Pathogenicity,sequence and phylogenetic analysis of <Emphasis Type="Italic">Malaysian Chicken anaemia virus</Emphasis> obtained after low and high passages in MSB-1 cells

Summary. Specific-pathogen-free (SPF) chickens inoculated with low passage Chicken anaemia virus (CAV), SMSC-1 and 3-1 isolates produced lesions suggestive of CAV infection. Repeated passages of the isolates in cell culture until passage 60 (P60) and passage 123 produced viruses that showed a significantly reduced level of pathogenicity in SPF chickens compared to the low passage isolates. Sequence comparison indicated that nucleotide changes in only the coding region of the P60 passage isolates were thought to contribute to virus attenuation. Phylogenetic analysis indicated that SMSC-1 and 3-1 were highly divergent, but their P60 passage derivatives shared significant homology to a Japanese isolate A2.     

Sequence analysis of segment a of a field virus isolate from an outbreak of Infectious bursal disease in India

Sequence analysis of genome segment A of an Indian Infectious bursal disease virus (IBDV) field isolate (KT1/99) revealed total 95 nucleotide substitutions, resulting in 17 amino acid changes. Of these, five amino acid changes, namely F60S, T137I, I374V, V519I and E682D were unique to the KT1/99 isolate. The amino acid change P222A and the proposed hot mutation spot 680Y, reported to be present in very virulent IBDV isolates were also found in KT1/99. This isolate had nucleotide divergence of 1.1% to 4.95% from the other reported serotype 1 IBDV isolates and 19.6% from serotype 2 strain OH in polyprotein gene sequence, while divergence at amino acid level was 0.6% to 2.9% and 11.4%, respectively. Based on both nucleotide and amino acid sequence analysis, KT1/99 was grouped phylogenetically with the reported Bangladesh isolate BD3/99 in one cluster along with other reported very virulent isolates in same lineage.     

Biological and molecular characterization of chicken anaemia virus isolates of Indian origin

In the present study, four chicken anaemia virus (CAV) isolates (CAV-A, -B, -E and -P) recovered from different geographical regions of India were characterized. CAV genome of 1,766 bp nucleotide region containing the complete coding region of VP2 and VP3 proteins, and partial coding region of VP1 protein were sequenced. The nucleotide and deduced amino acid sequence of the Indian CAV isolates were aligned and compared with CAV isolates of European, Asian, American and Australian origin. Phylogenetic analysis of the Indian CAV isolates were also carried out based on the nucleotide and deduced amino acid sequences. The results indicated that Indian isolates were genetically evolved from different parts of the world. Indian isolate, CAV-A was found closely related to European Cux-1 strain, CAV-B and -P were closely related to Bangladesh BD-3 strain and CAV-E was closely related to Australian 704 strain. The pathogenicity of the four CAV isolates was studied in day-old specific pathogen free (SPF) chicks. Day-old SPF chicks (n=50) were divided into five groups comprised of 10 chicks in each group. Group 1 was kept as control and groups 2-5 were infected with each CAV isolate separately. The chicks were infected at a dose rate of 1 ml cell culture fluid (10(4.5)TCID(50)/0.1 ml) per bird intramuscularly. The clinical signs, mortality and packed cell volume (PCV) and body weight gain were recorded on 5, 10 and 15 days post-infection. At 15th day, all the birds were sacrificed and various organs, viz., thymus, bone marrow, spleen, liver and bursa were examined for gross and microscopic changes. The pathogenicity study indicated that all the CAVs except CAV-B were able to produce clinical disease and immunosuppression in young chicks whereas the isolate CAV-B produced no clinical disease but only induced immunosuppression, which was revealed by microscopic examination of the lymphoid organs. The study showed valuable information on molecular epidemiological status of CAV isolates prevalent in India for the first time.     

Molecular characterization of Korean Pepper mottle virus isolates and its relationship to symptom variations

The symptom variations among Korean Pepper mottle virus (PepMoV) isolates infecting pepper, tomato and potato were described and the cause of variations in relation to molecular variability were investigated. In addition, the entire genome of the 13 PepMoV isolates, collected from five provinces (Kyonggi, Chungnam, Gyeongnam, Jeonbuk and Jeonnam) in Korea, were determined and compared including the previously reported Korean-Vb isolate and 2 other PepMoV isolates isolated from America (CA and FL). Our results showed that the nucleotide sequence of all Korean isolates tested were nearly identical (98–99%) and only 94% similar to American isolates. In general, the complete nucleotide sequences and deduced polyprotein sequences indicated low genetic variation among isolates showing 0.1–3% nucleotide changes per site. However, based on ratio between nucleotide diversity values in nonsynonymous and synonymous position (dN/dS ratio) surprisingly, P1 and 6K2 genes showed relatively high nucleotide substitution ratio (0.8 and 1.0 nucleotide, respectively). When the 6K2 amino acid were aligned, there were 15 amino acid substitutions found in PepMoV-infected potato and only 1 amino acid change from two isolates of PepMoV-infected bell pepper. Interestingly, three isolates including isolate numbers 731, 205135 and 205136 that possessed different aa changes at 6K2 region also showed distinct symptom differentiation in indicator hosts and cosegregated in the phylogenetic analysis. These results further proved previous studies that P1 and 6K2 genes with other proteins might have some involvement on host specificity and pathogenicity.     

Analysis of penicillin-binding protein lb and 2a genes from Streptococcus pneumoniae

Fifty clinical isolates (penicillin MICs, 0.03-8 microg/mL) of Streptococcus pneumoniae were randomly selected from hospitals throughout South Africa, together with seven strains isolated in Hungary (penicillin MICs, 16-32 microg/mL). Penicillin-binding protein (pbp) 1b and 2a genes were amplified by PCR, and the purified DNA was digested with HinfI, StyI, and MseI + DdeI restriction enzymes. The fragments were radioactively end-labeled and separated on polyacrylamide gels, and the DNA fingerprints were visualized following autoradiography. A collection of isolates was further selected for sequence analysis of pbp1b and 2a. DNA fingerprint analysis revealed a uniform profile amongst all isolates for both genes. All isolates revealed a maximum of only seven nucleotide substitutions in their pbp1b genes, resulting in a maximum of three amino acid substitutions in PBP 1B. In the case of the pbp2a gene, up to 13 nucleotide substitutions were observed randomly distributed amongst penicillin-susceptible and resistant isolates, revealing a maximum of five amino acid substitutions in PBP 2A. No amino acid substitutions were found to be common amongst all penicillin-resistant isolates. Transformation experiments with pbp1b and 2a genes isolated from two resistant strains (MICs, 4 and 16 microg/mL) failed to transform pneumococcal strains to increased levels of penicillin resistance. These results show that the pbp1b and 2a genes examined here do not display the typical mosaic gene patterns observed in the pbp2x, 2b, and 1a genes of penicillin-resistant pneumococci. In addition, the transformation studies suggest that PBPs 1B and 2A may not play a role in the development of penicillin resistance in some pneumococci.     

Molecular epidemiology of chicken anemia virus in Nigeria

Summary. Between February 2002 and May 2004, chicken anemia virus (CAV) was detected by PCR in organ samples from 14 flocks of poultry farms in Lagos, Ogun and Oyo States in Southwestern Nigeria. The farms reported low (<5%) to high mortalities (up to 100%) with various lesions at necropsy. The complete VP1 gene of 30 of these positive strains was sequenced. Strains that diverged by up to 4.4% on a nucleotide level differed only by up to 2.5% at the amino acid level (7 aa) as a result of clustered silent mutations. No amino acid substitutions specific for Nigerian strains were observed. Some birds had a CAV mixed infection. Genetic clustering of the VP1 gene did not correlate with differences in flock mortality but the co-infection of CAV with IBDV may be particularly lethal. This first molecular epidemiological study of CAV in Africa shows that the Nigerian strains cluster with viruses from very diverse geographic origins and were almost as diverse (4.4%) as all other strains combined (5.8%).     

Molecular analysis of the S1 subunit of the spike glycoprotein of respiratory and enteric bovine coronavirus isolates

It is unclear whether respiratory and enteric bovine coronavirus (BoCV) strains are distinctive in biological, antigenic and genetic characteristics. In the present study, we analyzed the nucleotide and amino acid sequence of the S1 subunit of the S glycoprotein, including the cleavage site, of both respiratory (n=5) and enteric (n=3) BoCV isolates including two paired isolates from the same feedlot animals and compared them with the prototype Mebus and two enteric and one respiratory BoCV strains from Quebec. A total of 75 polymorphic nucleotides were identified in the S1 subunit of the spike glycoprotein of BoCV isolates compared with the Mebus strain. These polymorphisms led to 42 amino acid changes at 38 distinct sites. The amino acid changes were distributed throughout the S1 subunit with clustering around residues 40-118, 146-179, and 458-531. Among these variations, only 19 amino acid substitutions altered the charge, hydrophobicity and surface probability of the protein. Based on phylogenetic analysis, our respiratory and enteric isolates clustered into two major groups with two subgroups. Although, there were only a few amino acid changes between the respiratory and enteric paired isolates, the other two respiratory isolates, one isolated from the same farm as a paired strain and the other from a different farm, showed more sequence diversity. Amino acid alterations in residues 113, 115, 118, 146, 148, 501, 510 and 531 of respiratory isolates conferred significant changes in the predicted secondary structure compared with the prototype winter dysentery (WD) and the calf diarrhea (CD) strains of BoCV. In conclusion, the data suggests that respiratory strains of BoCV may differ genetically from the classical calf enteric and adult WD strains.     

Comparative sequence analysis of open reading frames 2 to 7 of the modified live vaccine virus and other North American isolates of the porcine reproductive and respiratory syndrome virus

Summary.  To elucidate changes associated with the attenuated virulence in a modified live porcine reproductive and respiratory syndrome (PRRS) vaccine (Boehringer Ingelheim Animal Health, St. Joseph, MO), derived from an American prototype ATCC virus VR-2332, nucleotide sequence of 3′ genome covering open reading frames (ORFs) 2 to 7 coding regions from the vaccine virus was determined by RT-PCR with two overlapping fragments. Comparisons showed 98 base changes (94 substitutions, 3 deletions, and 1 addition) out of 3318 nucleotides between the vaccine virus and its parental virus. There were 15, 26, 17, 29, 9, and 6 base substitutions in ORFs 2, 3, 4, 5, 6, and 7, respectively, resulting in 5, 13, 8, 13, 2, and 3 amino acid (a.a.) substitutions in their deduced proteins, respectively. Most of these a.a. substitutions were also present in 17 known virulent/wild type PRRS virus isolates from North America. However, there were 1, 4, 1, and 1 unique a.a. substitutions in the vaccine virus ORFs 2, 3, 4, and 5 deduced proteins, respectively. These unique amino substitutions may be responsible for the attenuated virulence in the vaccine virus. Accepted October 6, 1997 Received August 7, 1997     

Identification of a new genotype of bovine leukemia virus

To investigate the degree of genetic variability of bovine leukemia virus (BLV) strains circulating in Croatia, 29 isolates from the six largest dairy farms were examined by PCR for a segment of the gp51 env gene, followed by DNA sequencing and phylogenetic analysis. The nucleotide sequences were compared with other previously characterized BLV strains from different geographical areas, comprising all seven known BLV genotypes. The Croatian sequences showed six to eight nucleotide substitutions: six silent substitutions and two amino acid changes. Four of those substitutions were within epitopes. In comparison to the sequences of other BLV genotypes, our isolates showed the closest relationship to genotype 1 isolates PL-3252 (FJ808585) and AL-148 (FJ808573) from Argentina. The degree of variation between our sequences and those of genotype 1 was 0.2- 4.6 %. In phylogenetic trees based on 400-nt and 519-nt sequences, all of the Croatian sequences clustered separately from the other sequences, revealing a new genotype.     

Divergence and conservation of the genomic RNAs of Taiwan and Hawaii strains of papaya ringspot potyvirus

Summary. The complete nucleotide sequence of the genome of a Taiwan isolate of papaya ringspot potyvirus (PRSV YK) was determined from three overlapping cDNA clones and by direct RNA sequencing. Comparison was made with the reported Hawaii isolate of PRSV HA. Both genomes are 10 326 nucleotides long, excluding the poly(A)-tail. They encode a polyprotein of 3 344 amino acids with a 5′ leader of 85 nucleotides and a 3′ non-translated region of 209 nucleotides. The two genomes share an overall nucleotide identity of 83.4% and an amino acid identity of 90.6%. The 3′ non-translated regions show 92.3% identity. The first 23 nucleotides of the leaders are identical, while the remaining parts of the leaders only show 51.6% identity. The P1 protein genes of the two isolates are very different, with 70.9% nucleotide identity and 66.7% encoded amino acids identity. However, the other viral proteins of the two virus isolates are similar, with a 82.5–89.8% nucleotide identity of their genes and 91.2–97.6% amino acid identity, indicating that they are strains of the same potyvirus. Analysis of the ratios of nucleotide differences to the actual amino acid changes revealed that there are only 2.63 nucleotide changes for each amino acid change in the P1 protein, whereas for the other proteins 4.0–16.4 nucleotide changes are required for each amino acid replacement. The P1 protein has 58% of all the differences of polyprotein. The unusual variation in the leader sequences and the P1 proteins suggests that the two PRSV strains were derived from different evolutionary pathways in different geographic areas. Received February 5, 1996 Accepted September 17, 1996     

Sequence variability and functional analysis of MutS of hypermutable Pseudomonas aeruginosa cystic fibrosis isolates

In this study, we investigated the variability of MutS among Pseudomonas aeruginosa recovered from cystic fibrosis (CF) patients. Sequencing of the mutS gene of 15 hypermutable P. aeruginosa isolates obtained from different patients revealed high rates of nucleotide substitutions as compared to that of strain PAO1. Significantly more synonymous than non-synonymous nucleotide substitutions have been found, indicating that generally MutS is highly conserved. The functional analysis of MutS variants by complementation of a PAO1 mutS mutant revealed 5 isolates with a defective MutS due to frameshift mutations or amino acid substitutions. This work supports the hypothesis that the respiratory tract of CF patients represents an environment that favors the selection of highly adaptive mutator phenotypes.     

Nucleotide sequence analysis of the nucleoprotein gene of an avian and a human influenza virus strain identifies two classes of nucleoproteins

The nucleotide sequences of RNA segment 5 of an avian influenza A virus, A/Mallard/NY/6750/78 (H2N2), and a human influenza A virus, A/Udorn/307/72 (H3N2), were determined and the deduced amino acid sequences of the nucleoprotein (NP) of these viruses were compared to two other avian and two other human influenza A NP sequences. The results indicated that there are separate classes of avian and human influenza A NP genes that can be distinguished on the basis of sites containing amino acids specific for avian and human influenza viruses and also by amino acid composition. The human influenza A virus NP genes appear to follow a linear pathway of evolution with the greatest homology (96.9%) between A/NT/60/68 (H3N2) and A/Udorn/72, isolated only 4 years apart, and the least homology (91.1%) between A/PR/8/34 (H1N1) and A/Udorn/72, isolated 38 years apart. Furthermore, 84% of the nucleotide substitutions between A/PR/8/34 and A/NT/60/68 are preserved in the NP gene of the A/Udorn/72 strain. In contrast, a distinct linear pathway is not present in the avian influenza NP genes since the homology (90.3%) between the two avian influenza viruses A/Parrot/Ulster/73 (H7N1) and A/Mallard/78 isolated only 5 years apart is not significantly greater than the homology (90.1%) between strains A/FPV/Rostock/34 and A/Mallard/78 isolated 44 years apart and only 49% of the nucleotide substitutions between A/FPV/34 and A/Parrot/73 are found in A/Mallard/78. A determination of the rate of evolution of the human influenza A virus NP genes suggested that there were a greater number of nucleotide substitutions per year during the first several years immediately following the emergence of a new subtype in 1968.     

Characterization of a genetic variant of human hepatitis A virus.

Human isolates of hepatitis A (HAV) are a single serotype; however, recent genetic surveys using limited nucleotide sequencing have provided evidence that more than one genotype is responsible for HAV infection in different parts of the world (Jansen et al. [1990]: Proc Natl Acad Sci USA 87:2867-2871; Robertson et al. [1991] J Infect Dis 163:286-292). One of these genotypes was originally isolated from Panamanian owl monkeys (strain PA21), but has subsequently been found associated with human cases of HAV from Sweden in 1979 (H-122) and the United States of America in 1976 (GA76). The nucleic acid sequence of the exposed capsid polypeptide region of GA76 differs from other human HAV sequences by approximately 20%, yet differs by only 2.4% when compared with P1 sequence of the PA21 strain. The 20% nucleic acid variability between GA76 and other human HAV results in limited amino acid changes (3%), while a comparison with PA21 revealed only four homologous amino acid substitutions within VP2, VP3, and VP1 polypeptides. HAV infected stool specimens from Nepal and northern India during 1989 and 1990 were found to contain virus whose genetic makeup was related to the PA21 and GA76 isolates. This genotype of HAV appears to be circulating in some parts of the world where HAV is hyperendemic, and is a potential cause of hepatitis A infection within a susceptible population.     

Glycoprotein evolution of vesicular stomatitis virus New Jersey

A T1 ribonuclease fingerprinting study of a large number of virus isolates had previously demonstrated that considerable genetic variability existed among natural isolates of the vesicular stomatitis virus (VSV) New Jersey (NJ) serotype [S.T. Nichol (1988) J. Virol. 62, 572-579]. Based on these results, 34 virus isolates were chosen as representing the extent of genetic diversity within the VSV NJ serotype. We report the entire glycoprotein (G) gene nucleotide sequence and the deduced amino acid sequence for each of these viruses. Up to 19.8% G gene sequence differences could be seen among NJ serotype isolates. Analysis of the distribution of nucleotide substitutions relative to nucleotide codon position revealed that third position changes were distributed randomly throughout the gene. Third base changes constituted 84% of the observed nucleotide substitutions and affected 89% of the third base positions located in the G gene. Only three short oligonucleotide stretches of complete sequence conservation were observed. The remaining nucleotide changes located in the first and second positions were not distributed randomly, indicating that most of the amino acids coded by the G gene cannot be altered without reducing the fitness of the VSV NJ serotype viruses. Despite these constraints, up to 8.5% amino acid differences were observed between virus isolates. These differences were located throughout the G protein including regions adjacent to defined major antibody neutralization epitopes. Apparent clusters of amino acid substitutions were present in the hydrophobic signal sequence, transmembrane domain, and within the cytoplasmic domain of the G protein. A maximum parsimony analysis of the G gene nucleotide sequences allowed construction of a phylogram indicating the evolutionary relationship of these viruses. The VSV NJ serotype appears to contain at least three distinct lineages or subtypes. All recent virus isolates from the United States and Mexico are within subtype I and appear to have evolved from an ancestor more closely related to the Hazelhurst historic strain than other older strains. The implications of these findings for the evolution, epizootiology, and classification of these viruses are discussed.     

Sequence and phylogenetic analysis of the L and VP1 genes of foot-and-mouth disease virus serotype Asia1

Most of the molecular epidemiological studies of foot-and-mouth disease virus (FMDV) are based on comparison of VP1 gene sequence. In this report, we determine the nucleotide (nt) sequence of the L (603 nt) and VP1 (633 nt) genes of 27 FMDV serotype Asia 1 isolates recovered from different outbreaks in India, and compared with each other and the vaccine strain, IND 63/72, used in the country. Independent phylogenetic analyses on both the aligned gene sequences identified two major lineages (designated A & B) in the Asia 1 isolates. Both L- and VP1-based trees were congruent with respect to the major branching pattern of the isolates. The lineage A is represented by the isolates of 1986-2000 including the vaccine strain IND 63/72, whereas, lineage B appeared to be dominant and responsible for most of the recent outbreaks. A correlation was observed between the clustering of the isolates in the phylogenetic tree and the amino acid changes at many of the positions in VP1 as well as in L protein. The annual rate of evolution in L and VP1 genes was found similar and estimated to be 4.0 x 10(-3) and 3.8 x 10(-3) substitutions per nucleotide, respectively. Our result, largely from the congruence in phylogenetic trees and the rate of evolution in both the genes, suggests the possibility for the use of L gene sequence in phylogenetic comparison of FMDV.     

Complete Nucleotide Sequence of a Mumps Virus Genotype I Strain Isolated in Korea

The complete nucleotide sequence of mumps virus isolated in Korea, Dg1062/Korea/98 (Dg1062), was determined. As other mumps viruses, its genome was to be 15,384 nucleotides (nts) in length and encoded seven proteins. The both 5' and 3' ends were confirmed to be 55 and 24 nts by RACE method, respectively. The full-length nucleotide sequence of Dg1062 isolate differed from other strains by 2.9-6.8% in the nucleotide sequence level, resulting in 206 nucleotide and 54 amino acid substitutions which were observed in only Dg1062 isolate relative to the consensus sequences of other strains. Despite the variations of amino acids over the full genome including HN gene, it might be considered that this isolate have no significant variations in the antigenic sites. This result is the first report of full-length genome of genotype I strain and provides an overview on the diversity of genetic characteristics of circulating mumps virus.     

Contributions of genetic drift and negative selection on the evolution of three strains of wheat streak mosaic tritimovirus

Summary.  Genome sequences of three Wheat streak mosaic virus (WSMV) strains were compared. The Type and Sidney 81 strains of WSMV from the American Great Plains were closely related, with sequence identities of 97.6% (nucleotide) and 98.7% (amino acid). In contrast, the El Batán 3 strain from central Mexico was divergent, and shared only 79.2–79.3% (nucleotide) and 90.3–90.5% (amino acid) sequence identity with Type and Sidney 81. All three WSMV strains were serologically related, however the El Batán 3 capsid protein (CP) had 15 fewer amino acid residues. Phylogenetic analysis of the CP cistron indicated that Type, Sidney 81, and nine other American isolates of WSMV were closely related and distinct from the El Batán 3 sequence. Nucleotide substitutions among the WSMV strains were not randomly distributed across the genome with more variation within P1, HC-Pro, and CP, and less within P3. One 400-nucleotide region of the genome, corresponding to the 3′-end of P3, was strikingly deficient in silent substitutions. Nonetheless, the ratio of synonymous to non-synonymous substitutions throughout the genome was essentially the same for all three WSMV strains. Collectively, our data indicate that both genetic drift and negative selection have contributed to the evolution of WSMV strains. Received April 10, 2000 Accepted August 2, 2000     

Genetic characterization of Aleutian mink disease viruses isolated in China

Aleutian mink disease virus (AMDV) is a parvovirus that causes an immune complex mediated disease in minks. To understand the genetic characterization of AMDV in China, the genomic sequences of three isolates, ADV-LN1, ADV-LN2, and ADV-LN3, from different farms in the Northern China were analyzed. The results showed that the lengths of genomic sequences of three isolates were 4,543, 4,566, and 4,566 bp, respectively. They shared only 95.5-96.3 % nucleotide identity with each other. The nucleotide and amino acid homology of genome sequence between the Chinese isolates and European or American strains (ADV-G, ADV-Utah1, and ADV-SL3) were 92.4-95.0 % and 92.1-93.8 %, respectively. The amino acid substitutions randomly distributed in the genome, especially NS gene. ADV-LN1 strain had a 9-amino-acid deletion at amino acid positions 70 and 72-79 in the VP1 gene, comparing with ADV-G strain; ADV-LN2 and ADV-LN3 strains had 1-amino-acid deletion at amino acid positions 70 in the VP1. Some potential glycosylation site mutations in VP and NS genes were also observed. Phylogenetic analysis results showed that the three strains belonged to two different branches based on the complete coding sequence of VP2 gene. However, they all were in the same group together with the strains from United States based on the NS1 sequence. It indicated that Chinese AMDV isolates had genetic diversity. The origin of the ancestors of the Chinese AMDV strains might be associated with the American strains.     

Genetic analysis of two porcine reproductive and respiratory syndrome viruses with different virulence isolated in China

The S1 and SY0608 strains of porcine reproductive and respiratory syndrome virus (PRRSV) were individually isolated and had different pathogenicity in pigs in 1997 and 2006. In order to understand their genomic characteristics, the full-length genome of S1 and SY0608 isolates were sequenced and analyzed. The results indicated that their genome composition differed significantly and shared only 88.5% nucleotide identity with each other. The genetic variation and amino acid substitutions were not randomly distributed in the genome, and mainly focused on ORF1a, ORF3 and ORF5. The SY0608 strain, with high pathogenicity, had a 30-amino-acid deletion at amino acid positions 480 and 532-560 in comparison with the S1 strain. The alignment of amino acid sequence of Nsp1-Nsp8, GP2-GP5, M and N of S1 and SY0608 with other PRRSV isolates demonstrated that variation was mainly found in the Nsp2, GP3 and GP5 proteins. In comparison with the S1 strain, the SY0608 strain showed some potential glycosylation site mutations in GP5 at amino acid positions between 26 and 39, which might be associated with viral antigenicity. Phylogenetic analysis showed that the two strains belonged to two different branches that do not indicate differences in pathogenicity. Interestingly, the deletion strains isolated recently in China formed a new minor branch, revealing the same evolutionary trend.