ORF2 Gene Research Articles

Development of a Quadruplex RT-qPCR for the Detection of Porcine Astrovirus, Porcine Sapovirus, Porcine Norovirus, and Porcine Rotavirus A

Porcine astrovirus (PoAstV), porcine sapovirus (PoSaV), porcine norovirus (PoNoV), and porcine rotavirus A (PoRVA) are newly discovered important porcine diarrhea viruses with a wide range of hosts and zoonotic potential, and their co-infections are often found in pig herds. In this study, the specific primers and probes were designed targeting the ORF1 gene of PoAstV, PoSaV, and PoNoV, and the VP6 gene of PoRVA. The recombinant standard plasmids were constructed, the reaction conditions (concentration of primers and probes, annealing temperature, and reaction cycle) were optimized, and the specificity, sensitivity, and reproducibility were analyzed to establish a quadruplex real-time quantitative RT-PCR (RT-qPCR) assay for the detection of these four diarrheal viruses. The results demonstrated that the assay effectively tested PoAstV, PoSaV, PoNoV, and PoRVA without cross-reactivity with other swine viruses, and had limits of detection (LODs) of 138.001, 135.167, 140.732, and 132.199 (copies/reaction) for PoAstV, PoSaV, PoNoV, and PoRVA, respectively, exhibiting high specificity and sensitivity. Additionally, it displayed good reproducibility, with coefficients of variation (CVs) of 0.09–1.24% for intra-assay and 0.08–1.03% for inter-assay. The 1578 clinical fecal samples from 14 cities in Guangxi Province, China, were analyzed via the developed assay. The results indicated that the clinical samples from Guangxi Province exhibited the prevalence of PoAstV (35.93%, 567/1578), PoSaV (8.37%, 132/1578), PoNoV (2.98%, 47/1578), and PoRVA (14.32%, 226/1578), and had a notable incidence of mixed infections of 18.31% (289/1578). Simultaneously, the 1578 clinical samples were analyzed with the previously established assays, and the coincidence rates of these two approaches exceeded 99.43%. This study developed an efficient and precise diagnostic method for the detection and differentiation of PoAstV, PoSaV, PoNoV, and PoRVA, enabling the successful diagnosis of these four diseases.

Effects of storage temperature on throat swabs preserved in different transport media for detection of SARS-CoV-2 by reverse transcriptase polymerase chain reaction

Objectives: The coronavirus disease 2019 (COVID-19) pandemic resulted in an increased need for molecular diagnostic testing. Delay in the specimen processing and storage of samples in laboratories leads to degradation of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) viral RNA. Inactivation transport medium (ITM) contains chaotropic agents that inactivate the virus and stabilize SARS-CoV-2 RNA for a longer duration, even at room temperature. The effect of different temperatures and duration of storage of samples in viral transport media (VTM) and ITM for detection of SARS-CoV-2 RNA was assessed. Materials and Methods: Samples from COVID-19 patients were aliquoted in ITM and VTM and kept at ambient temperature, 37°C and 45°C. SARS-CoV-2 viral RNA was extracted. Multiplex real-time polymerase chain reaction was done on days 0, 1, 3, and 5, and cycle threshold (Ct) values were noted. Statistical Analysis: Data were analyzed using the Statistical Package for the Social Sciences version 26.0. Linear variables were summarized as mean and standard deviations. One-way analysis of variance test with post hoc Tukey honestly significant difference was used to compare mean value between different loops and for pair-wise comparison. P < 0.05 was taken as significant. Results: The mean Ct values of both the Orf and E genes of the samples in VTM and ITM were stable across all temperature conditions on day 1. On day 5, the increase in Ct values for both E and Orf genes were significantly higher for VTM than ITM at ambient temperature, 37°C and 45°C. Ribonuclease P failure was significantly higher for VTM than ITM at ambient temperature and 37°C on day 3 and at all temperatures on day 5. Conclusions: ITM is a valuable transport media that can preserve SARS-CoV-2 for up to 5 days at ambient temperature and 37°C. As it renders the samples non-infectious, thus reducing the potential of biohazard events, this transport medium can be used effectively for the collection and transportation of SARS-CoV-2 samples, especially from remote or isolated healthcare facilities.

Hidden evolutionary constraints dictate the retention of coronavirus accessory genes.

Coronaviruses exhibit many mechanisms of genetic innovation, including the acquisition of accessory genes that originate by capture of cellular genes or through duplication of existing viral genes. Accessory genes influence viral host range and cellular tropism, but little is known about how selection acts on these variable regions of virus genomes. We used experimental evolution of mouse hepatitis virus (MHV) encoding a cellular AKAP7 phosphodiesterase and an inactive native phosphodiesterase, NS2, to model the evolutionary fate of accessory genes. After courses of serial infection, the gene encoding inactive NS2, ORF2, unexpectedly remained intact, suggesting it is under cryptic constraint uncoupled from the function of NS2. By contrast, AKAP7 was retained under strong selection but rapidly lost under relaxed selection. Experimental evolution also led to altered viral replication in a cell-type-specific manner and changed the relative proportions of subgenomic viral RNA in plaque-purified viral isolates, revealing additional mechanisms of adaptation. Guided by the retention of MHV ORF2 and similar patterns in related betacoronaviruses, we analyzed ORF8 of SARS-CoV-2, which is proposed to have arisen via gene duplication and contains premature stop codons in several globally successful lineages. As with MHV ORF2, the coding-defective SARS-CoV-2 ORF8 gene remained largely intact in these lineages, mirroring patterns observed during MHV experimental evolution, challenging assumptions on the dynamics of gene loss in virus genomes, and extending these findings to viruses currently adapting to humans.

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.

Molecular detection and genetic characteristics of porcine reproductive and respiratory syndrome virus in central China

Porcine reproductive and respiratory syndrome caused by porcine reproductive and respiratory syndrome virus (PRRSV) is one of the most economically devastating viral diseases in the global pork industry. To further clarify the epidemic characteristics of the virus, 365 clinical samples were collected from diseased pigs suffering from abortion and respiratory disease from 2018 to 2023 on 63 pig farms in Henan and Shanxi provinces, and screened for the presence of PRRSV using reverse transcription-polymerase chain reaction (RT-PCR). A total of 62 clinical samples (62/365, 16.99 %) were positive for PRRSV, and subsequently, full-length ORF5 gene sequences of 29 PRRSV strains and the complete genome sequence of one PRRSV HeN-HC isolate were obtained and analyzed. Phylogenetic analysis based on the ORF5 gene showed that 22 of the 29 PRRSV2 strains belonged to sublineage 1.8 (NADC30-like), 5 belonged to sublineage 8.5 (HP-PRRSV), and 2 belonged to sublineage 5.1 (VR-2332-like), indicating that both HP-PRRSV and NADC30-like strains were mainly circulating in Henan and Shanxi provinces. Compared to VR-2332 strain, different types of amino acid mutations were found in the GP5 protein of these 29 strains, and the amino acid deletions were displayed in the Nsp2 protein of the HeN-HC isolate, leading to the variation of protein structures. It is noteworthy that recombination events were identified in the HeN-Ping and HeN-B strains. In addition, a total of 60, 094 pig serum samples from Henan province were collected, and the positive rate of specific antibodies against PRRSV was 86.37 % from 2019 to 2022, and 86.66 %, 84.85 %, 87.54 % and 86.30 % in 2019, 2020, 2021 and 2022, respectively. Overall, this study provides valuable insights into the molecular epidemiology and evolution of PRRSV circulating in central China.

Development of a rapid LFA test based on direct RT-LAMP for diagnosis of SARS-CoV-2

IntroductionIn response to the rapid spread of the SARS-CoV-2 virus, we developed a rapid molecular approach to diagnose COVID-19 without the need for RNA extraction. MethodsThe study utilized two molecular methods, RT-qPCR and colorimetric RT-LAMP, to diagnose the RdRp and ORF8 genes, respectively, in oro-nasopharyngeal swabs. Due to the high sequence diversity of ORF8 in SARS-CoV and SARS-CoV-2, it has been identified as a suitable target for virus detection. The RT-LAMP method was also carried out directly on heat-treated swab samples. The strip tests were made using gold nanoparticles and combined with the RT-LAMP for further analysis. ResultsThe results showed that the isothermal amplification method had a sensitivity of 95 % (95 % C.I.: 86.08 %–98.96 %) and a specificity of 75 % (95 % C.I.: 19.41 %–99.37 %). The RT-LAMP-LFA method was able to distinguish positive and negative samples with 100 % sensitivity (95 % C.I.: 91.96–100) and 77.27 % specificity (95 % C.I.: 54.63–92.18). This method only required heating swab samples for 10 min at 65 °C before the RT-LAMP reaction. ConclusionBy utilizing the RT-LAMP in combination with the LFA, it is possible to diagnose SARS-CoV-2 rapidly without the need for RNA extraction. The entire process from sample collection to test interpretation takes only 75–90 min, and the results can be interpreted by untrained individuals with the naked eye. By employing the ORF8 gene as a diagnostic target and eliminating the need for RNA extraction, the direct RT-LAMP-LFA method achieves a significant breakthrough that was not previously reported.

Hidden evolutionary constraints dictate the retention of coronavirus accessory genes.

Coronaviruses exhibit many mechanisms of genetic innovation, including the acquisition of accessory genes that originate by capture of cellular genes or through duplication of existing viral genes. Accessory genes influence viral host range and cellular tropism, but little is known about how selection acts on these variable regions of virus genomes. We used experimental evolution of mouse hepatitis virus (MHV) encoding a cellular AKAP7 phosphodiesterase and an inactive native phosphodiesterase, NS2 to model the evolutionary fate of accessory genes. After courses of serial infection, the gene encoding inactive NS2, ORF2, unexpectedly remained intact, suggesting it is under cryptic constraint uncoupled from the function of NS2. In contrast, AKAP7 was retained under strong selection but rapidly lost under relaxed selection. Experimental evolution also led to altered viral replication in a cell type-specific manner and changed the relative proportions of subgenomic viral RNA in plaque-purified viral isolates, revealing additional mechanisms of adaptation. Guided by the retention of ORF2 and similar patterns in related betacoronaviruses, we analyzed ORF8 of SARS-CoV-2, which arose via gene duplication and contains premature stop codons in several globally successful lineages. As with MHV ORF2, the coding-defective SARS-CoV-2 ORF8 gene remains largely intact, mirroring patterns observed during MHV experimental evolution, challenging assumptions on the dynamics of gene loss in virus genomes and extending these findings to viruses currently adapting to humans.

A chimeric strain of porcine reproductive and respiratory syndrome virus 2 derived from HP-PRRSV and NADC30-like PRRSV confers cross-protection against both strains

Porcine reproductive and respiratory syndrome (PRRS) is one of the most significant swine viral infectious diseases worldwide. Vaccination is a key strategy for the control and prevention of PRRS. At present, the NADC30-like PRRSV strain has become the predominant epidemic strain in China, superseding the HP-PRRSV strain. The existing commercial vaccines offer substantial protection against HP-PRRSV, but their efficacy against NADC30-like PRRSV is limited. The development of a novel vaccine that can provide valuable cross-protection against both NADC30-like PRRSV and HP-PRRSV is highly important. In this study, an infectious clone of a commercial MLV vaccine strain, GD (HP-PRRSV), was first generated (named rGD). A recombinant chimeric PRRSV strain, rGD-SX-5U2, was subsequently constructed by using rGD as a backbone and embedding several dominant immune genes, including the NSP2, ORF5, ORF6, and ORF7 genes, from an NADC30-like PRRSV isolate. In vitro experiments demonstrated that chimeric PRRSV rGD-SX-5U2 exhibited high tropism for MARC-145 cells, which is of paramount importance in the production of PRRSV vaccines. Moreover, subsequent in vivo inoculation and challenge experiments demonstrated that rGD-SX-5U2 confers cross-protection against both HP-PRRSV and NADC30-like PRRSV, including an improvement in ADG levels and a reduction in viremia and lung tissue lesions. In conclusion, our research demonstrated that the chimeric PRRSV strain rGD-SX-5U2 is a novel approach that can provide broad-spectrum protection against both HP-PRRSV and NADC30-like PRRSV. This may be a significant improvement over previous MLV vaccinations.

Genetic characteristics associated with the virulence of porcine epidemic diarrhea virus (PEDV) with a naturally occurring truncated ORF3 gene

Porcine epidemic diarrhea virus (PEDV) has emerged in American countries, and it has reemerged in Asia and Europe, causing significant economic losses to the pig industry worldwide. In the present study, the 17GXCZ-1ORF3d strain, which has a naturally large deletion at the 172–554 bp position of the ORF3 gene, together with the 17GXCZ-1ORF3c strain, was serially propagated in Vero cells for up to 120 passages. The adaptability of the two strains gradually increased through serial passages in vitro. Genetic variation analysis of the variants of the two strains from different generations revealed that the naturally truncated ORF3 gene in the 17GXCZ-1ORF3d variants was stably inherited. Furthermore, the survival, viral shedding and histopathological lesions following inoculation of piglets demonstrated that the virulence of 17GXCZ-1ORF3d-P120 was significantly attenuated. These results indicate that the naturally truncated ORF3 gene may accelerate the attenuation of virulence and is involved in PEDV virulence together with mutations in other structural genes. Importantly, immunization of sows with G2b 17GXCZ-1ORF3d-P120 increased PEDV-specific IgG and IgA antibody levels in piglets and conferred partial passive protection against heterologous G2a PEDV strains. Our findings suggest that an attenuated strain with a truncated ORF3 gene may be a promising candidate for protection against PEDV.

Identification and biophysical characterization of epitope atlas of Porcine Reproductive and Respiratory Syndrome Virus

Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) have been a critical threat to swine health since 1987 due to its high mutation rate and substantial economic loss over half a billion dollar in USA. The rapid mutation rate of PRRSV presents a significant challenge in developing an effective vaccine. Even though surveillance and intervention studies have recently (2019) unveiled utilization of PRRSV glycoprotein 5 (GP5; encoded by ORF5 gene) to induce immunogenic reaction and production of neutralizing antibodies in porcine populations, the future viral generations can accrue escape mutations. In this study we identify 63 porcine-PRRSV protein-protein interactions which play primary or ancillary roles in viral entry and infection. Using genome–proteome annotation, protein structure prediction, multiple docking experiments, and binding energy calculations, we identified a list of 75 epitope locations on PRRSV proteins crucial for infection. Additionally, using machine learning-based diffusion model, we designed 56 stable immunogen peptides that contain one or more of these epitopes with their native tertiary structures stabilized through optimized N– and C–terminus flank sequences and interspersed with appropriate linker regions. Our workflow successfully identified numerous known interactions and predicted several novel PRRSV-porcine interactions. By leveraging the structural and sequence insights, this study paves the way for more effective, high-avidity, multi-valent PRRSV vaccines, and leveraging neural networks for immunogen design.

The mitochondrial genes orf113b and orf146 from Xinjiang wild rapeseed cause pollen abortion in alloplasmic male sterility

Nsa CMS, a type of cytoplasmic male sterility (CMS) in rapeseed, originates from the cross between Xinjiang wild rapeseed (Sinapis arvensis) and Xiangyou 15 (Brassica napus L.). Although this CMS variant shows promising applications, the factors contributing to its sterility and their underlying mechanisms remain unclear. To the best of our knowledge, we successfully assembled and analyzed the mitochondrial genome of 1258A (Nsa CMS) for the first time. This mitochondrial genome, spanning 263,010 bp, contains 91 genes, including 33 protein-coding and 36 orf genes. Our analysis identified a novel mitochondrial gene, orf113b, and a mutated, truncated gene, orf146, both likely linked to the sterility observed in 1258A. ORF113b and ORF146 were found to impede cell growth, disrupt gene expression associated with complexes I, III, and V of the mitochondrial oxidative phosphorylation pathway, and trigger reactive oxygen species (ROS) production. Additionally, transcriptome data analysis revealed key nuclear genes co-expressed with orf113b and orf146, suggesting that their aberrant expression may be influenced by retrograde signaling from the mitochondria. This signaling could lead to atypical programmed cell death (PCD) in the tapetum layer, resulting in pollen sterility. In conclusion, our study not only provides the first characterization of the Nsa CMS mitochondrial genome but also identifies orf113b and orf146 as crucial to pollen sterility. Furthermore, it suggests that ROS induced by these mitochondrial genes may play a central role in the abnormal regulation of nuclear genes essential for pollen development, offering new insights into the molecular mechanisms underlying Nsa CMS.

Comparison of PCR, Nested PCR, and RT-LAMP for Rapid Detection of Feline Calicivirus Infection in Clinical Samples.

Feline calicivirus (FCV) is a highly contagious virus that causes upper respiratory tract disease, commonly known as cat flu. It is widely distributed worldwide and poses a major threat to feline health. Therefore, it is essential to find an efficient and rapid method for detecting FCV. In this study, the colorimetric reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay, using neutral red as an indicator, was developed and validated to target the ORF2 gene of FCV for the first time. Additionally, the study compared the diagnostic abilities of polymerase chain reaction (PCR), nested PCR, and RT-LAMP assays for detecting FCV in clinical samples. The optimized RT-LAMP amplification was carried out at 56.3 °C. The technique visually detected FCV within 70 min, with a limit of detection of 14.3 × 101 copies/µL, and showed no cross-reactivity with other feline pathogens. Out of 54 oropharyngeal swab samples, 17 tested positive for FCV using both nested PCR and RT-LAMP, while only one tested positive using conventional PCR. The positivity rate was higher with nested PCR and RT-LAMP (31.48%) compared to conventional PCR (1.85%). Consequently, these results demonstrated the effectiveness of the colorimetric RT-LAMP assay developed in this study as an alternative for diagnosing FCV in cats.

Molecular and phylogenetic analysis of transmissible gastroenteritis virus strain VET-16, isolated from piglets in Vietnam.

Porcine transmissible gastroenteritis virus (TGEV) is a major pathogen that causes viral enteritis and severe diarrhea in newborn piglets. TGEV strains have been isolated in the USA, Europe, and China, and their molecular characteristics are well known. However, there have been few reports of molecular analysis of TGEV strains isolated in Southeast Asia. In 2016, we isolated TGEV strain VET-16 from fecal samples collected from piglets in Vietnam and determined its complete genome sequence by Sanger sequencing. We found that, while the full genome of the VET-16 strain was 92.4-99.9% identical to those of other TGEV strains, the ORF3 gene showed very little sequence similarity. Phylogenetic analysis suggested that the VET-16 strain belongs to the Purdue subgroup. Comparison of the predicted amino acid (aa) sequence of the spike protein of strain VET-16 with those of other TGEV strains revealed three aa substitutions (V378L, S379T, and D380N) and a 3-aa insertion (F383_F387insWEK) in antigenic site D of the VET-16 strain. Also, a single aa deletion (∆F1413) was found in the transmembrane domain of the spike gene of VET-16. Like the ORF3 gene from the TGEV Miller M60 vaccine strain, the VET-16 strain has a large deletion (∆725 nt) in the ORF3 gene. Previous studies have suggested that these mutations in the spike and ORF3 genes might be associated with a reduction in pathogenicity. The data from this study will facilitate further genetic analysis and research into the evolution of TGEV in pigs in Vietnam.

Establishment and application of quadruple fluorescence quantitative RT-PCR method for the identification of waterfowl astrovirus.

Avian astrovirus can infect a variety of poultry species and cause viral diarrhea, with a wide epidemic range strong pathogenicity and a high incidence. Among them, Duck astrovirus 3(DAstV-3), Duck astrovirus 4(DAstV-4), Goose astrovirus 1(GoAstV-1) and Goose astrovirus 2(GoAstV-2) are four types of astroviruses newly discovered in waterfowl in recent years. In order to realize the rapid detection of these four kinds of waterfowl stellate viruses, specific primers and probes were engineered to target a highly conserved region of ORF1b gene of DAstV-3, GoAstV-1 and GoAstV-2 and the ORF2 gene of DAstV-4, and a quadruple fluorescence quantitative RT-PCR method was developed. The results indicate that the method established in this study has good specificity and no cross reactivity with other pathogens. This method can detect viruses with a minimum concentration of 1 × 101 copies/μL for DAstV-4, GoAstV-1 and GoAstV-2, respectively, while the minimum concentration for DAstV-3 is 1 × 102 copies/μL. Compared with the routinely used RT-PCR method, the limit of detection by the multiplex RT-PCR lower. Both intra- and inter-assay variability tests revealed excellent reproducibility. This method was then used to analyze 269 field samples, and the results were verified by genome sequencing. In conclusion, this study presents a sensitive, accurate, and specific method for detecting DAstV-3, DAstV-4, GoAstV-1, and GoAstV-2 in a single reaction, enabling the monitoring and differential diagnosis of these four types of waterfowl astroviruses.

Molecular epizootiology of porcine reproductive and respiratory syndrome virus in the Xinjiang Uygur Autonomous Region of China.

Rapid evolution of porcine reproductive and respiratory syndrome virus (PRRSV) is the bottleneck for effective prevention and control of PRRS. Thus, understanding the prevalence and genetic background of PRRSV strains in swine-producing regions is important for disease prevention and control. However, there is only limited information about the epizootiological situation of PRRS in the Xinjiang Uygur Autonomous Region, China. In this study, blood or lung tissue samples were collected from 1,411 PRRS-suspected weaned pigs from 9 pig farms in Changji, Shihezi, and Wujiaqu cities between 2020 and 2022. The samples were first tested by RT-quantitative PCR, yielding a PRRSV-2 positive rate of 53.6%. Subsequently, 36 PRRSV strains were isolated through initial adaptation in bone marrow-derived macrophages followed by propagation in grivet monkey Marc-145 cells. Furthermore, 28 PRRSV-positive samples and 20 cell-adapted viruses were selected for high-throughput sequencing (HTS) to obtain the entire PRRSV genome sequences. Phylogenetic analysis based on the nucleotide sequences of the ORF5 gene of the PRRSV strains identified in this study grouped into sub-lineages 1.8 and 8.7 the former being the dominant strain currently circulating in Xinjiang. However, the NSP2 proteins of the Xinjiang PRRSV strains shared the same deletion patterns as sub-lineage 1.8 prototype strain NADC30 with the exception of 4 strains carrying 2-3 additional amino acid deletions. Further analysis confirmed that recombination events had occurred in 27 of 37 PRRSVs obtained here with the parental strains belonging to sub-lineages 1.8 and 8.7, lineages 3 and 5, with the recombination events having occurred most frequently in the 5' and 3' termini of ORF1a and 5' terminus of ORF1b.

Complete genome characterization and phylogenetic analysis of a novel polyomavirus detected in Eurasian beavers (Castor fiber)

The Eurasian beaver (Castor fiber), native to Hungary, faced local extinction in 1865 and was successfully reintroduced between mid-1980s and 2008. Despite screening programs focusing on animal health during reintroduction in other countries, information about viruses in the Hungarian beaver population remains limited. Polyomaviruses (PyVs) have been identified in various rodents, and have been detected just recently in beavers by us. In this paper we present the full genome analysis of the first PyV detected in Eurasian beaver. The novel PyV was discovered in the kidney tissues of two specimens. The genome is 5244 bp, and contains four genes. Small T-antigen (STAg) and alternative large T ORF (ALTO) genes are directly fused together forming the middle T-antigen (MTAg). VP3 is absent from the genome. Its large T-antigen (LTAg) coding sequence exhibited over 15% genetic divergence from known PyVs, supporting its classification into a new species within the genus Alphapolyomavirus, suggesting to be named Alphapolyomavirus castoris. Phylogenetic analysis, based on the LTAg gene showed, that the beaver PyV forms a distinct clade with primate PyVs within the genus Alphapolyomavirus, separate from other rodent PyVs. Phylogenetic study of the VP1 gene however showed this virus to belong in a distinct clade with the same primate PyVs, and additionally PyVs from rodents and a myocastor, which suggest host virus co-evolution. The virus detection of the euthanized beavers suggests an apathogenic persistent infections. The aquatic lifestyle of beavers may influence virus transmission, warranting further exploration of undiscovered viruses in beavers.

Evolution and virulence of porcine epidemic diarrhea virus following in vitro and in vivo propagation

Practice of inoculating porcine epidemic diarrhea virus (PEDV) in piglets generating feedback material might influence the genetic evolution and attenuation of PEDV. The study was conducted to evaluate evolutionary rate and attenuation following serial in vitro and in vivo propagation. In the study, PED-JPFP0-PJ, Passage 0 (P0), was isolated from infected pigs and serially passaged in Vero cells for 5 consecutive times, P1-P5. P0, P2 and P5 were then subjected to orally inoculate 3-day-old piglets. At 24 h post inoculation, intestines of each passage (F1), were collected, and subsequently sub-passaged in piglets for 2 additional passages (F2-F3). Virus titration, PEDV genomic copies number, VH:CD ratios, and immunohistochemistry were evaluated. S and ORF3 genes were characterized. The results of the study demonstrated that virus titer and virulence were negatively correlated with increased passages, both in vitro and in vivo. Increased substitution rate was observed in higher passages. The evolutionary rate of S gene was higher than that of ORF3. Seven aa changes at positions 223, 291, 317, 607, 694, 1114 and 1199, with reduced N-linked glycan were observed in P5F3. In conclusion, serial passage of PEDV, both in vitro and in vivo, influence the genetic development and the attenuation of PEDV.

Genetic substructure and host-specific natural selection trend across vaccine-candidate ORF-2 capsid protein of hepatitis-E virus.

Hepatitis E virus is a primary cause of acute hepatitis worldwide. The present study attempts to assess the genetic variability and evolutionary divergence among HEV genotypes. A vaccine promising capsid-protein coding ORF-2 gene sequences of HEV was evaluated using phylogenetics, model-based population genetic methods and principal component analysis. The analyses unveiled nine distinct clusters as subpopulations for six HEV genotypes. HEV-3 genotype samples stratified into four different subgroups, while HEV-4 stratified into three additional subclusters. Rabbit-infectious HEV-3ra samples constitute a distinct cluster. Pairwise analysis identified marked genetic distinction of HEV-4c and HEV-4i subgenotypes compared to other genotypes. Numerous admixed, inter and intragenotype recombinant strains were detected. The MEME method identified several ORF-2 codon sites under positive selection. Some selection signatures lead to amino acid substitutions within ORF-2, resulting in altered physicochemical features. Moreover, a pattern of host-specific adaptive signatures was identified among HEV genotypes. The analyses conclusively depict that recombination and episodic positive selection events have shaped the observed genetic diversity among different HEV genotypes. The significant genetic diversity and stratification of HEV-3 and HEV-4 genotypes into subgroups, as identified in the current study, are noteworthy and may have implications for the efficacy of anti-HEV vaccines.

The Development of a Sensitive Droplet Digital Polymerase Chain Reaction Test for Quantitative Detection of Goose Astrovirus

(1) Goose astrovirus (GAstV) is a novel emerging pathogen that causes significant economic losses in waterfowl farming. A convenient, sensitive, and specific detection method for GAstV in field samples is important in order to effectively control GAstV. Droplet digital polymerase chain reaction (ddPCR) is a novel, sensitive, good-precision, and absolute quantitation PCR technology which does not require calibration curves. (2) In this study, we developed a ddPCR system for the sensitive and accurate quantification of GAstV using the conserved region of the ORF2 gene. (3) The detection limit of ddPCR was 10 copies/µL, ~28 times greater sensitivity than quantitative real-time PCR (qPCR). The specificity of the test was determined by the failure of amplification of other avian viruses. Both ddPCR and qPCR tests showed good repeatability and linearity, and the established ddPCR method had high sensitivity and good specificity to GAstV. Clinical sample test results showed that the positive rate of ddPCR (88.89%) was higher than that of qPCR (58.33%). (4) As a result, our results suggest that the newly developed ddPCR method might offer improved analytical sensitivity and specificity in its GAstV measurements. The ddPCR could be widely applied in clinical tests for GAstV infections.

XBB.1, BQ1.1 and atypical BA.4.6/XBB.1 recombinants predominate current SARS-CoV-2 Wavelets with flu-like symptoms in Cameroon: A snapshot from genomic surveillance.

As of December 2022, Cameroon had observed a slight resurgence of COVID-19, raising concerns on genomic surveillance of related-SARS-CoV-2 variants under circulation. Following a laboratory-based survey, positive SARS-CoV-2 samples detected from December-2022 through March-2023 were processed for targeted sequencing at the Chantal BIYA International Reference Centre (CIRCB) in Yaoundé-Cameroon. From all positive cases detected, 13 were successfully sequenced (mean age 34 years, 70% female); the majority of the cases were unvaccinated (70%, 9/13) and symptomatic (92%, 12/13); all with flu-like symptoms (100%, 12/12). Following RT-PCR, the median cycle threshold was 22.23 [18-24] for the N gene; and 24.09 [20-26] for the ORF gene, underscoring high viral loads. Phylogenetic analysis of nucleotide sequences identified four major sub-variants in circulation, of which BA.5 (3/13), the recombinants BQ.1.1 (4/13), XBB.1 (4/13) and novel atypical variant of BA.4.6/XBB.1 (2/13). This snapshot surveillance indicates the introduction/emergence and circulation of new Omicron sub-variants, all accompanied by minor/mild symptoms. However, these new sub-variants and recombinants call for continuous genomic surveillance to prevent further resurgence of Covid-19 epidemiological wave.