Mariko Kohmoto

Predominance of Porcine Rotavirus G9 in Japanese Piglets with Diarrhea: Close Relationship of Their VP7 Genes with Those of Recent Human G9 Strains

ABSTRACT Type G9 of group A rotavirus (GAR) was shown to be predominant in a survey of VP7 (G) and VP4 (P) genotypes among porcine GARs associated with outbreaks of diarrhea in young pigs in Japan between 2000 and 2002. Comparison of the G9 VP7 gene sequences showed that the porcine G9 strains were more closely related to human G9 strains reemerging globally since the mid-1990s than to those from the mid-1980s. The VP7 gene sequences of porcine G9 strains from different farms were divergent (6.1 to 7.2% difference in nucleotides), suggesting that these G9 VP7 genes were not the result of recent introduction into the porcine population. Regarding the P genotype specificities of porcine G9 strains, while the majority of strains were close to unusual porcine P types (P[13] and P[23]), two strains were of the P[6] type, which has closer sequence identity with the human AU19 strain than with the porcine Gottfried strain. These unexpected results suggest that G9 GARs in the porcine population have spread more widely than previously thought and that the VP7 genes of porcine G9 strains and those of some human G9 strains detected recently may have a common progenitor.

Frequency of Enteropathogen Detection in Suckling and Weaned Pigs with Diarrhea in Japan

Fecal samples from suckling (n = 153) and weaned (n = 116) piglets with diarrhea in Japan were examined for shedding of viral, bacterial, and parasitic pathogens using culture, microscopic, and polymerase chain reaction methods. In suckling piglets, diarrhea was attributed to infection with a single etiologic agent in 60.8% of cases and with combinations of agents in 22.2%. In weaned piglets, diarrhea was attributed to a single etiologic agent in 43.1% and to combinations of agents in 47.4% of cases. Rotavirus was the most prevalent agent in suckling (67.3%) and weaned (65.5%) piglets. The detection of other pathogens was associated with age of the animals examined. Coccidia were predominantly isolated from suckling piglets, whereas Escherichia coli was found predominantly in weaned piglets. Although a relationship was not observed between detection rate of rotavirus and age of piglets, a single group of rotavirus was detected in 87.5% of suckling piglets whereas multiple groups were detected in 51.6% of weaned piglets. The results of this study confirm that diarrhea in piglets can, to a variable degree, be causally associated with multiple agents. Additionally, these results suggest reasons why this syndrome can be difficult to control.

Feline immunodeficiency virus can infect a human cell line (MOLT-4) but establishes a state of latency in the cells.

Infectivity of feline immunodeficiency virus (FIV) in feline and human lymphoblastoid cell lines was examined using homogeneous populations of FIV derived from infectious molecular clones of strains TMZ and Petaluma, and two recombinant chimeric clones carrying gag, pol, vif and ORF-A from the heterologous virus. FIV from the clones with the env region of the Petaluma strain was shown to infect and establish provirus in a human lymphoid cell line (MOLT-4), although the FIV-infected cells did not produce any infectious viruses. By treatment of the infected MOLT-4 cells with a phorbol ester, infectious virus was rescued. To examine which stage of the life-cycle of FIV is blocked in these cells, we analysed transcription of FIV-14 in the cells by RT-PCR. FIV-specific RNA expression could not be detected. These results strongly suggest that latency of the virus in MOLT-4 cells is due to a failure in transcription.

Antimicrobial resistance and genetic characterization of fluoroquinolone-resistant Mannheimia haemolytica isolates from cattle with bovine pneumonia.

Antimicrobial susceptibility and molecular characterization of quinolone-resistant Mannheimia haemolytica was conducted. The antimicrobial susceptibility of 229 M. haemolytica isolates which were obtained from cattle with bovine respiratory disease during the period 1984-2006, was determined using 14 antimicrobial agents. Of the 229 isolates, 114 (49.8%) were resistant to at least one agent and resistance rates ranged from 4.8% to 31.4%. Resistance rates for dihydrostreptomycin, oxytetracycline, doxycycline, ampicillin, amoxicillin, thiamphenicol, kanamycin chloramphenicol, nalidixic acid, enrofloxacin, and danofloxacin were 31.4%, 20.5%, 18.3%, 19.2%, 16.6%, 10.9%, 11.4%, 10.5%, 17.0%, 4.8% and 4.8%, respectively. The nucleotide sequences of the quinolone resistance-determining regions of the gyrA and parC genes of nalidixic acid-resistant M. haemolytica were determined. All nalidixic acid-resistant strains possessed at least one amino acid substitution in each of the GyrA and ParC fragments investigated. These results suggest that M. haemolytica require at least one amino acid substitution in both GyrA and ParC in order to attain significant levels of resistance to quinolones. All fluoroquinolone-resistant isolates belonged to serotype 6, and their genotype by PFGE analysis was identical. This result indicates that fluoroquinolone-resistant M. haemolytica strains have clonally expanded.

Feline CD 4 molecules expressed on feline non-lymphoid cell lines are not enough for productive infection of highly lymphotropic feline immunodeficiency virus isolates

SummaryTo investigate whether the feline CD 4 (fCD 4) molecules are involved in infections of highly lymphotropic feline immunodeficiency virus (FIV) isolates, we expressed fCD 4 stably on Crandell feline kidney cells andFelis catus whole foetus 4 cells by transfection of a cDNA encoding the fCD 4 glycoprotein, and then infected them with TM 1 and TM 2 strains of FIV, which are unable to infect these cells productively. In spite of fCD 4 being expressed on these cells, no virus production was observed. This result indicates that fCD 4 expression alone cannot induce a productive infection of the FIV TM 1 and TM 2 strains.

Production of feline immunodeficiency virus in feline and non-feline non-lymphoid cell lines by transfection of an infectious molecular clone

An infectious molecular clone of the TM1 strain of feline immunodeficiency virus (FIV) was transfected into each of one feline (CRFK), two simian (COS and Vero) and two human (SW480 and HeLa) non-lymphoid cell lines, and virus production was assayed on feline T lymphoblastoid MYA-1 cells by monitoring reverse transcriptase activity. Infectious virus was produced in CRFK, Vero and HeLa cells, but not in COS and SW480 cells. When the basal promoter activity of the FIV long terminal repeat (LTR) was examined in these cell lines by using a chloramphenicol acetyltransferase assay, the activity correlated with the virus production in each cell line. Furthermore, when the activity of the FIV LTR was compared with those of three primate lentivirus LTRs, the highest activity in all the cell lines examined was produced by the LTR of simian immunodeficiency virus from African green monkey (SIVAGM), suggesting that it has a wide expression range. In COS and SW480 cells, the activity of the FIV LTR was much lower than that of the SIVAGM LTR. These results indicate that, whereas the primary block to FIV infection of certain cells may occur at the cell surface, the FIV LTR may also participate in controlling virus replication, as an intracellular mechanism.

The AP-1 binding site in the feline immunodeficiency virus long terminal repeat is not required for virus replication in feline T lymphocytes

Sequences of 31 bp containing putative AP-1 and AP-4 binding sequences in the U3 region of the feline immunodeficiency virus (FIV) long terminal repeat (LTR) were deleted and the basal promoter activity of the LTR was measured by the chloramphenicol acetyltransferase (CAT) assay. The activity of the FIV LTR was reduced in Felis catus whole foetus 4 (fcwf-4) cells and Crandell feline kidney cells by this deletion. Cotransfection of murine c-Fos or c-Jun expression plasmids with the FIV LTR-CAT reporter plasmid into fcwf-4 cells revealed that FIV LTR could be activated by c-Fos but not c-Jun in the cells. The mutated LTR was introduced into an infectious molecular clone of FIV and the replication rate and the cytopathogenic activity of the mutant were compared with those of the wild-type in two feline CD4-positive T lymphoblastoid cell lines. It was found that the rate and activity of the mutant were almost the same as those of the wild-type. From these data, we conclude that the 31 bp fragment is important for achieving maximal expression of the FIV genome, but not required for the replication of FIV in feline T lymphocytes.

Phylogenetic analysis of feline immunodeficiency virus isolated from cats in Taiwan

SummaryFeline immunodeficiency virus was isolated from four cats from Taiwan. The isolates were designated TI-1, TI-2, TI-3 and TI-4. Each was isolated from PBMCs following co-cultivation of PBMCs with a feline T-lymphoblastoid cell line (MYA-1 cells). However, the Taiwanese isolates did not grow in a feline kidney cell line (CRFK cells). The nucleotide sequences of the V3-V5 region of the envelope gene of the Taiwanese isolates were determined and compared with those of previously described isolates. Phylogenetic analysis of this region indicates that Taiwanese isolates belong to subtype C.

EXPANSION OF CD8ALPHA +BETA - CELLS IN CATS INFECTED WITH FELINE IMMUNODEFICIENCY VIRUS

CD8+ lymphocytes have been subdivided into CD8alphabeta and CD8alpha alpha populations in the peripheral blood lymphocytes (PBL) of humans and in several animal species but have not yet been investigated in cats. Feline immunodeficiency virus (FIV) causes progressive immunological disorders similar to human AIDS. In this study, we analysed CD8+ cells in PBL of FIV-infected or uninfected cats by two-colour flow cytometric analysis. In specific pathogen-free adult cats, feline CD8alpha+beta(high) cells were observed but CD8alpha+beta- cells were not found in significant numbers. On the other hand, not only CD8alpha+beta(high) but also CD8alpha+beta- and CD8alpha+beta(low) cell populations were observed in cats chronically infected with FIV. The expansion of the CD8beta(low) or CD8beta- subpopulations resulted in the apparent differences in CD4/CD8 ratios depending on the anti-CD8 MAb used. These findings suggest a need to reconsider the CD4/CD8 ratio in studies of FIV infection. Furthermore, we found that the CD8alpha+beta- cell population expressed CD5 at a low level.

Annual changes in predominant genotypes of rotavirus A detected in the feces of pigs in various developmental stages raised on a conventional farm.

The goal of the present study was to improve understanding of the ecology of porcine rotavirus A (RVA) infection in pigs raised on a conventional farrow-to-finish farm. We collected 145 fecal samples over a 3-year period from suckling pigs and their dams, and pigs at 30, 60, 90, 120, and 150 days of age. Reverse transcriptase-polymerase chain reaction analysis revealed that 29 samples (20%) were positive for the viral VP7 gene. The detection rate of VP7 sequences was highest in 30-day-old pigs (67%), followed by suckling pigs (43%), lactating sows (17%), and 120-day-old pigs (7%). At least five different combinations of G and P genotypes were identified (G4P[13], G5P[6], G5P[13], G9P[6], and G9P[13]), and their appearance varied with time; three to four different combinations of G and P genotypes were detected in samples taken during each year, and predominant genotypes differed between suckling and 30-day-old pigs and changed annually. While the VP7 and VP4 sequences of isolates belonging to the same G or P genotype were highly similar with only two exceptions, some were combinations of different P or G genotypes, suggesting that gene reassortment occurred. Further, viral sequences carrying the same combinations of G and P genotypes were also identified in pigs of different ages in different years. Our findings here show a wide distribution of genetically diverse porcine RVA sequences that vary annually with respect to predominant genotype and according to developmental stage. These findings enhance our understanding of how RVA infections persist among farm-raised pigs.