Canine parvovirus type 2c in Vietnam continues to form distinct descendants with new mutations restricted to the Vietnamese variants

The complete sequence for viral protein 2 (VP2) of canine parvovirus (CPV) was obtained from 33 isolates collected from dogs with clinical symptoms and a vaccine in Vietnam from Mar 2017– June 2019. Molecular analysis revealed the descent evolution in CPV-2c-“new”, forming a “new var.” sub-lineage of the substantial 5G, 447M mutations restricted to the Vietnamese isolates in the epitope stretches. These were found besides the common 80R, 87L, 93N, 101T, 103A, 232I, 267Y, 297A, 300G, 305Y, 323N, 324I, 334A, 341P, 370R, 426E, 440T, 447I, 555V, 564S, and 568G typical to the “new” CPV-2c. The novel 447M mutation seemed to emerge in recent years in Vietnam and was present in 38 of 58 CPV2c strains isolated from December 2016 to June 2019 by our retrospective analysis, counting for 65.5%. With strong nodal support (98%), the topology of the phylogenetic tree revealed the substantially distinct Vietnamese sub-lineage of 2c-“new-var.” (5G/426E/447M) separated from the Vietnamese 2c-“new” (5G/426E/447I), within the 2c-(Asia)/Asia-2c. The Asia-2c strains of Vietnam were grouped with the recent Thai, Chinese, Mongolian, Taiwanese, Korean, and the Asian variants newly imported into Italy, Egypt, and Nigeria from Asia. The changes of amino acids in the epitope stretch in VP2 have effective alteration not only on the continual formation of substantial sub- or new genotypic variants but may be of subjects for antigenicity/immunogenicity and virulence to the CPV-2 strains and vaccine failure worldwide. of the individual fecal sample or the content of a vaccine vial was homogenized in sterile water and centrifuged at 13,000 rpm in 15 min. Total genomic DNA was extracted from the supernatant of the processed clinical samples or vaccine suspension using the GeneJET™ Genomic DNA Purication Kit (Thermo Scientic Inc., MA, USA) as instructed, eluted in 50 μL, and stored at −20°C until use. Primers for amplication of the entire VP2 gene were designed based on the conserved sequences of the available CPV genomes, including the Forward, CPF1: 5' AGCTAAAAAGGCAATTGCTCC 3' (2346–2366, coding frame numbering) and the Reverse, CPR4: 5’ TATAGACAGTATACGAGGCC 3’ (4555–4754); and two internal primers, CPF2: 5’ ACCAACAGATCCAATTGGAG 3’ and CPR3: 5’ GCATTTACATGAAGTCTTGG 3’ for sequencing. The PCR products (2,229 bp) were sequenced directly, or after cloning using pCR2.1-TOPO TA-cloning vector (Invitrogen, USA), by a service company. The complete VP2 sequence of 1,755 nucleotides obtained from each sample in this study (Table 1) and reference strains from previous studies and GenBank used for molecular analysis.

Viral protein 2 (VP2) is encoded by a large ORF at 3' end of the genomic sequence, accounting for 90% of the viral capsid proteins, and is highly responsible for virus-host cell interactions, tissue tropism, and immunogenicity [2,[27][28][29]. The VP2 protein of CPV-2 viruses exhibits a large extent of genetic and antigenic diversity, involving particular mutations at several key positions of inducing amino acid changes which are investigated for use for CPV host range, antigenic determination, phylogenetic analysis, and genotyping/ genogrouping of CPV-2 strains [2,29]. Amino acids at positions 93, 323, 324 in VP2 are responsible for transferrin receptor (TfR), at 87, 101, 297, 299, 300, 305 for host range, and changes of these residues have shifted the adaptivity from feline to canine hosts [27,28].
The VP2 structurally has two major epitope regions, namely B-cell epitope A (aa 1-23) containing peptide 2L21 (aa   Timeline analyses of new antigenic variants indicated that the restrictive mutations in the epitopes and loops in VP2 could lead to the very soon emergence of the new descendants comprising of amino acids distinct from the previous genogroups. CPV-2, characterized by K80R, K93N, V103A, D323N, N564S, and A568D in VP2 emerged from FPV in 1974-1978, and only 2-3 years later, in 1979, it was replaced by CPV-2a (M87L, I101T, A300G, D305Y, V505I); and in 1984 CPV-2a by CPV-2b (N426D and I555V); and then in the late 1990s to early 2000s CPV-2b was replaced by CPV-2c with the unique change of D426E to date [2]. Antigenic mutations in the epitope regions and b-barrels/loops of VP2, interspeci c recombination, incompatible antigenicity, and multi-lineage CPV-2c diversi cation in CPV strains demonstrate the need for a reappraisal of a new nomenclature for canine parvoviruses.
Within the new antigenic variant CPV-2c, however, strains have dynamically continued to evolve to form different lineages of distinct substitutions, particularly in Vietnam and Asia [12,13], and tentatively are divided into major lineages of Western (global strains, VT-I, -II, -III) and of Asian origins (Asia-I, -II, -III, -IV), respectively [18,20,22,26]. The substantially mutated Asian CPV-2c strains have been evidenced to appear in the European and African countries since 2018-2019 by any means, one of which, ie. via dog importation [18,22,23]. Severe parvoviral outbreaks in vaccinated populations with old/classical live attenuated CPV vaccines were worldwide documented [1,8,9].
In Vietnam, the FPV and CPV causative agents were rst reported in the late 1990s and early 2000s in cats and dogs in Ha Noi, Da Nang, and Ho Chi Minh City by few studies on clinical epidemiology, serosurvey, and some detections of the viral pathogens were made [12,36,37] (Fig. 1). Novel CPV-2 variants were revealed to appear in Vietnam in 1997, and at that time the new variant CPV-2c was detected by genetic retrospective analyses [12,13,[36][37][38]. In fact, after the early, intensive studies of feline and canine parvoviruses in Vietnam around the 2000s, there were no continual investigations until October 2013 when some CPV-2 sequences deposited in Genbank.
Imported attenuated CPV-2 vaccines have been used with boosted dose mainly for the imported breeds and mixed hybrid puppies between 6 weeks to 6 months of age in Vietnam. The indigenous Vietnamese dogs were rarely vaccinated and hence, they might be one of the most sustainable reservoirs for CPV-2 subsequent transmission and maintaining viruses for mutations. Despite the application of vaccination, severe canine parvoviral infections of novel variants have been reported throughout the country and also frequently been reported elsewhere in many other countries worldwide [1,8,9]. The reasons for vaccination failure were evidenced by the interference of maternally derived antibodies (MDA) and the emergence of new lineages with mutations in VP2 in the new antigenic variant CPV-2c. Some descendent mutations occurring in the CPV-2c genotype in recent years in Asian countries and in Vietnam tend to divide the currently circulating CPV-2c variants into "old" and "new" CPV-2c groups, of which many newly mutated strains originally emerged within the Vietnamese CPV2c population [12,13,20,26].
There remains a need for current and retrospective molecular analyses of the detected strains from 1997 to 2019 in Vietnam, including our study conducted from February 2017 to June 2019 for accurate understandings of diversifying CPV-2 and 2c genotypes, particularly from the clinically infected dogs. The timeline molecular analyses in this study provided evidence for descendent evolution in CPV-2c lineages in Vietnam that continually evolve to form distinct CPV-2c sub-genotypes or independent lineages of new variants (termed CPV-2c "new var.") with novel mutations originally restricted to the Vietnamese CPV-2c isolates.   Fig. 1; Table 1). Fresh fecal samples from diarrhea dogs were taken by a tampon tool and put into a tube and were stored at -20 o C until used for genomic DNA extraction. One commercial combined, multivalent vaccine, widely used in Vietnam, namely Vanguard®Plus5-CV (Zoetis, US), comprising an attenuated strain of prototype CPV-2 was also collected for molecular genotyping. The infected dogs were puppies of 6 weeks to 6 months old and the majority were of 3−4 months; including the breeds, ie. Fox, Rottweiler, Pomeranian, Corgi, Border Colli, Pit Bull, Dobermann, Malinois, service Berger (hybrid) or German shepherd, and several indigenous Vietnamese dogs ( Table 1). The detailed vaccination status was not exactly recorded, however, almost all the imported or mixed breeds (companion pets and service dogs) were vaccinated with the imported vaccines, mainly with the Vanguard®Plus5-CV multivalent vaccine.
A tampon of the individual fecal sample or the content of a vaccine vial was homogenized in sterile water and centrifuged at 13,000 rpm in 15 min. Total genomic DNA was extracted from the supernatant of the processed clinical samples or vaccine suspension using the GeneJET™ Genomic DNA Puri cation Kit (Thermo Scienti c Inc., MA, USA) as instructed, eluted in 50 μL, and stored at −20°C until use. Primers for ampli cation of the entire VP2 gene were designed based on the conserved sequences of the available CPV genomes, including the Forward, CPF1: 5' AGCTAAAAAGGCAATTGCTCC 3' (2346-2366, coding frame numbering) and the Reverse, CPR4: 5' TATAGACAGTATACGAGGCC 3' (4555-4754); and two internal primers, CPF2: 5' ACCAACAGATCCAATTGGAG 3' and CPR3: 5' GCATTTACATGAAGTCTTGG 3' for sequencing. The PCR products (2,229 bp) were sequenced directly, or after cloning using pCR2.1-TOPO TA-cloning vector (Invitrogen, USA), by a service company. The complete VP2 sequence of 1,755 nucleotides obtained from each sample in this study (Table 1) and reference strains from previous studies and GenBank were used for molecular analysis.  Table 1 and 2) were aligned for sequence and phylogenetic tree construction using GENEDOC 2.7 (http://iubio.bio.indiana.edu/soft/molbio/ibmpc/genedoc-readme.html) and MEGA X (www.megasoftware.net), with maximum-likelihood method tested by bootstrapping with 1000 replications [40]. The alignment of deduced amino acid sequences was further used for comparative genotypic and antigenic characterization. From the alignment, 10 Vietnamese strains of this and previous studies, and 3 reference strains representing CPV vaccines, CPV-2, 2a, 2b, and 2c variants were used for drawing a schematic presentation of CPV epitope stretches spanning b-strands and loops in the VP2 protein (Fig. 2).
Identical amino acid sequences were obtained from 4 groups of 16, 12, 3, and 2 isolates in each. The amino acid analysis of 34 VP2 sequences in this study (Mar 2017-June 2019) indicated that 33 belonged to the "new" CPV-2c and one was the Vanguard®Plus5-CV multivalent vaccine of CPV-2a (from Zoetis, US). No CPV-2a and CPV-2b variants were found. All such residues as 80R, 87L, 93N, 101T, 103A, 232I, 267Y, 297A, 300G, 305Y, 323N, 324I, 334A, 341P, 370Q/R, 426N/D/E, 440T, 555V, 564S, 568G, common to the global and typical to the Asian CPV-2c strains (summarized in [2]) were found, substantially con rming the status of the Asian "new" CPV-2c genogroup. However, the distinctness was revealed in these Vietnamese "new" CPV-2c strains. In these 33 "new" CPV-2c strains there was the presence of the descendants of A5G, P13S, and I447M mutations which are characteristically restricted to the Vietnamese CPV-2c-"new" strains sharply distinct from the global CPV-2c up-to-date known. We suggest naming these Vietnamese "new" CPV-2c strains as "new-var." CPV-2c viruses with the aim to discriminate them from the "old" or "common" and "new" global and Asian CPV-2c isolates (Table 1 and 2). The Vanguard®Plus5-CV multivalent vaccine used in Vietnam sequenced in August 2018 revealed to contain the CPV-2a typical residues completely different from those of the common and the "new"/"new var." CPV-2c in Vietnam ( Table 2). The mismatching antigenic residues essential for (sub)genotypic immune response re ected well-explained incompatibility in the complete protection of the Vietnamese pets/dogs against the current circulating virulent CPV-2c viruses, and vaccine failure or partial or incomplete protection has been observed in other countries as well [8-10, 39, 41].
In Vietnam, a strain of "mixed" CPV-2b and 2c antigenic variants was historically discovered in 2002 (ie., HNI-4-1, GenBank AB120727) which had residues of 5A/267F/324Y/370Q/426E/447I, and lacked such 5G/267Y/324I/370R typically to de ne the Asian and the Vietnamese CPV-2c [13,42]. This strain was not included in the phylogenetic tree with the Asia-2c majority clade (Fig. 3). However, this strain might be speculated as one of the transforming variants, being a representative for those experiencing genetic transition between the global CPV-2b and the Asian CPV-2c strains. The topology of the phylogenetic tree showed that other CPV-2c strains of Vietnam belonged to the "old" or "classical" CPV-2c which indicated the early introduction into Vietnam from the originally formed global lineage. They might play the role of an ancestral cluster for initiating the generation of the substantial "Asia-2c" and the Vietnamese "new" and "new-var." descendants during the timeline evolution (Fig. 3).
Currently, there are many reports regarding the circulation of the CPV-2c strains in many countries in Europe, Asia, North America, and South America, and tentatively divided into two global lineages of, ie. either the world/global (VT) and the Asian CPV-2c mutant strains (Asia-2c) [19,21,25,26]. The latter variants were evidenced to be originated in Asian countries and spread back to the European and African countries, ie., Italy, Egypt, Nigeria, respectively [19,22,23,43]. The Asian CPV-2c strains were reported circulating in India, China, Mongolia, Laos, Taiwan, Thailand, Korea, and Vietnam since 2010 [20,24,25,42,44,45]. These Asian CPV-2c strains geographically emerged in Asia, including those of the Vietnamese strains which were statistically grouped into a clade termed as Asia-IV originated from 2005, sharply distinguishable from the global VT-III CPV-2c strains [26]. Our latest data supports the two existing lineages of CPV-2 viruses in the world and the formation of the distinct Vietnamese sub-lineage of CPV-2c that continues to evolve to form novel, descendent mutations within the CPV-2c population in Vietnam. The changes of amino acids in the epitope stretch in VP2 have effective alteration not only on the continual formation of substantial sub-or new genogroups but may be of subjects for antigenicity/immunogenicity and virulence to the CPV-2 strains worldwide. The universal naming system proposed and used in [26] such as VT-I; VT-II and VT-III for global, and Asia-I, Asia-II, Asia-III, and Asia-IV have not yet been fully transparent by sequence analyses. However, new and separate two major lineages appeared on tree topology by the maximum-likelihood method with 1000 bootstrap resamplings and typical characteristics of the Asian and Vietnamese CPV-2c-"new" and "newvar." have proven to be reasonably reappraisal [20,26,42]. The Vietnamese clinical CPV-2c "new" and "new var." isolates of the period 2016-2019 of this and previous studies formed a well-de ned lineage, distinct from the typical global and previous Asian classes, indicating the continual evolution of multiple sub-lineages within the Asia-2c. The CPV-2c populations, particularly the "new" and "new var." strains that have been circulating in Vietnam since whatsoever time of their formation for two decades (1997-2019) appear to belong to a unique lineage, indicating the possibility that this evolved from a common ancestral source.
In conclusion, VP2 sequence and phylogenetic analyses, mutations of antigenic stretch, and topology of the phylogenetic tree revealed the two majorities of virulent "new" and "new-var." CPV-2c strains in Previous studies (54 strains)  M   54  HCM16  2a  MT106238  2017  A  P  V  L  T  Y  A  G  Y  I  Q  N  I   55  HN04  2c-new var  MT106229  2017  G  P  V  L  T  Y  A  G  Y  I  R  E  M   56  HN06  2c-new var  MT106235  2017  G  P  V  L  T  Y  A  G  Y  I  R  E  M   57  HN07  2c-new  MT106228  2017  G  P  V  L  T  Y  A  G  Y  I  R  E  I   58  HN08  2c-new var  MT106230  2017  G  P  V  L  T  Y  A  G  Y  I  R  E  M   59  HN30  2c-new var  MT106231  2017  G  P  V  L  T  Y  A  G  Y  I  R  E