Construction of an infectious plasmid clone of Muscovy duck parvovirus by TA cloning and creation of a partially attenuated strain.

Muscovy duck parvovirus (MDPV) infection is a highly contagious and fatal disease of Muscovy ducklings. The infectious clone methodology is a valuable tool to study the pathogenic mechanisms of viruses, but no infectious clone of MDPV is yet available. In this study, a plasmid clone containing the full-length genome of MDPV was constructed using the TA cloning methodology. This MDPV clone was found to be infectious after transfection of primary Muscovy duck embryo fibroblast cells and passage in embryonated Muscovy duck eggs. Site-directed mutagenesis showed that the K75N mutation in the VP1 protein of MDPV resulted in the partial attenuation of the virus. The availability of an MDPV infectious clone can facilitate investigation of the pathogenic mechanisms of MDPV and development of vaccines against diseases caused by MDPV.

Identification and functional analysis of the cytolethal distending toxin gene from Avibacterium paragallinarum.

Avibacterium paragallinarum is the causative agent of infectious coryza, an important respiratory disease of chickens. Cytolethal distending toxins (CDTs) are a family of protein cytotoxins that cause cell cycle arrest and apoptosis in eukaryotic cells. Whole-genome sequencing analysis showed that Av. paragallinarum contains cdtABC genes. Filter-sterilized lysates prepared from Av. paragallinarum or from recombinant Escherichia coli expressing cdtABC genes exhibited CDT activity on HeLa cells and chicken embryo fibroblast (DF-1) cells. In vitro DNase assays showed that purified recombinant CdtB has DNase activity. Polymerase chain reaction and sequencing analysis revealed that the cdtABC genes are present in all strains of Av. paragallinarum examined in this study. This is the first report of the identification and functional analysis of cdtABC genes from Av. paragallinarum. The gene products of cdtABC genes may be involved in the pathogenesis of the disease caused by Av. paragallinarum.

Identification of the lic1ABCD operon that controls the phase-variable expression of phosphorylcholine on lipopolysaccharide from Avibacterium paragallinarum.

Phosphorylcholine (ChoP) is an important virulence factor found on the surface of many mucosal pathogens and its expression enhances bacterial colonization on mucous membranes and reduces susceptibility to antimicrobial peptides. Whole-genome sequencing analyses showed that Avibacterium paragallinarum contained an operon with strong sequence similarity to the lic1ABCD operon from Haemophilus influenzae and the pcgDABC operon from Pasteurella multocida; both operons are involved in metabolism and addition of ChoP on bacterial lipopolysaccharide (LPS). Western immunoblot analysis with ChoP-specific monoclonal antibody showed that ChoP is present on LPS of Av. paragallinarum and the expression of ChoP is controlled by phase variation mediated by the number of 5'-CAAT-3' tetranucleotide tandem repeats within the coding region of the lic1A gene. The number of tetranucleotide repeats varied widely among strains, and variation in the number of repeats was observed following in vivo passage but not in vitro passage. Antimicrobial susceptibility assays showed that ChoP expression decreased susceptibility of Av. paragallinarum to chicken antimicrobial peptide fowlicidin-1. This is the first report showing that ChoP is present on LPS from Av. paragallinarum and that Av. paragallinarum contains a phase-variable gene. These results could be valuable for understanding the mechanism of pathogenicity of Av. paragallinarum.

Regions Important for Hemagglutination Activity and Serotypes of Avibacterium paragallinarum HMTp210 Protein.

Avibacterium paragallinarum is an important respiratory pathogen of domestic chickens. Avibacterium paragallinarum has been subtyped into three serogroups and nine serovars according to the Page and revised Kume schemes. The major hemagglutinin antigen of A. paragallinarum is HMTp210, which is a large protein of about 2000 amino acids (aa), including a 70-aa signal peptide at its N-terminal end. However, the regions important for the hemagglutination (HA) activity and serotypes of HMTp210 remain unclear. In this study we constructed a series of A. paragallinarum strains expressing HMTp210 in-frame deletion mutants and determined their HA titers to identify the regions important for the HA activity and serotypes of HMTp210. Two distinct types of HA activities were found in HMTp210. The type 1 HA activity resided in the region spanning the full-length HA (aa 71-2084), whereas the type 2 resided in the region spanning aa 1003-2084. The putative ligand binding of the type 1 HA activity was located at aa 176-360, which had a structure similar to YadA of Yersinia enterocolitica. The putative ligand binding site of the type 2 HA activity was located at aa 1003-1125, which had a structure similar to UspA1 from Moraxella catarrhalis. The type 1 HA activity appeared to be Page serogroup specific, whereas type 2 appeared to be Kume serovar specific. Finally, sequence analyses of the regions spanning aa 1-400 and aa 1100-1600 of HMTp210 could be useful for the molecular serotyping (the Page and revised Kume schemes) of A. paragallinarum isolates.

Regiones importantes para la actividad de hemaglutinación y serotipos de la proteína HMTp210 de Avibacterium paragallinarum. La bacteria Avibacterium paragallinarum es un patógeno respiratorio importante de los pollos domésticos. Avibacterium paragallinarum se subtipificó en tres serogrupos y nueve serovares de acuerdo con los esquemas revisados de Page y Kume. El principal antígeno de la hemaglutinina de A. paragallinarum es la proteína HMTp210, que es una proteína grande de unos 2000 aminoácidos (aa), que incluye un péptido señal de 70 aminoácidos en su extremo N-terminal. Sin embargo, las regiones importantes para la actividad de hemaglutinación (HA) y de los serotipos de la proteína HMTp210 siguen sin estar determinados. En este estudio, se construyó una serie de cepas de A. paragallinarum que expresaban mutantes de deleción en marco de lectura de HMTp210 y se determinaron sus títulos de hemaglutinación para identificar las regiones importantes para la actividad de hemaglutinación y de los serotipos de HMTp210. Se encontraron dos tipos distintos de actividades hemaglutinación en la proteína HMTp210. La actividad de hemaglutinación de tipo 1 residía en la región que abarcaba la longitud completa (aminoácidos 71­2084), mientras que la de tipo 2 residía en la región que abarcaba entre los aminoácidos 1003­2084. El sitio supuesto de unión al ligando de la actividad de hemaglutinación tipo 1 se ubicó entre los aminoácidos 176­360, que tenía una estructura similar a la proteína YadA de Yersinia enterocolitica. El supuesto sitio de unión del ligando de la actividad de hemaglutinación tipo 2 se ubicó entre los aminoácidos 1003­1125, que tenía una estructura similar a la proteína UspA1 de Moraxella catarrhalis. La actividad de hemaglutinación tipo 1 parecía ser específica del serogrupo Page, mientras que la hemaglutinación tipo 2 parecía ser específica del serovar Kume. Finalmente, los análisis de secuencias de las regiones que abarcan los aminácidos 1­400 y aminoácidos 1100­1600 de HMTp210 podrían ser útiles para la serotipificación molecular (por el esquema revisado de Page y Kume revisado) de aislamientos de A. paragallinarum.

Relationship Between the Serotypes and Hemagglutinin Gene Sequences of Avibacterium paragallinarum.

Avibacterium paragallinarum has been subtyped into three serogroups (A, B, and C) and nine serovars (A-1, A-2, A-3, A-4, B-1, C-1, C-2, C-3, and C-4) according to the Page and Kume schemes. Both schemes use the hemagglutination inhibition test for serotyping. However, the relationship between the hemagglutinin gene (HMTp210) sequences and serotypes of A. paragallinarum is still unclear. This problem is partly due to the lack of information on the complete HMTp210 sequence from the formal reference strain of Page serogroup B (strain 0222 or Spross). In this study, we determined the complete HMTp210 sequence of strain Spross. The sequence of Spross and those of other HMTp210 sequences retrieved from GenBank were used to conduct phylogenetic analyses to investigate the relationship between the serotypes and HMTp210 sequences of A. paragallinarum. Four phylogenetic clusters, designated clusters A-1, A-2, B, and C, were identified. Clustering based on complete HMTp210 sequences correlates with serotyping based on hemagglutination inhibition tests. Serovar A-2 was found to contain a chimeric HMTp210 gene that might have resulted from recombination between serovar A-1 and serovar C-1. In addition, phylogenetic analysis based on partial sequences (approximately nucleotides 1-1200) of HMTp210 was sufficient to discriminate between serogroups A, B, and C. These findings could be valuable for developing a molecular method for serotyping of A. paragallinarum.

Relación entre los serotipos y las secuencias génicas de hemaglutinina de Avibacterium paragallinarum. Avibacterium paragallinarum se ha subtipificado en tres serogrupos (A, B y C) y nueve serovares (A-1, A-2, A-3, A-4, B-1, C-1, C-2, C- 3 y C-4) de acuerdo con los esquemas Page y Kume. Ambos esquemas utilizan la prueba de inhibición de la hemaglutinación para la serotipificación. Sin embargo, la relación entre las secuencias del gene de la hemaglutinina (HMTp210) y los serotipos de A. paragallinarum aún no está clara. Este problema se debe en parte a la falta de información sobre la secuencia completa del gene HMTp210 de la cepa de referencia formal del serogrupo B de Page (cepa 0222 o Spross). En este estudio, se determinó la secuencia completa de HMTp210 de la cepa Spross. La secuencia de Spross y las de otras secuencias del gene HMTp210 obtenidas de GenBank se utilizaron para realizar análisis filogenéticos para investigar la relación entre los serotipos y las secuencias de HMTp210 de A. paragallinarum. Se identificaron cuatro agrupaciones filogenéticas, denominadas grupos A-1, A-2, B y C. La agrupación basada en las secuencias completas del gene HMTp210 se correlaciona con la serotipificación basada en pruebas de inhibición de la hemaglutinación. Se encontró que el serovar A-2 contenía un gene HMTp210 quimérico que podría haber resultado de la recombinación entre el serovar A-1 y el serovar C-1. Además, el análisis filogenético basado en secuencias parciales (aproximadamente nucleótidos 1-1200) del gene HMTp210 fue suficiente para discriminar entre los serogrupos A, B y C. Estos hallazgos podrían ser valiosos para desarrollar un método molecular para la serotipificación de A. paragallinarum.

Serotypes and Hemagglutinin Gene Sequences of Avibacterium paragallinarum Isolated in Taiwan.

Despite routine vaccine use, sporadic outbreaks of infectious coryza in poultry continue to occur in Taiwan. This study was designed to determine the serotypes and the complete nucleotide sequences of a hemagglutinin gene (HMTp210) of Avibacterium paragallinarum isolated in Taiwan between 1994 and 2017. Hemagglutination inhibition tests showed that these isolates belong to serogroups B and C. Sequence analyses of the HMTp210 gene showed that Taiwanese serogroup B isolates are most similar (94.7%-98.2% identity) to strain FARPER-174 isolated in Peru in 2015. In contrast, Taiwanese serogroup C isolates are most similar (96.3%-99.8% identity) to strain H-18 isolated in Japan in 1976. This is the first report showing the presence of A. paragallinarum of serogroup B in Taiwan. In addition, one Taiwanese isolate showed cross-reactivity with serogroup B and C antisera. This isolate contains a chimeric HMTp210 gene that might result from recombination between serogroups B and C. These findings could be valuable for the epidemiologic study and molecular serotyping of A. paragallinarum.

Serotipos y secuencias de genes de hemaglutinina de Avibacterium paragallinarum aislados en Taiwán. A pesar del uso rutinario de vacunas, en Taiwán continúan ocurriendo brotes esporádicos de coriza infecciosa en avicultura. Este estudio fue diseñado para determinar los serotipos y las secuencias de nucleótidos completas de un gene de hemaglutinina (HMTp210) de Avibacterium paragallinarum aislado en Taiwán entre 1994 y 2017. Las pruebas de inhibición de la hemaglutinación mostraron que estos aislamientos pertenecen a los serogrupos B y C. El análisis de secuencias del gene HMTp210 mostró que los aislamientos del serogrupo B taiwaneses son más similares (94.7% ­98.2% de identidad) a la cepa FARPER-174 aislada en Perú en el año 2015. En contraste, los aislamientos del serogrupo C taiwaneses son más similares (96.3% ­99.8% de identidad) a la cepa H -18 aislada en Japón en 1976. Este es el primer reporte que muestra la presencia de A. paragallinarum del serogrupo B en Taiwán. Además, un aislado taiwanés mostró reactividad cruzada con los antisueros del serogrupo B y C. Este aislado contiene un gene HMTp210 quimérico que podría resultar de la recombinación entre los serogrupos B y C. Estos hallazgos podrían ser valiosos para el estudio epidemiológico y la serotipificación molecular de A. paragallinarum.

Identification of sequence changes in live attenuated goose parvovirus vaccine strains developed in Asia and Europe.

Live attenuated vaccines have been used for control of the disease caused by goose parvovirus (GPV), but the mechanism involved in attenuation of GPV remains elusive. This report presents the complete nucleotide sequences of two live attenuated strains of GPV (82-0321V and VG32/1) that were independently developed in Taiwan and Europe, together with the parental strain of 82-0321V and a field strain isolated in Taiwan in 2006. Sequence comparisons showed that 82-0321V and VG32/1 had multiple deletions and substitutions in the inverted terminal repeats region when compared with their parental strain or the field virus, but these changes did not affect the formation of the hairpin structure essential for viral replication. Moreover, 82-0321V and VG32/1 had five amino acid changes in the non-structural protein, but these changes were located at positions distant from known functional motifs in the non-structural protein. In contrast, 82-0321V had nine changes and VG32/1 had 11 changes in their capsid proteins (VP1), and the majority of these changes occurred at positions close to the putative receptor binding sites of VP1, as predicted using the structure of adeno-associated virus 2 as the model system. Taken together, the results suggest that changes in sequence near the receptor binding sites of VP1 might be responsible for attenuation of GPV. This is the first report of complete nucleotide sequences of GPV other than the virulent B strain, and suggests a possible mechanism for attenuation of GPV.

Synergism between two amphenicol of antibiotics, florfenicol and thiamphenicol, against Staphylococcus aureus.

Synergistic effects between the same class of antibiotics are rarely reported. In the current study, two amphenicols, namely florfenicol and thiamphenicol, exhibited both in vitro and in vivo synergism against clinical isolates ofStaphylococcus aureusfrom chickens, cattle and pigs. Checkerboard assays on 21S. aureusisolates showed that in 80 per cent of methicillin-susceptibleS. aureus(MSSA) and 82 per cent of methicillin-resistantS. aureus(MRSA) isolates tested, the minimal inhibitory concentration (MIC) of florfenicol could be reduced by 75 per cent (1/4 MIC) or more (up to 1/16 MIC) when combined with 1/2 MIC of thiamphenicol to exhibit antimicrobial activity comparable to the respective drugs at original strength (1×MIC). A synergistic effect (fractional inhibitory concentration index ≤0.5 or ≥2-log10decrease in colony-forming unit/ml in time-kill study) was evident against 30 per cent of MSSA and 45 per cent of MRSA strains tested. A study in mice revealed that the florfenicol/thiamphenicol combination at reduced dosages provided sufficient protection againstS. aureuschallenge. The possible mechanism warrants further study but likely includes the facilitated uptake of thiamphenicol via florfenicol action, and this facilitation was not limited to amphenicol class. The present study may offer new strategy for combination therapy and provide potential alternatives for effective treatment againstS. aureusinfections.

The Fimbrial Protein is a Virulence Factor and Potential Vaccine Antigen of Avibacterium paragallinarum.

Fimbriae are recognized as virulence factors and potential vaccine antigens of several pathogenic bacteria, but the function of the fimbriae from Avibacterium paragallinarum is not well known. In this study, a gene encoding the fimbrial protein FlfA was identified in A. paragallinarum . Sequencing analysis of the putative promoter region of flfA suggests that flfA expression in A. paragallinarum might be controlled by phase variation. The flfA gene from A. paragallinarum was expressed as a recombinant protein (r-FlfA) in Escherichia coli . Immunization with r-FlfA conferred chickens protection against challenge infection with A. paragallinarum . Virulence assays showed that the flfA-deficient mutants of A. paragallinarum were less virulent than their parental wild-type strains. These results indicated that the fimbrial protein FlfA is a virulence factor and potential vaccine antigen from A. paragallinarum .

Comparison of the labeled avidin-biotin and the conventional enzyme-linked immunosorbent assay for detecting antibody to reovirus in chickens.

A labeled avidin-biotin enzyme-linked immunosorbent assay (LAB-ELISA) for detecting antibodies to avian reovirus (ARV) in chicken sera was developed and compared with conventional ELISA. Purified ARV, biotin-labeled rabbit anti-chicken IgG conjugate, and horseradish peroxidase-labeled avidin were used in the LAB-ELISA. The two types of ELISA had the similar ability to clearly distinguish ARV-positive and -negative sera. Furthermore, non-specific reactions with the two ELISAs against sera from ARV-negative chickens were reduced markedly. When sera from farm chickens were tested by the two ELISAs and serum neutralization (SN), the correlation rates between SN and conventional ELISA, and SN and LAB-ELISA were similar and were 93.4% and 92.3%, respectively.

Characterization of rabbit hemorrhagic disease virus field isolates in Taiwan.

Liver tissues from animals that were suspected to have died of rabbit hemorrhagic disease (RHD) were used for isolation and characterization of the causative agent. Three strains of RHD virus were isolated as the supernatants of liver homogenates reacted positively by hemagglutination (HA) assays and were infective for rabbits after second passage in animals. Following extraction of liver homogenates from animals infected with each of three isolates, each virus strain was purified by CsCl density gradient ultracentrifugation for further characterization. In negative-stained preparations, the purified virions were icosahedral, measured approximately 40 nm in diameter, and were without an envelope. Morphologically, the three isolates were identical. By immunoblotting, a protein with a molecular weight of 60,000 was identified as the major structural protein in each isolate. Furthermore, two sets of primer framed two different regions within RHD virus genome and could amplify two fragments of the expected size, respectively, from each isolate, whereas, none were obtained from uninfected control samples. The identity of the amplified products was confirmed further using different restriction endonucleases. Among three isolates of RHD virus, neither protein migration patterns of the virions nor cleavage patterns of the amplified product by restriction enzymes were found to differ.

Rapid differentiation of vaccine strains and field isolates of infectious laryngotracheitis virus by restriction fragment length polymorphism of PCR products.

A procedure was developed for differentiation of vaccine strains and field isolates of infectious laryngotracheitis virus (ILTV) by restriction fragment length polymorphism (RFLP) of DNA fragments amplified from the genome of ILTV by polymerase chain reaction (PCR). RFLP patterns of viral thymidine kinase (TK) gene, glycoprotein C (gC) gene, glycoprotein X (gX) gene and ICP4 gene amplified from different ILT viruses were compared. The results showed that the vaccine strain of tissue-culture-origin (TCO) could be readily distinguished from other ILT viruses. Moreover, two out of the four field isolates could be differentiated from vaccine strains of chicken embryo origin (CEO); but the remaining two field isolates were identical to the CEO vaccine strains. These results suggested that both vaccine-like and vaccine-unlike ILT viruses were involved in the field outbreak of this disease, and that the PCR/RFLP procedure could serve as a fast and sensitive method for the detection and differentiation of vaccine strains and field isolates of ILT viruses.

Analysis of the double-stranded RNA genome segments among avian reovirus field isolates.

Nine isolates of avian reovirus (ARV) from both healthy birds and birds with different clinical illness and one commercially available vaccine strain were selected and characterized by analysis of the migration pattern of their genomic double-stranded RNA (dsRNA) segments following separation by polyacrylamide gel electrophoresis. Different electropherotypes were observed and analyzed. The results show that the dsRNA segments of ARV were markedly polymorphic among isolates within the same serotype as well as among different serotypes. The results also show no correlation between electropherotype and disease state.

Identification and subtyping of avian influenza viruses by reverse transcription-PCR.

Avian influenza viruses have 15 different hemagglutinin (HA) subtypes (H1-H15). We report a procedure for the identification and HA-subtyping of avian influenza virus by reverse transcription-PCR (RT-PCR). The avian influenza virus is identified by RT-PCR using a set of primers specific to the nucleoprotein (NP) gene of avian influenza virus. The HA-subtypes of avian influenza virus were determined by running simultaneously 15 RT-PCR reactions, each using a set of primers specific to one HA-subtype. For a single virus strain or isolate, only one of the 15 RT-PCR reactions will give a product of expected size, and thus the HA-subtype of the virus is determined. The result of HA-subtyping was then confirmed by sequence analysis of the PCR product. A total of 80 strains or isolates of avian influenza viruses were subtyped by this RT-PCR procedure, and the result of RT-PCR gave an excellent (100%) correlation with the result of the conventional serological method. The RT-PCR procedure we developed is rapid and sensitive, and could be used for the identification and HA-subtyping of avian influenza virus in organ homogenates.

Phylogenetic analysis of parvoviruses isolated in Taiwan from ducks and geese.

Two major outbreaks of parvovirus infection occurred in domestic waterfowls in Taiwan in the last two decades; the first was in 1982 and the second in 1989/1990. Parvoviruses isolated in the two outbreaks were sequenced between nucleotides 142 and 680 of the VP3 gene. Sequence comparisons reveal that these viruses could be divided into two groups respectively related to goose parvovirus (GPV) and Muscovy duck parvovirus (MDPV). Nucleotide differences between ''GPV'' and ''MDPV'' groups range from 16.2% to 19.4%. In comparison, the differences within the ''GPV'' group are only 0-6.5%, while those within the ''MDPV'' group are only 0.2-1.7%. Phylogenetic analysis reveals that parvoviruses isolated in the 1982 outbreak in Taiwan are all GPV-related, whereas those isolated in the 1989/1990 outbreak are all MDPV-related. GPV-related isolates from Taiwan were separated into two groups, Thai group and European group. In comparison, all MDPV-related isolates from Taiwan are clustered in a single group that is closely related to a French MDPV isolate. The MDPV-related infection in Taiwan occurred at almost the same time in 1989 as the MDPV outbreak in France. The close phylogenetic relationship between the ''MDPV'' Taiwanese isolates and the French MDPV isolate exhibited on the VP3 fragment investigated suggests that they should be compared more deeply, to look for a possible common origin. The MDPV-related 1985 isolate might be a candidate.

A homopolymer stretch composed of variable numbers of cytidine residues in the terminal repeats of infectious laryngotracheitis virus.

A homopolymer stretch composed of variable numbers of cytidine residues was found within the inverted terminal repeats of infectious laryngotracheitis virus (ILTV). A polymerase chain reaction procedure was developed to amplify a 750-bp fragment containing this homopolymer stretch. This fragment was then sequenced directly to determine the number of repeated cytidine residues in this homopolymer stretch, which could be used for strain differentiation. By this procedure, vaccine strains of tissue culture origin could be differentiated into two types: type I contains eight repeated cytidine residues, whereas type II contains 10 such residues. Vaccine strains of chicken embryo origin could also be divided into two types: type I contains mainly 11 repeated cytidine residues, whereas type II contains 15-21 such repeats. In comparison, two of the five field isolates examined contain 12-13 repeats; the other three field isolates contain 15-19 repeats, which were similar to the type II chicken-embryo-origin vaccines. The number of repeated cytidine residues described here could serve as a marker for the strain differentiation and epidemiologic study of ILTV.

An enzyme-linked immunosorbent assay for the detection of antibody to avian reovirus by using protein sigma B as the coating antigen.

An enzyme-linked immunosorbent assay using the expressed protein sigma B as the coating antigen (sigma B-ELISA) for detecting antibody to avian reovirus (ARV) in chickens was developed and compared with a conventional ELISA. Both ELISA s and a serum neutralisation (SN) test were used to test the sera from experimentally vaccinated and farm chickens. The sigma B-ELISA could clearly distinguish the SN-positive and -negative sera in 38-week-old chickens. The correlation rate between SN and a sigma B-ELISA was 100 per cent (65/65), and that between SN and conventional ELISA was 84 per cent (55/65). With the sigma B-ELISA, all SN-negative sera had low absorbance values (below 0.06), and the absorbance values correlated closely with the SN titres. However, the sera which were antibody-negative by SN had various absorbance values, ranging from 0.07 to 0.39 in the conventional ELISA. Hence, the sigma B-ELISA had lower non-specific binding reactions than the conventional ELISA against sera from ARV -negative birds. Antibody against ARV could be detected by sigma B-ELISA after vaccination. Absorbance values peaked 4 weeks after vaccination at 2 weeks of age and were maintained until the birds were 27 weeks old. The results suggest that the presence of antibody against viral protein sigma B in birds may be used as a good indicator by the sigma B - ELISA for detecting immune status of a chicken flock or to detect chickens infected with ARV.

Detection of avian reovirus RNA and comparison of a portion of genome segment S3 by polymerase chain reaction and restriction enzyme fragment length polymorphism.

A reverse transcription-polymerase chain reaction (RT-PCR) was established to amplify a 672-base pairs fragment on the segment S3 of avian reovirus (ARV). The amplified fragments were detected in nine strains of ARV as well as three tendon tissue specimens, indicating that the primer regions were well conserved. The RT-PCR was able to detect as low as 0.2 pg using an ethidium bromide stained gel. The detection limit could be enhanced further to 0.04 pg by hybridisation after southern transfer. The amplified DNA fragments from nine ARV strains and two tissue specimens showed different restriction enzyme cleavage patterns. Analysis of the data revealed that these 11 strains were classified into four groups. The results suggest that PCR followed by restriction enzyme analysis may provide a simple and rapid method for the characterisation of ARV isolates.

Experimental infections of rabbits with rabbit haemorrhagic disease virus monitored by polymerase chain reaction.

Adult and 4-5-week-old rabbits were inoculated subcutaneously with rabbit haemorrhagic disease virus (RHDV). Samples were prepared from various tissues at intervals postinoculation (PI) for the detection of viral RNA and antigens. Using a haemagglutination test (HAT), viral antigens were detected in the liver, bile and spleen of the adult rabbits at and after 36 h PI. The reverse transcription-polymerase chain reaction (RT - PCR) showed that RHDVRNA was present in the liver, bile and spleen as early as 18 hours PI, whereas lung, kidney, thymus, mesenteric lymph node and buffy coat were found to be positive after more than 26 hours PI. In addition, viral RNA in urine and faeces showed a variable positivity at and after 36 hours PI. In the young rabbits, RT - PCR showed that RHDVRNA was present as early as 1 day PI in the liver, bile, spleen and buffy coat; whereas lung, kidney, thymus, mesenteric lymph node and faeces were found to be positive at and after 2 days PI. Bile and spleen were the only samples in which viral RNA was detected throughout the length of the experiment. Virus was not reactivated in six recovered virus-inoculated rabbits treated with dexamethasone or a classical swine fever virus vaccine. Using a haemagglutination inhibition test and an ELISA, antibody titres increased rapidly from one week PI onwards, peaked at approximately three weeks of age, and were maintained throughout the length of the experiment.

Susceptibility of piglets to rabbit hemorrhagic disease virus following experimental infection.

The possibility exists that rabbit hemorrhagic disease virus (RHDV) can be transmitted to swine, through lapinized hog cholera virus (HCV) vaccine. To investigate the infectivity of RHDV in swine, 16 four- to six-week-old piglets were inoculated subcutaneously with RHDV, and samples of liver, lung, spleen, kidney, bile, adrenal gland, tonsil, mesenteric lymph node, thymus, urine, buffy coat, and feces were collected from each of 2 animals on Days 0, 1, 2, 3, 5, 7, 14, and 28 post infection. Using reverse transcription-polymerase chain reaction, viral RNA was detected in most tissues by Day 3 and was absent after Day 5, except in lung and liver tissues, in which viral RNA was detected up to Day 14. Viral RNA was not detected in kidney, urine, feces or bile. Antibody responses, as detected by hemagglutination inhibition, were of low titer and short duration, and were similar in animals inoculated with viable RHD and in those given formalin-inactivated RHDV (n = 2). Neither viral RNA nor antibody were detected in the negative control or in the uninfected, in-contact animals.