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To study the quasispecies diversity of porcine reproductive and respiratory syndrome virus (PRRSV), open reading frame 5 (ORF5) of strain SD0612 was amplified and cloned. Sixty clones of ORF5 were sequenced and analyzed with DNAStar software. Nucleic acid sequence homology was 97.7%-100%, with 78 mutations observed. Among these 60 clones, the sequences of 17 clones were identical and recognized as the dominant quasispecies of strain SD0612. Evolution of SD0612 quasispecies diversity under antibody selective pressure was also studied. SD0612 was passed continuously in the Marc-145 cell line over 40 passages in 6 independent lineages. SD0612 antiserum was not added to lineage A, B, and C cultures; however, antiserum was added to culture medium for lineages D, E, and F. PRRSV ORF5 was then amplified, cloned, and sequenced from each of the 6 lineages, designated as A40-F40. F40 was further passed in Marc-145 cells using 6 independent lineages with or without F40 antiserum for another 40 passages. ORF5 from the 6 newly-derived virus lineages, which we designated as a40-f40, were amplified, cloned and sequenced. The proportion of dominant quasispecies increased with passage number in cell cultures supplemented with antibodies, but decreased when antibodies were lacking. Our work has demonstrated a diversity of quasispecies for ORF5 in PRRSV SD0612. Antibody selective pressure was able to significantly influence quasispecies diversity and promote a dominant quasispecies that was able to evade immune reactions.

To study the correlation between ELISA and IFA tests in detection of ALV-A/B antibody in chicken sera, ELSA S/P values and IFA titers for different serum samples were measured and statistically analyzed. The results indicated that there was a strong positive correlation between ELISA S/P values and IFA titers (r = 0.97435, P0.001). Because the positive correlation between ELISA and IFA was so strong and antibody positive rates were identical in two tests, it suggested that IFA could be used as a alternative method to replace ELISA kit when only limited numbers of samples to be tested to reduce the cost and increase the sensitivity.

In chicken fibroblast cell (CEF) cultures with antiserum against Newcastle disease virus (NDV) strain TZ060107, the virus was passed serially for 50 passages in 3 independent lineages. HN and F genes were amplified and sequenced every 10 passages. The derived virus A1-50 with most mutations among 3 lineages was further passed for another 50 passages in CEF with or without antiserum against A1-50, each in 3 independent lineages. Sequence comparisons for HN and F genes of 60, 70, 80, 90 and 100 passages indicated that the ratio of nonsynonymous mutations (NS) vs synonymous mutations (S) for HN genes in the lineages passed with antiserum against A1-50 was 5.25, which was obviously higher than 2. 375 of NS/ S in the lineages without the antiserum. The stable NS mutations occurred in the first 50 passages with the antiserum against the original TZ060107 were still maintained and one more new stable NS mutation appeared. For the F gene, 3 new stable NS mutations occurred during the second 50 passages in lineages with antiserum against A1-50 when the original NS mutations obtained in the first 50 passages with antiserum against TZ060107 still existed. Cross hemagglutination inhibition (HI) between original virus and its derivative viruses indicated that the more continuous passages in cell culture with antiserum passed, the bigger difference of antigenicity between the virus and the original virus had.

LG1 strain of avian influenza virus H9N2 was passaged continuously for 40 generations in chicken embryos with anti-LG1 maternal antibodies in 4 parallel experiments, of which 3 experiments had a stable mutation of "G" to "A" at #99 of the neuraminidase gene(NA)from the 20th passage resulting in a change of Met to Ile and 2 had a stable mutation of "A" to "G" at #473 of the NA gene from the 30th passage resulting in a change of Asn to Ser which occurred in the 50th passage of another experiment. Eighty continuous passages in chicken embryos without antibody did not have the same mutation, indicating that the mutations of the 2 positions were associated with selective pressure of antibodies. Analysis of the ratios of nonsynonium (NS) vs synonium (S) mutations of nucleic acids demonstrated that NS/S of 4 parallel experiments with antibodies was 4.6 (32/7) compared with 2.0 (16/8) of the 2 experiments without antibodies and this significant difference implied the selective pressure of antibodies.

The purpose of this study was to compare the whole genome sequences and replication dynamics in cell cultures of two Avian leukosis viruses of subgroup B (ALV) isolates, SDAU09E3 and SDAU09C2. Comparison of the amino acid sequences indicated that the gp85 identity of these two subgroup B isolates was 95.4%, the identity with other three ALV-B reference strains was 91.0%-94.9%, and less than 87.9% with ALV subgroup A, C, D, E and J. Comparison of the nucleotide sequence of gag and pol genes indicated that homologies of gag gene and pol gene of these two ALV-B isolates with all compared reference strains of different subgroups were above 93%. Homologies of LTR sequence of these two ALV-B isolates with other exogenous ALVs subgroups A, B, C, D and J were 72.6%-88.3%, but only 51.5% when compared with endogenous ALV subgroup E. The identity of LTR between these two ALV-B strains was only 74.8%, which was far lower than the identity of other genes. The identity of U3 region of LTR between these two ALV-B isolates was only 68.8% and there were obvious differences in the number CAAT Boxes. Replication dynamics in DF-1 cell indicated that the value of TCID50 was similar between 2 isolates but the concentration of nucleocapsid protein p27 antigen of SDAU09E3 was significantly higher than SDAU09C2 in cell culture supernatant, which indicated there was no parallel relationship between p27 antigen concentration and infectious virus particles. Whether such difference was resulted from the diversity of U3 region of LTR, further studies with their recombinant infectious clones is necessary.

The influences of avian reovirus (ARV) infection at 1 day of age on bursa development, antibody responses after vaccinations to avian influenza virus (AIV), Newcastle disease virus (NDV) and infectious bursal disease virus (IBDV), and pathogenecity of virulent IBDV (v-IBDV) were studied in chickens of SPF-origin. The results indicated that LY strain ARV infection in 1-day-old chickens caused atrophy of the Bursa and decreased lymphocyte numbers in the bursa, but it gave no significant negative effects on growth rates and antibody titers to AIV and NDV after vaccination. LY strain ARV infection decreased antibody titers to IBDV in B87 vaccinated birds but all vaccinated birds infected with ARV were still full protected from death or clinical syndromes after v-IBDV challenge. Although all B87-vaccinated birds were full protected from death after v-IBDV challenges, the antibody titers to AIV or NDV after vaccinations with inactivated vaccines were significantly lower in v-IBDV challenged birds than controls. Supprisingly, ARV infection at ages of 1-7 days could compromise the immune suppression induced by v-IBDV in B87-vaccinated birds as HI antibody titers to AIV and NDV in ARV-infected groups were significantly higher than chicknes with no ARV infection. A discussion was made on the interactions between ARV infection and vaccine IBDV or v-IBDV to explain such sophisticated phenomena in the bird experiments.

A recombinant baculovirus expressing reticuloendotheliosis virus env gene was constructed with Bac-to-Bac Baculovirus Expression Systems. After transfecting the recombinant virus into Sf9 cells for 3 days, REV env can be detected by indirect immunofluorescence antibody assay (IFA) and Western blot with specific monoclonal antibodies of REV. The oil-water emulsion vaccine was then produced using this infected Sf9 cells lycates and inoculated SPF chickens to validate the immunogenicity for REV. The results show that special anti-REV antibody can maintain more than 45 days and resist the infection of REV viruses. This is the first success to induce anti-REV antibody in chickens by none-live viruses.