Abstract Reticulophagy reduces Zaire Ebolavirus (EBOV) glycoprotein GP 1,2 expression, and thus increases viral fitness. Reticulophagy degrades endoplasmic reticulum (ER) subdomains containing misfolded proteins to restore the ER proteostasis, but whether degraded cargo proteins are polyubiquitinated is still unknown. Here, we show that the calnexin-calreticulin cycle promoted EBOV-GP 1,2 misfolding via PDIA3/ERp57, and that misfolded GP 1,2 was degraded via reticulophagy dependent on ATG3, ATG5, and SQSTM1/p62. EBOV-GP 1,2 degradation also required its lysine 673 (K673), which served as a substrate for K27-linked ubiquitination. Although E3 ubiquitin ligases RNF26, RNF185, MARCH8, and TRIM25 either degraded or polyubiquitinated EBOV-GP 1,2 , only RNF185 had both activities. Thus, RNF185 polyubiquitinates misfolded EBOV-GP 1,2 in the ER and then SQSTM1/p62 recruits polyubiquitinated GP 1,2 into autophagosomes for autophagic degradation. We conclude that ubiquitin is required for reticulophagy, which has played a major role in other autophagic pathways. In addition, EBOV infection should be blocked by enhanced GP 1,2 degradation via reticulophagy.

Porcine bocavirus (PBoV) was first identified in Sweden in 2009. Due to its association with healthy as well as diseased pigs, its role in clinical disease has not been reported yet. In the present study, bocavirus was identified from the intestinal content of a 30-day-old piglet and its whole genome was constructed and phylogenetic analysis was carried on. The pathogenesis of bocavirus was investigated following orogastric inoculation of the colostrum-deprived newborn piglet with bacteria free intestinal content. The bocavirus-inoculated piglets developed diarrhea, shed virus in the rectal swabs from 18 h post inoculation and developed macroscopic and microscopic lesions in small intestine with virus confirmed by conventional PCR. This study experimentally confirmed pathogenicity and characterized bocavirus as the etiological agent of diarrhea in the colostrum deprived newborn piglets. On phylogenetic analysis, it was observed that this virus has long evolutionary history with subsequent mutation as well as better host adaptation. This study highlights the importance of identifying bocavirus as the etiological agent of viral diarrhea that could threaten livestock, public health as well as economic loss.

Virus infection affects cellular proteostasis and provides an opportunity to study this cellular process under perturbation. The proteostasis network in the endoplasmic reticulum (ER) is composed of the calnexin cycle, and the two protein degradation pathways ER-associated protein degradation (ERAD) and ER-to-lysosome-associated degradation (ERLAD/ER-phagy/reticulophagy). Here we show that calnexin and calreticulin trigger Zaire Ebolavirus (EBOV) glycoprotein GP1,2 misfolding. Misfolded EBOV-GP1,2 is targeted by ERAD machinery, but this results in lysosomal instead of proteasomal degradation. Moreover, the ER Ub ligase RNF185, usually associated with ERAD, polyubiquitinates EBOV-GP1,2 on lysine 673 via ubiquitin K27-linkage. Polyubiquinated GP1,2 is subsequently recruited into autophagosomes by the soluble autophagy receptor sequestosome 1 (SQSTM1/p62), in an ATG3- and ATG5-dependent manner. We conclude that EBOV hijacks all three proteostasis mechanisms in the ER to downregulate GP1,2 via polyubiquitination and show that this increases viral fitness. This study identifies linkages among proteostasis network components previously thought to function independently.

Ebola viruses (EBOV) express cytotoxic structural glycoprotein GP1,2 to mediate virus entry. To explore a novel viral fitness mechanism, we used mass spectrometry to investigate how EBOV-GP1,2 expression is regulated in the endoplasmic reticulum (ER). We found that ER protein 57 (ERp57), a member of protein disulfide isomerases (PDIs) that promote protein folding, targets EBOV-GP1,2 cysteine residues required for correct cross-linking, thus adverselytriggering misfolding, which in turn activates the unfolded protein response. Misfolded EBOV-GP1,2 is targeted by ER-associated protein degradation machinery and, unexpectedly, is degraded in lysosomes via autophagosomes, but not proteasomes. Five other PDIs also have similar inhibitory activity. In contrast, the expression of Marburg virus and some other enveloped virus glycoproteins is increased by ERp57. Thus, PDIs can negatively regulate EBOV glycoprotein expression, which enables optimum viral infectivity and minimizes cytotoxicity. Importantly, PDIs emerge as potential targets for inhibition of Ebola virus infection.

Secretory IgA is critical to prevent the invasion of pathogens via mucosa. However, the key factors and the mechanisms of IgA generation in the porcine gut are not well understood. In this study, a panel of factors, including BAFF, APRIL, CD40L, TGF-β1, IL-6, IL-10, IL-17A, and IL-21, were employed to stimulate IgM+ B lymphocytes from porcine ileum Peyer’s patches. The results showed that IL-21 significantly upregulated IgA production of B cells and facilitated cell proliferation and differentiation of antibody-secreting cells. In addition, three transcripts in porcine IgA class switch recombination (CSR), germ-line transcript α, post-switch transcript α, and circle transcript α, were first amplified by (nest-)PCR and sequenced. All these key indicators of IgA CSR were upregulated by IL-21 treatment. Furthermore, we found that IL-21 predominantly activated JAK1, STAT1, and STAT3 proteins and confirmed that the JAK-STAT signaling pathway was involved in porcine IgA CSR. Thus, IL-21 plays an important role in the proliferation and differentiation of IgA-secreting cells in porcine Peyer’s patches through the JAK-STAT signaling pathway. These findings provide insights into the mucosal vaccine design by regulation of IL-21 for the prevention and control of enteric pathogens in the pig industry.

The intestinal microbiota is crucial to intestinal homeostasis. Porcine epidemic diarrhea virus (PEDV) is high pathogenic to intestines, causing diarrhea, even death in piglets. To investigate the detailed relationship between PEDV infection and intestinal microbiota, the composition and distribution of intestinal microbiota from pigs were first analyzed using 16S rRNA sequencing technology. The results demonstrated that the composition and distribution of microbes in different intestinal segments were quite similar between 1-week-old and 2-week-old piglets but different from 4-week-old (weaned) piglets. Then piglets at different ages were inoculated with PEDV. The results showed that the 1-week-old piglets exhibited the most severe pathogenicity comparing to the other age groups. Further investigations indicated that Lactobacillus, Escherichia coli, and Lactococcus in the intestinal microbiota of piglets were significantly changed by PEDV infection. These results strengthen our understanding of viruses influencing intestinal microbes and remind us of the potential association between PEDV and intestinal microbes.

Glycosylated Gag (glycoGag) is an accessory protein expressed by most gammaretroviruses, including murine leukemia virus (MLV). MLV glycoGag not only enhances MLV replication and disease progression but also increases human immunodeficiency virus type 1(HIV-1) infectivity as Nef does. Recently, SERINC5 (Ser5) was identified as the target for Nef, and the glycoGag Nef-like activity has been attributed to the Ser5 antagonism. Here, we investigated how glycoGag antagonizes Ser5 using MLV glycoMA and murine Ser5 proteins. We confirm previous observations that glycoMA relocalizes Ser5 from plasma membrane to perinuclear punctated compartments and the important role of its Y36XXL39 motif in this process. We find that glycoMA decreases Ser5 expression at steady-state levels and identify two other glycoGag crucial residues, P31 and R63, for the Ser5 downregulation. The glycoMA and Ser5 interaction is detected in live cells using a bimolecular fluorescence complementation assay. Ser5 is internalized via receptor-mediated endocytosis and relocalized to Rab5(+) early, Rab7(+) late, and Rab11(+) recycling endosomes by glycoMA. Although glycoMA is not polyubiquitinated, the Ser5 downregulation requires Ser5 polyubiquitination via the K48- and K63-linkage, resulting in Ser5 destruction in lysosomes. Although P31, Y36, L39, and R63 are not required for glycoMA interaction with Ser5, they are required for Ser5 relocalization to lysosomes for destruction. In addition, although murine Ser1, Ser2, and Ser3 exhibit very poor antiviral activity, they are also targeted by glycoMA for lysosomal destruction. We conclude that glycoGag has a broad activity to downregulate SERINC proteins via the cellular endosome/lysosome pathway, which promotes viral replication. IMPORTANCE MLV glycoGag not only enhances MLV replication but also increases HIV-1 infectivity similarly as Nef. Recent studies have discovered that both glycoGag and Nef antagonize a novel host restriction factor Ser5 and promote viral replication. Compared to Nef, the glycoGag antagonism of Ser5 is still poorly understood. MLV glycoGag is a transmembrane version of the structural Gag protein with an extra 88-amino-acid leader region that determines its activity. We now show that glycoGag interacts with Ser5 in live cells and internalizes Ser5 via receptor-mediated endocytosis. Ser5 is polyubiquitinated and relocalized to endosomes and lysosomes for massive destruction. In addition to the previously identified tyrosine-based sorting signal, we find two more important residues for Ser5 relocalization and downregulation. We also find that the Ser5 sensitivity to glycoGag is conserved in the SERINC family. Together, our findings highlight the important role of endosome/lysosome pathway in the enhancement of viral replication by viral proteins.

The present study aimed to investigate the compensatory effect of early protein restriction followed by a realimentation on growth performance of lamb and to explore the transcriptomic changes in liver. Thirty-two lambs with an initial birth weight of 2.3 ± 0.20 kg that were weaned on day 15 were randomly divided into two groups. The lambs were fed a basal diet with normal protein level (NPL, protein level in the milk replacer and starter, 25 and 21%, respectively) or low protein level (LPL, protein level in the milk replacer and starter, 19 and 15%, respectively) from 15 to 60 d, after which all lambs consumed the same diet with a normal protein level from 61 to 90 d. Protein restriction led to a significant decrease in average daily gain (ADG), body weight and liver weight (P < 0.05). Transcriptome analysis showed that 302 or 12 differentially expressed genes (DEGs) were identified during the restriction or recovery periods, respectively (P < 0.05). The Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis indicated that DEGs enriched in nutrient metabolism and antioxidant capacity were down-regulated, while vessel development and immunity response-related genes up-regulated. The genes involved in metabolism of tyrosine were still down-regulated in the realimentation phase. Studies in this area indicated the accelerated growth effect of early protein restriction followed by a realimentation on growth performance of lambs and explored the transcriptomics change of liver which can help to develop feeding strategies to optimize the use of feedstuffs and in providing a new perspective for the study of early nutrition and epigenetics in later life.

The primate lentiviral accessory protein Nef downregulates CD4 and major histocompatibility complex class I (MHC-I) from the cell surface via independent endosomal trafficking pathways to promote viral pathogenesis. In addition, Nef antagonizes a novel restriction factor, SERINC5 (Ser5), to increase viral infectivity. To explore the molecular mechanism of Ser5 antagonism by Nef, we determined how Nef affects Ser5 expression and intracellular trafficking in comparison to CD4 and MHC-I. We confirm that Nef excludes Ser5 from human immunodeficiency virus type 1 (HIV-1) virions by downregulating its cell surface expression via similar functional motifs required for CD4 downregulation. We find that Nef decreases both Ser5 and CD4 expression at steady-state levels, which are rescued by NH4Cl or bafilomycin A1 treatment. Nef binding to Ser5 was detected in living cells using a bimolecular fluorescence complementation assay, where Nef membrane association is required for interaction. In addition, Nef triggers rapid Ser5 internalization via receptor-mediated endocytosis and relocalizes Ser5 to Rab5+ early, Rab7+ late, and Rab11+ recycling endosomes. Manipulation of AP-2, Rab5, Rab7, and Rab11 expression levels affects the Nef-dependent Ser5 and CD4 downregulation. Moreover, although Nef does not promote Ser5 polyubiquitination, Ser5 downregulation relies on the ubiquitination pathway, and both K48- and K63-specific ubiquitin linkages are required for the downregulation. Finally, Nef promotes Ser5 colocalization with LAMP1, which is enhanced by bafilomycin A1 treatment, suggesting that Ser5 is targeted to lysosomes for destruction. We conclude that Nef uses a similar mechanism to downregulate Ser5 and CD4, which sorts Ser5 into a point-of-no-return degradative pathway to counteract its restriction.IMPORTANCE Human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV) express an accessory protein called Nef to promote viral pathogenesis. Nef drives immune escape in vivo through downregulation of CD4 and MHC-I from the host cell surface. Recently, Nef was reported to counteract a novel host restriction factor, Ser5, to increase viral infectivity. Nef downregulates cell surface Ser5, thus preventing its incorporation into virus particles, resulting in disruption of its antiviral activity. Here, we report mechanistic studies of Nef-mediated Ser5 downregulation in comparison to CD4 and MHC-I. We demonstrate that Nef binds directly to Ser5 in living cells and that Nef-Ser5 interaction requires Nef association with the plasma membrane. Subsequently, Nef internalizes Ser5 from the plasma membrane via receptor-mediated endocytosis, and targets ubiquitinated Ser5 to endosomes and lysosomes for destruction. Collectively, these results provide new insights into our ongoing understanding of the Nef-Ser5 arms race in HIV-1 infection.

We report the effects of a protein-restricted diet and realimentation with a milk replacer (MR) and starter on the growth, digestibility, fermentation, and expression of the genes involved in volatile fatty acid (VFA) absorption in the lamb ruminal epithelium. Sixteen newborn twin Hu lamb pairs were used. Each pair was fed the MR and starter with a normal protein level (NPL, 250 and 210 g/kg, respectively) or a low protein level (LPL, 190 and 150 g/kg, respectively) from 15 to 60 days of age (restriction period). Then, all lambs were fed the starter containing 210 g crude protein/kg diet from 61 to 90 days of age (realimentation period). Intake was measured daily, and body weight was measured at 15, 60, and 90 days of age. Digestibility trials were carried out from 51 to 60 and from 81 to 90 days of age with four lambs randomly selected from each group. Eight lambs from each group were slaughtered on 60 and 90 days of age before morning feeding, and their ruminal fermentation parameters were measured. The expression levels of the genes involved in VFA absorption in ruminal epitheliums were measured. During the restriction period, feed intake did not differ between the groups (P = 0.116). The final body weight (P = 0.006) and the average daily gain (ADG; P = 0.003) was greater, while the feed conversion ratio (P = 0.011) was lower in the NPL group. No difference was observed in dry matter (P = 0.894) and organic matter (P = 0.495) digestibility, or the N retention/N intake (P = 0.607). The ruminal pH of the lambs fed the LPL diets was greater (P = 0.012). A decrease in ammonia content (P = 0.023) and the total VFA concentration (P < 0.001) was found in the LPL lambs. The molecular acetate content was greater in the LPL group (P = 0.002), but that of propionate was lower (P = 0.034). Protein restriction decreased the mRNA abundance of the transporters, anion exchanger 2 (P = 0.019) and was downregulated in adenoma (P = 0.025), Na+/H+ exchanger 1 (P = 0.017), 2 (P = 0.025), and 3 (P = 0.037). After realimentation, body weight (P = 0.006) and ADG (P = 0.013) were lower in the lambs fed the LPL diet. The feed conversion ratio (P = 0.301) was the same in the two groups. Lambs fed a LPL diet had a greater N retention/N intake (P = 0.031). No difference was determined in the ruminal pH or the total VFA concentration between the groups (P = 0.572). A greater mRNA level of monocarboxylic acid transporter 4 (P = 0.021) and Na+/H+ exchanger 2 (P = 0.049) was observed in the LPL group. These results suggest that realimentation partially recovers nutrient requirements and ruminal function, but lambs subjected to protein restriction still physically lag behind those continuously fed the NPL diet, indicating a permanent effect on later growth owing to nutrient restriction during very early life.

ABSTRACT Innate immunity provides an immediate defense against infection after host cells sense danger signals from microbes. Endoplasmic reticulum (ER) stress arises from accumulation of misfolded/unfolded proteins when protein load overwhelms the ER folding capacity, which activates the unfolded protein response (UPR) to restore ER homeostasis. Here, we show that a mechanism for antiviral innate immunity is triggered after the ER stress pathway senses viral glycoproteins. When hemagglutinin (HA) glycoproteins from influenza A virus (IAV) are expressed in cells, ER stress is induced, resulting in rapid HA degradation via proteasomes. The ER-associated protein degradation (ERAD) pathway, an important UPR function for destruction of aberrant proteins, mediates HA degradation. Three class I α-mannosidases were identified to play a critical role in the degradation process, including EDEM1, EDEM2, and ERManI. HA degradation requires either ERManI enzymatic activity or EDEM1/EDEM2 enzymatic activity when ERManI is not expressed, indicating that demannosylation is a critical step for HA degradation. Silencing of EDEM1, EDEM2, and ERManI strongly increases HA expression and promotes IAV replication. Thus, the ER stress pathway senses influenza HA as “nonself” or misfolded protein and sorts HA to ERAD for degradation, resulting in inhibition of IAV replication. IMPORTANCE Viral nucleic acids are recognized as important inducers of innate antiviral immune responses that are sensed by multiple classes of sensors, but other inducers and sensors of viral innate immunity need to be identified and characterized. Here, we used IAV to investigate how host innate immunity is activated. We found that IAV HA glycoproteins induce ER stress, resulting in HA degradation via ERAD and consequent inhibition of IAV replication. In addition, we have identified three class I α-mannosidases, EDEM1, EDEM2, and ERManI, which play a critical role in initiating HA degradation. Knockdown of these proteins substantially increases HA expression and IAV replication. The enzymatic activities and joint actions of these mannosidases are required for this antiviral activity. Our results suggest that viral glycoproteins induce a strong innate antiviral response through activating the ER stress pathway during viral infection.

The Ebola virus (EBOV) trimeric envelope glycoprotein (GP) precursors are cleaved into the receptor-binding GP1 and the fusion-mediating GP2 subunits and incorporated into virions to initiate infection. GP1 and GP2 form heterodimers that have 15 or two N-glycosylation sites (NGSs), respectively. Here we investigated the mechanism of how N-glycosylation contributes to GP expression, maturation, and function. As reported before, we found that, although GP1 NGSs are not critical, the two GP2 NGSs, Asn563 and Asn618, are essential for GP function. Further analysis uncovered that Asn563 and Asn618 regulate GP processing, demannosylation, oligomerization, and conformation. Consequently, these two NGSs are required for GP incorporation into EBOV-like particles and HIV type 1 (HIV-1) pseudovirions and determine viral transduction efficiency. Using CRISPR/Cas9 technology, we knocked out the two classical endoplasmic reticulum chaperones calnexin (CNX) and/or calreticulin (CRT) and found that both CNX and CRT increase GP expression. Nevertheless, NGSs are not required for the GP interaction with CNX or CRT. Together, we conclude that, although Asn563 and Asn618 are not required for EBOV GP expression, they synergistically regulate its maturation, which determines its functionality.

This study investigated the effects of tea saponins (TSP) on milk performance, milk fatty acids, and blood immune function in dairy cows. A total of 20 early-lactation Holstein cows (days in milk = 66.4 ± 16.8 d; parity = 1.75 ± 0.91; and milk yield = 36.3 ± 7.32 kg/d; mean ± standard deviation) were randomly divided into 4 homogeneous treatment groups, with TSP added at 0, 20, 30, and 40 g/d per head, respectively. All cows had 2 wk of adaptation and 6 wk of treatments. Feed, milk, and blood were sampled and analyzed weekly. At the end of the experimental period (wk 6), the dry matter intake and yields of energy-corrected milk, milk, and milk protein, fat, and lactose in the cows fed TSP showed a quadratic response, with the lowest values in cows fed TSP at 40 g/d. The milk fat content of cows fed TSP increased linearly. Significant interactions for treatment by week were found in milk C16:1 cis-9 and C18:1 cis-9, with the highest values at wk 2, 3, and 4 in the cows fed TSP at 40 g/d. The levels declined quickly after 4 wk of feeding to values similar to those for other TSP treatments and the control at wk 5 and 6. Plasma malondialdehyde concentration decreased as the supplement level of TSP increased. The concentration of superoxide dismutase increased as the supplement level of TSP increased. The plasma concentration of tumor necrosis factor-α increased as the supplement level of TSP increased. In summary, this study showed that an intermediate dose of TSP (20 and 30 g/d) had no significant effect on feed intake, but the supplementation of 40 g/d TSP decreased feed intake, resulting in a lower milk yield. The energy-corrected milk of cows fed 40 g/d TSP declined at first but increased after 3 wk of feeding, indicating the potential adaptation to high doses of TSP supplements in dairy cows. The supplementation of TSP could reduce oxidative stress in cows and improve the immunity of dairy cows during 6 wk of feeding.

Eighty Dorper × thin-tailed Han cross-bred non-castrated male lambs [mean body weight (BW), 25.87 ± 1.06 kg] were randomly allocated to one of five different concentrations of slow-release urea (urea phosphate, UP). The feed consisted of an equal amount of concentrate diet and roughage; the concentrate feed was formulated to be isoenergetic and isonitrogenic and contained 0%, 1%, 2%, 4% and 8% UP (UP0.0, UP1.0, UP2.0, UP4.0 and UP8.0, respectively) as a replacement for soya bean meal. Feed intake, BW, average daily gain (ADG), feed utilisation efficiency (FUE), absolute and relative organ weights and biochemical and histopathological parameters were measured. Feed intake, BW, ADG and FUE significantly decreased in the group receiving UP8.0 (p < 0.05), but no difference was found among the other groups (p > 0.05). Quadratic equations were developed between the UP dosage in the concentrate feed and ADG or FUE (r2 = 0.973 for ADG and r2 = 0.761 for FUE) to determine the appropriate dosage of UP given the desire to maximise either ADG or FUE, the appropriate dosage (feed concentration) was calculated as 2.01% UP to achieve the greatest ADG or 2.13% UP to achieve the best FUE. The relative weight of the liver (% BW) in the UP2.0 groups was significantly greater than that of UP0.0 (p < 0.05), and the relative weight of the intestine in the UP8.0 was significantly greater than that of UP0.0 (p < 0.05); the relative weight of the carcass, heart, spleen, lung, kidney, rumen, reticulum, omasum and abomasum did not differ among treatments (p > 0.05). The UP8.0 treatment significantly increased serum phosphorus levels (p < 0.05) and decreased the levels of alkaline phosphatase, glucose and calcium (Ca) compared with the lower UP dosage (p < 0.05). No histopathological differences were found in either hepatic tissues or renal tissues among treatments. Dietary UP as a replacement for soya bean in concentrate feeds for mutton sheep should not exceed 4%, as higher dosing may cause malnutrition and mineral disorders.

This study was conducted to evaluate the efficacy and safety of dietary urea in sheep rations having a 50:50 concentrate:roughage ratio. Sixty-four Dorper × thin-tailed Han crossbred ram lambs with an average body weight of 30.8 (±0.02) kg were randomly divided into four groups of 16 sheep each, and each group was fed one of the following diets: a basal diet (CON), or CON supplemented with 0.5% (0.5UTM), 1.5% (1.5UTM) or 2.5% (2.5UTM) urea. Growth performance, carcass characteristics, non-carcass offals, meat quality and peptic tissue lesions were assayed. The average daily weight gains for CON, 0.5UTM, 1.5UTM and 2.5UTM were 216, 218, 200 and 170 g, respectively, with the CON and 0.5UTM groups higher than 2.5UTM group (p < 0.05). Sheep from the 2.5UTM treatment had a significantly lower dry matter intake (1.29 kg/day) than those from the CON and 0.5UTM treatments (1.42 and 1.43 kg/day, p < 0.05), and the feed conversion ratio in the 2.5UTM group was the highest (p < 0.05). Carcass characteristics, including shrunk body weight, empty body weight, hot carcass weight, dress percentage, and the absolute or relative weight (% body weight) of heart, liver, spleen, lung and kidney, were not altered by the treatments (p > 0.05). The muscular pH of 2.5UTM was higher than that of CON (5.68 vs. 5.52, p < 0.05), and shear force in 0.5UTM was lower compared with CON and 2.5UTM (p < 0.05). The anatomical structure lesions in kidneys became more serious with the increasing dietary urea concentrations, with the 2.5UTM animals showing the most severe lesions compared with CON animals. Therefore, supplementary urea as a non-protein nitrogen source for sheep should not exceed 1.5% of ration having a 50:50 concentrate:roughage ratio to ensure efficacy and safety.

Glycosylated protein 3 (GP3) of PRRSV is variable between different PRRSV strains, so it is helpful for subtype classifying by using distinct epitopes. In this study, two dominant linear GP3 epitopes that were recognized by highly dilute serum in an enzyme-linked immunosorbent assay (ELISA) were identified. Sequence alignments of 36 North American (NA) PRRSV isolates revealed that the epitope H(87)DELGFMV(94) is well conserved, whereas the epitope T(59)RQAAAEILE(68) differs in other low-virulence NA-type strains, which have at least one amino acid mutation in this region. A mutational analysis revealed that none of these mutations could be recognized by the purified antibodies directed against the corresponding epitope, indicating that the genetic variations altered the antigenicity of the antigenic region. Using ELISA, we also found that antibodies directed against the two epitopes were present in more than 45 of 50 HP-PRRS-positive pig sera, suggesting that their antigenicity is excellent in vivo.

Loop-mediated isothermal amplification (LAMP) is a sensitive method for DNA amplification. In the present report, the development of a single-tube, one-step, real-time accelerated reverse transcription (RT)-LAMP for the detection of Porcine teschovirus (PTV) is described. Six designed primers amplified target gene sequences successfully at constant temperature (65°C) within 1 hr, and the amplification results could be visualized directly by the naked eye. The sensitivity of the LAMP was 10 times higher than that of conventional polymerase chain reaction, and no cross-reactivity was found when the genomes of other common swine pathogens were subjected to the RT-LAMP system. When 43 clinical samples were tested by the RT-LAMP method, results indicated that the test is simple, rapid, accurate, and sensitive for the detection of PTV.