Intrahost variation and evolutionary dynamics of adenoviruses correlate to neutrophils in infected patients.

With deep sequencing of virus genomes within the hosts, intrahost single nucleotide variations (iSNVs) have been used for analyses of virus genome variation and evolution, which is indicated to correlate with viral pathogenesis and disease severity. Little is known about the features of iSNVs among DNA viruses. We performed the epidemiological and laboratory investigation of one outbreak of adenovirus. The whole genomes of viruses in both original oral swabs and cell-cultured virus isolates were deeply sequenced. We identified 737 iSNVs in the viral genomes sequenced from original samples and 46 viral iSNVs in cell-cultured isolates, with 33 iSNVs shared by original samples and cultured isolates. Meanwhile, we found these 33 iSNVs were shared by different patients, among which, three hot spot areas 6367-6401, 9213-9247, and 10 584-10 606 within the functional genes of the adenovirus genome were found. Notably, the substitution rates of iSNVs were closely correlated with the clinical and immune indicators of the patients. Especially a positive correlation to neutrophils was found, indicating a predictable biomarker of iSNV dynamics. Our findings demonstrated the neutrophil-correlated dynamic evolution features of the iSNVs within adenoviruses, which indicates a virus-host interaction during human infection of a DNA virus.

A Bat-Derived Putative Cross-Family Recombinant Coronavirus with a Reovirus Gene.

The emergence of severe acute respiratory syndrome coronavirus (SARS-CoV) in 2002 and Middle East respiratory syndrome coronavirus (MERS-CoV) in 2012 has generated enormous interest in the biodiversity, genomics and cross-species transmission potential of coronaviruses, especially those from bats, the second most speciose order of mammals. Herein, we identified a novel coronavirus, provisionally designated Rousettus bat coronavirus GCCDC1 (Ro-BatCoV GCCDC1), in the rectal swab samples of Rousettus leschenaulti bats by using pan-coronavirus RT-PCR and next-generation sequencing. Although the virus is similar to Rousettus bat coronavirus HKU9 (Ro-BatCoV HKU9) in genome characteristics, it is sufficiently distinct to be classified as a new species according to the criteria defined by the International Committee of Taxonomy of Viruses (ICTV). More striking was that Ro-BatCoV GCCDC1 contained a unique gene integrated into the 3'-end of the genome that has no homologs in any known coronavirus, but which sequence and phylogeny analyses indicated most likely originated from the p10 gene of a bat orthoreovirus. Subgenomic mRNA and cellular-level observations demonstrated that the p10 gene is functional and induces the formation of cell syncytia. Therefore, here we report a putative heterologous inter-family recombination event between a single-stranded, positive-sense RNA virus and a double-stranded segmented RNA virus, providing insights into the fundamental mechanisms of viral evolution.

Hepatitis C virus envelope glycoproteins complementation patterns and the role of the ecto- and transmembrane domains.

We separated E1 and E2 of hepatitis C virus (HCV) genotypes 1a, 1b, and 2a into two individual expression plasmids and replaced the transmembrane domains of 1b and 2a E1 and E2 with that of genotype 1a. The complementation features of E1 and E2 as well as the contributions of both the ecto- and transmembrane domains to the formation of the E1E2 complex were evaluated using the HCV pseudoparticle(s) (HCVpp(s)) system. We demonstrated that 1aE2 could not only complement its native 1aE1, but could also complement 1bE1 as well; in genotype 1b, glycoprotein complex formation is primarily dependent on the overall biological characteristics of the intact native E1 and E2; in genotype 2a, although the interaction of intact native E1 and E2 is critical for the formation of the glycoprotein complex, the ectodomain made a greater contribution than that of the transmembrane domain. Our study provides valuable findings regarding HCV E1 and E2 biology and will be of use in both anti-HCV strategy and understanding on the mechanisms of coinfection of different HCV strains.

The persistent prevalence and evolution of cross-family recombinant coronavirus GCCDC1 among a bat population: a two-year follow-up.

Bats are connected with the increasing numbers of emerging and re-emerging viruses that may break the species barrier and spread into the human population. Coronaviruses are one of the most common viruses discovered in bats, which were considered as the natural source of recent human-susceptible coronaviruses, i.e. SARS-COV and MERS-CoV. Our previous study reported the discovery of a bat-derived putative cross-family recombinant coronavirus with a reovirus gene p10, named as Ro-BatCoV GCCDC1. In this report, through a two-year follow-up of a special bat population in one specific cave of south China, we illustrate that Ro-BatCoV GCCDC1 persistently circulates among bats. Notably, through the longitudinal observation, we identified the dynamic evolution of Ro-BatCoV GCCDC1 in bats represented by continuously recombination events. Our study provides the first glimpse of the virus evolution in one longitudinally observed bat population cohort and underlines the surveillance and pre-warning of potential interspecies transmittable viruses in bats.

Detection of anti-MHAV and anti-MHEV IgM in patients with sporadic acute hepatitis in Beijing between 1995-2000.

OBJECTIVE: To investigate the age range, liver function damage and prognosis of patients with sporadic acute hepatitis A and E in Beijing. METHODS: Enzyme immunoassay (EIA) was used to detect anti-HAV and anti-HEV immunoglobulin M (IgM). Serum samples were collected from the patients with sporadic acute viral hepatitis in Beijing from January 1995 to June 2000. RESULTS: The total Positive rate for anti-HAV and anti-HEV IgM was 55.2% (112) in 203 patients with acute hepatitis, of whom 22.2% (45 patients) and 33.0% (67) were positive for anti-HAV and anti-HEV respectively. The duration of anti-HEV IgM was 45-60 days and that of anti-HAV IgM was at least 2-3 months. The patients with acute hepatitis A and hepatitis E all experienced jaundice and a rising of liver enzyme, but did not develop chronic hepatitis or died. CONCLUSION: Acute hepatitis A as well as acute hepatitis E plays an important role in sporadic enterically transmitted hepatitis in Beijing.

[Comparison of ELISA diagnostic kits used in China for hepatitis C virus IgG antibody detection].

OBJECTIVE: To compare the commercial diagnostic reagent available in China for hepatitis C virus ( HCV) IgG antibody detection in order to provide some useful information for HCV prevention. METHODS: The HCV-IgG detection reagents produced by six different Enterprises named A, B, C, D, E and F were chosen and applied to detect 160 HCV specious sera samples. HCV-IgG reagent from ABBOTT was adopted as gold-standard and the samples in gray zone were determined by RIBA method finally. RESULTS: 160 sera samples comprised 88 positive samples and 72 negative samples. The total conformity ranged from 88.13% to 98.13% and the Youden indexes ranged from 0.74 to 0.96 when the reagents from six different Enterprises were compared with gold-standard. The highest conformity was 98.13%, observed in D reagent with the highest Youden index of 0.96. CONCLUSION: The total conformity rates were more than 88% when the HCV-IgG antibody detection reagents from six different Enterprises were compared with ABBOTT reagent. It was highly conformable. However, some reagent proved to be less conformable in negative samples detection.

[Antibody detection of hepatitis E virus in human population of different national in China].

OBJECTIVE: To investigate the seroprevalence of HEV infection in different national human population in Han, Hui and Zang in China. METHODS: EIA was used for detecting anti-HEV IgG of the serum samples. All samples were collected in 2006-2008 in Sichuan, Beijing, Heilongjianin, Sandong, Gansuo, Ningxia and Qinghai areas. RESULTS: The total positive rate of anti-HEV IgG was 17.97% (1878/ 10448), 24.32% (1794/7376) in Han national, 3.59% (81/2258) of Hui national and 0.37% (3/814) of Zang national, respectively. The positive rate of Han human at different age group, was 5.19% of < or = 10 year, 11.64% of 11-20 year, 20.08% of 21-30 year, 34.17% of 31-40 year, 41.75% of 41-50 year, 48.58% of 51-60 year, 57.43% of > or = 61 year. The positive rate of Hui human at different age group, was 3.11%, 3.96%, 2.11%, 3.98, 2.52%, 4.57% and 6.67%, respectively. Three positive of Zang human was between 21-60 year. CONCLUSIONS: The HEV infection in Han national population was higher than the Hui and Zang national, significantly. The HEV infection was correlation with age significantly, the infection rate was increased with age.

[Establishing a high-titer infectious avian influenza A (H5N1) pseudotyped viral particle].

A transient four-plasmid cotransfection system was used to construct avian influenza A (H5N1) pseudotyped viral particle (H5N1Pp) by incorporating hemagglutinin (HA) protein and neuraminidase (NA) protein from H5N1 avian influenza virus onto Murine leukemia virus pseudotyped viral particles, the transmission electron microscopy, infectivity titer assay, hemagglutination assay, neutralization assay of H5N1Pp were studied. We established a pseudotyped H5N1 viral particle at a high titer of 10(8) Pp/mL, the morphology,the hemagglutination activity and neutralization specificity of H5N1Pp is simililar to wild H5N1 virus. The research result sets a platform for studying this virus, including its receptors, the functional analysis of HA and NA, neutralizing antibodies and anti-H5N1 drug development.

[Study on the effect of realgar nanoparticles on reducing the respiratory syncytial virus type A (RSV-a) replication in vitro].

This study was to establish a model to explore anti- RSV effect of different administration method of Chinese medicine realgar on respiratory syncytial virus type A (RSV-A) replication in Hep-2 cells. Using high-energy ball milling with distilled water to prepare realgar nanoparticles,the concentration of nanometer realgar was tested by molybdenum blue staining method and the size of realgar nanoparticles was tested on Nano Series. Cell culture with ribavirin as a positive control was applied to observe the effect of anti-respiratory syncytial virus type A replication through prevention, treatment or direct inactivation of three different drug administration methods. Realgar nano-particles was found to be a potential inhibitor of RSV-A in a concentration-dependent manner with the median toxic concentration(TC50) of 0.649 microg/mL in Hep-2 cell culture. The median inhibition concentration (IC50) was 0.20 microg/mL when drug was added before virus infection. The IC50 was 0.13 microg/mL when drug was added after virus infection,and it was 0.16 microg/mL when the drug was mixed with virus and added. The therapeutic index (TI) was 3.18, 4.99 and 4.11, respectively. The results showed realgar nanoparticles could inhibit the replication of the RSV and inactivate the RSV in vitro.

[Hepatitis C viruses infection situation in the human population of six provinces in China].

OBJECTIVE: To investigate the seroprevalence of hepatitis C viruse infection in the human population in six regions of Beijing, Heilongjiang, Shandong, Ningxia, Gansu and Sichuan in China. METHODS: ELISA was used for detecting anti-HCV IgG of the serum samples. All sample were collected in 2006-2008 in six areas. RESULTS: 9538 samples were detected. The total positive rate of anti-HCV was 0.39% (37), 0.23% (3/1328) in Beijing, 0.74% (12/1629) in Heilongjiang, 0.26% (5/1962) in Shandong, 0.1% (1/1000) in Ningxia, 0.44% in Gansu (9/2 037) and Sichuan (7/1582), respectively. The 37 positive samples at the sex were 19 (51.35%) of man and 18 (48.65%) of female. At the age group were 1 (2.70%) of < 10 years old, 5 (13.51%) of 10-19 years old, 4 (10.81%) of 20-29 years old, 6 (16.22% ) of 30-39 years old, 9 (24.32%) of 4049 years old, 12 (32.43%) of > or = 50 years old. The positive samples were detected anti-HAV-IgG, anti-HEV-IgG and HBsAg/ HBcAb /HBeAb. The positive Number was 35 (94.59%), 10 (27.03%) and 2 (5.41%) respectively. CONCLUSIONS: HCV infection rate in the population was 0.1% -0.74%, 1/3 of > or = 50 years old in HCV positive. Hepatitis C virus co-infection with HAV, HEV and HBV.

[The characterization analysis of HAV recombinant antigen was expressed by vaccinia virus vector].

OBJECTIVE: To find a suitable cell line for hepatitis A antigen expressed by vaccinia virus vector and to find a way of inactivation and preservation of the HAV recombinant antigen. Methods Series of cell lines such as K4,143, HEL, Hep-2 and Vero were inoculated with vaccinia virus that can express HAV recombinant antigen. ELISA was used to determine the contents of expression antigen. The characterization of the HAV antigen expressed by vaccinia virus was then analyzed after it was treated with different methods. RESULTS: The expression of HAV recombinant antigen in K4,143 and HEL cell lines was a little more than expression in Hep-2 and Vero cell lines. The antigenicity is obviously higher when HAV recombinant antigen was inactivated by beta-propiolactone other than it was inactivated by formalin. It was best to preserve the prepared HAV recombinant antigen under -40 degrees C condition. CONCLUSIONS: The application of vaccinia virus vector in hepatitis A antigen preparation was very useful and promising.

Characterization of neuraminidases from the highly pathogenic avian H5N1 and 2009 pandemic H1N1 influenza A viruses.

To study the precise role of the neuraminidase (NA), and its stalk region in particular, in the assembly, release, and entry of influenza virus, we deleted the 20-aa stalk segment from 2009 pandemic H1N1 NA (09N1) and inserted this segment, now designated 09s60, into the stalk region of a highly pathogenic avian influenza (HPAI) virus H5N1 NA (AH N1). The biological characterization of these wild-type and mutant NAs was analyzed by pseudotyped particles (pseudoparticles) system. Compared with the wild-type AH N1, the wild-type 09N1 exhibited higher NA activity and released more pseudoparticles. Deletion/insertion of the 09s60 segment did not alter this relationship. The infectivity of pseudoparticles harboring NA in combination with the hemagglutinin from HPAI H5N1 (AH H5) was decreased by insertion of 09s60 into AH N1 and was increased by deletion of 09s60 from 09N1. When isolated from the wild-type 2009H1N1 virus, 09N1 existed in the forms (in order of abundance) dimer>>tetramer>monomer, but when isolated from pseudoparticles, 09N1 existed in the forms dimer>monomer>>>tetramer. After deletion of 09s60, 09N1 existed in the forms monomer>>>dimer. AH N1 from pseudoparticles existed in the forms monomer>>dimer, but after insertion of 09s60, it existed in the forms dimer>>monomer. Deletion/insertion of 09s60 did not alter the NA glycosylation pattern of 09N1 or AH N1. The 09N1 was more sensitive than the AH N1 to the NA inhibitor oseltamivir, suggesting that the infectivity-enhancing effect of oseltamivir correlates with robust NA activity.

Neuraminidase and hemagglutinin matching patterns of a highly pathogenic avian and two pandemic H1N1 influenza A viruses.

BACKGROUND: Influenza A virus displays strong reassortment characteristics, which enable it to achieve adaptation in human infection. Surveying the reassortment and virulence of novel viruses is important in the prevention and control of an influenza pandemic. Meanwhile, studying the mechanism of reassortment may accelerate the development of anti-influenza strategies. METHODOLOGY/PRINCIPAL FINDINGS: The hemagglutinin (HA) and neuraminidase (NA) matching patterns of two pandemic H1N1 viruses (the 1918 and current 2009 strains) and a highly pathogenic avian influenza A virus (H5N1) were studied using a pseudotyped particle (pp) system. Our data showed that four of the six chimeric HA/NA combinations could produce infectious pps, and that some of the chimeric pps had greater infectivity than did their ancestors, raising the possibility of reassortment among these viruses. The NA of H5N1 (A/Anhui/1/2005) could hardly reassort with the HAs of the two H1N1 viruses. Many biological characteristics of HA and NA, including infectivity, hemagglutinating ability, and NA activity, are dependent on their matching pattern. CONCLUSIONS/SIGNIFICANCE: Our data suggest the existence of an interaction between HA and NA, and the HA NA matching pattern is critical for valid viral reassortment.

[Expression of hepatitis delta antigen Inner Mongolian strain in prokaryotic cell and analysis of its antigenicity].

OBJECTIVE: To obtain high yield and good antigenic activity of HDV L-Ag and to detect different regional patients' sera to test the purified antigen's antigenicity. METHODS: Hepatitis delta virus' sequence was obtained from Inner Mongolian patient by using RT-PCR and PCR methods, PET43a was used and His-tag was added at the HDV L-Ag 5' and 3' to construct the recombinant expression plasmid, transform the plasmid into host bacterium BL21 and induce it with IPTG. The expression supernatant was purified by saturated (NH4)2SO4 and affinity chromatography. The activity and antigenicity of the expressed product were analyzed by using EIA. RESULTS: Comparison of results obtained with detection by using the expressed protein coated plate and ABBOTT Murex anti-Delta (total) of 15 positive and 10 negative sera, the consistency was good (100%). CONCLUSION: EIA proved that the purified antigen had good antigenicity, no serological difference was found in detection between different region's sera, therefore the purified delta antigen may be useful in diagnostic and other research.

[Therapeutic effect of autologous cytokine-induced killer cells on patients with liver cirrhosis caused by HBV infection].

OBJECTIVE: To observe the therapeutic effect of autologous cytokine-induced killer cells (CIK) on HBV DNA positive patients with liver cirrhosis. METHODS: HBV DNA positive 33 patients with cirrhosis were treated with CIK. Before and after cultured in vitro and post-treatment, CD3+, CD3+CD4+, CD3+CD8+, CD3+CD56+ cells, mDC and pDC were detected by flow cytometry. The indexes of virus and liver function were compared between pre- and post-treatment. RESULTS: CD3+, CD3+CD8+ cells and CD3+CD56+ cells were higher after cultured in vitro and after transfused back than those before culture (91.5 +/- 10.3, 74.4 +/- 9.9 vs. 67.9 +/- 12.8; 60.9 +/- 15.5, 37.3 +/- 15.1 vs. 27.9 +/- 10.9; 18.4 +/- 11.7, 14.5 +/- 7.5 vs. 10.6 +/- 7.1). The percentages of mDC and pDC also increased after-treatment vs. pre-treatment (0.54 +/- 0.18 vs. 0.70 +/- 0.29; 0.26 +/- 0.13 vs. 0.41 +/- 0.25). HBV DNA became undetectable in 12 patients and decrease exceeded 100 times in 4 patients after treatment. HBeAg became undetectable in 10 of 14 patients who were HBeAg positive pretreatment patients, among them 2 patients had HBeAb sero conversion. The liver function was improved after treatment. All patients tolerated the treatment. CONCLUSION: CIK treatment can increase immune effector cells and has some antiviral effect and is safe.

[Influence of the mutants of hepatitis B surface antigen on the cell immunity].

OBJECTIVE: To study the influence of the mutants of hepatitis B surface antigen on the cell immunity. METHODS: The recombinant plasmids of NS2Swt, NS2S126, NS2S133, NS2S141 and NS2S145 were transfected into Chinese hamster ovary (CHO) cells and the expressed proteins were detected by means of ELISA. Following PHA-activated lymphoblasts proliferation assay and IFN-gamma, IL-2, IL-10 induction assay were done with these proteins. RESULTS: It was identified that these proteins of HBsAg could stimulate human lymphoblasts proliferation. Besides, there were no significant difference between the mutants and the wild. It was deserved to point out that the HBsAg with T126S mutation could increase the expression of IFN-gamma in the culture medium while the HBsAg with M133T mutation induced more expression of IL-10. CONCLUSION: The results suggested that the cellular immune response to mutants of HBV might not be strengthened or weakened. But it should not be ignored that HBV T126S or M133T mutation may assert a potential impact on the cell immunity.