Molecular analysis of partial VP-2 gene amplified from rectal swab samples of diarrheic dogs in Pakistan confirms the circulation of canine parvovirus genetic variant CPV-2a and detects sequences of feline panleukopenia virus (FPV)

Background The infection in dogs due to canine parvovirus (CPV), is a highly contagious one with high mortality rate. The present study was undertaken for a detailed genetic analysis of partial VP2 gene i.e., 630 bp isolated from rectal swab samples of infected domestic and stray dogs from all areas of district Faisalabad. Monitoring of viruses is important, as continuous prevalence of viral infection might be associated with emergence of new virulent strains. Methods In the present study, 40 rectal swab samples were collected from diarrheic dogs from different areas of district Faisalabad, Pakistan, in 2014–15 and screened for the presence of CPV by immunochromatography. Most of these dogs were stray dogs showing symptoms of diarrhea. Viral DNA was isolated and partial VP2 gene was amplified using gene specific primer pair Hfor/Hrev through PCR. Amplified fragments were cloned in pTZ57R/T (Fermentas) and completely sequenced. Sequences were analyzed and assembled by the Lasergene DNA analysis package (v8; DNAStar Inc., Madison, WI, USA). Results The results with immunochromatography showed that 33/40 (82%) of dogs were positive for CPV. We were able to amplify a fragment of 630 bp from 25 samples. In 25 samples the sequences of CPV-2a were detected showing the amino acid substitution Ser297Ala and presence of amino acid (426-Asn) in partial VP2 protein. Interestingly the BLAST analysis showed the of feline panleukopenia virus (FPV) sequences in 3 samples which were already positive for new CPV-2a, with 99% sequence homology to other FPV sequences present in GenBank. Conclusions Phylogenetic analysis showed clustering of partial CPV-VP-2 gene with viruses from China, India, Japan and Uruguay identifying a new variant, whereas the 3 FPV sequences showed immediate ancestral relationship with viruses from Portugal, South Africa and USA. Interesting observation was that CPV are clustering away from the commercial vaccine strains. In this work we provide a better understanding of CPV prevailing in Pakistan at molecular level. The detection of FPV could be a case of real co-infection or a case of dual presence, due to ingestion of contaminated food.


Background
Canine parvovirus-2 (CPV-2) is a single stranded (ss) DNA virus that is the most dreadful enteropathogen causing myocarditis and acute hemorrhagic gastroenteritis in dogs and young pups while Feline panleukopenia is a disease of cats caused by a highly contagious virus i.e., feline panleukopenia virus (FPV) [1].
CPV belongs to family Parvoviridae within subfamily Parvovirinae under the genus Protoparvovirus, as a viral variant of Carnivore protoparvovirus 1 species along with other variant like FPV, mink enteritis virus (MEV) and raccoon parvovirus (RaPV) [2]. After CPV-2 emergence occurred, its further antigenic variants arose quickly in the field, CPV-2a (showing Met87Leu, Asn375Asp, Ile101Thr and Val555Ile amino acid variations) and CPV-2b (VP2 protein showing residue 426 changed from Asn426Asp and then from Asp426Glu in the CPV-2b and CPV-2c antigenic variant strains, respectively. However, as the strains CPV-2b and CPV-2c differ from CPV-2a by only one amino acid position (VP2 residue 426), they are not considered as distinct subtypes but only dominant variants of CPV-2a [3,4]. Antigenic variants, CPV-2a and CPV-2b divergence occurred from the CPV-2 strain due to changes in 5-6 amino acids in the antigenically important VP2 capsid region and in regaining the ability to infect feline cells in vitro and cats in-vivo again, while providing better adaptation to the canine host. CPV and FPV are two closely related viruses, causing disease in respective hosts, but new variants of CPVs have acquired the feline host range allowing them to infect both cats and dogs [5,6], whereas the original CPV-2 does not replicate in cats. There are many studies showing that parvoviruses have a considerable degree of variability and that several viral variants can be detected simultaneously in an infected animal i.e., co-infection [1,[7][8][9][10]. Amplification of partial VP2 gene and subsequent sequencing of the PCR products, where many important informative amino acids reside, would give definitive antigenic and genetic difference between the original CPV-2, its variants, and the vaccine strains [11]. As mutation and recombination in VP2 gene influence the host range and affinity of receptor binding [12], it becomes important for researchers to sequence VP2 encoding gene for better and precise identification of emerging strains, their relationship with vaccine strains, and also to understand the evolutionary pattern of CPV strains in the field. For the purpose of identification of CPV, there are quite a few published papers where researchers have used partial VP2 gene sequence to report CPV [13,14].
There is a recent study involving partial VP2 gene and amino acid analysis, where phylogenetic analysis had shown the circulation of heterogeneous CPV-2a strains in dog population in Pakistan. These strains shared high level of identity with Chinese strains [14]. The present study was undertaken for more detailed genetic analysis of partial VP2 gene sequences obtained from dogs and for possible co-infection of different parvoviruses, covering all areas of district Faisalabad. In this study we have reported phylogenetic and sequence analysis of two viruses i.e., CPV and FPV detected in dogs. The presence of a new CPV-2a variant was identified. The main objective of the study was to detect and genetically characterize prevalent parvoviruses circulating in Faisalabad, Pakistan.

Clinical samples
A total of 40 fecal samples/rectal swabs were collected from suspected dogs i.e., domestic as well as stray dogs from Veterinary Teaching Hospital, Department of Clinic Medicine and Surgery, University of Agriculture Faisalabad Pakistan and Civil Veterinary Hospital Faisalabad regardless of their vaccination status, as it was not available. In Pakistan more than 90% dogs are stray dogs with no vaccination what so ever. The samples were collected from dogs from different areas of Faisalabad district over the period of two years, (2014 and 2015). Five fecal samples/ rectal swabs were collected from healthy dogs as negative control. The collected samples were emulsified in 0.1 M PBS and centrifuged at 3000 g for 10 min at 4C. The supernatant was used for further analysis.

Qualitative detection of CPV antigens in feces of dogs
All the collected samples were checked for CPV Ag with the help of FASTest PARVO Strip (https://jfa.no/produkt/ fastest-parvo-strip-2stk/), according to the instructions of manufacturer).
DNA extraction and PCR amplification of partial VP2 gene Viral DNA from fecal samples of infected and healthy dogs was extracted as described by [15]. To reduce the residual effect of DNA polymerase activity inhibitors the supernatants were diluted to a ratio of 1:10 using distilled water. A single step polymerase chain reaction (PCR) was performed to amplify viral DNA from clinical specimens. Gene specific primer pair Hfor/Hrev [16] were used to amplify 630 bp fragment of the capsid protein gene (VP2). The PCR-amplification of partial VP2 gene was carried out using 100 ng template DNA, in PCR mix as described earlier [16]. The PCR amplified products were run on 1.0% agarose gel containing ethidium bromide in Tris acetate EDTA (TAE) buffer and then visualized under UV transilluminator (Syngene, UK).

Sequence and phylogenetic analysis
PCR amplified fragments were cloned in pTZ57R/T vector (Fermentas) and completely sequenced by chain termination method of sequencing on automated sequencer Applied Biosystem 3100. Sequences were analyzed and assembled by the Lasergene DNA analysis package (v8; DNA-STAR Inc., Madison, WI, USA). Phylogenetic trees were generated, first by aligning the molecules using CLUSTAL-W, followed by Maximum likelihood method of phylogenetic tree construction with 1000 bootstrap replications in MEGA7 program [17]. MegAlign program in DNA STAR was used to produce pairwise comparisons for amino acid sequence similarities. The accession numbers for CPV and FPV isolated in this study are provided in Tables 1 and 2. The other viral sequences used for amino acid sequence comparisons and phylogenetic analyses were downloaded from GenBank. The branch lengths were measured in terms of the number of substitutions per site.

CPV antigen detection and PCR amplification
From 40 CPV suspected cases, fecal samples were collected and with the help of rapid immunochromatographic technique, 33 were found positive (82%) while 7 were negative (17%), 5 samples taken as negative control were found negative when analyzed using immunochromatographic technique. Of the total 33 positive samples, 25 samples, 75% (25/33), were confirmed positive for CPV as a fragment of 630 bp was amplified in all 25 samples. Amplified product from all samples were successfully cloned in pTZ57R/T (Fermentas) vector and completely sequenced.

Sequencing analysis of CPV from Pakistan
VP2 gene amplified from representative isolates (total of 25) selected from diverse geographical locations of   [19]. Twenty out of twenty-five isolates had an additional amino acid change 440 replacing Thr with Ala and further two out of those 20 isolates showed amino acid substitution Asp434Gly. Residue 440 gains the importance antigenically as it is present in GH loop of the VP2 protein which makes the surface of the virus capsid [20]. Residue 440 is present at the top of the three fold spike, which is considered to be the main antigenic site of the virus and also counts as one of the few VP sites that undergo positive selection [21]. Three isolate were found as Feline panleukopenia virus (Table 2), which mostly infects cats and cause gastroenteritis, was detected in three infected canine fecal samples. Previously it was known that two of CPV specific sequence amino acid at position 93-Asn and 323-Asn if introduced in FPV will allow it to multiply invitro canine cell line. These two amino acids along with 103-Val allow CPV to efficiently multiply in canine cells [19], but in the present study residue 323 remain FPV specific (Asp) and the remaining two amino acids were not determined in present sequence analysis. In addition, among the five amino acids in Table 2, the only difference between FPV and CPV from Pakistan as well as other regions is at the amino acid residue 297 (Ala in FPV and Ser in CPV isolate). On the other side, a study have shown that in-vivo inoculation of the FPV in dogs, unexpectedly enabled the virus to replicate in thymus and bone marrow cells but not in the intestine or mesenteric lymph nodes [22]. But, a recombinant virus of CPV and FPV was shown to multiply in high titers in canine intestine and mesenteric lymph nodes [23]. Two non-synonymous mutations, found in the present study, resulting in amino acid change Thr390Ala in (FPV sequence MF182903), Asp434Gly in (New CPV-2a sequence MF182909, MF182911, MF182917), which were previously not been reported in any other country.

Phylogenetic analysis
Maximum likelihood method was used to construct phylogenetic tree to determine the evolutionary relationship of our analyzed new CPV-2a strains with other strains of different geological areas along with vaccine strains. Three strains of FPV comprising 630 bp of partial VP2 gene from Pakistan along with other FPV strains of different geological areas were also included in the tree (Fig. 1). The parvoviruses sequenced in this study clustered in three different monophyletic clades in the phylogenetic tree at the antigenic region of VP2 gene though there is a very high sequence similarity among these sequences (Fig. 1

Conclusions
To date, there are no studies available on the clinical course and prognosis of cases presenting CPV-FPV dual infection. In this study CPV and FPV are detected in rectal swab samples of three dogs. We are not sure that whether presence of these two viruses together is just by chance or have some scientific relevance. If this is a true co-infection then we need to be very careful to study the epidemiology of these viruses. Analogous to what happens in the presence of multiple human infections [24], the clinical course of parvovirus infection could be altered and CPV could accelerate the progression of panleukopenia infection, since FPV represents a novel pathogen for dogs or vice versa. Alternatively, CPV-FPV co-infection could have a direct implication for virus persistence, host-viral interaction and pathogenesis [25]. As already discussed presence of these viruses together in rectal swab samples could be due to coprophagous behavior of dogs and FPV detection was due to faeces ingestion and they are of no clinical relevance. However, further sophisticated experiments are needed to support this observation.