Global distribution, dissemination and overexpression of potent multidrug efflux pump RE-CmeABC in Campylobacter jejuni.

OBJECTIVES: To investigate the global distribution, dissemination and overexpression of RE-CmeABC in Campylobacter jejuni. METHODS: WGS information for 433 RE-cmeABC-positive C. jejuni isolates (including 18 isolates sequenced in this study and 415 isolates from GenBank) was used for the generation of minimum-spanning trees with STs. WGS information for 95 representative RE-cmeABC-positive C. jejuni isolates was used for phylogenetic analysis. RT-PCR was conducted to evaluate the association between inverted repeat (IR) sequence diversity in the RE-CmeABC promoter region and RE-cmeABC gene expression. RESULTS: WGS analysis revealed the global distribution of RE-cmeABC among C. jejuni from more than 10 countries. MLST results indicated that various STs were involved in the dissemination of RE-cmeABC, with ST2109 being the most predominant ST. Phylogenetic analysis revealed the close relationship between RE-cmeABC-carrying C. jejuni isolates from poultry and humans. The IR polymorphism in the RE-CmeABC promoter region is associated with the overexpression of RE-cmeABC, which was demonstrated experimentally by RT-PCR. CONCLUSIONS: To the best of our knowledge, our analysis represents the first view of the global distribution of RE-CmeABC, which is horizontally transferable and diffused regionally in a clonal manner. The close relationship of RE-cmeABC-positive C. jejuni from poultry and humans supports the potential of these isolates for zoonotic transmission. Overexpressed RE-CmeABC in C. jejuni will increase the fitness of the corresponding bacteria and be of advantage under antimicrobial selection.

Detection and genetic characteristics of porcine bocavirus in central China.

To investigate the epidemic profile and genetic diversity of porcine bocavirus (PBoV), 281 clinical samples, including 236 intestinal tissue samples and 45 fecal samples were collected from diarrheic piglets on 37 different pig farms in central China, and two SYBR Green I-based quantitative PCR assays were developed to detect PBoV1/2 and PBoV3/4/5, respectively. One hundred forty-eight (52.67%) of the 281 clinical samples were positive for PBoV1/2, 117 (41.63%) were positive for PBoV3/4/5, 55 (19.57%) were positive for both PBoV1/2 and PBoV3/4/5, and 86.49% (32/37) of the pig farms were positive for PBoV. Overall, the prevalence of PBoV was 74.73% (210/281) in central China. Subsequently, nearly full-length genomic sequences of two PBoV strains (designated CH/HNZM and PBoV-TY) from two different farms were determined. Phylogenetic analysis demonstrated that the two PBoV strains obtained in this study belonged to the PBoV G2 group and had a close relationship to 10 other PBoV G2 strains but differed genetically from PBoV G1, PBoV G3, and seven other bocaviruses. CH/HNZM and PBoV-TY were closely related to the PBoV strain GD18 (KJ755666), which may be derived from the PBoV strains 0912/2012 (MH558677) and 57AT-HU (KF206160) through recombination. Compared with reference strain ZJD (HM053694)-China, more amino acid variation was found in the NS1 proteins of CH/HNZM and PBoV-TY. These data extend our understanding of the molecular epidemiology and evolution of PBoV.

Studies on the role of IS1216E in the formation and dissemination of poxtA-carrying plasmids in an Enterococcus faecium clade A1 isolate.

OBJECTIVES: To analyse the role of IS1216E in the dissemination of the phenicol-oxazolidinone-tetracycline resistance gene poxtA in an Enterococcus faecium clade A1 isolate. METHODS: MICs were determined by broth microdilution. The poxtA-positive isolate was typed by MLST. The two plasmids were characterized by PCR, conjugation, S1-PFGE, Southern blot hybridization and WGS analysis. The presence of translocatable units (TUs) was examined by PCR and sequencing. RESULTS: Isolate E1077 contains the 217661 bp conjugative plasmid pE1077-217 and the 23710 bp mobilizable plasmid pE1077-23. pE1077-217 harbours erm(B), aac(A)-aph(D), aadE, spw, lsa(E), lnu(B), aphA3 and dfrG, whereas pE1077-23 carries a Tn6657-like transposon containing poxtA and fexB. pE1077-23 was apparently formed by an IS1216E-mediated composite transposon-plasmid fusion event, involving a replicative transposition process. Conjugation experiments showed that pE1077-23 is mobilizable by pE1077-217. Moreover, a novel 31742 bp plasmid, pT-E1077-31, was found in a transconjugant. WGS analysis indicated that pT-E1077-31 was formed by the integration of a Tn6657-derived, IS1216E-based translocatable unit, which carried fexB and poxtA, into a copy of pE1077-23. CONCLUSIONS: This study showed the presence of two cointegrate formation events in the formation and spread of a poxtA/fexB-carrying plasmid in E. faecium. One was the integration of a transposon into a plasmid while the other was the integration of a TU into a different site of the same type of plasmid-borne transposon from which it originated. In both events, IS1216E played a major role, suggesting that IS1216E-mediated transposition and translocation processes aid the dissemination and persistence of important antimicrobial resistance genes, such as poxtA, among enterococci.

A novel multiresistance gene cluster located on a plasmid-borne transposon in Listeria monocytogenes.

OBJECTIVES: To identify the genetic context and the transferability of the multiresistance gene lsa(E) in Listeria monocytogenes. METHODS: MICs were determined by broth microdilution. Transferability of lsa(E) was investigated by conjugation, electrotransformation and natural transformation. The lsa(E)-carrying plasmid was sequenced using the Illumina MiSeq and PacBio RSII platforms. The presence of translocatable units (TUs) was examined by PCR. RESULTS: The 85 555 bp non-conjugative multiresistance plasmid pNH1 from L. monocytogenes harboured nine antimicrobial resistance genes including a multiresistance gene cluster, consisting of the genes aphA3, erm(B), aadE, spw, lsa(E) and lnu(B), and in addition the genes dfrG, tet(S) and catA8 were also located on plasmid pNH1 The multiresistance gene cluster, and each of the genes tet(S), catA8 and cadA were flanked by IS1216 elements. PCR identified four types of TUs, consisting of either the multiresistance gene cluster and one copy of IS1216, the catA8 gene and one copy of IS1216, or both, but also the tet(S) gene and one copy of IS1216, respectively. Natural transformation into Streptococcus mutans UA159 yielded transformants that harboured a novel 13 208 bp transposon, designated Tn6659. This transposon consisted of the multiresistance gene cluster bounded by IS1216 copies. All transformants displayed elevated MICs of the respective antimicrobial agents. At the integration site in the transformants, 8 bp direct target duplications (5'-ATTCAAAC-3') were found immediately up- and downstream of Tn6659. CONCLUSIONS: To the best of our knowledge, this is the first report of this novel multiresistance gene cluster and the gene catA8, flanked by IS1216 elements located on a plasmid of L. monocytogenes. Moreover, a novel functionally active multiresistance transposon was identified.

Characteristics of the spike and ORF3 genes of porcine epidemic diarrhea virus in Henan and Shanxi provinces of China.

To investigate the epidemic characteristics of porcine epidemic diarrhea virus (PEDV), 135 clinical samples (including intestinal tissues and feces) were collected from diseased piglets during outbreaks of diarrhea from 2015 to 2019 on farms in Henan and Shanxi provinces of China where swine had been immunized with attenuated PEDV (CV777). A total of 86 clinical samples (86/135, 63.7%) were positive for PEDV by RT-PCR, and subsequently, the complete spike (S) and ORF3 genes of 32 PEDV samples were sequenced. Phylogenetic analysis showed that the 32 PEDV strains obtained in this study belonged to group 2 (pandemic variant strains) and had a close relationship to 17 Chinese strains after 2010, two South Korean strains (KNU-1305 and KNU-1807), three American strains (PC22A-P140.BI, USA/Colorado/2013, and USA/OK10240-6/2017) and a Mexican strain (PEDV/MEX/QRO/02/2017), but differed genetically from a South Korean strain (SM98), a European strain (Br1/87), a Chinese strain (LZC), and a vaccine strain (CV777). G2-a subgroup strains were the dominant pandemic variant strains circulating in Henan and Shanxi provinces of China. Furthermore, a cross-recombination event was identified in the S region of the SX/TY2/2017 strain, and the putative parental strains were the epidemic strains CH/GDGZ/2012 and CH/YZ1/2015, identified in China in 2012 and 2015, respectively. These results provide further information about PEDV evolution, which could improve our understanding of the circulation of PEDV in Henan and Shanxi provinces. This information will also be helpful for developing new strategies for prevention and control of variant strains.

Analysis of a poxtA- and optrA-co-carrying conjugative multiresistance plasmid from Enterococcus faecalis.

OBJECTIVES: To investigate the presence and transferability of the poxtA gene and identify the genetic context of poxtA in two enterococcal plasmids from swine. METHODS: MICs were determined by broth microdilution. A total of 114 porcine enterococci with florfenicol MICs of ≥16 mg/L were screened for the presence of the poxtA gene by PCR. Transferability of poxtA was investigated by conjugation and transformation. The poxtA-carrying plasmids were completely sequenced using the Illumina Miseq and PacBio RSII platform. The presence of circular intermediates was examined by inverse PCR. RESULTS: The poxtA gene was present in 57.9% (66/114) of the florfenicol-resistant porcine enterococci. Two poxtA-carrying plasmids, pE035 and pE076, were identified. The conjugative 121524 bp plasmid pE035 carried poxtA and optrA along with the resistance genes erm(A), erm(B), aac(A)-aph(D), lnu(G), fexB, dfrG and bcrABDR. Three mobile elements, comprising a mobile dfrG locus, a mobile bcrABDR locus and an unconventional circularizable structure containing aac(A)-aph(D), were located on this plasmid and all proved to be active by inverse PCR. The non-conjugative 19832 bp plasmid pE076 only carried poxtA and fexB. After transfer, both the transconjugant and the transformant displayed elevated MICs of the respective antimicrobial agents. CONCLUSIONS: To the best of our knowledge, this is the first report of the co-location of the oxazolidinone resistance genes poxtA and optrA on a conjugative multiresistance plasmid from a porcine enterococcal strain. In addition, the presence of three mobile elements in such a plasmid will aid in the persistence and dissemination of poxtA and optrA among enterococci.

A prophage and two ICESa2603-family integrative and conjugative elements (ICEs) carrying optrA in Streptococcus suis.

OBJECTIVES: To investigate the presence and transfer of the oxazolidinone/phenicol resistance gene optrA and identify the genetic elements involved in the horizontal transfer of the optrA gene in Streptococcus suis. METHODS: A total of 237 S. suis isolates were screened for the presence of the optrA gene by PCR. Whole-genome DNA of three optrA-positive strains was completely sequenced using the Illumina MiSeq and Pacbio RSII platforms. MICs were determined by broth microdilution. Transferability of the optrA gene in S. suis was investigated by conjugation. The presence of circular intermediates was examined by inverse PCR. RESULTS: The optrA gene was present in 11.8% (28/237) of the S. suis strains. In three strains, the optrA gene was flanked by two copies of IS1216 elements in the same orientation, located either on a prophage or on ICESa2603-family integrative and conjugative elements (ICEs), including one tandem ICE. In one isolate, the optrA-carrying ICE transferred with a frequency of 2.1 × 10-8. After the transfer, the transconjugant displayed elevated MICs of the respective antimicrobial agents. Inverse PCRs revealed that circular intermediates of different sizes were formed in the three optrA-carrying strains, containing one copy of the IS1216E element and the optrA gene alone or in combination with other resistance genes. CONCLUSIONS: A prophage and two ICESa2603-family ICEs (including one tandem ICE) associated with the optrA gene were identified in S. suis. The association of the optrA gene with the IS1216E elements and its location on either a prophage or ICEs will aid its horizontal transfer.

Expression of bioactive porcine interferon-alpha in Lactobacillus casei.

In this study, we constructed an expression cassette containing the inducible lac promoter and the secretion signal from an S-layer protein of Lactobacillus brevis for the expression of porcine interferon-alpha (IFN-α) in Lactobacillus casei (Lb. casei). Reverse-transcriptase PCR verified the presence of porcine IFN-α mRNA in the recombinant Lb. casei. The porcine IFN-α protein expressed in the recombinant Lb. casei was identified by both Western blot analysis and ELISA. We used various pH values and induction times to optimize the yield of IFN-α, and found that induction with 0.8% lactose for 16 h under anaerobic conditions produced the highest concentrations of IFN-α. Furthermore, the activity of porcine IFN-α in the cultural supernatant was evaluated on ST cells infected with pseudorabies virus. The results revealed that porcine IFN-α inhibited virus replication in vitro. The findings of our study indicate that recombinant Lb. casei producing porcine IFN-α has great potential for use as a novel oral antiviral agent in animal healthcare.

Genomic sequence analysis of a new reassortant infectious bursal disease virus from commercial broiler flocks in Central China.

We report the complete nucleotide sequence of a reassortant infectious bursal disease (IBD) virus (IBDV) HN isolate from commercial broiler flocks in central China. The genome consisted of 3,232 and 2,652 nucleotides in the coding regions of segments A and B, respectively. Alignment of both nucleotide and deduced amino acid sequences and phylogenetic analysis revealed that the genome segments A and B of HN were derived from the attenuated strain B87 and the VV strain OKYM. This is a new reassortant IBDV strain that has emerged in nature, involving segment A of a cell-culture-adapted attenuated vaccine strain B87.

A systematic review of tea pigments: Prevention of major diseases, protection of organs, and potential mechanisms and applications.

With the growing awareness of a healthy life, tea pigments (TPGs) are in focus for their health benefits. TPGs not only provide specific color to tea liquor but also possess health benefits such as anti-obesity, anti-tumor, anti-inflammatory, anti-viral, anti-oxidative, and bacteriostatic properties. Also, TPGs can benefit bone, liver, kidney, cardiovascular, gut microbiome, and sleep health. Based on previous reports, this review provides a brief introduction to the health benefits of TPGs, focusing on the prevention of human diseases and the protection of organs. Also, the latest research on the functional mechanism(s), practical application, and development strategies of TPGs is discussed.

Synthesis of functionalized 2,3-dihydroisoxazoles by domino reactions in water and unexpected ring-opening reactions of 2,3-dihydroisoxazoles.

α,α-Dicyanoolefins react with hydroxylamine to afford 2,3-dihydroisoxazoles (2,3-dihydroisoxazoles can be easily isolated by filtration) in excellent yields under mild and environmentally benign conditions. A one-pot reaction in tandem with an unexpected ring-opening of 2,3-dihydroisoxazoles has been developed as well.

Angiolymphoid hyperplasia with eosinophilia and Kimura's disease coexisting in the same patient: evidence for a spectrum of disease.

The relationship between angiolymphoid hyperplasia with eosinophilia (ALHE) and Kimura's disease has always been contentious. Initially, ALHE and Kimura's disease were thought to be conditions within the same disease spectrum, but it is now widely accepted that they are two separate disease entities. The two lesions may coexist in one patient. Thus, ALHE and Kimura's disease may be different manifestations of the one disease.

Plasmid-Assisted Horizontal Transfer of a Large lsa(E)-Carrying Genomic Island in Enterococcus faecalis.

The horizontal transfer of genomic islands is essential for the adaptation and evolution of Enterococcus faecalis. In this study, three porcine E. faecalis strains, each harboring a large lsa(E)-carrying genomic island, were identified. When using the E. faecalis OG1RF as the recipient, the horizontal transfer of the lsa(E)-carrying genomic island occurred only from E. faecalis E512, which also harbored a pheromone-responsive conjugative plasmid, but not from the other two E. faecalis strains, E533 and E509, which lacked such a plasmid. Subsequently, through plasmid curing of E. faecalis E512 and plasmid introduction into E. faecalis E533, the pheromone-responsive conjugative plasmid was identified to be indispensable for the horizontal transfer of the lsa(E)-carrying genomic island and a subsequent homologous recombination between the chromosomal DNA of the donor and the recipient. In addition, the presence of a chromosomally-located conjugative transposon, Tn916, in E. faecalis E509 could not mediate the horizontal transfer of the lsa(E)-carrying genomic island, although Tn916 itself could transfer by conjugation. Thus, these data highlight the role of the pheromone-responsive conjugative plasmid in the transfer of the lsa(E)-carrying genomic island in E. faecalis, thereby establishing the dual role of pheromone-responsive conjugative plasmids in contributing to the dissemination of both plasmid-borne resistance genes and chromosomally-located genomic islands. IMPORTANCE In this study, it was shown that a pheromone-responsive conjugative plasmid played an indispensable role in the horizontal transfer of a lsa(E)-carrying genomic island. This finding indicates a dual role of the pheromone-responsive conjugative plasmid in disseminating both plasmid-borne resistance genes and chromosomally-located genomic islands. The role of the pheromone-responsive conjugative plasmid in disseminating chromosomal genomic islands is suggested to be essential in the genomic evolution of E. faecalis, which has become one of the leading nosocomial pathogens worldwide.

Water-regulated and bioinspired one-step pyrolysis of iron-cobalt nanoparticles-capped carbon nanotubes/porous honeycombed nitrogen-doped carbon composite for highly efficient oxygen reduction.

To achieve commercial applications of green fuel cells and rechargeable metal-air batteries, rational design and synthesis of low-cost, high-efficiency and ultra-stable transition metal-based electrocatalysts are of significant importance for alkaline oxygen reduction reaction (ORR). In this work, iron-cobalt (FeCo) nanoparticles-capped carbon nano-tubes/-porous nitrogen-doped honeycombed carbon composite (FeCo-CNTs/NHC-800) is synthesized by a water-regulated and bioinspired one-step pyrolysis method at 800 °C, where l-histidine behaves as the C and N sources combined by working as a chelating agent of Fe/Co. The formation mechanism is discussed by adjusting the pyrolysis temperature and water amount. The resultant FeCo-CNTs/NHC-800 exhibits a positive onset potential (Eonset = 1.091 V) and half-wave potential (E1/2 = 0.88 V), showing promising ORR activity, outperforming home-made controls and many lately reported catalysts. The hierarchically porous honeycombed structures have fascinating open porous spaces for fast diffusion of active species, large specific surface area, high conductivity, and stable sites for anchoring FeCo nanoparticles (NPs). Moreover, the N-doped carbon nanotubes coupling with homogeneous FeCo NPs greatly improve the catalytic activity and stability of ORR. This work provides some valuable insights to prepare hierarchical, reliable, and high-efficiency carbon-based ORR catalysts for new energy-correlated devices.

Excision and integration of unconventional circularizable structures involving the erm(B) gene in enterococci.

Traditionally, insertion sequences (ISs) play a major role in disseminating antimicrobial resistance genes (ARGs) in bacteria through transposition and translocation, forming regions that contain multiple ARGs flanked by single or multiple copies of IS. In addition, unconventional circularizable structures (UCSs), lacking recombinase genes but being surrounded by directly repeated sequences (DRs) of various sizes which do not contain transposase genes, were reported to be involved in the dissemination of ARGs. In this study, a novel UCS was identified on plasmid pE508-2 in E. faecalis E508, which carried a 24,411 bp multiresistance gene cluster, consisting of the resistance genes aphA3, lnu(B), lsa(E), spw, aac(A)-aph(D), lnu(B), dfrG, and two copies of aadE flanked by copies of erm(B). PCR assays revealed that three types of UCSs with lengths of 7235, 16,437, and 23,673 bp were formed, each of which contained the respective resistance genes and one copy of erm(B). Using erm(B)-negative and -positive strains, we demonstrated that erm(B)-carrying UCSs failed to transfer into an erm(B)-negative strain, but could integrate into an erm(B)-positive strain in a new site adjacent to a pre-existing erm(B) gene by natural transformation. Database searches revealed that erm(B)-flanked multiresistance gene regions, which might be able to form the respective UCSs, are present among various bacteria from different sources in various countries. In summary, this study experimentally demonstrated the excision and integration of UCS involving structures that include erm(B). The widespread presence of these UCSs in various Gram-positive bacteria highlights its role in the dissemination of ARGs among bacterial pathogens.

Theophylline-regulated pyrolysis synthesis of nitrogen-doped carbon nanotubes with iron-cobalt nanoparticles for greatly boosting oxygen reduction reaction.

At present, construction of economical, efficient and stable electrocatalysts for oxygen reduction reaction (ORR) is critical to alleviate the energy shortage. Theophylline (THP) can be easily extracted from natural plants, whose nitrogen atoms can chelate with metal ions. With assistance of THP, FeCo alloy was confined in N-doped carbon nanotubes (FeCo/NCNTs-800) by one-step pyrolysis of a mixture of the metal precursors, g-C3N4 and THP. The resulting FeCo/NCNTs-800 showed a better ORR performance (onset potential, Eonset = 1.09 V; half-wave potential, E1/2 = 0.87 V) than commercial Pt/C (50 wt%) in a 0.1 M KOH solution, with a limiting current density as high as -5.54 mA cm-2. This work offers a feasible strategy for developing transitional bimetal-based carbon catalysts in alkaline fuel cells.

Well entrapped platinum-iron nanoparticles on three-dimensional nitrogen-doped ordered mesoporous carbon as highly efficient and durable catalyst for oxygen reduction and zinc-air battery.

The high-performance and durable oxygen reduction reaction (ORR) catalyst on air cathode is a key component in assembly of Zn-air batteries. Herein, three-dimensional N-doped ordered mesoporous carbon (3D N-OMC) was first prepared with silica as a template via pyrolysis with assistance of dicyandiamide as a N-doping agent, combined by full adsorption of platinum (II) acetylacetonate (Pt(acac)2) and iron (II) phthalocyanine (FePc) via π-π interactions. After further pyrolysis of the resulting mixture, many PtFe nanoparticles were efficiently incorporated in 3D N-OMC (termed as PtFe@3D N-OMC for simplicity). Control experiments were certificated the important role of the pyrolysis temperature played in this synthesis. The resultant composite synergistically combines advantages of hierarchically accessible surfaces, highly open structure, and well-dispersed PtFe particles, which endow the PtFe@3D N-OMC with onset and half-wave potentials of 0.98 and 0.86 V in alkaline media, respectively, showing appealing catalytic activity for the ORR. Most significantly, the PtFe@3D N-OMC based Zn-air battery has a high power density of 80.57 mW cm-2 and long-term durability (220 h, 660 cycles). This work opens a new avenue for design of high-efficiency and durable ORR electrocatalysts in energy conversion and storage systems.

Cross-species transmission of an emerging porcine circovirus (PCV4): First molecular detection and retrospective investigation in dairy cows.

Porcine circovirus 4 (PCV4), a novel porcine circovirus identified in pigs, has recently been proved to be pathogenic to piglets. However, little is known about its cross-species transmission, and demonstration of PCV4 in dairy cows is lacking. To explore whether the PCV4 genome exists in dairy cows, 1170 fecal samples were collected from dairy farms in 7 cities in Henan Province of China during 2012-2021, and screened by qPCR for the presence of PCVs (PCV2-PCV4). The detection results showed that the positive rate of PCV4 in dairy cows was 2.22 % (26/1170), but all fecal samples were negative for PCV2 and PCV3. Three full-length and five partial genomes of PCV4 strains were acquired, of which two PCV4 strains (NY2012-DC and XC2013-DC) were achieved from 2012 and 2013, indicating that PCV4 has been circulating in dairy cows in Henan Province of China for at least 10 years. The three PCV4 strains sequenced in this study shared high identity (97.5-99.5 %) with reference strains at the genome level. In phylogenetic analysis, three genotypes (PCV4a, PCV4b and PCV4c) were temporarily confirmed by analyzing 44 strains, and one amino acid variation in Rep (V239L) and three amino acid variations in Cap (N27S, R28G and M212L) were considered as a conserved genotype specific molecular marker. Analyzed from three perspectives (cross-time, cross-species and transboundary), the high nucleotide homology of PCV4 strains indicated the PCV4 evolutionary rate might be slow. Overall, this study was the first to report the detection of PCV4 in dairy cows and conducted a long-term retrospective investigation of PCV4 in Henan Province of China, which has important implications for understanding the genetic diversity and cross-species transmission of the ongoing PCV4 cases.

Isolation and Phylogenetic Analysis of Reemerging Pseudorabies Virus Within Pig Populations in Central China During 2012 to 2019.

To understand the biological characteristics of the reemerging pseudorabies virus (PRV) strains, a total of 392 tissue samples were collected from diseased pigs during reemerging PR outbreaks between 2012 and 2019 on farms in central China where swine had been immunized with Bartha-K61 and 51 (13. 01%) were positive for the gE gene by PCR. Sixteen PRV strains were isolated and caused clinical symptoms and death in mice. Subsequently, gE, gC, gB, and gD complete genes were amplified from the 16 PRV isolates and sequenced. Phylogenetic analysis based on these four gene sequences shows that the 16 PRV isolates were more closely related to the Chinese PRV variants (after 2012) but genetically differed from early Chinese PRV isolates (before 2012). Sequence analysis reveals that PRV isolates exhibited amino acid insertions, substitutions, or deletions compared with early Chinese PRV isolates and European-American PRV strains. In addition, this is the first report that eight isolates (8/16) in this study harbor a unique amino acid substitution at position 280 (F to L) of the gC protein, and six isolates have an amino acid substitution at position 338 (A to V) of the gD protein compared with the Chinese PRV variants. The emulsion containing inactivated PRV NY isolate could provide complete protection against the NY isolate. This study might enrich our understanding of the evolution of reemerging PRV strains as well as pave the way for finding a model virus to develop a novel vaccine based on reemerging PRV strains.