Distribution of gene segments of the pandemic A(H1N1) 2009 virus lineage in pig populations.

Swine influenza viruses (SIVs) are important not only for pig farming, but also for public health. In fact, pandemic A(H1N1) 2009 viruses [A(H1N1)pdm09] were derived from SIVs. Therefore, timely characterization of locally circulating SIVs is necessary for understanding the global status of SIVs. To genetically characterize SIVs circulating in Japanese pig populations, we isolated 24 SIVs of three subtypes (17 H1N1, four H1N2 and three H3N2 strains) from 14 pig farms in Japan from 2013 to 2016. Genetic analyses revealed that the haemagglutinin (HA) and neuraminidase (NA) genes of the 17 H1N1 and the HA gene of one H1N2, A/swine/Aichi/02/2016 (H1N2), SIVs belonged to the A(H1N1)pdm09 lineage. More importantly, all of the remaining six gene segments (i.e., PB1, PB1, PA, NP, M and NS) of the 24 SIVs, regardless of the HA and NA subtype, were also classified as belonging to the A(H1N1)pdm09 lineage. These results indicate that gene segments of A(H1N1)pdm09 lineage are widely distributed in SIVs circulating in Japanese pig populations In addition, the NA gene of A/swine/Aichi/02/2016 (H1N2) shared less than 88.5% nucleotide identity with that of the closest relative A/swine/Miyagi/5/2003 (H1N2), which was isolated in Japan in 2003. These results indicate the sustained circulation of classical H1N2-derived SIVs with remarkable diversity in the NA genes in Japanese pig populations. These findings highlight the necessity of both intensive biosecurity systems and active SIV surveillance in pig populations worldwide for both animal and public health.

Simultaneous analysis of the nasal shedding kinetics of field and vaccine strains of Bordetella bronchiseptica.

Groups of four two-week-old puppies were administered serial dilutions of an intranasal vaccine containing live Bordetella bronchiseptica and canine parainfluenza virus vaccine and housed individually in isolator cages. Three vaccinated groups and one unvaccinated control group were exposed to virulent B bronchiseptica four weeks after vaccination and evaluated. Nasal swabs for bacterial culture and sera for agglutination tests were taken from all the dogs every week for four weeks. The bacteria isolated were identified by growth on specific agar and by specific PCR to distinguish between vaccine and challenge strains. The vaccine strain persisted in the nasal cavity after vaccination but no adverse reactions were observed. Serum agglutination titres were raised in the vaccinated dogs at challenge. Vaccine strains were not isolated after the challenge from most of the vaccinated dogs. The challenge strain was shed in the dogs vaccinated with the lowest dose (10(6.0) cfu/dose) for two to three weeks but the other vaccinated groups (10(7.0) and 10(8.0) cfu/dose) shed the challenge strain transiently or not at all. Only the group vaccinated with 10(6.0) cfu/dose exhibited clinical signs after challenge.

Genogrouping of vaccine breakdown strains (VBS) of feline calicivirus in Japan.

Although prevention of feline calcivirus (FCV) infection by vaccination has been attempted, and isolation of FCV, development of the disease, and a few fatal cases in vaccinated cats have been reported. Fifteen FCV strains isolated from cats that had been vaccinated with commercially available FCV vaccines (F9, FCV-255, and FC-7) were genogrouped. Molecular analysis of viral genomes involved the construction of a phylogenetic tree of capsid genes using the NJ method. Cat anti-F9 serum and rabbit anti-FCV-255 serum were used for virus neutralization tests. Molecular phylogenetic analysis of the amino acid sequences of 15 virus isolates and those of the previously published and GenBank-deposited 9 global and 14 Japanese strains showed that 8 (53%) of the 15 virus isolates as well as the vaccine strains F9 and FCV-255 belonged to genogroup I (G(A)I), and 7 (47%) belonged to genogroup II (G(A)II). Of the 8 G(A)I strains, 2 were isolated from cats that had been vaccinated with an F9 strain live vaccine, 5 from cats vaccinated with an FCV-255-derived vaccine, and 1 from a cat vaccinated with an FC-7-derived vaccine. Of the 7 GAll strains, 5 were isolated from cats that had been vaccinated with the F9 strain live vaccine, 1 from a cat vaccinated with the FCV-255-derived vaccine, and 1 from a cat vaccinated with the FC-7-derived vaccine. These results indicate that more vaccine breakdown strains isolated from the cats vaccinated with the F9 strain-derived vaccine belong to G(A)II than to G(A)I, whereas more vaccine breakdown strains isolated from the cats vaccinated with the FCV-255 strain-derived vaccine belong to G(A)I than to G(A)II, and that when the FC-7 strain-derived vaccine is used, the vaccine breakdown strains belong almost equally to G(A)I and G(A)II. Thus, the genogroups of virus isolates varied with the vaccine strain used (p < 0.05). On the other hand, the neutralizing titres of feline anti-F9 serum and rabbit anti-FCV-255 serum against the 15 isolates were very low, showing no relationships between neutralizing antibody titres and genogroups. The DNA sequence identities between the virus isolates and the vaccine strains were low, at 70.6-82.9%, and no strains were found to have sequences derived from the vaccine strains. Alignment of amino acid sequences showed that the G(A)I or G(A)II virus isolates from the F9-vaccinated cats differed at position 428 of the 5' hypervariable region (HVR) of capsid region of the F9 strain, whereas those from the FCV-255-vaccinated cats differed at positions 438, 453, and 460 of the 5'HVR of capsid region E of the F9 strain. We speculate that these differences influence genogrouping. The amino acid changes within the F9 linear epitopes common to G(A)I and G(A)II were noted at positions 450, 451, 457 of 5'HVR of the capsid region E in the isolates from F9-derived vaccine-treated cats, and 449, 450, and 451 of 5'HVR of capsid region E in the isolates from FCV-255-derived vaccine-treated cats, suggesting that these amino acid changes are involved in escapes. These results suggest that alternate vaccination with the F9 and FCV-255 strains or the use of a polyvalent vaccine containing GAll strains serves to inhibit development.

Detection of feline calicivirus (FCV) from vaccinated cats and phylogenetic analysis of its capsid genes.

We analysed genogroups of four feline calcivirus (FCV) isolates (FCV-S, H10, Ao198-1 and ML89) obtained from cats that experienced FCV infection after having been vaccinated against FCV. New PCR primer sets (8F/8R, Ao-S/Ao-A, cp-S/cp-A) were also designed, since the conventional Seal primer failed to amplify the target sequences in two samples. The genogroups of the four isolates as well as eight global and 17 domestic strains were determined by phylogenetic analysis of their amino acid sequences. One out of the four strains (25%) isolated in this study, H10, was grouped into genogroup I, along with the vaccine strains F9 and FCV-255. The other three isolates (75%) belonged to genogroup II. Thus, there were more isolates in genogroup II than in genogroup I. However, the antibody values of the four isolates against cat anti-F9 antisera were significantly decreased. There may be no relationship between the neutralizing antibody titre and genogroup. Amino acid sequence alignment of the four isolates showed that only a single amino acid in region C, which is involved in neutralization epitopes, was different in ML89 strain from that of F9. The other three strains, H10, Ao198-1 and FCV-B, shared the same amino acid sequence with F9. Alignment of amino acids for linear epitopes in the F9 strain, which are located at regions D and E, showed variations in 5' hypervariable region (HVR) of E, whereas D and conE had only synonymous substitutions i.e. no change in the amino acid sequence. This mutation in 5' HVR of region E suggested a vaccine breakdown, as the region is known to be essential for antigenicity. The genogroup II FCV is likely to be the cause of the FCV infection in this study, while the vaccine strains belong to genogroup I. Thus, the existing vaccine may need reevaluation for its effectiveness.

Capsid protein genetic analysis and viral spread to the spinal cord in cats experimentally infected with feline calicivirus (FCV).

We investigated primitively the molecular basis of the neural spread of a feline calcivirus isolate (FCV-S) from the spinal cord of a cat that died after manifesting excitation. Experimental infections of cats with three clones from parent virus isolate FCV-S, isolated based on plaque size, were performed, and virus recovery from the spinal cord and the nucleotide and predicted amino acid sequences of the viral capsid protein region (ORF2) were compared. In the experimental infection with the one-time cloned virus (C1L1) isolated from a large plaque, the C1L1 was recovered from the spinal cord. In contrast, seven-times cloned C6L7 (from large plaque) and five-times cloned C5S2 (isolated from small plaque) were not recovered from the spinal cord. Genetic analysis of the capsid protein gene of the three viral clones revealed that four bases were different and two amino acids were different at positions 34 (Val in C6L7 and Ala in C1L1 and C5S2) and 46 (Leu in C6L7 and Pro in C1L1 and C5S2) between C6L7 (with large plaque) and C5S2 (with small plaque). The amino acid at position 434 of C1L1 was different from those of C6L7 and C5S2 (Gly in C1L1, D (Asp) in C6L7 and C5S2). From these results, the plaque size seemed not to be related to the spread of virus to the spinal cord. Clone C1L1, which spread to the spinal cord, had a difference of one amino acid from the other two clones, which may be related to the ability to spread to the spinal cord.

Properties of a calicivirus isolated from cats dying in an agitated state.

In June 1993, two of five pet cats kept in Yokohama city in Japan suddenly became agitated and died. Feline calicivirus (FCV) was isolated from them. One strain (FCV-S) was isolated from the spinal cord, lung and tonsil of cat 1, another (FCV-B) from the ileum, medulla oblongata and cervical spinal cord of cat 2, and a third (FCV-SAKURA) from the oral cavity of one of the three surviving cats which showed no clinical signs. These three strains were equally resistant to pH 3.0 and serologically similar to each other, but distinct from strain F9. A genetic analysis, using a 208 base pair fragment from region E of the capsid, showed that FCV-Ari had a 70.4 per cent nucleotide and 77.3 per cent amino acid homology and FCV-F9 had a 68.6 per cent nucleotide and 73.9 per cent amino acid homology with the three strains, indicating that these two strains were genetically distinct from the three new isolates. Unvaccinated cats and cats which had been vaccinated against FCV-F9 developed watery diarrhoea but did not become agitated after the administration of FCV-S. The FCV-S strain did not induce signs of excitability after it was administered orally to specific pathogen-free cats.

Detection of canine distemper virus antigen in canine serum and its application to diagnosis.

Canine distemper virus (CDV) antigen was detected in the serum of dogs by an ELISA and the results of this assay were compared with an anti-CDV immunoglobulin M (IgM) antibody test. In paired sera from 26 naturally infected dogs, the antigen-positive rate was 26.9 per cent at the first examination and 11.5 per cent at the second examination two to three weeks later. The antigen was detected in three of the 10 dogs which were negative for anti-CDV IgM antibody at the first examination. It could also be detected in the serum of between eight and two of 40 specific pathogen-free dogs vaccinated against CDV, for up to four weeks after they were vaccinated.

Phylogenetic analysis of field isolates of feline calcivirus (FCV) in Japan by sequencing part of its capsid gene.

The molecular epidemiology of the infectious disease caused by feline calcivirus (FCV) in Japan was investigated by analysing the phylogenetic relationship among 21 Japanese field isolates, including the F4 strain, and 30 global isolates. Parts of the capsid gene (B-F) of the isolates were amplified by RT-PCR, and the amino acid sequences were compared with those from the global isolates. Thirty-seven and 14 out of a total of 51 isolates were clustered into two distinct genogroups, I and II respectively, by UPGMA and NJ analysis. Seven of the 21 Japanese isolates (33%) fell into group I together with 30 global isolates, while the other 14 Japanese isolates (67%) belonged to group II. The bootstrap repetition analysis of groups I and II formed by the NJ method gave a value of 99.00%. The 14 latter Japanese isolates were clearly separated from the isolates in group I, and they were different from any previously known FCV, forming a new genogroup, which implies that this lineage has been confined to Japan. Comparing the amino acid sequences shared by groups I and II, the amino acid at position 377 in B region was asparagine (Asn or Asp (NH2)) in group I, while it was lysine (Lys) in all the strains in group II. Similarly, the amino acid at position 539 in the F region was alanine (Ala) or proline (Pro) in group I, while it was valine (Val) in group II; glycine (Gly) at position 557 in group I was serine (Ser) in Group II; and phenylalanine (Phe) or leucine (Leu) at position 566 in genogroup I was tyrosine (Tyr) in group II.

Molecular cloning of an Actinobacillus pleuropneumoniae outer membrane lipoprotein (OmlA) from serotype 5a.

The gene encoding an outer membrane lipoprotein (OmIA) was cloned from Actinobacillus pleuropneumoniae strain NG-8 (serotype 5a). The deduced amino acid sequence of OmIA from strain NG-8 showed 61% identity to the OmIA from serotype 1 strain, which confers protective immunity to pigs. Southern blot analysis showed the presence of a sequence highly homologous to the omIA gene of strain NG-8 in strains of serotype 5a, 5b and 10. A specific serum against OmIA of NG-8 also detected a homologous protein in the strains of these serotypes. These data shows the presence of antigenic variability among A. pleuropneumoniae OmIA proteins.

The role of cell-mediated immunity in chickens inoculated with the cell-associated vaccine of attenuated infectious laryngotracheitis virus.

The cell-mediated immune responses of the chickens inoculated with the cell-associated (CA) ILT vaccine were studied. Lymphocyte blastogenic response was tested by MTT assay with spleen, thymus, bursa of Fabricius and peripheral blood lymphocytes of the vaccinated chickens. The blastogenic response of peripheral blood and spleen lymphocytes was enhanced by a T-cell mitogen. The CA vaccine induced an immunity satisfactorily in bursectomized chickens. To study the role of immune cells in the vaccinated chickens, the bursectomized chickens were inoculated intravenously with the splenic, thymic, bursal and peripheral blood lymphocytes of the vaccinated chickens. Protective effects were shown in the chickens received the splenic cells and peripheral blood lymphocytes, indicating that these play an important role in eliciting the protective effects of the CA vaccine. From these results, it was found that the immune mechanism with CA vaccine against ILT involves mainly cell-mediated immunity.

Immune response and in vivo distribution of the virus in chickens inoculated with the cell-associated vaccine of attenuated infectious laryngotracheitis (ILT) virus.

Chickens inoculated with the cell-associated (CA) vaccine acquired higher protective immunity to ILT. In chickens vaccinated with CA or cell-free (CF) vaccine, respectively, virus-neutralizing and IgG- and IgM-ELISA antibodies were detected in the serum, but no antibody was detected in the tracheal washes of the vaccinated chickens. More apparent antibody response was seen in chickens vaccinated with the CA vaccine than with the CF vaccine. The antibody titers did not correlate closely with the protection against challenge with ILT virus. After subcutaneous injection of either CA or CF vaccine, ILT virus was isolated from the liver, spleen, thymus, lungs and other organs of the chickens from 1 to 6 days after injection, and there was no correlation of the isolation rate for the CA-vaccinated and the CF-vaccinated chickens.

Construction of recombinant infectious laryngotracheitis virus expressing the LacZ gene of E. coli with thymidine kinase gene.

We constructed the recombinant infectious laryngotracheitis virus (ILTV), CE strain, containing the LacZ gene of E. coli in the thymidine kinase gene. The growth property of the recombinant virus was almost the same as parental CE strain in chicken embryo fibroblasts. The recombinant CE strain of ILTV could be used as a live vaccine vector.

Safety and efficacy of the cell-associated vaccine prepared by an attenuated infectious laryngotracheitis virus.

The safety and efficacy of the cell-associated (C-A) vaccine prepared by chicken embryo fibroblast (CEF) cells infected with the tissue-culture-modified strain of infectious laryngotracheitis (ILT) virus were studied in chickens. Over seventy percent of chickens inoculated with the C-A vaccine by the subcutaneous (S.C.) or intramuscular (I.M.) route at 1 day of age was protected against challenge with a virulent strain of ILT virus without any clinical signs. Chickens vaccinated with the C-A vaccine at 1 day of age acquired immunity within 6 days after vaccination, and the protection rate maintained more than 60% until 10 weeks post-vaccination. The C-A vaccine was invariably effective for chickens at various age. There was no evidence that the development of immunity was hindered by further vaccination with Newcastle disease and infectious bronchitis combined live vaccine. In addition, the C-A vaccine was safe when chickens were inoculated with 10 doses. In the field trials of the C-A vaccine, no adverse reaction was observed, and over 65% of vaccinated chickens was protected against the challenge of the virulent ILT virus at 8 weeks after vaccination.