​Pathological and Molecular Investigation of Porcine Kobuvirus (PKV) from Indian Pigs

The Kobuvirus is a single-stranded, positive-sense, RNA virus of the family Picornaviridae which composed of small, non-enveloped viruses (Reuter et al., 2009; ICTV, 2019). The genus Kobuvirus is consist of six species viz. Aichivirus A, Aichivirus B, Aichivirus C, Aichivirus D, Aichivirus E and Aichivirus F, infecting a variety of hosts like pigs, sheep, goats, cattle, ferrets, bats, dogs, cats and humans (Reuter et al., 2011; Khamrin et al., 2014 and Lu et al., 2018). The Aichivirus C is the only Kobuvirus species reported to infect pigs, previously known as “porcine kobuvirus (PKV)” (Khamrin et al., 2014). The PKV was first identified in Hungary from faecal samples of pig in 2008 followed by China (Yu et al., 2009; Reuter et al., 2010). There after, PKV was subsequently reported from Asia, Africa, Europe and United states (Khamrin et al., 2009; Barry et al., 2011; Verma et al., 2013; Amimo et al., 2014 and Zhou et al., 2016). The virus has been detected from both diarrhoeic (Khamrin et al., 2009; Park et al., 2010; Cromeans et al., 2014; Van Dung et al., 2016 and Almeida et al., 2018) and non-diarrhoeic pigs (Dufkova et al., 2013; Goecke et al., 2017 and Jackova et al., 2017). Prevalence of PKV in domestic pigs was reported from 13-99% (CFSPH, 2015), which decreases as age advances (Shan et al., 2011; Chen et al., 2013). Furthermore, PKV is suspected to cause large outbreaks of diarrhoea and dehydration along with vomition in suckling piglets with a morbidity rate of 80-100% and mortality of 50-90% in China (Cao et al., 2012). The large outbreaks of diarrhoea with highly significant mortality rate may contribute to great economic losses. The pathological studies of PKV in piglets have not been conducted in India and unavailability of a vaccine for protection of piglets from PKV together raises the need for further studies.
       
The present study has been conducted for pathological and molecular characterization of PKV infection in Indian pigs. The pathological characterization of natural PKV infection in Indian pigs is likely being attempted for the very first time.

Sample collection
 
A total of 41 intestinal content and tissue samples (brain, tonsil, lung, liver, spleen, heart and intestine) from dead pigs (Table 1) were collected from post-mortem facility of Division of Pathology, ICAR-Indian Veterinary Research Institute (ICAR-IVRI) during April, 2019 to July 2020. The clinical history of all the presented carcasses was recorded. Thereafter, the carcasses were subjected to detailed systematic necropsy examination and gross abnormalities were recorded carefully. Appropriate representative tissue samples were collected in 10% neutral buffered formalin and stored at -80°C for histopathological examination and molecular investigation respectively.
 

 
Histopathological examination
 
For histopathological processing, the thin pieces of formalin fixed tissue samples were washed overnight under running tap water followed by dehydration through ascending grades of alcohol. After dehydration, clearing was done with acetone and benzene followed by embedding in paraffin wax blocks by automatic tissue processor. The 4-5 μm thick paraffin sections were trimmed by microtome and stained with routine haematoxylin and eosin stain using standard protocol (Bancroft and Gamble, 2008). The stained sections were examined microscopically and the histopathological lesions were recorded carefully and photographed digitally (Olympus BX41, USA).
 
Molecular examination
 
Total RNA form all the intestinal content and tissue samples was extracted as per manufacture’s protocol from all PKV
suspected samples using commercial TRIzol® Reagent (Thermo Fisher Scientific, USA). All extracted RNA samples were subjected for quantification by NanoVue plus (Thermo Fisher Scientific, USA) and the purity of RNA was also checked by A260/230 and A260/280 ratio. The cDNA synthesis was performed as described earlier by Ray et al., (2018) and stored at -20°C for further use. The amplification of 3D region of PKV was carried out via RT-PCR using the primer forward: 5¢-TGGAC GACCAGCTCTTCCTTAAACAC-3¢ and reverse: 5¢-AGTGCAAGTGCAAGTCTGGGTTGC AGCCAACA-3¢  (Yu et al., 2009). The samples from all dead animals were also screened for porcine teschovirus (PTV) and porcine sapelovirus (PSV) using specific primers targeting 5¢ UTR gene.
       
PCR reaction (12 μL) was carried out in 0.2 ml PCR tubes containing 6.0 μL of DreamTaq^TM Green PCR Master Mix (2X), 0.5 μL of forward primer (10 pmol/μL), 0.5 μL of reverse primer (10 pmol/μL), 2.0 μL of cDNA (100 ng/μL) and 3.0 μl of nuclease-free water by using a thermocycler (S1000TM Thermal Cycler, BIO-RAD Laboratories India). The PCR was carried out with an initial denaturation of 94°C for 4 minutes; 35 cycles of 94°C for 30 seconds (denaturation), 60°C for 30 seconds (annealing) and 72°C for 50 seconds (extension) and a final extension at 72°C for 7 minutes. The visualization of amplified PCR products was done by agarose gel (1.5% w/v) electrophoresis after staining with 0.5 μg/mL ethidium bromide at 90 V for 1 h under UV transilluminator (Geldoc, USA).   
 
Phylogenetic analysis
 
The amplified PCR products were further confirmed by commercial DNA sequencing facility (Genotypic Technology Pvt. Ltd., Bangalore, India) via employing Sanger sequencing method using specific forward and reverse primers for the target gene used in the study. The 3D gene based phylogenetic analysis was performed using sequence data received in form of coloured electropherograms and text files to establish the genotypes of the sequenced PKV strains. The sequences of various isolates corresponding to other genotypes from different countries were retrieved from Gen Bank and used as input sequences for multiple sequence alignment. The sequence alignment was carried out using ClustalW programme of MEGA v.6 software followed by construction of a phylogenetic tree (Tamura et al., 2013).

In the present study a total of six out of 41 samples were found positive for PKV whereas all the PKV positive samples were found negative for PTV and PSV. The history of all PCR positive PKV cases revealed diarrhoea as the common clinical sign as discussed by Cao et al., (2012). Among PKV positive piglets the consistent lesions noticed were clouding and thickening of brain meninges, congestion of brain, severe to moderate degree of pneumonia along with congestion of lungs, presence of frothy exudate in trachea, congestion of mesenteric lymph nodes and thickening of intestinal mucosa along with catarrhal enteritis (Fig 1). Variable degree of consolidation and emphysema was observed in the different lobes of the lungs. Microscopic lesions in brain mainly include congestion of cerebral and meningeal vessels, infiltration of mononuclear cells, neuronophagia, mild to severe perivascular cuffing and gliosis. Mild to severe interstitial pneumonia along with congestion and haemorrhages was observed in the lungs. Moreover, infiltration of mononuclear cells in the interalveolar septa and accumulation of protein rich fluid was also observed. Intestines revealed congestion of mucosal and submucosal vessels, sloughing of villous epithelium along with infiltration of mononuclear cells in lamina propria. Lymphoid depletion in the Peyer’s patches was also observed in the ileum along with congestion of vessels
 

 
(Fig 2). However, the above lesions were found associated with PKV positive cases with the history of diarrhoea and the possibility of involvement of other porcine enteric picornaviruses (PTV and PSV) has also been ruled out but presence of other pathogens and their role in the development of pathology cannot be ignored. Therefore, the above lesions may only be strongly considered as the speculative lesions associated with PKV infection. Further studies with inoculation of experimental animals and natural hosts are highly warranted to ascertain the PKV specific lesions.

       
In RT-PCR specific amplicons of 495 bp were obtained from PKV positive samples (Fig 3). Among six PKV positive cases, 2 were 6 day old, 2 were 10 day old, 1 was 11 days old and 1 piglet was 20 days of age suggesting the prevalence of the virus in piglets of less than 1 month of age (Table 1). The 3D regions of 12 PKV sequences were retrieved from NCBI and used for the construction of phylogenetic tree along with the Indian isolates detected in the present study. The Indian isolates of the present study were named as PKV/India/IVRI-184A/2020 (Accession no. MW018696) and PKV/India/IVRI-188A/2020 (Accession no. MT946377). Phylogenetic analysis of the 3D region revealed that Indian isolate of this study is closely related to other Indian isolates MH394282/India/2015 and MH394279/India/2015. Indian isolates PKV/India/IVRI-188A/2020 and PKV/India/IVRI-184A/2020 were clustered together in a single node with a bootstrap value of 100. On Blast analysis of 3D gene maximum identity of 91-93% with query coverage of 74%-94% with Indian isolate MH394282/PKV/India/IVRI-187/2015 and Chinese strain FJ459905/swine/2007/CHN isolate Z324 was observed (Fig 4).
 

 

The present pathological and molecular investigation of PKV in naturally infected piglets concluded that PKV primarily infected intestine, brain and lungs. Moreover, mild to severe encephalitis, interstitial pneumonia and severe catarrhal and haemorrhagic enteritis was observed as major pathological findings. Although very limited information on pathology of PKV is available it is hoped that this study will add to the understanding of PKV infection in pigs. Moreover, further studies in suitable animal model will definitely provide better insights of the pathology and pathogenesis of the PKV.

The authors are thankful to the Director and Joint Director (Research) of the Institute (ICAR- IVRI, Izatnagar, Bareilly) for providing the funds (Institute funded project) and facilities to carry out this work.

The authors declare that they have no conflict of interest.