Roles of conserved residues in the receptor binding sites of human parainfluenza virus type 3 HN protein.

Human parainfluenza virus type 3 (hPIV-3) entry and intrahost spread through membrane fusion are initiated by two envelope glycoproteins, hemagglutinin-neuraminidase (HN) and fusion (F) protein. Binding of HN protein to the cellular receptor via its receptor-binding sites triggers conformational changes in the F protein leading to virus-cell fusion. However, little is known about the roles of individual amino acids that comprise the receptor-binding sites in the fusion process. Here, residues R192, D216, E409, R424, R502, Y530 and E549 located within the receptor-binding site Ⅰ, and residues N551 and H552 at the putative site Ⅱ were replaced by alanine with site-directed mutagenesis. All mutants except N551A displayed statistically lower hemadsorption activities ranging from 16.4% to 80.2% of the wild-type (wt) level. With standardization of the number of bound erythrocytes, similarly, other than N551A, all mutants showed reduced fusogenic activity at three successive stages: lipid mixing (hemifusion), content mixing (full fusion) and syncytium development. Kinetic measurements of the hemifusion process showed that the initial hemifusion extent for R192A, D216A, E409A, R424A, R502A, Y530A, E549A and H552A was decreased to 69.9%, 80.6%, 71.3%, 67.3%, 50.6%, 87.4%, 84.9% and 25.1%, respectively, relative to the wt, while the initial rate of hemifusion for the E409A, R424A, R502A and H552A mutants was reduced to 69.0%, 35.4%, 62.3%, 37.0%, respectively. In addition, four mutants with reduced initial hemifusion rates also showed decreased percentages of F protein cleavage from 43.4% to 56.3% of the wt. Taken together, Mutants R192A, D216A, E409A, R424A, R502A, Y530A, E549A and H552A may lead to damage on the fusion activity at initial stage of hemifusion, of which decreased extent and rate may be associated with impaired receptor binding activity resulting in the increased activation barrier of F protein and the cleavage of it, respectively.

Research progress on coronavirus S proteins and their receptors.

Coronaviruses are a large family of important pathogens that cause human and animal diseases. At the end of 2019, a pneumonia epidemic caused by a novel coronavirus brought attention to coronaviruses. Exploring the interaction between the virus and its receptor will be helpful in developing preventive vaccines and therapeutic drugs. The coronavirus spike protein (S) plays an important role in both binding to receptors on host cells and fusion of the viral membrane with the host cell membrane. This review introduces the structure and function of the S protein and its receptor, focusing on the binding mode and binding region of both.

Pathogenic New World Relapsing Fever Borrelia in a Myotis Bat, Eastern China, 2015.

We identified Candidatus Borrelia fainii, a human pathogenic bacterium causing New World relapsing fever in a Myotis bat in eastern China. This finding expands knowledge about the geographic distribution of Borrelia spp. and the potential for infection with New World relapsing fever in China.

Pathogenic Leptospira Species in Insectivorous Bats, China, 2015.

PCR amplification of the rrs2 gene indicated that 50% (62/124) of insectivorous bats from eastern China were infected with Leptospira borgpetersenii, L. kirschneri, and several potentially new Leptospira species. Multilocus sequence typing defined 3 novel sequence types in L. kirschneri, suggesting that bats are major carriers of Leptospira.

Contribution of 3CD Region to the Virulence of Enterovirus 71.

Enterovirus 71 is a neuroinvasive virus that is associated with severe neurological complications. We had earlier suggested that the replication capacity of a severe strain was higher than that of a mild strain. The recombinant 3CRV and 3CDRV virus strains were successfully rescued in our previous study. In the present study, we found no difference in virulence between 3CRV and severe strains. However, the capacity of replication and to cause cell injury of 3CDRV strain decreased in vitro, especially at 39.5 °C. Replacement of 3CD region in the severe strain led to milder symptoms, less body weight loss, and lower viral load in ICR mice. Histopathological findings indicated less severe injury in mice infected with 3CDRV strain. This study suggests that the 3CD region contributes to the attenuation of the severe strain, including its replication capacity and temperature sensitivity.

Neutralizing Antibodies to Severe Fever with Thrombocytopenia Syndrome Virus 4 Years after Hospitalization, China.

Severe fever with thrombocytopenia syndrome is an emerging hemorrhagic fever disease in eastern Asia, caused by a tickborne bunyavirus. Of 25 patients hospitalized with this disease in China, 100% produced and maintained neutralizing antibodies to severe fever with thrombocytopenia syndrome virus for the study period of 4 years.

Haemaphysalis longicornis Ticks as Reservoir and Vector of Severe Fever with Thrombocytopenia Syndrome Virus in China.

Severe fever with thrombocytopenia syndrome (SFTS) is an emerging hemorrhagic fever in East Asia caused by SFTS virus (SFTSV), a newly discovered phlebovirus. The Haemaphysalis longicornis tick has been suspected to be the vector of SFTSV. To determine whether SFTSV can be transmitted among ticks, from ticks to animals, and from animals to ticks, we conducted transmission studies between developmental stages of H. longicornis ticks and between ticks and mice. Using reverse transcription PCR, we also analyzed the prevalence of SFTSV infection among H. longicornis ticks collected from vegetation in Shandong Province, China. Our results showed a low prevalence of SFTSV among collected ticks (0.2%, 8/3,300 ticks), and we showed that ticks fed on SFTSV-infected mice could acquire the virus and transstadially and transovarially transmit it to other developmental stages of ticks. Furthermore, SFTSV-infected ticks could transmit the virus to mice during feeding. Our findings indicate ticks could serve as a vector and reservoir of SFTSV.

Severe fever with thrombocytopenia syndrome, Shandong Province, China, 2011.

Severe fever with thrombocytopenia syndrome (SFTS) is an emerging infectious disease in China. The incidence and clinical and laboratory characteristics of SFTS are not clearly defined. During May 22-October 2, 2011, a total of 24 patients with fever, thrombocytopenia, and leukopenia were clinically diagnosed as having SFTS in Yiyuan County, Shandong Province, China. We conducted laboratory tests for these SFTS patients. SFTS virus (SFTSV) infection was confirmed in 22 patients by using reverse transcription PCR and ELISA by acute-phase and convalescent-phase serum samples. Clinical and laboratory manifestations included fever (100%), gastrointestinal symptoms (91%), myalgia (55%), chills (41%), thrombocytopenia (100%), and leukopenia (95%).

Prevalence of SFTSV among Asian house shrews and rodents, China, January-August 2013.

To evaluate the role of small mammals as hosts of severe fever with thrombocytopenia syndrome virus (SFTSV), we tested serum samples from rodents and shrews in China, collected in 2013. SFTSV antibodies and RNA were detected, suggesting that rodents and shrews might be hosts for SFTSV.

Effects of HIV-1 tat on secretion of TNF-α and IL-1ß by U87 cells in AIDS patients with or without AIDS dementia complex.

OBJECTIVE: To explore the role of HIV-1 tat gene variations in AIDS dementia complex (ADC) pathogenesis. METHODS: HIV-1 tat genes derived from peripheral spleen and central basal ganglia of an AIDS patient with ADC and an AIDS patient without ADC were cloned for sequence analysis. HIV-1 tat gene sequence alignment was performed by using CLUSTAL W and the phylogentic analysis was conducted by using Neighbor-joining with MEGA4 software. All tat genes were used to construct recombinant retroviral expressing vector MSCV-IRES-GFP/tat. The MSCV-IRES-GFP/tat was cotransfected into 293T cells with pCMV-VSV-G and pUMVC vectors to assemble the recombinant retrovirus. After infection of gliomas U87 cells with equal amount of the recombinant retrovirus, TNF-α, and IL-1ß concentrations in the supernatant of U87 cells were determined with ELISA. RESULTS: HIV-1 tat genes derived from peripheral spleen and central basal ganglia of the AIDS patient with ADC and the other one without ADC exhibited genetic variations. Tat variations and amino acid mutation sites existed mainly at Tat protein core functional area (38-47aa). All Tat proteins could induce U87 cells to produce TNF-α and IL-1ß, but the level of IL-1ß production was different among Tat proteins derived from the ADC patient's spleen, basal ganglia, and the non-ADC patient's spleen. The level of Tat proteins derived from the ADC patient's spleen, basal ganglia, and the non-ADC patient's spleen were obviously higher than that from the non-ADC patient's basal ganglia. CONCLUSION: Tat protein core functional area (38-47aa) may serve as the key area of enhancing the secretion of IL-1ß. This may be related with the neurotoxicity of HIV-1 Tat.

Complete genome sequencing and analysis of six enterovirus 71 strains with different clinical phenotypes.

BACKGROUND: Hand, foot and mouth diseases (HFMD) caused by enterovirus 71(EV71) presents a broad spectrum of clinical manifestations ranging from mild febrile disease to fatal neurolocal disease. However, the mechanism of virulence is unknown. METHODS: We isolated 6 strains of EV71 from HFMD patients with or without neurological symptoms, and sequenced the whole genomes of the viruses to reveal the virulence factors of EV71. RESULTS: Phylogenetic tree based on VP1 region showed that all six strains clustered into C4a of C4 sub-genotype. In the complete polypeptide, 298 positions were found to be variable in all strains, and three of these positions (Val(P814)/Ile(P814) in VP1, Val(P1148)/Ile(P1148) in 3A and Ala(P1728)/Cys)/Val(P1728) in 3C) were conserved among the strains with neurovirulence, but variable in strains without neurovirulence. In the 5'-UTR region, it showed that the first 10 nucleotides were mostly conserved, however from the 11th nucleotide, nucleotide insertions and deletions were quite common. The secondary structure prediction of 5'-UTR sequences showed that two of three strains without neurovirulence (SDLY11 and SDLY48) were almost the same, and all strains with neurovirulence (SDLY96, SDLY107 and SDLY153) were different from each other. SDLY107 (a fatal strain) was found different from other strains on four positions (C(P241)/T(P241), A(P571)/T(P571), C(P579)/T(P579) in 5'-UTR and T(P7335)/C(P7335) in 3'-UTR). CONCLUSIONS: The three positions (Val(P814)/Ile(P814) in VP1, Val(P1148)/Ile(P1148) in 3A and Ala(P1728)/Cys(P1728)/Val(P1728) in 3C), were different between two phenotypes. These suggested that the three positions might be potential virulent positions. And the three varied positions were also found to be conserved in strains with neurovirulence, and variable in strains without neurovirulence. These might reveal that the conservation of two of the three positions or the three together were specific for the strains with neurovirulence. Varation of secondary structure of 5'-UTR, might be correlated to the changes of viral virulence. SDLY107 (a fatal strain) was found different from other strains on four positions, these positions might be related with death.

'a'-Position-mutated and G4-mutated hemagglutinin-neuraminidase proteins of Newcastle disease virus impair fusion and hemagglutinin-neuraminidase-fusion interaction by different mechanisms.

OBJECTIVES: To determine the effects of heptad repeat regions (HRs) and N-linked carbohydrate sites of the Newcastle disease virus hemagglutinin-neuraminidase (HN) protein on fusion of HN and fusion (F) proteins and HN-F interaction. METHODS: We mutated six 'a' residues in the HRs and four asparagines in N-linked carbohydrate sites to alanine in the HN protein. A vaccinia-T7 RNA polymerase expression system was used to express HN cDNAs in BHK-21 cells to determine the HN functions. Deglycosylation was treated with PGNase F digestion. The formation of HN-F protein complexes was determined by the coimmunoprecipitation assay. RESULTS: Each HR-mutated protein interfered with fusion and the HN-F interaction. The G4-mutated protein not only impaired fusion and HN-F interaction but also decreased activities in cell fusion promotion, hemadsorption and neuraminidase. CONCLUSIONS: It is assumed that two different mechanisms for mutations in these two regions are responsible for the decreased fusion promotion activity and the reduced ability of interaction with F protein. Mutations in the HRs impair fusion and HN-F interaction by altering the transmission of a signal from the globular domain to the F-specific region in the stalk, but the G4 mutation modulates fusion and HN-F interaction by the misfolding of some important structures.

Identification of differentially expressed HERV-K(HML-2) loci in colorectal cancer.

Colorectal cancer is one of the malignant tumors with the highest mortality rate in the world. Survival rates vary significantly among patients at various stages of the disease. A biomarker capable of early diagnosis is required to facilitate the early detection and treatment of colorectal cancer. Human endogenous retroviruses (HERVs) are abnormally expressed in various diseases, including cancer, and have been involved in cancer development. Real-time quantitative PCR was used to detect the transcript levels of HERV-K(HML-2) gag, pol, and env in colorectal cancer to systematically investigate the connection between HERV-K(HML-2) and colorectal cancer. The results showed that HERV-K(HML-2) transcript expression was significantly higher than healthy controls and was consistent at the population and cell levels. We also used next-generation sequencing to identify and characterize HERV-K(HML-2) loci that were differentially expressed between colorectal cancer patients and healthy individuals. The analysis revealed that these loci were concentrated in immune response signaling pathways, implying that HERV-K impacts the tumor-associated immune response. Our results indicated that HERV-K might serve as a screening tumor marker and a target for tumor immunotherapy in colorectal cancer.

Significant Upregulation of HERV-K (HML-2) Transcription Levels in Human Lung Cancer and Cancer Cells.

Lung cancer is the second most common cancer worldwide and the leading cause of cancer death in the world. Therefore, there is an urgent need to develop new and effective biomarkers for diagnosis and treatment. Under this circumstance, human endogenous retroviruses (HERVs) were recently introduced as novel biomarkers for cancer diagnosis. This study focused on the correlation between lung cancer and HERV-K (HML-2) transcription levels. At the cellular level, different types of lung cancer cells and human normal lung epithelial cells were used to analyze the transcription levels of the HERV-K (HML-2) gag, pol, and env genes by RT-qPCR. At the level of lung cancer patients, blood samples with background information from 734 lung cancer patients and 96 healthy persons were collected to analyze the transcription levels of HERV-K (HML-2) gag, pol, and env genes. The results showed that the transcriptional levels of the HERV-K (HML-2) gag, pol, and env genes in lung cancer cells and lung cancer patient blood samples were significantly higher than those in the healthy controls, which was also verified by RNAScope ISH technology. In addition, we also found that there was a correlation between the abnormal transcription levels of HERV-K (HML-2) genes in lung cancer patients and the clinicopathological parameters of lung cancer. We also identified the distribution locations of the gag, pol, and env primer sequences on each chromosome and analyzed the function of these loci. In conclusion, HERV-K (HML-2) genes may be a potential biomarker for the diagnosis of lung cancer.

Severe fever with thrombocytopenia syndrome virus infection during pregnancy in C57/BL6 mice causes fetal damage.

BACKGROUND: Severe fever with thrombocytopenia syndrome virus (SFTSV) is a novel tick-borne phlebovirus, which is listed in the most dangerous pathogens by the World Health Organization, and has 12-30% fatality rates. SFTSV antibodies were reported in minks that experienced abortion or reproductive failure. The aim of this study was to determine whether SFTSV infection causes an adverse pregnancy outcome in the fetus using a pregnant mouse model. METHODOLOGY/PRINCIPAL FINDINGS: We found SFTSV in the fetus after infection in pregnant mice, and some dams showed adverse pregnancy outcomes after infection with SFTSV including placental damage, fetal reabsorption, and fetal intrauterine growth restriction (IUGR). SFTSV had obvious tropism characteristics in the placenta, especially in the labyrinth. In early-gestation, pregnant mice infected with SFTSV had fetal IUGR and a high viral load in the fetus. The virus widely spread in infected fetuses, including the hindbrain, thymus, heart, spinal cord, and liver. CONCLUSIONS: Our study demonstrated that SFTSV was vertically transmitted to the fetus through the placental barrier of immunocompetent mice, and resulted in adverse pregnancy outcomes.

The untranslated regions of EV-A71 contribute to its pathogenicity and virulence.

Enterovirus A71 (EV-A71) is known for its manifestation as hand foot and mouth disease (HFMD), which has caused countless large-scale epidemic outbreaks throughout the world. However, the molecular pathogenesis of EV-A71 infection is still elusive. Previous studies found that the biological characteristics of a mild EV-A71 strain (SDLY1) and a severe EV-A71 strain (SDLY107) are significantly different, and sequence analysis showed that there are several differences in nucleotide sites of UTRs (88 nt, 123 nt, 143 nt, 154 nt, 187 nt, 241 nt, 243 nt, 253 nt, 291 nt, 438 nt, 440 nt, 571 nt, 579 nt, 602 nt, 658 nt, 664 nt, 690 nt, 696 nt, 7328 nt, 7335 nt, 7367 nt, and 7395 nt). The aim of this study was to determine whether these amino sites in UTRs are associated with the pathogenesis of EV-A71 and are responsible for different clinical manifestations. Based on the reverse genetics technology, we rescued two chimeric viruses SDLY107(1-5'UTR) and SDLY107(1-3'UTR) by replacing 5'UTR/3'UTR gene fragments of an infectious cDNA clone. Replication kinetics and cytotoxicity assays showed that the virulence of the two chimeric strains significantly changed in vitro. The viral loads of the two chimeric strains in infected ICR mice were reduced and pathological damage in the brains, lungs, intestinal tissues, and muscles were lightened. Our findings suggest that some nucleotide sites in UTRs may have a function in the pathogenicity and virulence of EV-A71.

Trypanosoma dionisii in insectivorous bats from northern China.

Although bats were considered as a major host of trypanosomatid flagellates, information of trypanosomes in bats is unknown in China. We collected bats in 2015 from Shandong Province of China and used PCR to amplify the Trypanosoma glycosomal glyceraldehyde phosphate dehydrogenase (gGAPDH) gene and 18S rRNA gene from the bat blood samples and heart tissues. The results showed that 10.3% (13/126) of bats (Eptesicus serotinus and Myotis pequinius) were positive for trypanosomatid DNA and DNA sequencing showed that all PCR amplified Trypanosoma DNA belonged to T. dionisii. We concluded that T. dionisii had a infection rate in bats from China. For the first time, Trypanosoma infections were detected in bats from China, providing valuable information on the prevalence of these parasites in Asia. This is also the first report of Trypanosoma dionisii in Myotis pequinius, suggesting that Trypanosoma dionisii has a broad host species.

Enterovirus 71 VP1 Protein Regulates Viral Replication in SH-SY5Y Cells via the mTOR Autophagy Signaling Pathway.

BACKGROUND: Enterovirus 71 (EV71) is the main pathogen that causes severe hand, foot, and mouth disease with fatal neurological complications. However, its neurovirulence mechanism is still unclear. Candidate virulence sites were screened out at structural protein VP1, but the function of these candidate virulence sites remains unclear. Several studies have shown that autophagy is associated with viral replication. However, the relationship between VP1 and autophagy in human neurons has not been studied. METHODS: A recombinant virus-SDLY107-VP1, obtained by replacing the VP1 full-length gene of the SDLY107 strain with the VP1 full-length gene of the attenuated strain SDJN2015-01-was constructed and tested for replication and virulence. We then tested the effect of the recombinant virus on autophagy in nerve cells. The effect of autophagy on virus replication was detected by western blot and plaque test. Finally, the changes of mTOR signaling molecules during EV71 infection and the effect of mTOR on virus replication at the RNA level were detected. RESULTS: Viral recombination triggered virulence attenuation. The replication ability of recombinant virus SDLY107-VP1 was significantly weaker than that of the parent strain SDLY107. The SDLY107 strain could inhibit autophagic flux and led to accumulation of autophagosomes, while the SDLY107-VP1 strain could not cause autophagosome accumulation. The synthesis of EV71 RNA was inhibited by inhibiting mTOR. CONCLUSIONS: Replacement of VP1 weakened the replication ability of virulent strains and reduced the level of autophagy in nerve cells. This autophagy facilitates the replication of virulent strains in nerve cells. VP1 is an important neurovirulence determinant of EV71, which affects virus replication by regulating cell autophagy. mTOR is a key molecule in this type of autophagy.

Nonstructural protein 2A modulates replication and virulence of enterovirus 71.

Enterovirus 71 (EV71) can cause hand, foot, and mouth disease in children, and severe infections can induce neurological complications and even death. However, the pathogenesis of EV71 remains unknown. The 2A proteinase (2Apro) of EV71 plays an important role in segmenting the precursor polyprotein during viral replication, inhibiting host protein synthesis, and evading innate immunity. This study was to determine the function of EV71 2Apro in replication and virulence. A chimeric strain (SDLY 107-2A-1) was recombined by replacing 2Apro of a severe strain (SDLY107) with that of a mild strain (SDLY1) based on an infectious cDNA clone. The replication kinetics of the chimeric strain in vitro and in vivo were determined by qRT-PCR, which showed that the chimeric strain replicated slower and generated less viral RNA than the severe strain. The pathological change and viral load of chimeric strain infected mice were intermediate between severe strain infected mice and mild strain infected mice. Cellular cytotoxicity assays revealed that 2Apro was associated with the neurotoxicity of EV71. Histopathological and immunohistochemical assays detected tissue pathological damage in the lungs, muscles, brain, and intestinal tissues. Together, these results suggest that 2Apro modulates replication and virulence of EV71. This provides a theoretical basis for virulence determination of EV71.