Hepatitis E virus (HEV) is a major cause of acute viral hepatitis globally. Genotype 1 HEV (HEV-1) is responsible for multiple outbreaks in developing countries, causing high mortality rates in pregnant women. However, studies on HEV-1 have been hindered by its poor replication in cultured cells. The JE04-1601S strain recovered from a Japanese patient with fulminant hepatitis E who contracted HEV-1 while traveling to India was serially passaged 12 times in human cell lines. The cell-culture-generated viruses (passage 12; p12) grew efficiently in human cell lines, but the replication was not fully supported in porcine cells. A full-length cDNA clone was constructed using JE04-1601S_p12 as a template. It was able to produce an infectious virus, and viral protein expression was detectable in the transfected PLC/PRF/5 cells and culture supernatants. Consistently, HEV-1 growth was also not fully supported in the cell culture of cDNA-derived JE04-1601S_p12 progenies, potentially recapitulating the narrow tropism of HEV-1 observed in vivo. The availability of an efficient cell culture system for HEV-1 and its infectious cDNA clone will be useful for studying HEV species tropism and mechanisms underlying severe hepatitis in HEV-1-infected pregnant women as well as for discovering and developing safer treatment options for this condition.

The chromatin environment at origins of replication is thought to influence DNA replication initiation in eukaryotic genomes. However, it remains unclear how and which chromatin features control the firing of early-efficient (EE) or late-inefficient (LI) origins. Here, we use site-specific recombination and single-locus chromatin isolation to purify EE and LI replication origins in Saccharomyces cerevisiae. Using mass spectrometry, we define the protein composition of native chromatin regions surrounding the EE and LI replication start sites. In addition to known origin interactors, we find the microtubule-binding Ask1/DASH complex as an origin-regulating factor. Strikingly, tethering of Ask1 to individual origin sites advances replication timing (RT) of the targeted chromosomal domain. Targeted degradation of Ask1 globally changes RT of a subset of origins, which can be reproduced by inhibiting microtubule dynamics. Thus, our findings mechanistically connect RT and chromosomal organization via Ask1/DASH with the microtubule cytoskeleton.

There are four major porcine reproductive and respiratory syndrome virus 2 (PRRSV2) lineages circulating in China based on classification system, including lineages 1 (NADC30-like), 3 (QYYZ-like), 5.1 (VR2332-like) and 8 (JXA1-like/CH-1a-Like), which leads to the potential recombination. In the present study, a novel variant of PRRSV2 strain named JS18-3 was isolated from piglets suffering severe breathing difficulties in Jiangsu Province of China in 2018. Full-length genome analysis indicated that JS18-3 shared 86.5%, 87.9%, 84.2%, 82.2% and 86.4% nucleotide similarity with PRRSVs CH-1a, JXA1, VR2332, QYYZ and NADC30, respectively. 4871-6635 of JS18-3 shared the highest identity of 99.3% in nucleotide sequence with HP-PRRSV representative strain JXA1 indicating ongoing evolution to HP-PRRSV. JS18-3 was classified into classical lineage 8 of PRRSV2 based on phylogenetic analysis of complete genome and ORF5. Genomic break points in structural (ORF3) and non-structural (NSP2, NSP3) regions of genomes were detected in recombination analysis. JS18-3 is a recombinant isolate from lineages 8, 1 and 3. Replication enhancement and severe cytopathic effects caused by JS18-3 were observed in Marc-145 cells and porcine alveolar macrophages (PAMs) as compared to JX07, a typical strain of lineage 8. Pathogenicity results indicated that piglets inoculated with JS18-3 presented persistent fever, dyspnoea, serious microscopic lung lesions and lymph node congestion. The study suggests that lineage 8 of PRRSV2 is involved in continuing evolution by genetic recombination and mutation leading to outbreaks in vaccinated pigs in China.

BACKGROUND: PRRSV is an infectious illness causing lung injury and abortion in sows. Cells apoptosis in the interface between the endometrium and fetal placenta is a crucial factor causing abortion. Previous study confirmed PRRSV could cause apoptosis of macrophages but rarely produced an obvious change in porcine endometrial epithelial cells (PECs). Recently, PRRSV-induced abortion was attributed to fetal placental and endometrium epithelial cells (Sn+ and CD163+) apoptosis. However, the mechanism of abortion is still unrevealed because of the limit of porcine endometrium epithelial cells (PEC). The aim of this study was to establish a stable immortalized PECs lines and use it to reveal the abortion mechanism.
RESULTS: In this study, highly purified primary PECs were harvested through differential digestion, and their characteristics were confirmed by CK18, ERɑ and PR staining. Cells were then immortalized by transfecting a lentiviral vector that expressed SV40 large T antigen. PECs lines were obtained after puromycin screening. Proliferation of cell line was evaluated by cell growth curve and cell cycle assays. Cell lines exhibited faster proliferation capacity than primary cells. Biological characteristics of cell line were assessed by Western blot, karyotype analysis and staining, which confirmed that the cell line retained the endometrium characteristics. Finally, PRRSV sensitivity was assessed; expression of Sn and CD163 indicated that primary PECs and cell lines were all potentially sensitive to PRRSV. PRRSV infection tests showed an obvious increase in apoptotic rate in the infected PEC cell line, which suggested its susceptibility.
CONCLUSIONS: The newly constructed cell line is a useful tool for studying the mechanism of abortion caused by PRRSV.

Genotype 3 hepatitis E virus (HEV) infection is generally associated with mild disease. However, recently eight genotype 3 HEV isolates were identified from patients with severe hepatitis. Importantly, three mutations (S605P, I978V and V1213A) in these genotype 3 isolates were found to be typical of genotype 4 HEV, which is sometime associated with more severe hepatitis. Therefore in this study we seek to determine if these unique mutations contribute to enhanced virus replication and thus potentially severe disease.
In the lack of an efficient cell culture system to study the effect of mutations on HEV replication, we developed a genotype 3 HEV replicon with Renilla luciferase (Rluc) as reporter and subsequently used it to construct numerous mutants, including swMu-1 (V1213A), swMu-2 (Q1246H), swMu-3 (V1213A and Q1246H), swMu-4 (S605P and I978V), and swMu-5 (V1213A, S605P and I978V). RNA transcripts from mutant replicons were transfected into Huh7 S10-3 liver cells to measure the effect of mutations on HEV replication efficiency.
The results showed that the V1213A mutant had the highest reduction in HEV replication efficiency than other mutants. The V1213A and S605P + I978V mutations have a cumulative, if not synergistic, effect on HEV replication. The Q1246H mutant decreased HEV replication compared to the wild-type HEV Rluc replicon but replicated better than the V1213A mutant. The amino acid residue V1213 favors the replication of both genotypes 3 and 4 HEV strains, but not genotype 1 HEV.
The results suggested that the V1213A mutation reduced HEV replication, but is likely not associated with the reported severe hepatitis caused by genotype 3 HEV isolates containing this mutation.

The highly pathogenic porcine reproductive and respiratory syndrome virus (HP-PRRSV) has caused huge economic losses to the swine industry in China. Understanding the molecular basis in relation to the virulence of HP-PRRSV is essential for effectively controlling clinical infection and disease. In the current study, we constructed and rescued a serial of mutant viruses in nsp9 and nsp10 based on the differential amino acid sites between HP-PRRSV JXwn06 and LP-PRRSV HB-1/3.9. The replication efficiency in pulmonary alveolar macrophages (PAMs) and the pathogenicity of the mutant viruses for piglets were analyzed. Our results showed that the mutation of Thr to Ala in 586 and Ser to Thr in 592 of nsp9 decreased the replication efficiency of HP-PRRSV in PAMs, and could attenuate its virulence for piglets, suggesting that the residues 586 and 592 of nsp9 are critical sites natively in determining the fatal virulence of the Chinese HP-PRRSV for piglets.