High prevalence of torque teno sus virus in China and genetic diversity of the 5' non-coding region.

Members of the family Anelloviridae are emerging circular DNA viruses infecting many species of vertebrates including pigs. To date, members of two distinct genera, Iotatorquevirus, including torque teno sus virus 1a and torque teno sus virus 1b (TTSuV1a and TTSuV1b), and Kappatorquevirus, including torque teno sus virus k2a and torque teno sus virus k2b (TTSuVk2a and TTSuVk2b), have been identified in domestic pigs and wild boars. The goal of this study was to evaluate the prevalence and genetic diversity of these viruses based on 5' non-coding genes in Chinese swine herds experiencing clinical symptoms. One hundred eighty-five clinical samples from 11 different regions, collected during 2008-2009, were analyzed using a PCR method, and the results revealed a high TTSuV-positive rate of 78.9 % (146/185) in pigs. Moreover, we detected co-infection with multiple TTSuV strains in the same pig. Nucleotide sequencing results revealed greater genetic diversity within the genus Kappatorquevirus than within the genus Iotatorquevirus. In addition, TTSuVk2b, a novel virus discovered in New Zealand in 2012, was also identified in this study. In summary, the present work helps us obtain more knowledge about the epidemiology and genetic diversity of TTSuVs.

Co-existence of multiple strains of porcine circovirus type 2 in the same pig from China.

Pigs are often co-infected by different viral strains from the same virus. Up to now, there are few reports about co-existence of different porcine circovirus type 2 (PCV2) strains in China. The aim of this study was to evaluate it in Chinese swine herds. 118 PCV2 positive DNAs isolated from diseased pigs identified by classic PCR were re-detected using a modified differential PCR assay. The results indicated that co-existence rates of PCV2 were 32.2% (38/118) in diseased pigs and 0% (0/41) in asymptomatic pigs. Four PCV2 complete genomes were cloned from two co-infected samples and their nucleotide (nt) identities were 95%-97.3%. The phylogenetic analysis showed that four PCV2 strains were divided into different genotypes, PCV2a, PCV2b, PCV2d and PCV2e, respectively. In addition, co-existence were not detected in 41 serum samples from healthy pigs but PCV2 single infection (31.7%, 13/41) existed. These data revealed that the co-existence of different strains of PCV2 might contribute to the development of more severe clinical symptoms for pigs. This is the first report confirming the co-existence of different PCV2 strains in Chinese swine herds. Meanwhile, this study could help us to understand new infection and prevalence forms of PCV2 clinically.

The RNA profile of porcine parvovirus 4, a boca-like virus, is unique among the parvoviruses.

PPV4 transcribes its genome from a single promoter, and the RNAs are generated via alternate splicing coupled with alternate polyadenylation, a strategy similar to that of the bocaviruses; however, several differences were detected. The PPV4 ORF1 codes for four NS proteins, while the bocavirus ORF1 codes for 1-3 NS proteins. Whereas the VP1/VP2 capsid proteins of bocavirus are encoded by a single RNA, VP1 and VP2 of PPV4 are encoded by two separate RNAs. While ORF3 of PPV4 encodes two NP proteins, ORF3 of bocavirus codes for only one NP polypeptide. Taken together, PPV4 is unique among the parvoviruses.

Detection of a novel porcine parvovirus, PPV4, in Chinese swine herds.

To determine whether the novel porcine parvovirus type 4 (PPV4) recently reported in America is prevalent in China, a set of specific primers was designed and used for molecular survey of PPV4 among the clinical samples collected from various provinces of China between 2006 and 2010. The results showed that PPV4 is present in Chinese swine herds at a rate of 2.09% (12/573) among the clinical samples examined and 0.76% (1/132) among the samples taken from healthy animals. We also noted that PPV4 was not detected in samples taken prior to 2009. Analysis of the coding sequences showed that the Chinese and American PPV4 genome sequences are closely related with greater than 99% nucleotide sequence identity. Similar to a previous study, viral genomes in head-to-tail configuration of various lengths of the non-coding region were detected. Our findings confirmed that PPV4 is a unique recently discovered virus in pigs. Phylogenetically, PPV4 is most closely related to bovine parvovirus 2 (BPV2, which is not a Bocavirus and is not assigned to any Parvovirinae genus) and shares limited ORF1 (33.6%) and ORF2 (24.5%) amino acid identity. With respect to genome structure and organization, PPV4 encodes an ORF3 in the middle of the viral genome that resembles the Bocavirus genus. However, the PPV4 ORF3 encoded protein shares minimal amino acid identity with the ORF3 encoded proteins of the Bocavirus genus.

[The deletion of nucleotides of NS gene from 263 to 277 of H5N1 increases viral virulence in chicken].

Since 2000, more and more NS fragment of H5N1 subtype influenza A virus had a unique deletion of nuleotides from 263 to 277. In order to investigate the biological significance of the mutation, four recombinants, RWSN-848, RWSN-m848, RWSN-248 and RWSN-m248, were generated via eight-plasmid reverse genetic system. These reassortants had the same inner gene and outer gene derived from A/WSN/33(H1N1) and A/D/SD/04(H5N1), respectively. But their NS genes were different. RWSN-m248 and RWSN-848 were isogenic with RWSN-248 and RWSN-m848 respectively, except for the NS gene, the formers encoded a mutant NS with a deletion from 263 - 277 nucleotides. All of the four recombinants could grow well in embryonated chicken eggs and had the similar viral titer, EID50 and MDT. Infections were done with the same Multiplicity of infection (MOI, 0.001), and the viruses in supernatants from infected cells were titrated at 12, 24, 36, and 48 h post-infection by plaque assay in fresh MDCK. The results show that four viruses grown well and had the same viral title in Vero cell, a non-IFN-responsive cell system. But in MDCK and COS-1, the cell lines can produce IFN, RWSN-248 and RWSN-m848 had a higher viral titer (two fold) than RWSN-m248 and RWSN-848. It revealed that the deletion of nucleotides of NS from 263 - 277 of H5N1 influences decreases viral growth ability in IFN producing cell line. IVPI were done for the virulence test of reassorted H5N1 viruses. Ten six-week-old SPF White Leghorn chickens were inoculated intravenously with 0.2mL of a 1:10 dilution of allantoic fluid containing each of the four recombinant viruses. The chickens were daily monitored for disease signs and death for 10 days post-inoculation. Finally, IVPI was counted for each virus. RWSN-m248 caused 4 chickens died in 10 days and its index was 1.63. RWSN-248 only caused one chicken died and its index was 0.45. RWSN848 caused 3 chickens died in 10 days and its index was 0.81. RWSN-m848 caused one of the ten chickens died and its' index was only 0.175. The results revealed that the deletion of nucleotides of NS gene from 263 to 277 sites increases H5N1 pathogenesis in chicken.

[Biological significance of amino acids deletion in NA stalk of H5N1 avian influenza virus].

It has been reported that NA gene of some H1N1 Influenza A virus strains isolated since 1933 is characterized by a deletion of 11 to 16 amino acids in the stalk. The spontaneous mutant in NA stalk of H1N1 virus lacks enzyme activity with large substrate (fetuin) but not with small substrate (sialyllactose). Recently, H5N1 virus also has been found that NA has the same unique mutation in the stalk, a deletion of 15 to 20 amino acids. However, biological significance of this mutation has not yet been reported. In order to investigate biological significance of the amino acids deletion in NA stalk of H5N1, five reassorted H5N1/PR8 viruses were generated via eight-plasmid based reverse genetics system. These five viruses were named 506, m506-, 646, m646+ and 196, respectively. The six internal genes of recombinants were all from A/PR8/34(H1N1), and HA gene was from A/G/JS/03(H5N1), however, they had different NA genes. 506 and m506- held NA fragments derived from A/G/HD/00(H5N1), and the former was distinguished with a longer NA which had no 20 amino acids deletion in the stalk. 646 and m646+ held NA fragments from A/G/JS/03(H5N1), and the NA stalk of m646+ was 20 amino acids longer than that of 646. The NA of 196 was derived from A/PR8/34 which had 15 amino acids deletion in its stalk. Biological characteristics of these viruses showed that recombinants with different NA length could grow well in embryonated SPF eggs, and their EID50, MDT, and viral titers were similar. However, the length of NA was related to the capacity of eluting viruses from erythrocytes for 506 and 646+ which holding longer NA stalks eluted from erythrocytes more quickly than m506-, 646 and 196 did. Moreover, 15 or 20 amino acids deletion in NA stalk had a pronounced effect on virus growth ability in MDCK cells. Viral titers in supernatant of MDCK infected with m506- or 646 were 10 to 100 folds higher than those infected by 506 or m646+. And the plaque size of m506- and 646 were larger than that of 506 and m646+. The results reveals that H5N1 AIV with amino acids deletion in NA stalk would expand its host range. The unique amino acids deletion in NA molecule of H5N1 may be associated with the adaptation of virus to terrestrial poultry or the increasing ability of interspecies transmission.

[Establishment of reverse genetics system for H5N1 subtype AI].

H5N1 subtype influenza virus A/Duck/Shandong/093/2004 (A/SD/04) strain was chosen as the master strain for rescue research. 11 sets of primers for 8 plasmids construction were designed base on the sequencing of the full-length of A/SD/04. Eleven fragments of A/SD/04 were amplified by the designed primers and were ligated with PHW2000 for rescue plasmid construction. Eight transcription/expression plasmids were obtained, which encoded the eight segments of A/SD/04, and designated as 241, 242, 243, 244, 245, 246, 247 and 248, respectively. The COS-1 cell was cotransfected with eight plasmids with different combination of A/SD/04 and PR8. The eight reassortants shared the same HA (from A/SD/04) but contained different internal genes and NA. All of the eight reassorted viruses had some similar bio-characteristics, such as the viral title in fertilized eggs was range from 256 to 1024, the EID50 were between 10(-8.5) to approximately 10(-9), and MDT were between 34 to approximately 46h. But the IVPI of the eight reassortants was differently and all were lower than the wild-type A/SD/04. These results confirmed that different recombination of internal genes of H5N1 has influence on viral virulence to 6-week SPF chicken but not on viral replication ability in embryonated chicken eggs. The establishment of eight-plasmid rescue system for A/SD/04 is the base for farther research on genes function of H5N1. And A/SD/04 can be used as a backbone to replace PR8 entirely in generation of H5 AIV vaccine candidate.

[Cloning of full-length genes of H5N1 subtype Avian influenza virus strain A/duck/Shandong/093/2004 and analysis of the sequences].

The eight full-length genes, including the 5' and 3' ends of H5N1 subtype Avian influenza virus (A/duck/ Shandong/093/2004) were amplified by using the universal primers and H5 specific primers. The method used for the amplification of Avian influenza virus's full-length sequence was more easily and rapidly than that of rapid amplification of cDNA ends assay (RACE). The amplified segments were cloned into the T vector PCR 2.1, respectively. Three to five positive clones of each gene were sequenced and the same two sequencing results of the full-length genes were obtained. The phylogenetic analysis results showed that all the eight segments of the A/duck/Shandong/093/2004 were different from the A/Quail/Hongkong/G1/97 and A/Chicken/Beijing/1/94, but showed highly similarity (99% and above) to that of four H5N1 strains, which were isolated in 2002 in duck. It revealed that this strain was resulted from re-assortment of H5N1 rather than H9N2. The NA sequence of A/D/SD/04 was analyzed and the result demonstrated that there are 20 amino acids missing in 48 - 68 sites, however, there was no residue lost in NS gene in 263 to 277 sites. The motif of HA cleavage site is PQRERRRKKR/G, which is the characteristic of HPAIV. The 226 amino acid residue was Met (M), which can react with both Aalpha-2, 3Gal and SAalpha-2, 6Gal receptor. And the 627 residue of PB2 was Glutamic acid (E). The result mentioned above confirmed that H5N1 subtype AIV has multiple determinants in its virulence. A/D/SD/04 is the mid-strain evolving from HPAIV to a virulent strain of mammal.

[Generation of attenuated H5N1 and H5N2 subtypes of influenza virus recombinants by reverse genetics system].

The HA connecting peptide at cleavage site, PQRERRKKR / GL, of an H5N1 subtype avian influenza virus (AIV) was replaced with PQRESR / GL, and then the modified HA gene was cloned into the transcription/expression vector, pHW2000, constructing a plasmid named pHW524-HA. The NA (N1) gene from the H5N1 virus and the NA (N2) gene from an H9N2 AIV were also cloned into pHW2000 separately, resulting in plasmids pHW506-NA and pHW206-NA. With the organization of pHW524-HA, pHW506-NA or pHW206-NA, and six plasmids containing internal genes from A/WSN/33 backbone virus, two transfectants, H5N1/WSN and H5N2/WSN, were subsequently generated by eight-plasmid system. After 15 consecutive passages in embryonated eggs, the two recombinants grew up to high titers of 1:2(9) in hemagglutination test with no changes in nucleotide sequences of the surface genes detected. Both the recombinant viruses belonged to mildly pathogen when evaluated by the pathogenicity test in six-week-old SPF chickens. H5N2/WSN recombinant virus was obviously less pathogenic than H5N1/WSN virus for embryonated chicken eggs. This presentation showed that the reverse genetics system is a very useful tool for studying the construction and function of individual genes and for the generation of virus as vaccine candidate.

[Generation of newcastle disease virus strain ZJI isolated from an outbreak in the goose using reverse genetics technique].

The full-length cDNA clone, NDV3GM122, and the three helperplasmids pCI-NP, pCI-P and pCI-L of Newcastle disease virus strain ZJI isolated from an outbreak in the goose were cotransfected into BSR-T7/5 cell expressing T7 RNA polymerase. Meanwhile, the full-length cDNA clone NDV3GM122 and the three helperplasmids, pCIneoNP, pCIneoP and pCIneoL which were derived from NDV strain La Sota, were also cotransfected into the cell, respectively. Indiect immunofluorescence assay (IFA) was performed 48 to 96 hours post-transfection using NDV HN-specific monoclonal anbtibody (McAb) 6B1 and bright stainings were found in the transfectants, indicating that the full-length clone was functional and the HN protein was expressed. The transfected cell and the supernatant were mixed well and thereafter the mixture was inoculated into specific pathogen free (SPF) chicken eggs. The allanotoic fluid of the injected eggs gave a positive hemagglutinin( HA) titer ranging from 16 to 32 in the secondary passage and increased to 128 in the third passage, which was same to the level of parent wild-type virus. The allantoic fluid containing the recovered NDV was analyzed in hemagglutination inhibition( HI) test by using McAb 6B1 and the specific inhibition was found. The typical morphology of the produced NDV was detected in the electronic microscope. The results mentioned above demonstrated that infectious NDV of strain ZJI was successfully generated, which laid good foundation for the further related research.

Virulence of H5N1 avian influenza virus enhanced by a 15-nucleotide deletion in the viral nonstructural gene.

More and more H5N1 subtype avian influenza viruses possessing a 15-nucleotide (15-nt) deletion in the viral nonstructural protein (NS) gene from position 263 to 277 have emerged since 2000. In order to investigate the biological significance of this deletion, two pairs of H5N1 reassortants designated as rWSN-SD versus rWSN-mSD and rWSN-YZ versus rWSN-mYZ were generated by reverse genetics technique. These recombinant viruses shared the same inner genes of PB1, PB2, PA, NP, and M from strain A/WSN/33(H1N1) and outer genes of HA and NA from strain A/Duck/Shandong/093/2004 (H5N1) (A/D/SD/04), whereas they bore different NS gene. Recombinant rWSN-SD carried the full sequence NS gene from A/D/SD/04 in the natural state without deletion, whereas rWSN-mSD carried the same NS gene, but with an artificial 15-nt deletion from position 263 to 277. On the other hand, rWSN-YZ contained the NS gene in the natural state with a deletion from A/Duck/Yangzhou/232/2004 (H5N1) (A/D/YZ/04), while rWSN-mYZ bore the same NS gene but with an artificial insertion of 15-nt in site 263-277. All the four reassortants grew well in embryonated chicken eggs with similar mean death time (MDT) and viral titer of EID50 or HA. However, the virulence of these reassortant viruses in chickens and mice was different. Reassortant viruses with deletion in their NS gene (rWSN-mSD and rWSN-YZ) had much higher intraveneous pathogenicity index (IVPI) in chickens and lower MLD50 in mice than their counterparts without the deletion (rWSN-SD and rWSN-mYZ). Furthermore, rWSN-mSD and rWSN-YZ caused significantly more deaths in infected chickens and higher virus titers in tissues of inoculated mice than did rWSN-SD and rWSN-mYZ respectively. Sequence analysis also showed that H5N1 viruses carrying the 15-nt deletion in the NS gene invariably had the D92E shift in their NS1 protein. The results indicated that the 15-nucleotide deletion of NS gene from site 263 to 277 associated with D92E shift in NS1 protein contributes to the virulence increase of H5N1 viruses in chickens and mice.

[Role of amino acid residues at positions 322 and 329 of hemagglutinin in virulence of H5N1 avian influenza virus].

Two H5N1 avian influenza viruses (AIV), A/mallard/Huadong/S/2005 (S, IVPI = 2.65, in mallard) and A/mallard/Huadong/Y/2003 (Y, IVPI = 0, in mallard), were capable of distinct in pathogenicity to non-immunized mallards (Anas platyrhynchos). There were two amino acid residues difference in the HA cleavage site between two viruses, 322 (S, Leu; Y, Gln) and 329 (S, deletion; Y, Lys). Based on the variation, a series of recombinant viruses carrying HA gene either from S or Y virus with mutation at 322 and/or 329 were constructed via reverse genetics system to explore the influence of the two amino acid residues on viral pathogenicity in mallards. Recombinant viruses with S virus backbone were completely attenuated in terms of their virulence to ducks when position 322 (L322Q) and/or position 329 (-329K) of HA gene had been mutated. The critical role that L322 and -329 of HA protein from S virus play in the high virulence to ducks were influenced by the entire background of that protein because the recombinant virus with HA gene from Y and other seven genes from S were completely attenuated even if Q322L and K329- mutations of HA gene had been achieved. Recombinant viruses with Y virus backbone significantly increased their virulence to ducks when position 322 (Q322L) and/or position 329 (K329-) of HA gene had been mutated. All recombinant viruses carrying HA gene from Y with Q322L and/or K329-mutations and other seven genes from S were completely attenuated in terms of virulence to ducks whereas all recombinant viruses carrying HA gene from Y with same mutations and other seven genes from Y gained significant virulence. It seems that the compatibility among eight genes might be an important factor for HA to exert its functions. Results indicated that the mutation at amino acid position 322 and deletion at 329 in HA cleavage site significantly influence the pathogenicity of S and Y viruses in mallard, the compatibility among eight genes also contribute to the pathogenicity of both viruses in mallard.