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Immunosenescence is a result of progressive decline in immune system function with advancing age. Epidermal Langerhans cells (LCs), belonging to the dendritic cell (DC) family, act as sentinels to play key roles in the skin immune responses. However, it has not been fully elucidated how aging affects development and function of LCs. Here, we systemically analyzed LC development and function during the aging process in C57BL/6J mice, and performed global microRNA (miRNA) gene expression profiles in aged and young LCs. We found that the frequency and maturation of epidermal LCs were significantly reduced in aged mice starting at 12 months of age, while the Langerin expression and ability to phagocytose Dextran in aged LCs were increased compared to LCs from < 6 month old mice. The migration of LCs to draining lymph nodes was comparable between aged and young mice. Functionally, aged LCs were impaired in their capacity to induce OVA-specific CD4+ and CD8+ T cell proliferation. Furthermore, the expression of miRNAs in aged epidermal LCs showed a distinct profile compared to young LCs. Most interestingly, aging-regulated miRNAs potentially target TGF-β-dependent and non- TGF-β-dependent signal pathways related to LCs. Overall, our data suggests that aging affects LCs development and function, and that age-regulated miRNAs may contribute to the LC developmental and functional changes in aging.

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 THE EDITOR Epidermal Langerhans cells (LCs) are professional antigen-presenting dendritic cells (DCs) that reside in the epidermis and form the first immunological barrier to the external environment (Romani et al., 2010). Although considerable progress has been made in identifying the developmental requirement of LCs, the mechanisms that regulate LC development and homeostasis are still not fully elucidated. TGF-β1 is one of key regulators to control LC development and homeostasis. LCs are absent in mice that lack TGF-β1, owing to a failure in LC differentiation, survival or both (Borkowski et al., 1996; Kaplan et al., 2007; Zahner et al., 2011). This finding is further supported by the dependence of LCs development on other TGFβ1-related molecules, including inhibitor of DNA binding 2 (ID2), a TGFβ1-induced inhibitor of helix–loop–helix transcription factors (Hacker et al., 2003), and Runx3, specifically expressed by mature DCs and mediates their response to TGFβ1 (Fainaru et al., 2004). Furthermore, TGF-β1 is also required to maintain the pool of immature LCs in the epidermis (Kel et al., 2010). Although TGF-β1 is expressed by both keratinocytes and LCs, an autocrine source of TGF-β1 from LCs is required for LC development (Kaplan et al., 2007). Taken together, TGF-β1 is essential for the ontogeny, homeostasis, and function of epidermal LCs. However, the intracellular signaling pathway of TGF-β1 in epidermal LCs remains elusive. In a well-defined classical TGF-β linear signaling pathway, once activated, TGF-β1 signals through its two cell surface receptors, TGF-β receptor 1 (TβR1) and TGF-β receptor 2 (TβR2), leading to Smad-mediated transcriptional regulation. There are eight Smads: Smad1 to Smad8. Smad2 and Smad3 are activated through carboxy-terminal phosphorylation by TGFβ1. These receptor-activated Smads (R-Smads) are released from the receptor complex to form a heterotrimeric complex with a common Smad4, and translocate into the nucleus to regulate the transcription of target genes (Derynck and Zhang, 2003; Tsukazaki et al., 1998). In addition, TGF-β1 also can activate other signaling cascades, including MAPK pathways (Itoh et al., 2000; Massague, 2000; Moustakas et al., 2001). Unlike mice with the conventional disruptions of Smad2 and Smad4 that are lethal, mice with Smad3 deficiency are viable and can survive. However, Smad3 knockout (KO) mice develop progressive diseases, including leucocytosis and massive inflammation. The loss of Smad3 results in multiple cell defects, including T cells, neutriphils and macrophage (Werner et al., 2000; Yang et al., 1999). Here we used Smad3 KO mice (Datto et al., 1999) to directly test if TGFβ/Smad3 signaling pathway is involved in the ontogeny and homeostasis of epidermal LCs. During LC early development, a single wave of LC precursors recruited in the epidermis around embryonic day 18, which subsequently differentiate into LCs after birth, and LCs then undergo a massive burst proliferation during the first week of life to form a typical LC network (Chorro et al., 2009). Given that TGF-β signals are required for ontogeny and maintaining of LCs in the epidermis, we first assessed if Smad3 is required for LC homeostasis in vivo. Epidermal cell suspensions prepared from ears and dorsal skin of Smad3KO mice and WT littermates, on day 0, 1, 3, 8 after birth, as well as at 3 and 5-week old, were stained with anti-Langerin and anti-MHC-II antibodies. As shown in Figure 1 a & b, we did observe a 3–4-fold expansion of LCs from 0.78 ± 0.11% on day 0 to 2.72 ± 0.14% on day 3 in WT mice. Langerin expression was undetectable in MHCII+ LCs in the epidermis on day 0 and 1, but upregulated Langerin expression was detected in about 70%–80% LCs by day 3, which is consistent with recent report (Chorro et al., 2009; Kel et al., 2010). However, there were no significant differences on the rations of epidermal LCs between Smad3 KO and WT mice at any time points by flow cytometry (Figure 1a & b) and immunohistological staining at day 0 after born and 5 weeks old (Figure 1c & d). Thus, Smad3 is not required in the TGF-β signal pathway for oncogeny and homeostasis of epidermis LCs. Figure 1 Smad3 is not required for ontogeny and homeostasis of epidermis LCs. (a) and (b) Epidermal suspensions prepared from ears and trunk skin of Smad3 KO and WT littermates on day 0, 1, 3, 8 after birth, as well as at 3 and 5 weeks old, were stained with anti-Langerin ... Upon activation by various stimuli, immature LCs residing in epidermis collect antigen and increase their MHC II and costimulatory molecules, including CD80 and CD86, and then migrate to T cells areas of draining lymph nodes, leading to immune responses (Romani et al., 2010). However, loss of TβR1 in LCs makes LC spontaneous maturation, suggesting the TGF-β signal as an essential pathway to control the immature state of LCs (Kel et al., 2010). We next tested if TGF-β/Smad3 signaling pathway is required for maintaining the immature state of epidermal LCs. As shown in Figure 2a, the frequencies of CD80+ LCs and CD86+LCs were comparable between Smad3 KO and WT mice. Furthermore, the expression levels of MHCII, CD80 and CD86 on LCs, represented by mean fluorescence intensity (MFI), were not changed (Figure 2b). LC maturation in Smad3 KO mice were further evaluated after in vitro epidermal culture. As shown in Figure 2c, after 60h culture, both the percentages of CD86- and CD80-positive LCs and MHCIIhigh LCs as well as the relative CD80, CD86, and MHCII expression levels on LCs (Figure 2d) were significantly increased in Smad3KO and WT mice compared to unstimulation condition (Figure 2a & b), but there was no significant difference between Smad3 KO and WT mice (P > 0.05). Thus, the lack of Smad3 signaling does not affect the immature state of LCs as well as LC maturation. Figure 2 Smad3 is not an essential factor to control epidermis LC maturation and LC uptaking-antigen ability. (a) and (b) Epidermal suspensions freshly isolated from ears and trunk skin of Smad3 KO and WT littermates at 5 weeks old, were stained with anti-Langerin, ... Due to their physical location, LCs acquire and process antigens. To evaluate the role of Smad3 in antigen phagocytic function of LCs, freshly isolated epidermal cells from KO and WT mice were incubated at 37°C or 4°C (as control) with FITC-Dextran for 45 minutes and then stained with anti-mouse MHC-II and CD45.2 antibodies. As shown in Figure 2e, the phagocytic capacity of LCs in Smad3 KO mice had no significant difference (P > 0.05) compared to the WT LCs, based on the ratio of FITC-positive LCs (Figure 2e) or MFI expression levels (Figure 2f). Thus, lack of Smad3 signal pathway does not affect LC phagocytosis. In summary, lack of Smad3 surprisingly does not significantly interrupt the development and immature state of epidermal LCs, and Smad3-deficient LCs have normal maturation and phagocytosis. Our data suggest that Smad3 is not required in the TGF-β signal pathway for ontogeny, homeostasis, and function of epidermal LCs. Recent report indicated that Smad2 and Smad3 were redundantly essential for TGF-β–mediated induction of Foxp3-expressing regulatory T cells and suppression of IFN-γ production in CD4+ T cells (Takimoto et al., 2010). This raises the possibility that Smad2/Smad3 redundancy may also exist in TGF-β/Smads pathways in LCs. In addition, non-TGF-β/Smads pathways may also regulate LC ontogeny and homeostasis (Figure 2g). Further investigations are warranted to clarify the TGF-β signaling pathways through which TGF-β controls LC ontogeny and homeostasis.

An exogenous avian leukosis virus (ALV) strain SDAU09C1 was isolated in DF-1 cells from one of 240 imported 1-day-old white meat-type grand parent breeder chicks. Inoculation of SDAU09C1 in ALV-free chickens induced antibody reactions specific to subgroup A or B. But gp85 amino acid sequence comparisons indicated that SDAU09C1 fell into subgroup A; it had homology of 88.8%-90.3% to 6 reference strains of subgroup A, much higher compared to other subgroups including subgroup B. This is the first report for ALV of subgroup A isolated from imported breeders.

Radix astragali, Radix codonopis, Herba epimedii and Radix glycyrrizae are 4 plants commonly used in Chinese traditional medicine or veterinary medicine to improve immune functions against chronic diseases in humans and animals.We compared immunological enhancement by 4 herbal extracts in clinical healthy chickens or immunosuppressed chickens singly and in combination.Water extracts of 4 herbs individually and in different combinations were supplemented in drinking water. Hemagglutination inhibition (HI) antibody titers against Newcastle disease virus (NDV) and H5 avian influenza virus (H5-AIV) after vaccination were measured as indicators to evaluate immunological stimulation across groups supplemented with different herbal extracts. The experiments were conducted in both clinically healthy chickens and chickens with immunosuppression induced by reticuloendotheliosis virus (REV) infection.In clinically healthy chickens HI antibody titers against NDV and H5-AIV after vaccination were not influenced by supplementation with the herbal extracts of Radix astragali, Radix codonopis, Herba epimedii and Radix glycyrrizae in drinking water. In chicks with REV-induced immunosuppression, however, supplementation of some herbal extracts significantly increased HI antibody titers to NDV and H5-AIV when compared to the immunosuppressed control group (P0.01), but the titers were still lower than those in chicks not infected by REV. The 4 herbal mixtures produced the best enhancement among various combinations. The components of the herbal extract were water soluble and treatment by ether had no influence on immunological enhancement. The molecular weights of the active components of the herbal extracts were in the range of 10,000-100,000 Da.Our results show that the herbal extract supplementation in drinking water can induce an immune stimulation response in immunosuppressed chickens. It suggests that chickens with REV infection-induced immunosuppression could be used as an experiment model for determination of immunological enhancement effects of some herbal components.

Three cases of Newcastle disease virus (NDV) found in nature had the lentogenic motif 112 G-R-Q-G-R-L 117 in their fusion protein cleavage sites. However, both intracerebral pathogenicity and intravenous pathogenicity indexes showed that these NDV isolates were virulent. In comparison with the LaSota live virus vaccine, these viruses had significant genetic variations in the hemagglutinin-neuraminidase gene.

To determine if the maternal antibody from breeders vaccinated with cell culture-adapted reticuloendotheliosis virus (REV) could protect chicks from early REV infection, one-day-old chicks with or without anti-REV maternal antibodies were inoculated with REV, and then their growth rates and antibody titers to Newcastle disease virus (NDV) and avian influenza virus (AIV), after vaccination with inactivated vaccines, were compared. This study indicated that REV infection could cause growth retardation and severely inhibit immune reactions to inactivated vaccines against NDV and Avian influenza virus (AIV, H9 and H5) in one-day-old broilers without maternal antibodies specific to REV. Maternal antibody from breeders vaccinated with an attenuated REV vaccine effectively protected REV-challenged birds from growth retardation and immunosuppression on antibody reactions to NDV and AIV vaccines. Four weeks after vaccination, the HI titers to NDV, AIV-H9, and AIV-H5 in maternal antibody positive and negative groups were 3.36±2.04 versus 1.58±1.69 (P

In order to clarify Avian leukosis virus (ALV) characteristics from Chinese native chicken breeds, three ALV JS11C1, JS11C2 and JS11C3 were isolated from Chinese native breed "luhua" by inoculation of DF1 cell culture and detection of p27 antigen. Using PCR amplification of env gene, the amplified gp85 genes were analyzed and compared to all six chicken ALV subgroups reported. The gp85 genes of these three viruses were 1 005bp in length and encoded 335 amino acids, and the gp37 genes were 609bp and encoded 203 amino acids. The homology of gp85 among these three isolated strains was 91.9%-97.0%. Comparing to 18 stains of subgroup A, B, C, D, E published in GenBank, the homology was only in the range of 77.7%-84.6%, significantly lower than the gp85 homology observed within the common chicken subgroups A (88.2%-98.5%), B (91.6%-98.8%), and E (97.9%-99.4%). The gp85 homology compared with subgroup J was only 34.2%-36.5%. These results suggested that three isolated strains from Chinese native breed "luhua" belong to a new subgroup different from all six known subgroups from Chickens, and thus designated as subgroup K.

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.

To study the correlation between 50% tissue-culture infective dose (TCID50) value and p27 antigen S/P value of Avian leukosis virus subgroup J and discuss their significance, chicken embryo fibroblast (CEF) cells were inoculated with Avian leukosis virus subgroup J strain NX0101 and samples were tested continuously for ten days after changing maintenance media. The correlation between TCID50 and p27 antigen S/P value of ten days were then analysized. Simultaneously, DF-1 cells were inoculated with NX0101 and passaged to 20 generations. Samples taken from 1st generation, 5th generation, 10th generation, 15th generation and 20th generation were tested for the TCID50 titer and the p27 antigen S/P value separately. A significant Pearson correlation was found between them in CEF cells (r = 0.85277; P0.0001) and in DF-1 cells (r = 0.93000; P = 0.0220). This study provided an important parameter for predicting TCID50 by detecting the p27 antigen S/P value.

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 predominant field strains of Marek’s disease virus in Guangxi were clearly different from the vaccine strain CVI988/Rispens based on sequencing of the envelope glycoprotein I (gI), glycoprotein E (gE) and oncogenic meq genes. These differences may be partly responsible for the most recent outbreaks in Guangxi.

By inoculation of blood samples in DF-1 (C/E) cell culture, an exogenous avian leukosis virus (ALV) strain SDAU09C2 was isolated from a breeder farm of Chinese native breed "Luhua" in Shandong province. Comparisons of the amino acid sequence of env gene gp85 from the isolate with those from other ALV reference strains of different subgroups indicated that SDAU09C2 had the highest gp85 identity to two reference strains of subgroup B of 92.5%. Its gp85 identity to other chicken ALV subgroups A, C, D, E was in the range of 73.2%-87.9%. The identity to subgroup J was only 30.3%-32.4%. This is the first report on isolation and identification of ALV-B and its gp85 from Chinese native breed chickens.

Two Avian leukosis viruses of subgroup J (ALV-J) were isolated from layers by inoculating the sample into chicken embryo fibroblast (CEF) cells and by indirect fluorescent assay (IFA) with ALV-J specific monoclonal antibody JE-9. Sequence comparison indicated the gp85 identities were only in the ranges of 83.4%-87.3% compared with five international reference strains and 86.4%-89.6% compared with eight Chinese strains isolated from white meat-type chickens. The gp37 identities were in the range of 91.8-96.4% compared with the five international strains and 93.4-95.9% compared with the eight domestic strains. When compared with the above thirteen strains, two layers isolates were more close to the prototype HPRS-103 and had less deletion in 3'-Ter than those strains. All strains isolated from white meat-type chickens in China had a deletion in the "E" region of 3'-Ter except these two isolates, suggesting these two ALV-J isolates from layers have different evolution origins from other Chinese isolates from white meat-type chickens.

A field strain C14 of chicken infectious anemia virus (CAV) was isolated from a 14 day-old broiler flock with growth runting syndromes. Antibody reactions to inactivated vaccines to avian influenza viruses (AIV) were suppressed in SPF chickens inoculated with C14 strain CAV at 1 day-old. Also C14 strain CAV and reticuloendotheliosis Virus demonstrated a synergism in immunosuppression when chickens were infected with both virus. The viral genomic DNA was amplified by PCR in 3 overlapped fragments and PCR products were cloned into T-vector plasmid for sequencing. The sequencing results indicated that the total genome of C14 strain CAV was 2298bp, it contained 3 overlapped ORF and 1 non-coding regulation fragment. Its whole genome had 97.2% - 99.2% of homogeneity to other several published CAV reference strains. Sequence data indicated that there are many motifs in the non-coding area of about 400bp as the binding sites for transcriptional factors. All these motifs were very conservative. There were some mutations in 3 genes VP1, VP2 and VP3. Relatively, VP1 was less conservative than VP2 and VP3. Among different strains, mutations in these 3 genes were not correlated.

Twenty-four isolates of Newcastle disease virus (NDV) prevailing during 1997 -- 2005 in China were collected. These isolates were purified by CEF plaque assay and replicated in SPF chicken embryos. The hemagglutinin-neuraminidase (HN) genes of these viruses were cloned and sequenced. The HN gene sequences of thirty-six NDV reference strains in GenBank were also used in this study. The amino acid homologing of these viruses were compared and analyzed. The correlations among different fragments of HN gene were also analyzed. The results indicated that the homology of Chinese field NDV strains was 94.4%-99.4%, but 86.9%-89% compared with LaSota and Clone30, 87.9%-89.9% to F48E9, and 87.2%-96.2% to foreign NDV strains. There had the nearest distances among Chinese NDV isolates as compared with that of the LaSota, Clone30 and F48E9 by the phylogenetic tree. However, the distances of seven foreign NDV isolates were very close to Chinese NDV isolates as compared with these of the other foreign NDV isolates. We also found that all the Chinese field isolates were devoid of glycosylation site in position 538 -- 540. There were good correlations between different length amino acid fragments and the genomes of HN, especially the 5'-terminus first 80aa.

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 velogenic Newcastle disease virus (NDV) strain, designated as SRZ03, was isolated from an egg layer flock with NDV vaccine immunization failure in China in 2003. Recombination was found in the F gene of SRZ03. Complete genome sequences analysis indicated that the N-terminal of SRZ03 F gene originated from a genotype II NDV strain, whereas the C-terminal of F gene and the rest of the genes originated from a prevalent velogenic genotype VII NDV strain. It provides us valuable information for understanding the recombination of nonsegmented negative-sense RNA viruses.

Newcastle disease virus (NDV) field strain SQZ04 was isolated from a broiler flock with typical symptoms and lesions, and cloned by plaque-purification three times. NDV SQZ04 was determined as a virulent strain with MDT of 50.5h and 51.2h, ICPI of 2.0 and 1.92, IVPI of 2.8 and 2.68 respectively before and after plaque-purification. Analysis of F gene indicated that SQZO4 was determined as a virulent gene type II , and its protein amino acid sequence has homologies of 99.3% , 98.7% and 96.9% with published gene type II vaccine strains LaSota, B1, virulent strain Taxas48,much higher than homologies of 88.3% - 88.6% or 91.3% - 92.1% with published gene types VI and IX. This is the first virulent field strain of gene type UI reported in China. Further more, the amino acid sequence 111 GGRQGRL117 in the F protein cleavage site in SQZ04 strain is identical to lentogenic strains of NDV, such as vaccine strains LaSota, B1. This is the first report that virulent NDV could have lentogenic amino acid sequence in the cleavage site of F protein, where HN genes was compared SQZ04 has a higher homologies of 95.3%- 97.3% with known velogenic strains, but lower homologies of 87.8% - 89.5% with published lentogenic strains.

By use of genomic DNA prepared from 5 fowlpox virus (FPV) vaccines made in China (from Shandong, Beijing, Liaoning, Zhejing and Shanghai respectively) as the templates, reticuloendotheliosis virus (REV) LTR was amplified in PCR with a pair of primers synthesized according to the sequences flanking the integrated REV-5'LTR in FPV genomes published in US and Australia. Sequence analysis indicated that the REV-5'LTR integration sites in genomes of all 5 Chinese PFV vaccine products were identical to American and Australian FPV vaccines with integrated REV-5' LTR. Among the 5 FPV vaccine products made in China, 3 productes Vac-B-Ch, Vac-D-Ch and Vac-E-Ch had the integrated REV-5' LTR sequences of 223bp with 100% homology to that in American vaccine Vac-3-Am and Australian vaccine Vac-M3-Au. The integrated 505bp REV-LTR sequences in another Chinese products Vac-A-Ch and Vac-C-Ch had 99.6% homology to the integrated REV-LTR of American vaccine Vac-1-Am and Australian vaccine Vac-S-Au. However, REV-5'LTR integrated in all 5 FPV vaccines made in China gave only 75.4%-91.5% homology to LTR of a Chinese field strain HA9901 of REV. Based on the above results, it is reasonable to speculate that the virus stocks for the 5 Chinese FPV vaccine products were rather originally imported with their integrated REV LTR than recombinated with LTR of REV local field strains in China.

Myeloid leukosis (ML) cases were first diagnosed in a chicken flock of Chinese local breed in Shan dong province. The main symptom included wasting, weight loss, anemia. It caused about 10% mortality of about 15000 birds at the age of 120-day. In the necropsy, gray-white nodules and protrusions in various sizes were commonly observed on the surface of the sternum, intestine and trachea. Almost all viscera tissues showed moderate to severe enlargement with diffuse gray-white nodules. Histological examination indicated that the tumor cells proliferated in tissues were myelocytes with eosinophilic granules in cytoplasm. In PCR with a pair of ALV-J-specific primers, 15 of 17 liver samples were positive. PCR product of one positive sample was sequenced and demonstrated 98.05% and 97.4% identity with ALV-J HPRS-103 strain at nuclei acid and amino acids level, respectively. By immunohistochemistry (IHC) technique with ALV-J monoclonal antibody, the most intense staining was in the tumor tissue, liver, spleen, kidney, bone marrow, and proventriculus. The results indicate that ALV-J already caused chickens infection and dead in Chinese local breed.

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.

The bi-directional promoter between pp38 gene and 1.8kb mRNA transcripts of Marek's disease viruses (MDV) was divided into two single-direction promoters from the replication of MDV genomic DNA. The pp38 gene was cloned into pUC18 vector for plasmid pUC-pp38. Then the complete bi-directional promoter was cloned into pUC-pp38 in two directions to form plasmids pPro(f)pp38 and pPro(r)pp38, and the divided two single directional promoters were cloned in pUC-pp38 for plasmids pdPro(f)pp38 and pdPro(r)pp38. 24 to 48 hours after transfection to chicken embryo fibroblast (CEF) cells, the expression of pp38 could be detected in above 4 samples with Indirect Immuno-fluorescent Assay (IFA). In order to analysis the activity of the promoter quantificationally, CAT was used as the report gene. The complete or divided promoters were cloned into pCAT-Basic vector for plasmids pPro(f)CAT, pPro(r)CAT, pdPro(f)CAT and pdPro(r)CAT. The activity of CAT was measured from the lysed CEF cells, when they were transfected for 48 hours by the above four plasmids, respectively. The results showed the activity of the divided promoters reduce on both directions, especially for the direction of 1.8kb mRNA transcript, nearly down to 1/41.

PCV-2 (Porcine circovirus type-2, PCV-2) and PRRSV (Porcine reproductive and respiratory syndrome virus, PRRSV) were detected by PCR from 253 porcine pleuropneumonia samples and 125 clinically healthy lung samples were collected from different districts of Shandong province. The results showed that 171 samples for PCV-2 and 101 samples for PRRSV were positive. The positive ratio were 67.5% and 40%, respectively. The co-infection number of PCV-2 and PRRSV was 68 in 253 samples, the positive ratio was 26.8%. While in the clinically healthy samples, only 21 samples for PCV-2 and 12 samples for PRRSV were detected positive, the positive ratio were 16.8% and 9.6%, respectively, no co-infection samples were found. Statistical result showed significant difference between positive ratio for PCV-2 and PRRSV in porcine pleuropneumonia samples and that of in clinically healthy samples. The above results demonstrated that there maybe some relationship between the infection of porcine pleuropneumonia and PCV-2 and PRRSV in pigs.

By using PCR, 3 fragments of provirus cDNA of avian leukosis virus (ALV-J) strain NXO101 were amplified from the genomic DNA of ALV-J infected cells,and then combined in the right direction and sequences into recombinant plasmid pALV-J-NX, containing the whole genome of NX0101. After transfection of chicken embryo fibroblast (CEF) cells with plasmid pALV-J-NX DNA, the rescued virus was identified in CEF by indirect fluorescence antibody test with ALV-J specific monoclonal antibody JE9. The rescued virus could replicate in CEF at a titer of 10(5.6)/mL. The chicken experiment demonstrated that the rescued virus was still able to induce tumors in commercial meat-type broilers.

In recent years, the prevention and cure of infectious bursal disease (IBD) have become more and more difficult due to the emergence of very virulent strains of infectious bursal disease virus (vvIBDV) and the variant strains of IBDV. In this research, the hybridoma cell lines which secretes anti-idiotypic antibodies against anti-IBDV IgG were established. According to the Jerne's theory of immune network, the use of the anti-idiotypic antibodies as a vaccine will be a new method for the prevention of IBD. In this study, the SPF chickens were inoculated with the IBDV- SD strain, and the bursal was obtained from the died chickens. The bursal was then homogenized and frozen-thawed 3 cycles, and the virus samples were prepared by cane sugar density gradient centrifugation and dialysis. Typical IBDV particles were observed under an electron microscope, and the concentration of the virus protein measured by ultraviolet absorbance spectrophotometry was 10.8 mg/mL. SPF chickens were immunized with the virus and the highly immunized sera were prepared and purified by Sulfuric acid ammonia salt out and Sephadex G-25 chromatography. Then, Balb/C mice of six or eight weeks old were immunized interapertoneally(I. P.) with purified antibodies to IBDV at regular intervals. SP2/0 myeloma cells were fused with the spleencytes from the immunized mice at a ratio of 10:1, in 50% polyethylene glycol (1540) and were then cultured in HAT until all the SP2/0 cells died. The hybridoma cells were selected by ELISA and the highly positive holes were cloned 3 times with the method of limited dilution. Two strains (2B6 strain,5F4 strain) of hybridoma cells were obtained, which were shown by ELISA to steadily secrete anti-IBDV idiotypic antibodies. The chromosome number of the two hybridoma cells were about 88 - 106, 95 in average, and the antibodies secreted belonged to the types of IgG1 and Kappa. Balb/c mice of 3 months old were inoculated I.P. with about 10(7) hybridoma cells per capita, and the ascites were collected 12 days later and the titre of anti-IBDV idiotypic antibodies measured by ELISA was 1 :25600 (for 2B6) and 1:12800 (for 5F4) . The ascites containing the anti-IBDV idiotypic antibodies were emulsified with complete or incomplete Freund's adjuvants, and the anti-IBDV idiotypic antibody vaccine was obtained. SPF and common Jingbai chickens were immunized with the vaccine obtained. The immunized chickens with the vaccine were inoculated with IBDV-SD strain at a dose of 2000 ELD50 after twoimmunizations. All the 10 SPF chickens in the non-immunized group were sick, and 8 of them died; and 5 out of the 50 SPF chickens immunized group got sick and 2 died. All the 10 common Jingbai chickens in the control group were sick, and 6 died; 7 of the 30 immunized common Jingbai chickens got sick and only 1 died. Chi2 analysis showed that the difference between the immunized and the non-immunized groups in both the SPF and the common Jingbai chickens were significant (P0.01). Our result indicated that the anti-IBDV idiotypic antibody vaccine well protected chickens and had a great potential in both research and clinical application.

The capsid protein (VP60) gene of RHDV was subcloned into the Pichia expressin vector pPICZ B to express the VP60 protein intracellularly. The recombinant plasmid was initially transformed into a E. coli strain TOP10 F'. After verification of the construct by sequencing, the recombinant plasmid was linearized by Sac I in the 5' AOX1 region and then transformed into Pichia pastoris strain GS115 using the Pichia EasyComp Kit. After selecting and verifing for the insertion of VP60 gene in the genome, two clones of Pichia transformants were select for expression test. The recombinant clones were first inoculate with BMGY in baffled flask at 28-30 degrees C in a shaking incubator (250-300 r/min) until culture reaches an OD600 = 2-6, then resuspend the cell pellet to an OD6oo of 1.0 in BMMY medium to induce expression for 5 days by methanol at a concentration of 0.5% in a 1 liter baffled flask covered with 2 layers of sterile gauze. Collect the cell pellets and break it by acid-washed 0.5 mm glass beads. The expression of recombinant Pichia strains was detected by SDS-PAGE and Western analysis with a polyclonal serum which showed a specific protein band of 60kD. Theses results indicates that the recombinant VP60 produced in Pichia was antigenically similar to the viral polypeptide. Electron microscopic observation of the recombinant Pichia-derived protein revealed the presence of virus-like particles similar in size and appearance to native virus capsids. In the haemagglutination test, the recombinant VLPs, like the native RHDV, also agglutinated human blood type O erythrocytes and could be inhibited by the anti-RHDV polyclonal serum.

The RHDV capsid protein (VP60) gene was first subcloned into the transfer plasmid pBLUEBACHIS2B located downstream of a 6 * HIS tag, then the recombinant transfer plasmid DNA were cotransfected Sf 9 cells with Bac-N-Blue DNA and purified for cloned recombinant baculovirus by plaque assay. The expression of fused-VP60 gene was analyzed by SDS-PAGE and Western blot. A specific 69 kD protein band was obtained. Observed under electron micrography, the recombinant baculovirus expressed VP60 protein assembled into viruslike particles which were morphologically and antigenically similar to native RHD virus but did not package RNA. The close-to-native conformation of the VLPs was also supported by the haemagglutination test, in which recombinant VLPs, like RHDV, agglutinated human blood type O erythrocytes.

The way of life and mode of thinking of mankind is being changed by modern biological technology. It may be come true again that coexist and evolution of man and nature because the development of modern biological technology, but it also cannot avoid produce some new problem which made people have a think deeply to biological warfare, ethics and morals, law, society, food safety, production of industry and agriculture, energy resources, environment.