies, respectively. This test was rapid, specific, and sensitive for the detection of ARVs and will be useful in veterinary diagnostic laboratories and for the quantitation of vaccine viruses for pharmaceutical companies.