Genetic Polymorphism and mRNA Expression Studies Reveal IL6R and LEPR Gene Associations with Reproductive Traits in Chinese Holsteins

Genetic selection of milk yield traits alters the energy distribution of high producing cows, resulting in gene-induced negative energy balance, and consequently, poor body condition scores and reduced reproductive performances. Here, we investigated two metabolic-syndrome pathway genes, IL6R (Interleukin 6 receptor) and LEPR (Leptin receptor), for their polymorphism effects on reproductive performance in dairy cows, by applying polymorphism association analyses in 1588 Chinese Holstein cows (at population level) and gene expression analyses in granulosa cells isolated from eight cows (at cell level). Among the six single nucleotide polymorphisms we examined (two SNPs for IL6R and four SNPs for LEPR), five were significantly associated with at least one reproductive trait, including female fertility traits covering both the ability to recycle after calving and the ability to conceive and keep pregnancy when inseminated properly, as well as calving traits. Notably, the identified variant SNP g.80143337A/C in LEPR is a missense variant. The role of IL6R and LEPR in cattle reproduction were further confirmed by observed differences in relative gene expression levels amongst granulosa cells with different developmental stages. Collectively, the functional validation of IL6R and LEPR performed in this study improved our understanding of cattle reproduction while providing important molecular markers for genetic selection of reproductive traits in high-yielding dairy cattle

Genome-wide association mapping for dominance effects in female fertility using real and simulated data from Danish Holstein cattle

Exploring dominance variance and loci contributing to dominance variation is important to understand the genetic architecture behind quantitative traits. The objectives of this study were i) to estimate dominance variances, ii) to detect quantitative trait loci (QTL) with dominant effects, and iii) to evaluate the power and the precision of identifying loci with dominance effect through post-hoc simulations, with applications for female fertility in Danish Holstein cattle. The female fertility records analyzed were number of inseminations (NINS), days from calving to first insemination (ICF), and days from the first to last insemination (IFL), covering both abilities to recycle and to get pregnant in the female reproductive cycle. There were 3,040 heifers and 4,483 cows with both female fertility records and Illumina BovineSNP50 BeadChip genotypes (35,391 single nucleotide polymorphisms (SNP) after quality control). Genomic best linear unbiased prediction (BLUP) models were used to estimate additive and dominance genetic variances. Linear mixed models were used for association analyses. A post-hoc simulation study was performed using genotyped heifers' data. In heifers, estimates of dominance genetic variances for female fertility traits were larger than additive genetic variances, but had large standard errors. The variance components for fertility traits in cows could not be estimated due to non-convergence of the statistical model. In total, five QTL located on chromosomes 9, 11 (2 QTL), 19, and 28 were identified and all of them showed both additive and dominance genetic effects. Among them, the SNP rs29018921 on chromosome 9 is close to a previously identified QTL in Nordic Holstein for interval between first and last insemination. This SNP is located in the 3' untranslated region of gene peptidylprolyl isomerase like 4 (PPIL4), which was shown to be associated with milk production traits in US Holstein cattle but not known for fertility-related functions. Simulations indicated that the current sample size had limited power to detect QTL with dominance effects for female fertility probably due to low QTL variance. More females need to be genotyped to achieve reliable mapping of QTL with dominance effects for female fertility

Genetic Polymorphism and mRNA Expression Studies Reveal <i>IL6R</i> and <i>LEPR</i> Gene Associations with Reproductive Traits in Chinese Holsteins

Genetic selection of milk yield traits alters the energy distribution of high producing cows, resulting in gene-induced negative energy balance, and consequently, poor body condition scores and reduced reproductive performances. Here, we investigated two metabolic-syndrome pathway genes, IL6R (Interleukin 6 receptor) and LEPR (Leptin receptor), for their polymorphism effects on reproductive performance in dairy cows, by applying polymorphism association analyses in 1588 Chinese Holstein cows (at population level) and gene expression analyses in granulosa cells isolated from eight cows (at cell level). Among the six single nucleotide polymorphisms we examined (two SNPs for IL6R and four SNPs for LEPR), five were significantly associated with at least one reproductive trait, including female fertility traits covering both the ability to recycle after calving and the ability to conceive and keep pregnancy when inseminated properly, as well as calving traits. Notably, the identified variant SNP g.80143337A/C in LEPR is a missense variant. The role of IL6R and LEPR in cattle reproduction were further confirmed by observed differences in relative gene expression levels amongst granulosa cells with different developmental stages. Collectively, the functional validation of IL6R and LEPR performed in this study improved our understanding of cattle reproduction while providing important molecular markers for genetic selection of reproductive traits in high-yielding dairy cattle

RPS5 interacts with the rabbit hemorrhagic disease virus 3' extremities region and plays a role in virus replication

Rabbit hemorrhagic disease virus (RHDV), a member of Caliciviridae family, causes a highly contagious disease in rabbits. The RHDV replication mechanism is poorly understood due to the lack of a suitable culture system in vitro. This study identified RHDV 5' and 3' extremities (Ex) RNA binding proteins from the rabbit kidney cell line RK-13 based on a pull-down assay by applying a tRNA scaffold streptavidin aptamer. Using mass spectrometry (MS), several host proteins were discovered which interact with RHDV 5' and 3' Ex RNA. The ribosomal protein S5 (RPS5) was shown to interact with RHDV 3' Ex RNA directly by RNA-pulldown and confocal microscopy. To further investigate the role of RPS5 in RHDV replication, small interfering RNAs for RPS5 and RPS5 eukaryotic expression plasmids were used to change the expression level of RPS5 in RK-13 cells and the results showed that the RHDV replication and translation levels were positively correlated with the expression level of RPS5. It was also verified that RPS5 promoted RHDV replication by constructing RPS5 stable overexpression cell lines and RPS5 knockdown cell lines. In summary, it has been identified that RPS5 interacted with the RHDV 3' Ex RNA region and played a role in virus replication. These results will help to understand the mechanism of RHDV replication

Variance components and correlations of female fertility traits in Chinese Holstein population

Abstract Background The objective of the present study was to estimate (co)variance components of female fertility traits in Chinese Holsteins, considering fertility traits in different parities as different traits. Data on 88,647 females with 215,632 records (parities) were collected during 2000 to 2014 from 32 herds in the Sanyuan Lvhe Dairy Cattle Center, Beijing, China. The analyzed female fertility traits included interval from calving to first insemination, interval from first to last insemination, days open, conception rate at first insemination, number of inseminations per conception and non-return rates within 56 days after first insemination. Results The descriptive statistics showed that the average fertility of heifers was superior to that of cows. Moreover, the genetic correlations between the performances of a trait in heifers and in cows were all moderate to high but far from one, which suggested that the performances of a trait in heifers and cows should be considered as different but genetically correlated traits in genetic evaluations. On the other hand, genetic correlations between performances of a trait in different parities of cows were greater than 0.87, with only a few exceptions, but variances were not homogeneous across parities for some traits. The estimated heritabilities of female fertility traits were low; all were below 0.049 (except for interval from calving to first insemination). Additionally, the heritabilities of the heifer interval traits were lower than those of the corresponding cow interval traits. Moreover, the heritabilities of the interval traits were higher than those of the threshold traits when measuring similar fertility functions. In general, estimated genetic correlations between traits were highly consistent with the biological categories of the female fertility traits. Conclusions Interval from calving to first insemination, interval from first to last insemination and non-return rates within 56 days after first insemination are recommended to be included in the selection index of the Chinese Holstein population. The parameters estimated in the present study will facilitate the development of a genetic evaluation system for female fertility traits to improve the reproduction efficiency of Chinese Holsteins

Divergence Analyses of Sperm DNA Methylomes between Monozygotic Twin AI Bulls

Semen quality is critical for fertility. However, it is easily influenced by environmental factors and can induce subfertility in the next generations. Here, we aimed to assess the impacts of differentially methylated regions and genes on semen quality and offspring fertility. A specific pair of monozygotic (MZ) twin artificial insemination (AI) Holstein bulls with moderately different sperm qualities (Bull1 &gt; Bull2) was used in the study, and each twin bull had produced ~6000 recorded daughters nationwide in China. Using whole genome bisulfite sequencing, we profiled the landscape of the twin bulls&rsquo; sperm methylomes, and we observed markedly higher sperm methylation levels in Bull1 than in Bull2. Furthermore, we found 528 differentially methylated regions (DMR) between the MZ twin bulls, which spanned or overlapped with 309 differentially methylated genes (DMG). These DMG were particularly associated with embryo development, organ development, reproduction, and the nervous system. Several DMG were also shown to be differentially expressed in the sperm cells. Moreover, the significant differences in DNA methylation on gene INSL3 between the MZ twin bulls were confirmed at three different age points. Our results provided new insights into the impacts of AI bull sperm methylomes on offspring fertility

Corrigendum to “Nucleolin interacts with the rabbit hemorrhagic disease virus replicase RdRp, nonstructural proteins p16 and p23, playing a role in virus replication”<[Virologica Sinica 37 (2022) 48–59]>, (S1995820X22000049), (10.1016/j.virs.2022.01.004)

Due to our negligence, the original version of this article, published online on 12 January 2022, contained a mistake in Fig. 4A. The lane of β-actin in Western blotting was misused. The correct Fig. 4 is given below. We apologize for our oversight when preparing the figure and state that this does not change the scientific conclusions of the article in any way.[Formula presented] Fig. 4. Identification of interactions between RHDV nonstructural proteins and host factors of RCs. A NCL siRNA inhibited the formation of the RHDV RC. After HA tag affinity purification, the eluted proteins were resolved by SDS-PAGE. The protein bands were visualized with silver staining. PBS acted as a negative control; β-actin acted as an internal control and was detected by IB with mAb against β-actin. B Identification of these interactions by M2H assays. Bait and prey plasmids were co-transfected with pG5luc plasmids into subconfluent HEK-293T cells at a molar ratio of 1:1:1 for the pACT:pBIND:pG5luc vector. At 48 h post-transfection (hpt), the HEK-293T cells were lysed, and Rluc and Fluc activities were evaluated using the Promega Dual-Luciferase Reporter Assay System. All experimental groups were compared with the negative control group (ACT-Bind). Statistical analysis was performed by Student t-tests. ∗P < 0.05 and ∗∗P < 0.01. Data are shown as mean with SD. Replicate 1, 2, 3 means three independent experiments, and each experiment contains three technical replicate values. The number of cells used in all replicate experiments was similar. C These interactions were verified using Co-IP assays. RK-13 cells were co-transfected with bait and prey plasmids. Cell lysates were prepared 48 hpt and the proteins were subjected to IP followed by IB analysis. Myc fusion proteins acted as bait proteins and Flag fusion proteins acted as prey proteins. RHDV, rabbit hemorrhagic disease virus; RC, replication complex; IP, immunoprecipitation; IB, immunoblotting; mAb, monoclonal antibody; SD, standard deviation

Construction and immunogenicity of novel bivalent virus-like particles bearing VP60 genes of classic RHDV(GI.1) and RHDV2(GI.2)

Rabbit hemorrhagic disease (RHD) is an acute, inflammatory, septic, and devastating infectious disease caused by Rabbit hemorrhagic disease virus (RHDV), which poses a serious threat to the rabbit industry. RHDV2 (GI.2/RHDVb), a recently reported new variant could cause RHD in wild populations, but also RHDV-vaccinated rabbits. For now, both RHDV and RHDV2 are the main causes of RHD. To develop a new subunit vaccine that could protect rabbits against both classic RHDV and RHDV2 infections, we constructed a recombinant baculovirus (Bac-classic RHDV VP60-RHDV2 VP60) containing the VP60 genes of classic RHDV and RHDV2. Both VP60 genes were well expressed simultaneously in Spodoptera frugiperda cells (Sf9) after infection with the recombinant baculovirus. Transmission electron microscopy showed that the recombinant VP60 self-assembled into virus-like particles (VLPs). The antigenicity and immunogenicity of the bivalent VLPs vaccine were examined with animal experiments. Our results demonstrated that both the humoral and cellular immune responses were efficiently induced in rabbits by a subunit vaccine based on the recombinant baculovirus. In addition, all rabbits immunized with the bivalent VLPs vaccine survived after challenged with classic RHDV, and showed no clinical signs of RHD, whereas all the rabbits in the negative control group died from classic RHDV infection and showed typical clinical signs of RHD. In summary, our results indicated that the recombinant baculovirus carrying two VP60 genes is a candidate construct from which to develop a bivalent VLPs vaccine against both classic RHDV and RHDV2 infections.</p

Genetic Polymorphism and mRNA Expression Studies Reveal IL6R and LEPR Gene Associations with Reproductive Traits in Chinese Holsteins

Genetic selection of milk yield traits alters the energy distribution of high producing cows, resulting in gene-induced negative energy balance, and consequently, poor body condition scores and reduced reproductive performances. Here, we investigated two metabolic-syndrome pathway genes, IL6R (Interleukin 6 receptor) and LEPR (Leptin receptor), for their polymorphism effects on reproductive performance in dairy cows, by applying polymorphism association analyses in 1588 Chinese Holstein cows (at population level) and gene expression analyses in granulosa cells isolated from eight cows (at cell level). Among the six single nucleotide polymorphisms we examined (two SNPs for IL6R and four SNPs for LEPR), five were significantly associated with at least one reproductive trait, including female fertility traits covering both the ability to recycle after calving and the ability to conceive and keep pregnancy when inseminated properly, as well as calving traits. Notably, the identified variant SNP g.80143337A/C in LEPR is a missense variant. The role of IL6R and LEPR in cattle reproduction were further confirmed by observed differences in relative gene expression levels amongst granulosa cells with different developmental stages. Collectively, the functional validation of IL6R and LEPR performed in this study improved our understanding of cattle reproduction while providing important molecular markers for genetic selection of reproductive traits in high-yielding dairy cattle

Pathogenicity and immunogenicity of gI/gE/TK-gene-deleted Felid herpesvirus 1 variants in cats

Abstract Background Felid herpesvirus 1 (FHV-1) is a major pathogenic agent of upper respiratory tract infections and eye damage in felines worldwide. Current FHV-1 vaccines offer limited protection of short duration, and therefore, do not reduce the development of clinical signs or the latency of FHV-1. Methods To address these shortcomings, we constructed FHV ∆gIgE-eGFP, FHV ∆TK mCherry, and FHV ∆gIgE/TK eGFP-mCherry deletion mutants (ΔgI/gE, ΔTK, and ΔgIgE/TK, respectively) using the clustered regularly interspaced palindromic repeats (CRISPR)/CRISP-associated protein 9 (Cas9) system (CRISPR/Cas9), which showed safety and immunogenicity in vitro. We evaluated the safety and efficacy of the deletion mutants administered with intranasal (IN) and IN + subcutaneous (SC) vaccination protocols. Cats in the vaccination group were vaccinated twice at a 4-week interval, and all cats were challenged with infection 3 weeks after the last vaccination. The cats were assessed for clinical signs, nasal shedding, and virus-neutralizing antibodies (VN), and with postmortem histological testing. Results Vaccination with the gI/gE-deleted and gI/gE/TK-deleted mutants was safe and resulted in significantly lower clinical disease scores, fewer pathological changes, and less nasal virus shedding after infection. All three mutants induced virus-neutralizing antibodies after immunization. Conclusions In conclusion, this study demonstrates the advantages of FHV-1 deletion mutants in preventing FHV-1 infection in cats