VP1 Gene Research Articles

Genetic Diversity and Recombination Analysis of Canine Parvoviruses Prevalent in Central and Eastern China, from 2020 to 2023

Canine parvovirus type-2 (CPV-2), the primary causative agent of serious canine enteric diseases, is highly contagious and associated with high fatality rates worldwide. To comprehend the current emergence of CPV-2 in central and eastern China, 130 rectal swabs from domestic or stray dogs with gastroenteritis symptoms were collected during 2020–2023. A total of 118 positive samples were detected via polymerase chain reaction, and further used to amplify and sequence the VP2 gene. Sequence analysis of the deduced amino acids of VP2 protein indicated that CPV-2c was the most prevalent variant (n = 106, 89.83%), followed by the novel CPV-2a (n = 10, 8.47%) and CPV-2b (n = 2, 1.69%) variants. The VP2 protein from the obtained and reference strains showed 86.95% (AH2103 and HB2108) to 99.94% identity. Based on the nine predicted recombination events, some prevalent CPV-2c strains were highly similar to previously isolated strains, indicating their complex evolution and recombination. The predicted analysis suggested that mutations in the antigen epitope (Val219Ile, Phe267Tyr, and Asn426Glu) and other mutations (Met87Leu, Ile101Thr, and Ser297Ala) affect the tertiary structure of the VP2 protein. This research will help us understand the recent evolution and mutation of Chinese CPV-2 and provide suggestions for updating the CPV-2 vaccine.

McHDV VP60 Virus-like Particles Elicit Protective Immunity Against Moschus chrysogaster Hemorrhagic Disease in Rabbits

Moschus chrysogaster viral hemorrhagic disease (McVHD), caused by the Moschus chrysogaster hemorrhagic disease virus (McHDV), is an acute and highly fatal infectious disease of musk deer. At present, there is no prevention or treatment for this disease. In this study, we constructed a recombinant bacmid containing the McHDV VP60 gene and obtained the recombinant baculovirus rBac-McHDV VP60 by transfection into Sf9 (Spodoptera frugiperda) insect cells. The McHDV VP60 protein was successfully expressed in the insect cell-baculovirus expression system; furthermore, it was released in the supernatant of infected insect cells and spontaneously assembled to form virus-like particles (VLPs), which were structurally and immunologically indistinguishable from the Moschus chrysogaster viral hemorrhagic disease virion. Hypodermic vaccination of rabbits with the VLPs conferred complete protection in 14 days; this protection was found to be effective from the seventh day after VLP injection and was accompanied by a strong humoral response. This study is the first attempt to express the VP60 gene of McHDV using an insect baculovirus system, which provides an experimental basis for the virus-like particle vaccine of McVHD.

Development of a Quadruplex RT-qPCR for the Detection of Feline Kobuvirus, Feline Astrovirus, Feline Bufavirus, and Feline Rotavirus

Feline kobuvirus (FeKoV), feline astrovirus (FeAstV), feline bufavirus (FeBuV), and feline rotavirus (FRV) are important pathogens for gastroenteritis, which is characterized by vomiting, diarrhea, and dehydration. Four pairs of primers and probes were designed to target the FeKoV VP1, FeAstV ORF2, FeBuV VP2, and FRV NSP4 genes, and a quadruplex real-time quantitative RT-PCR (RT-qPCR) assay capable of the simultaneous detection of four feline enteroviruses was developed after optimization of reaction conditions. The established quadruplex RT-qPCR assay showed high specificity, sensitivity, and reproducibility. The assay could detect and discriminate FeKoV, FeAstV, FeBuV, and FRV, but not other feline-related pathogens. The limits of detection (LODs) of FeKoV, FeAstV, FeBuV, and FRV were 109.761, 115.834, 125.481, and 113.875 copies/reaction, respectively. The intra- and inter-assay coefficients of variation (CV) were 0.15–1.61% and 0.15–1.59%, respectively. In all, 1869 clinical samples from Guangxi province in Southern China were tested using the developed assay, and the positivity rates of FeKoV, FeAstV, FeBuV, and FRV were 1.93%, 9.36%, 0.32%, and 0.75%, respectively. These samples were also tested using reference assays, and the coincidence rates of the results between the developed and reference methods were 99.63% (FeKoV), 98.72% (FeAstV), 100% (FeBuV), and 100% (FRV), respectively. The results indicated that the developed assay could provide a new detection method for these four viruses associated with feline gastroenteritis.

Development and application of a quadruplex real-time PCR method for Torque teno sus virus 1, Porcine circovirus type 2, pseudorabies virus, and porcine parvovirus.

In clinical diagnosis of porcine diseases, co-infection with multiple viruses often leads to similar clinical symptoms. Postweaning multisystemic wasting syndrome (PMWS) can be caused by infections with TTSuV or PCV2, while PCV2, PRV, and PPV can cause respiratory and reproductive disorders in pigs. The overlapping clinical and pathological features of these infections necessitate the development of a rapid and specific method for differentiating and detecting these four DNA viruses. In this study, four pairs of primers and TaqMan probes were designed targeting the conserved sequence of TTSuV, the Rep gene of PCV2, the gE gene of PRV, and the VP2 gene of PPV. After optimizing reaction conditions, including annealing temperature, primer concentration, and probe concentration, a quadruplex real-time PCR method was developed. This method can specifically detect TTSuV1, PCV2, PRV, and PPV simultaneously, with no cross-reactivity with ASFV, CSFV, PRRSV, PEDV, PSV, and TGEV. The minimum detection limit for each virus was 10 copies/μl, and the inter-assay and intra-assay coefficients of variation ranged from 0.33% to 1.43%. Subsequently, 150 clinical samples were tested to evaluate the practical applicability of this method. The positive rates for TTSuV1, PCV2, PRV, and PPV were 8.6% (13/150), 10.67% (16/150), 14% (21/150), and 11.33% (17/150), respectively. The results indicate that the established quadruplex real-time PCR method can assist in the accurate and rapid diagnosis of TTSuV1, PCV2, PRV, and PPV in clinical settings, providing robust support for the prevention and control of these infections.

Comparative pathogenicity of duck hepatitis A virus genotype 3 in different duck breeds: Implications of the diagnosis and prevention of duck viral hepatitis

Duck hepatitis A virus (DHAV) infection in ducklings causes acute hepatitis with considerable economic losses. In this study, Pekin and Muscovy duckling flocks (n=9) suffering from high mortality and hepatic lesions were examined by RT-PCR for DVHA. 44.4 % (4/9) of samples were positive for DHAV (5′ UTR region), of which 100 % (4/4) were DVHA-3 (VP1 gene). VP1 sequencing and phylogenetic analysis of an isolate originated from Muscovy ducklings showed that it shared 96.8 % −100 %, 88.5–89.2 %, and 86.5–88.2 % nucleotide similarity (ns) with the Egyptian, Korean-Vietnamese, and Chinese DVHA-3 strains, respectively. It was distinguished from the DHAV-1 vaccine (67.6 % ns). The sequenced DVHA-3 isolate was used to experimentally infect 5-day-old Pekin and Muscovy ducklings vs. control groups. No apparent clinical signs or deaths were reported in the experimentally-infected groups. Pekin ducklings showed greater cloacal viral shedding than Muscovy until the 6th dpi (P<0.05). DVHA-3 induced a significant rise in IFN-β and IL-1β serum levels, where Muscovy ducklings' levels were higher than Pekin ducklings. Among the biochemical parameters, AST was only increased on the 4th dpi in both breeds vs. control (P<0.05). Compared to Muscovy ducklings at 2, 4, and 6 day post infection (dpi), the infected Pekin group had lower lipase levels (P≥0.05, p<0.05, and p<0.05, respectively), while ALT was higher at 4 and 6 dpi (P<0.05). The histopathological lesions supported the gross lesions, and their scores were dominant at 2 and 4 dpi in both breeds. At 6 and 8 dpi, Pekin showed more severe histopathological changes compared to Muscovy for the liver, heart, brain, and intestines; the pancreas, kidney, and lung showed the opposite pattern. In conclusion, Pekin ducklings displayed distinct DHAV-3 infection results from Muscovy ducklings, and more research utilizing a variety of DHAV-3 strains has to be carried out.

Immunogenicity analysis based on VP1 and VP2 proteins of bovine enterovirus

Bovine enterovirus (BEV) infection manifests as a spectrum of clinical signs affecting the respiratory, gastrointestinal, and reproductive systems in cattle. Outbreaks of this disease results in large economic losses to the bovine industry worldwide. Currently there are no efficacious vaccines and medicines to prevent BEV infection. In the present study, reverse transcription-polymerase chain reaction was used to amplify the VP1 and VP2 genes of BEV, enabling the synthesis of a chimeric recombinant protein which contained partial sequences from both proteins. Subsequently, the emulsified purified proteins with Freund’s adjuvant were used for triple-fold immunization of 4-week-old Institute of Cancer Research (ICR) mice. The mice were subsequently subjected to a challenge assay which elicited an immune response that was characterized by elevated titers of BEV-specific neutralizing antibodies. Notably, the results showed that the purification of pET32a-VP1 and pET32a-VP2 proteins markedly curtailed virus excretion and mitigated the histopathological damage usually associated with BEV infections. These were observed in the small intestines, kidneys and brain in infected animals. It also alleviated clinical symptoms such as hypothermia and weight loss. In summary, this study offers a theoretical and practical basis for BEV vaccine development.

Molecular Detection and Phylogenetic Tree of Rotavirus from Human and Sheep, Iraq

Group A rotaviruses (RVA) have been linked to acute gastroenteritis in children, as well as young domestic and wild animals. In Iraq few molecular studies involved the genotype profile of rota virus and this is the first study target VP1 gene. Data were collected from January to February 2018 and subsequently examined utilizing a PCR and because of low viral concentration we utilized Nested PCR. Sequenced PCR result at 327 bp fragment in 4 A rota virus-positive samples deposited in GenBank under accession number (MH11809.1, MH118097.1) for human and (MH118095.1, MH118094.1) to sheep. Children samples were identical to the other human A rota virus at (99-100%), with Japan feline-like human G6P and G6P[14] isolated from cattle as well as, animal rota virus (99-100%) similarity with cross relation with human Wa-Like and porcine, moreover Morocco characterized caprine, bovine rotavirus and Slovenian Roe deer ARV.

Whole Genome Sequences of the Wildtype AU-1 Rotavirus A Strain: The Prototype of the AU-1-like Genotype Constellation.

Most human rotaviruses belong to the Wa-like, DS-1-like, or AU-1-like genotype constellation. The AU-1-like constellation, albeit minor, captured attention because its prototype strain AU-1 originated from feline rotavirus, leading to the concept of interspecies transmission of rotavirus. The AU-1 genome sequence determined by various laboratories over the years has documented two conflicting VP7 sequences in the GenBank. As culture-adaptation may introduce changes in the viral genome, the original fecal (wild-type) and the seed stock of culture-adapted AU-1 genomes were sequenced using the Illumina's MiSeq platform to determine the authentic AU-1 sequence and to identify what mutational changes were selected during cell-culture adaptation. The wild-type and culture-adapted AU-1 genomes were identical except for one VP4-P475L substitution. Their VP7 gene was 99.9% identical to the previously reported AU-1 VP7 under accession number AB792641 but only 92.5% to that under accession number D86271. Thus, the wild-type sequences determined in this study (accession numbers OR727616-OR727626) should be used as the reference. The VP4-P475L mutation was more likely incidental than inevitable during cell-culture adaptation. This was the first study in which the whole genomes of both wild-type and cultured RVA strains were simultaneously determined by deep sequencing.

Wide circulation of type 1 vaccine-derived poliovirus strains in clinical specimens from suspected cases of poliomyelitis, their contacts and in wastewater in Madagascar since late 2020

Madagascar has faced three major outbreaks of vaccine-derived polioviruses (VDPVs) in recent decades, with VDPV type 1 reemerging in late 2020. Here, we report the molecular characterization of these cVDPV1 strains. WHO protocols were used for poliovirus detection in stool and wastewater samples. Molecular genotyping was based on the 5′ non-coding (5′NC), VP1, and 3Dpol regions. From 2020 to 2022, 92 of 5690 stool samples and 129 of 1046 wastewater samples tested positive for cVDPV1. Genetic analysis of the VP1 gene revealed 1.3%–6.1% variability compared to the Sabin strain. Most sequences showed mutations at neurovirulence attenuation sites. Phylogenetic analysis distributed strains into four genogroups originating from Southern Madagascar. All analyzed cVDPV1 strains were recombinant, containing mutated oral polio vaccine sequences in VP1 and type C enterovirus sequences in other regions. This study demonstrated that all strains were closely related during this epidemic.

Establishment and Application of Mismatch Amplification Mutation Assay-PCR for Rapid Detection and Differentiation of Duck Hepatitis A Virus-1 Attenuated Vaccine and Wild Strains.

Duck hepatitis A virus type 1 (DHAV-1) is the main pathogen causing viral hepatitis in ducks, marked by high contagion and acute mortality. Live attenuated DHAV-1 vaccines are widely used to control the disease. This study aims to develop a mismatch amplification mutation assay (MAMA)-PCR for the rapid detection and differentiation of Korean DHAV-1 wild-type strains from vaccine strains. A MAMA primer was designed to target a single nucleotide polymorphism (SNPs) at position 2276 within the VP1 gene, allowing differentiation in a single PCR reaction. The MAMA-PCR accurately identified both strains, with detection limits of 100.5 ELD50/mL and 102.3 ELD50/mL, respectively. The MAMA-PCR demonstrated specificity, showing no cross-reactivity with 12 other viral and bacterial pathogens. The MAMA-PCR was applied to 89 farms, yielding results consistent with nested-PCR and sequence determination, identifying four positive farms for DHAV-1 vaccine strains. In conclusion, this study is the first to employ the MAMA-PCR method to distinguish between DHAV-1 wild-type and vaccine strains. The developed method is rapid, simple, specific, and sensitive, thereby serving as an effective tool for clinical diagnostics in identifying and differentiating between Korean DHAV-1 wild-type and vaccine strains.

Full genome sequence analysis of the predominant and uncommon G9P[4] rotavirus strains circulating in Tehran, Iran, 2021–2022: Evidence for inter and intra-genotype recombination

Group A rotaviruses (RVAs) are a major cause of acute gastroenteritis in children under 5 years of age worldwide. Herein, the genetic sequences of 11 RNA segments from three uncommon G9P[4] RVA strains found in the stool samples of children under 5 years of age in Iran were analyzed using next-generation sequencing (NGS) technology. The genomic constellations of these three uncommon G9P[4] strains indicated the presence of the double and quadruple reassortants of two G9P[4] strains, containing the VP7/NSP2 and VP7/VP2/NSP2/NSP4 genes on a DS-1-like genetic background, respectively. The genome of one strain indicated a Wa-like genetic backbone in a single-reassortant with the VP4 of the DS1-like human strains. With the exception of VP1, VP2, VP7, NSP2, NSP3, and NSP4 genes, which clustered with RVA of human origins belonging to cognate gene sequences of genogroup 1/2 genotypes/lineages, the remaining five genes (VP8/VP4, VP3, VP6, NSP1, NSP5) displayed direct evidence of recombination. It is presumed that the presence of uncommon G9P[4] strains in Iran is not linked to vaccination pressure, but rather to the high prevalence of RVA co-infection or the direct import of these uncommon RVA reassortants strains from other countries (especially those that have implemented RV vaccination).

Screening high-efficiency promoter to construct trans-vp28 gene Anabaena sp. PCC7120 against white spot syndrome virus of Litopenaeus vannamei

This study aimed to select high-quality promoters to construct trans-vp28 gene Anabaena sp. PCC7120 and feed Litopenaeus vannamei to assess the effect of L.vannamei against white spot syndrome virus (WSSV). Transgenic algae were created using five plasmids containing PrbcL, Pcpc560, Ptrc, Ptac, and PpsbA. According to the gene expression efficiency and the growth index of transgenic algae, Pcpc560 was determined to be the most efficient promoter. Shrimps were continuously fed trans-vp28 gene Anabaena sp. PCC7120 for one week and then challenged with WSSV. After the challenge, the transgenic algae group (vp28-7120 group) was continuously immunized [continuous immunization for 0 days (vp28-7120-0d); continuous immunization for 2 days (vp28-7120-2d); continuous immunization for 4 days (vp28-7120-4d)]. After seven days, the daily survival rate of each experimental group was continuously tracked. Following the viral challenge, the hepatopancreas samples were assayed for their levels of superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GSH-Px), thioredoxin peroxidase (TPX), acid phosphatase (ACP), and alkaline phosphatase (AKP) at varying time intervals. In comparison to the positive control group (challenge and no vaccination) and the wild-type group (challenge, fed wild-type Anabaena sp. PCC7120), the vp28-7120 group (challenge, fed trans-vp28 gene Anabaena sp. PCC7120) exhibited a remarkable increase in survival rates, reaching 50 % (vp28-7120-0d), 76.67 % (vp28-7120-2d), and 80 % (vp28-7120-4d). Furthermore, the vp28-7120 group consistently displayed significantly higher activities of SOD, CAT, GSH-Px, ACP, and AKP, while exhibiting notably lower TPX activity, when compared to the control group. These results indicate that the Pcpc560 promoter effectively elevated the expression level of the exogenous vp28 gene and spurred the growth of the trans-vp28 gene Anabaena sp. PCC7120. Consequently, trans-vp28 gene Anabaena sp. PCC7120 significantly bolstered the immunity of L.vannamei. Therefore, utilizing the Pcpc560 promoter to develop trans-vp28 gene Anabaena sp. PCC7120 based oral vaccine is highly beneficial for industrial-scale cultivation, advancing its commercialization prospects.

Comparative Analysis of Ligninolytic Potential among Pleurotus ostreatus and Fusarium sp. with a Special Focus on Versatile Peroxidase

Lignocellulosic biomass is contemplated to be an inexpensive and copious feedstock that can be used for numerous industrial applications. However, lignin forms the lignin sheath and provides a physical barrier to enzymatic hydrolysis. In addition, lignin physically blocks cellulase, preventing it from being combined with the substrate in a process known as non-productive binding. Therefore, the depletion of lignin is a crucial method for obtaining fermentable sugars from the lignocellulosic biomass. Different white-rot fungi secrete different sets of lignin-mineralizing enzymes and each fungus secretes one or more of the three enzymes essential for lignin degradation. Among efficient redox enzymes, versatile peroxidase is extensively studied for its ability to degrade aromatics without the need for a mediator or polyvalent catalytic site. However, the presence of versatile peroxidase in F. spp. has not been studied. This study was planned with the objective of screening and comparing the production of versatile peroxidase enzymes from F. spp. and a standard culture of Pleurotus ostreatus MTCC-142. These fungal strains were first screened on solid media containing tannic acid, malachite green, or bromocresol green. The potency index for the tannic acid, malachite green, and bromocresol green on the 16th day of incubation was reported to be 1.28, 1.07, 1.09, and 1.10, respectively. Versatile peroxidase production patterns were investigated under solid state fermentation conditions for a period of 25 days at different temperatures ranging from 10 to 35 °C. The highest versatile peroxidase activity (592 UL−1) in F. sp. was observed at 30 °C after the 7th day of incubation. The molecular confirmation showed the presence of the vp gene in F. sp. along with Pleurotus ostreatus MTCC-142. The results determined that F. sp. possesses a versatile peroxidase enzyme and is able to degrade lignin efficiently, and thus it could be utilized as an alternative to other ligninolytic enzyme-producing fungi.

Genomic surveillance reveals low-level circulation of two subtypes of genogroup C coxsackievirus A10 in Nanchang, Jiangxi Province, China, 2015-2023.

In recent years, coxsackievirus (CV) A10 has been associated with increasing sporadic hand, foot, and mouth disease (HFMD) cases and outbreaks globally. In addition to mild symptoms such as pharyngitis and herpangina, CVA10-related complications or even fatality can occur. Currently, systematic phylogenetic studies of CVA10 are limited. In this study, we first explored the epidemiological and genetic characteristics of CVA10 in Nanchang, an inland southeastern city of China, based on the HFMD surveillance network from 2015-2023. Among 3429 enterovirus-positive cases, 110 (3.04%) were associated with CVA10, with a male-to-female ratio of 1.62. The median age of the CVA10 patients was 2.3 years (interquartile range, IQR 1.0-4.0), with 94.55% (104/110) of the patients aged less than 5 years. Phylogenetic analyses using the full-length VP1, 5'UTR, P1, P2, P3 sequences and near full-length genomes indicated that CVA10 strains (n = 57) isolated in Nanchang belonged to genogroup C; two strains identified in 2017 belonged to C1 subtypes clustered with strains from Vietnam, Madagascar, France and Spain; and the others belonged to C2 subtypes interdigitating with CVA10 isolates from mainland China, the United States and Australia. Through extensive analysis, we identified a rare F168Y mutation in epitope 4 of VP1 in a Madagascar strain of genogroup F and a Chinese strain of genogroup C. Based on Bayesian evolutionary analyses, the average nucleotide substitution rate for the VP1 gene of CV10 strains was 3.07×10-3 substitutions/site/year. The most recent common ancestor (tMRCA) of genogroup C was dated 1990.84, and the tMRCA of CVA10 strains from Nanchang was dated approximately 2003.16, similar to strains circulating in other regions of China, suggesting that the viruses were likely introduced and cryptically circulated in China before the establishment of the HFMD surveillance network. Recombination analysis indicated intertypic recombination of the Nanchang strain with the genogroup G strain in the 3D region. Given the shifting dominance of viral genotypes and frequent recombination events, the existing surveillance system needs to be regulated to enhance genomic surveillance efforts on a more diverse spectrum of genotypes in the future.

Epidemiological survey of calf diarrhea related viruses in several areas of Guangdong Province.

Bovine torovirus (BToV), Bovine enterovirus (BEV), Bovine norovirus (BNoV), Bovine coronavirus (BCoV), Bovine rotavirus (BRV), and Bovine viral diarrhea virus (BVDV) are significant pathogens causing diarrhea in calves, characterized by their high prevalence and challenging prevention and control measures. We analyzed 295 calf diarrhea samples, amplifying the M gene from BToV-positive samples, the 5'UTR gene from BEV-positive samples, the RdRp gene from BNoV-positive samples, the VP7 gene from BRV-positive samples, the S gene from BCoV-positive samples, and the 5'UTR gene from BVDV-positive samples. Subsequent homology analysis and phylogenetic tree construction were performed. The overall viral positive rate in Guangdong Province was 21.36%. Specific detection rates were as follows: Foshan City at 50.00% (18/36), Guangzhou City at 43.90% (36/82), Huizhou City at 21.21% (7/33), Yangjiang City at 2.08% (1/48), Meizhou City at 1.39% (1/72), and Heyuan City at 0.00% (0/24). The detection rates for BToV, BEV, BNoV, BCoV, BRV, and BVDV were 0.34% (1/295), 6.10% (18/295), 0.68% (2/295), 1.36% (4/295), 10.85% (32/295), and 2.03% (6/295), respectively. Notably, the highest overall virus detection rate was observed in the Guangzhou-Foshan region, with BRV and BEV showing the highest detection rates among the six viruses. This study marks the first report of BToV and BNoV in Guangdong Province. Phylogenetic analysis revealed that the BToV strain belonged to type II, sharing genetic similarities with epidemic strains from various provinces in China. The BEV strains were categorized into E and F types, with the F type being the predominant strain in Guangdong Province and exhibiting the closest genetic relationship to strains from Heilongjiang and Guangxi. The BNoV strains, along with Hebei strains, were identified as GIII.2 subgenotype. BCoV strains showed the highest genetic similarity to strains from Sichuan. All BRV strains were classified under the G6 subtype and had the closest genetic relationship with human rotavirus strains. BVDV strains were identified as subtype 1b, closely related to the Beijing strain. In conclusion, this study investigated the prevalence and evolutionary characteristics of diarrhea-associated viruses in calves in specific areas of Guangdong Province, providing a valuable reference for establishing effective prevention and control measures in cattle farms.

Construction of Recombinant Marek's Disease Virus Co-Expressing VP1 and VP2 of Chicken Infectious Anemia Virus.

The chicken infectious anemia virus (CIAV) has been reported in major poultry-producing countries and poses a significant threat to the poultry industry worldwide. In this study, two Marek's disease virus (MDV) recombinants, rMDV-CIAV-1 and rMDV-CIAV-2, were generated by inserting the CIAV VP1 and VP2 genes into the MDV vaccine strain 814 at the US2 site using the fosmid-based rescue system. For rMDV-CIAV-1, an internal ribosome entry site was inserted between VP1 and VP2, so that both proteins were produced from a single open reading frame. In rMDV-CIAV-2, VP1 and VP2 were cloned into different open reading frames and inserted into the MDV genome. The recombinant viruses simultaneously expressed VP1 and VP2 in infected chicken embryo fibroblasts and exhibited growth kinetics similar to those of the parent MDV. The two recombinant viruses induced antibodies against CIAV in chickens. A single dose of the recombinant viruses provided strong protection against CIAV-induced anemia in chickens. These recombinant VP1- and VP2-expressing MDVs are potential vaccines against CIAV in chickens.

Long Amplicon Nanopore Sequencing for Dual-Typing RdRp and VP1 Genes of Norovirus Genogroups I and II in Wastewater

Noroviruses (NoVs) are the leading cause of non-bacterial gastroenteritis with societal costs of US$60.3 billion per annum. Development of a long amplicon nanopore-based method for dual-typing the RNA-dependent RNA polymerase (RdRp) and major structural protein (VP1) regions from a single RNA fragment could improve existing norovirus typing methods. Application to wastewater-based epidemiology (WBE) and environmental testing could enable the discovery of novel types and improve outbreak tracking and source apportionment. Here, we have developed such a method with a consensus-based bioinformatics pipeline and optimised reverse transcription (RT) and PCR procedures. Inhibitor removal and LunaScript® RT gave robust amplification of the ≈ 1000 bp RdRP + VP1 amplicon for both the GI and GII PCR assays. Platinum™ Taq polymerase showed good sensitivity and reduced levels non-specific amplification (NSA) when compared to other polymerases. Optimised PCR annealing temperatures significantly reduced NSA (51.3 and 42.4% for GI and GII), increased yield (86.5% for GII) and increased taxa richness (57.7%) for GII. Analysis of three NoV positive faecal samples showed 100% nucleotide similarity with Sanger sequencing. Eight GI genotypes, 11 polymerase types (p-types) and 13 combinations were detected in wastewater along with 4 GII genotypes, 4 p-types and 8 combinations; highlighting the diversity of norovirus taxa present in wastewater in England. The most common genotypes detected in clinical samples were all detected in wastewater while we also frequently detected several GI genotypes not reported in the clinical data. Application of this method into a WBE scheme, therefore, may allow for more accurate measurement of norovirus diversity within the population.

Molecular characterization of a Porcine Group A rotavirus of VP6 gene of by Reverse Transcriptase–Polymerase Chain Reaction

Molecular characterization of a Porcine Group A rotavirus of VP6 gene of by Reverse Transcriptase–Polymerase Chain Reaction

Characterization of cross-reactivity of coxsackievirus A2 VP1-specific polyclonal antibodies with enterovirus A71, coxsackievirus A16, and coxsackievirus A6

Coxsackievirus A2 (CVA2) is associated with multiple diseases in children. Currently, there is limited research on immunological detection methods for CVA2. Herein, the VP1 gene of CVA2 strain 201711, belonging to cluster 2 within genotype D, was analyzed. The structures of VP1 from CVA2 strains 201711, 7-1 and 12-1, enterovirus A71 (EV-A71) strain 201713, coxsackievirus A16 (CVA16) strain 201717, and coxsackievirus A6 (CVA6) strain JLS10 were compared. The Escherichia coli BL21(DE3)/pET vector system was employed to express the recombinant protein containing the entire VP1 of CVA2 strain 201711. Mice were immunized with the purified protein, and the sera were collected and used to specifically identify the VP1 in CVA2-infected RD cells by Western blot and immunofluorescence assay. There was no evident cross-reactivity of the sera with the VP1 of EV-A71, CVA16, and CVA6 strains mentioned above. Therefore, this study provided mouse-specific anti-CVA2 VP1 polyclonal antibodies for CVA2 detection.

Whole-genome sequence analysis of canine parvovirus reveals replacement with a novel CPV-2c strain throughout India.

Canine parvovirus (CPV) infection causes severe gastroenteritis in canines, with high mortality in puppies. This virus evolved from feline panleukopenia virus by altering its transferrin receptor (TfR), followed by the emergence of CPV-2 variants in subsequent years with altered immunodominant amino acid residues in the VP2 protein. While previous studies have focused on the VP2 gene, there have been fewer studies on non-structural protein (NS1 and NS2) genes. In the present study, CPV genome sequences from clinical samples collected from canines throughout India in 2023, previous Indian CPV isolates from 2009-2019, and the current Indian CPV vaccine strain were compared. The study showed that the CPV-2c (N426E) variant had almost completely replaced the previously dominant CPV-2a variant (N426) in India. The Q370R mutation of VP2 was the most common change in the recent CPV-2c strain (CPV-2c 370Arg variant). Phylogenetic analysis showed the existence of three clades among the recent CPV-2c strains, and sequence analysis identified several new sites of positive selection in the VP1 (N-terminus), VP2, NS1, and NS2 protein-encoding genes in recent CPV strains, indicating the emergence of new CPV-2c variants with varied antigenic and replication properties. The predominant 'CPV-2c 370Arg variants' were grouped with the Chinese and Nigerian CPV-2c strains but were separate from the CPV vaccine strain and earlier isolates from our repository. VP2 epitope analysis predicted nine amino acid variations (including two new variations) in four potential linear B-cell epitopes in the CPV-2c 370Arg variants that might make vaccine failure more likely. This pan-Indian study lays the foundation for further research concerning the dynamics of virus evolution and understanding genetic mutations.