[Analysis of a child with CLN1 neuronal ceroid lipofuscinosis in conjunct with Hereditary hyperferinemia cataract syndrome].

OBJECTIVE: To analyze the clinical data and genetic characteristics of a child with CLN1 neuronal ceroid lipofuscinosis in conjunct with Hereditary hyperferritinemia cataract syndrome (HHCS). METHODS: A child who was admitted to the PICU of the First Affiliated Hospital of Zhengzhou University in November 2020 was selected as the study subject. Clinical data of the child was collected. Genetic testing was carried out for the child, and the result was analyzed in the light of literature review to explore the clinical and genetic characteristics to facilitate early identification. RESULTS: The patient, a 3-year-old male, had mainly presented with visual impairment, progressive cognitive and motor regression, and epilepsy. Cranial magnetic resonance imaging revealed deepened sulci in bilateral cerebral hemispheres, and delayed myelination. The activity of palmitoyl protein thioesterase was low (8.4 nmol/g/min, reference range: 132.2 ~ 301.4 nmol/g/min), whilst serum ferritin was increased (2417.70 ng/mL, reference range: 30 ~ 400 ng/ml). Fundoscopy has revealed retinal pigment degeneration. Whole exome sequencing revealed that he has harbored c.280A>C and c.124-124+3delG compound heterozygous variants of the PPT1 gene, which were respectively inherited from his father and mother. Neither variant has been reported previously. The child has also harbored a heterozygous c.-160A>G variant of the FTL gene, which was inherited from his father. Based on the clinical phenotype and results of genetic testing, the child was diagnosed as CLN1 and HHCS. CONCLUSION: The compound heterozygous variants of the PPT1 gene probably underlay the disorders in this child. For children with CLN1 and rapidly progressing visual impairment, ophthalmological examination should be recommended, and detailed family history should be taken For those suspected for HHCS, genetic testing should be performed to confirm the diagnosis.

Prevalence of Infectious Hypodermal and Hematopoietic Necrosis Virus (IHHNV) in Farmed Procambarus clarkii of the Middle and Lower Reaches of the Yangtze River in China.

Procambarus clarkii is an important economic aquaculture species worldwide. Infectious hypodermal and hematopoietic necrosis virus (IHHNV) infects numerous crustacean hosts, including P. clarkii. However, there have been few reports on the prevalence of IHHNV in P. clarkii. In this study, 200 farmed P. clarkii were collected from Anhui, Jiangsu, Zhejiang, Hunan, Hubei, and Sichuan provinces in China. PCR detection was employed per the protocol by the World Organization for Animal Health (WOAH) to identify and detect the presence of IHHNV. The positive rate of IHHNV in different provinces ranged from 16.7 to 56.7%, and the overall IHHNV-positive rate was 38.5%. IHHNV strains isolated in this study related closely to infectious IHHNV and split into two major distinct branches. Besides, the IHHNV strains shared a high homology (93.4-99.4%). These findings suggest that a high prevalence of IHHNV was established in farmed P. clarkii in the middle and lower reaches of the Yangtze River.

Recovery of Siniperca chuatsi rhabdovirus from cloned cDNA.

Siniperca chuatsi rhabdovirus (SCRV) is an important pathogen that infects mandarin fish. A reverse genetics system is an important technical platform for virus research. In this study, the minigenome in which the enhanced green fluorescent protein gene is flanked by the viral genomic ends of SCRV and transcribed using a T7 promoter-terminator cassette was constructed. Co-transfection of the minigenome construct with SCRV-supporting plasmids of N, P, and L in BSRT7 cells resulted in the expression of the reporter gene. Transcription of a positive-strand RNA copy from cDNA of the SCRV genome along with the viral N, P, and L proteins resulted in the recovery of infectious SCRV in cells. Viral titre up to 108 PFU/ml was achieved. Recombinant SCRV was verified by the detection of a unique restriction site engineered into the SCRV genome. The phenotypes of the recombinant SCRV and the parental virus were evaluated by plaque size, replication kinetics in vitro, and pathogenicity in vivo. The recovered SCRV from cDNA showed similar phenotypes compared to the parental virus. The established reverse genetics system is of great significance and value for the functional genome study of SCRV and for laying a foundation for the development of the viral vector and SCRV vaccine.

Discovery of a novel Piscanivirus in yellow catfish (Pelteobagrus fulvidraco) in China.

A bacilliform virus was isolated from yellow catfish in China. This virus can directly adapt in cultures of EPC cells. The virus particles, which were rod-shaped approximately 120 nm long and 20 nm wide, were visible in the cytoplasm of EPC cells. The full-length genome of this virus is 26,985 nt. The genome contains four open reading frames that encode polyprotein1ab, spike glycoprotein, M protein, and N protein. There was a putative slippery sequence 14861UUUAAAC14867, which could be modeled into an RNA pseudoknot structure. The predicted amino acid sequence of pp1ab, S, M, and N genes shares 8.7%-40.2% homology with those of the two known Bafinivirus strains-WBV and FHMNV. Based on the viral morphology, genome organization, and sequence homology, this newly identified bacilliform virus appears to be Piscanivirus. To the best of our knowledge, this is the first report of Piscanivirus in yellow catfish and Piscanivirus in China.

A Reverse Genetics Approach for the Design of Methyltransferase-Defective Live Attenuated Avian Metapneumovirus Vaccines.

Avian metapneumovirus (aMPV), also known as avian pneumovirus or turkey rhinotracheitis virus, is the causative agent of turkey rhinotracheitis and is associated with swollen head syndrome in chickens. aMPV belongs to the family Paramyxoviridae which includes many important human pathogens such as human respiratory syncytial virus (RSV), human metapneumovirus (hMPV), and human parainfluenza virus type 3 (PIV3). The family also includes highly lethal emerging pathogens such as Nipah virus and Hendra virus, as well as agriculturally important viruses such as Newcastle disease virus (NDV). For many of these viruses, there is no effective vaccine. Here, we describe a reverse genetics approach to develop live attenuated aMPV vaccines by inhibiting the viral mRNA cap methyltransferase. The viral mRNA cap methyltransferase is an excellent target for the attenuation of paramyxoviruses because it plays essential roles in mRNA stability, efficient viral protein translation and innate immunity. We have described in detail the materials and methods used to generate recombinant aMPVs that lack viral mRNA cap methyltransferase activity. We have also provided methods to evaluate the genetic stability, pathogenesis, and immunogenicity of live aMPV vaccine candidates in turkeys.

Development of an overlapping PCR method to clone the full genome of infectious hypodermal and hematopoietic necrosis virus (IHHNV).

Decapod Penstyldensovirus 1, previously named as infectious hypodermal and hematopoietic necrosis virus (IHHNV), is an economically important pathogen that causes shrimp diseases worldwide. However, a rapid method for cloning full-length IHHNV genome sequences is still lacking, which makes it difficult to study the genomics and molecular epidemiology of IHHNV. Here, a novel and rapid PCR technique was developed to determine the complete genomic sequences of IHHNV. The IHHNV genome was amplified in two overlapping fragments which each yielded a 2kb PCR product covering the first half or the second half of IHHNV genome, respectively. Using this method, six complete genomic sequences of IHHNV, which were collected from different regions of Zhejiang province in China, were cloned and sequenced successfully. The new cloning method will greatly facilitate the study on the genomics and molecular epidemiology of IHHNV.

Methyltransferase-defective avian metapneumovirus vaccines provide complete protection against challenge with the homologous Colorado strain and the heterologous Minnesota strain.

UNLABELLED: Avian metapneumovirus (aMPV), also known as avian pneumovirus or turkey rhinotracheitis virus, is the causative agent of turkey rhinotracheitis and is associated with swollen head syndrome in chickens. Since its discovery in the 1970s, aMPV has been recognized as an economically important pathogen in the poultry industry worldwide. The conserved region VI (CR VI) of the large (L) polymerase proteins of paramyxoviruses catalyzes methyltransferase (MTase) activities that typically methylate viral mRNAs at guanine N-7 (G-N-7) and ribose 2'-O positions. In this study, we generated a panel of recombinant aMPV (raMPV) Colorado strains carrying mutations in the S-adenosyl methionine (SAM) binding site in the CR VI of L protein. These recombinant viruses were specifically defective in ribose 2'-O, but not G-N-7 methylation and were genetically stable and highly attenuated in cell culture and viral replication in the upper and lower respiratory tracts of specific-pathogen-free (SPF) young turkeys. Importantly, turkeys vaccinated with these MTase-defective raMPVs triggered a high level of neutralizing antibody and were completely protected from challenge with homologous aMPV Colorado strain and heterologous aMPV Minnesota strain. Collectively, our results indicate (i) that aMPV lacking 2'-O methylation is highly attenuated in vitro and in vivo and (ii) that inhibition of mRNA cap MTase can serve as a novel target to rationally design live attenuated vaccines for aMPV and perhaps other paramyxoviruses. IMPORTANCE: Paramyxoviruses include many economically and agriculturally important viruses such as avian metapneumovirus (aMPV), and Newcastle disease virus (NDV), human pathogens such as human respiratory syncytial virus, human metapneumovirus, human parainfluenza virus type 3, and measles virus, and highly lethal emerging pathogens such as Nipah virus and Hendra virus. For many of them, there is no effective vaccine or antiviral drug. These viruses share common strategies for viral gene expression and replication. During transcription, paramyxoviruses produce capped, methylated, and polyadenylated mRNAs. Using aMPV as a model, we found that viral ribose 2'-O methyltransferase (MTase) is a novel approach to rationally attenuate the virus for vaccine purpose. Recombinant aMPV (raMPV) lacking 2'-O MTase were not only highly attenuated in turkeys but also provided complete protection against the challenge of homologous and heterologous aMPV strains. This novel approach can be applicable to other animal and human paramyxoviruses for rationally designing live attenuated vaccines.

Rational design of human metapneumovirus live attenuated vaccine candidates by inhibiting viral mRNA cap methyltransferase.

UNLABELLED: The paramyxoviruses human respiratory syncytial virus (hRSV), human metapneumovirus (hMPV), and human parainfluenza virus type 3 (hPIV3) are responsible for the majority of pediatric respiratory diseases and inflict significant economic loss, health care costs, and emotional burdens. Despite major efforts, there are no vaccines available for these viruses. The conserved region VI (CR VI) of the large (L) polymerase proteins of paramyxoviruses catalyzes methyltransferase (MTase) activities that typically methylate viral mRNAs at positions guanine N-7 (G-N-7) and ribose 2'-O. In this study, we generated a panel of recombinant hMPVs carrying mutations in the S-adenosylmethionine (SAM) binding site in CR VI of L protein. These recombinant viruses were specifically defective in ribose 2'-O methylation but not G-N-7 methylation and were genetically stable and highly attenuated in cell culture and viral replication in the upper and lower respiratory tracts of cotton rats. Importantly, vaccination of cotton rats with these recombinant hMPVs (rhMPVs) with defective MTases triggered a high level of neutralizing antibody, and the rats were completely protected from challenge with wild-type rhMPV. Collectively, our results indicate that (i) amino acid residues in the SAM binding site in the hMPV L protein are essential for 2'-O methylation and (ii) inhibition of mRNA cap MTase can serve as a novel target to rationally design live attenuated vaccines for hMPV and perhaps other paramyxoviruses, such as hRSV and hPIV3. IMPORTANCE: Human paramyxoviruses, including hRSV, hMPV, and hPIV3, cause the majority of acute upper and lower respiratory tract infections in humans, particularly in infants, children, the elderly, and immunocompromised individuals. Currently, there is no licensed vaccine available. A formalin-inactivated vaccine is not suitable for these viruses because it causes enhanced lung damage upon reinfection with the same virus. A live attenuated vaccine is the most promising vaccine strategy for human paramyxoviruses. However, it remains a challenge to identify an attenuated virus strain that has an optimal balance between attenuation and immunogenicity. Using reverse genetics, we generated a panel of recombinant hMPVs that were specifically defective in ribose 2'-O methyltransferase (MTase) but not G-N-7 MTase. These MTase-defective hMPVs were genetically stable and sufficiently attenuated but retained high immunogenicity. This work highlights a critical role of 2'-O MTase in paramyxovirus replication and pathogenesis and a new avenue for the development of safe and efficacious live attenuated vaccines for hMPV and other human paramyxoviruses.

[Metapneumovirus expands the understanding of Paramyxovirus cell fusion--a review].

For most viruses in Paramyxoviridae, cell fusion requires both attachment protein and fusion protein. The attachment protein is responsible for the binding to its cognate receptors, while the interaction between fusion protein and attachment protein triggers the fusion protein which is responsible for the fusion. However, the Metapneumovirus fusion in Pneumovirinae subfamily displayed different mechanism where the attachment protein is not required. The cell fusion is accomplished by fusion protein alone without the help of the attachment protein. Recent studies indicate that low pH is required for cell fusion promoted by some hMPV strains. The fusion protein of aMPV type A is highly fusogenic, whereas that of type B is low. The original fusion models for Paramyxovirus cannot explain the phenomenon above. The mechanism to regulate the cell fusion of Metapneumovirus is poorly understood. It is becoming a hot spot for the study of cell fusion triggered by Paramyxovirus where it enlarged the traditional scope of Paramyxovirus fusion. In this review, we discuss the new achievements and advances in the understanding of cell fusion triggered by Metapneumovirus.

Heat shock protein 70 enhances mucosal immunity against human norovirus when coexpressed from a vesicular stomatitis virus vector.

UNLABELLED: Human norovirus (NoV) accounts for 95% of nonbacterial gastroenteritis worldwide. Currently, there is no vaccine available to combat human NoV as it is not cultivable and lacks a small-animal model. Recently, we demonstrated that recombinant vesicular stomatitis virus (rVSV) expressing human NoV capsid protein (rVSV-VP1) induced strong immunities in mice (Y. Ma and J. Li, J. Virol. 85:2942-2952, 2011). To further improve the safety and efficacy of the vaccine candidate, heat shock protein 70 (HSP70) was inserted into the rVSV-VP1 backbone vector. A second construct was generated in which the firefly luciferase (Luc) gene was inserted in place of HSP70 as a control for the double insertion. The resultant recombinant viruses (rVSV-HSP70-VP1 and rVSV-Luc-VP1) were significantly more attenuated in cell culture and viral spread in mice than rVSV-VP1. At the inoculation dose of 1.0 × 10(6) PFU, rVSV-HSP70-VP1 triggered significantly higher vaginal IgA than rVSV-VP1 and significantly higher fecal and vaginal IgA responses than rVSV-Luc-VP1, although serum IgG and T cell responses were similar. At the inoculation dose of 5.0 × 10(6) PFU, rVSV-HSP70-VP1 stimulated significantly higher T cell, fecal, and vaginal IgA responses than rVSV-VP1. Fecal and vaginal IgA responses were also significantly increased when combined vaccination of rVSV-VP1 and rVSV-HSP70 was used. Collectively, these data indicate that (i) insertion of an additional gene (HSP70 or Luc) into the rVSV-VP1 backbone further attenuates the VSV-based vaccine in vitro and in vivo, thus improving the safety of the vaccine candidate, and (ii) HSP70 enhances the human NoV-specific mucosal and T cell immunities triggered by a VSV-based human NoV vaccine. IMPORTANCE: Human norovirus (NoV) is responsible for more than 95% of acute nonbacterial gastroenteritis worldwide. Currently, there is no vaccine for this virus. Development of a live attenuated vaccine for human NoV has not been possible because it is uncultivable. Thus, a live vector-based vaccine may provide an alternative vaccine strategy. In this study, we developed a vesicular stomatitis virus (VSV)-based human NoV vaccine candidate. We constructed rVSV-HSP70-VP1, coexpressing heat shock protein (HSP70) and capsid (VP1) genes of human NoV, and rVSV-Luc-VP1, coexpressing firefly luciferase (Luc) and VP1 genes. We found that VSVs with a double gene insertion were significantly more attenuated than VSV with a single VP1 insertion (rVSV-VP1). Furthermore, we found that coexpression or coadministration of HSP70 from VSV vector significantly enhanced human NoV-specific mucosal immunity. Collectively, we developed an improved live vectored vaccine candidate for human NoV which will be useful for future clinical studies.

Roles of the putative integrin-binding motif of the human metapneumovirus fusion (f) protein in cell-cell fusion, viral infectivity, and pathogenesis.

UNLABELLED: Human metapneumovirus (hMPV) is a relatively recently identified paramyxovirus that causes acute upper and lower respiratory tract infection. Entry of hMPV is unusual among the paramyxoviruses, in that fusion is accomplished by the fusion (F) protein without the attachment glycoprotein (G protein). It has been suggested that hMPV F protein utilizes integrin αvß1 as a cellular receptor. Consistent with this, the F proteins of all known hMPV strains possess an integrin-binding motif ((329)RGD(331)). The role of this motif in viral entry, infectivity, and pathogenesis is poorly understood. Here, we show that α5ß1 and αv integrins are essential for cell-cell fusion and hMPV infection. Mutational analysis found that residues R329 and G330 in the (329)RGD(331) motif are essential for cell-cell fusion, whereas mutations at D331 did not significantly impact fusion activity. Furthermore, fusion-defective RGD mutations were either lethal to the virus or resulted in recombinant hMPVs that had defects in viral replication in cell culture. In cotton rats, recombinant hMPV with the R329K mutation in the F protein (rhMPV-R329K) and rhMPV-D331A exhibited significant defects in viral replication in nasal turbinates and lungs. Importantly, inoculation of cotton rats with these mutants triggered a high level of neutralizing antibodies and protected against hMPV challenge. Taken together, our data indicate that (i) α5ß1 and αv integrins are essential for cell-cell fusion and viral replication, (ii) the first two residues in the RGD motif are essential for fusion activity, and (iii) inhibition of the interaction of the integrin-RGD motif may serve as a new target to rationally attenuate hMPV for the development of live attenuated vaccines. IMPORTANCE: Human metapneumovirus (hMPV) is one of the major causative agents of acute respiratory disease in humans. Currently, there is no vaccine or antiviral drug for hMPV. hMPV enters host cells via a unique mechanism, in that viral fusion (F) protein mediates both attachment and fusion activity. Recently, it was suggested that hMPV F protein utilizes integrins as receptors for entry via a poorly understood mechanism. Here, we show that α5ß1 and αv integrins are essential for hMPV infectivity and F protein-mediated cell-cell fusion and that the integrin-binding motif in the F protein plays a crucial role in these functions. Our results also identify the integrin-binding motif to be a new, attenuating target for the development of a live vaccine for hMPV. These findings not only will facilitate the development of antiviral drugs targeting viral entry steps but also will lead to the development new live attenuated vaccine candidates for hMPV.

mRNA cap methylation influences pathogenesis of vesicular stomatitis virus in vivo.

UNLABELLED: One role of mRNA cap guanine-N-7 (G-N-7) methylation is to facilitate the efficient translation of mRNA. The role of mRNA cap ribose 2'-O methylation is enigmatic, although recent work has implicated this as a signature to avoid detection of RNA by the innate immune system (S. Daffis, K. J. Szretter, J. Schriewer, J. Q. Li, S. Youn, J. Errett, T. Y. Lin, S. Schneller, R. Zust, H. P. Dong, V. Thiel, G. C. Sen, V. Fensterl, W. B. Klimstra, T. C. Pierson, R. M. Buller, M. Gale, P. Y. Shi, M. S. Diamond, Nature 468:452-456, 2010, doi:10.1038/nature09489). Working with vesicular stomatitis virus (VSV), we previously showed that a panel of recombinant VSVs carrying mutations at a predicted methyltransferase catalytic site (rVSV-K1651A, -D1762A, and -E1833Q) or S-adenosylmethionine (SAM) binding site (rVSV-G1670A, -G1672A, and -G4A) were defective in cap methylation and were also attenuated for growth in cell culture. Here, we analyzed the virulence of these recombinants in mice. We found that rVSV-K1651A, -D1762A, and -E1833Q, which are defective in both G-N-7 and 2'-O methylation, were highly attenuated in mice. All three viruses elicited a high level of neutralizing antibody and provided full protection against challenge with the virulent VSV. In contrast, mice inoculated with rVSV-G1670A and -G1672A, which are defective only in G-N-7 methylation, were attenuated in vivo yet retained a low level of virulence. rVSV-G4A, which is completely defective in both G-N-7 and 2'-O methylation, also exhibited low virulence in mice despite the fact that productive viral replication was not detected in lung and brain. Taken together, our results suggest that abrogation of viral mRNA cap methylation can serve as an approach to attenuate VSV, and perhaps other nonsegmented negative-strand RNA viruses, for potential application as vaccines and viral vectors. IMPORTANCE: Nonsegmented negative-sense (NNS) RNA viruses include a wide range of significant human, animal, and plant pathogens. For many of these viruses, there are no vaccines or antiviral drugs available. mRNA cap methylation is essential for mRNA stability and efficient translation. Our current understanding of mRNA modifications of NNS RNA viruses comes largely from studies of vesicular stomatitis virus (VSV). In this study, we showed that recombinant VSVs (rVSVs) defective in mRNA cap methylation were attenuated in vitro and in vivo. In addition, these methyltransferase (MTase)-defective rVSVs triggered high levels of antibody responses and provided complete protection against VSV infection. Thus, this study will not only contribute to our understanding of the role of mRNA cap MTase in viral pathogenesis but also facilitate the development of new live attenuated vaccines for VSV, and perhaps other NNS RNA viruses, by inhibiting viral mRNA cap methylation.

[Effectiveness of anti-CD47 antibody and Ara-C combination targeting therapy NOD/SCID mouse of myeloid leukemia].

OBJECTIVE: To study the prognostic significance of CD47 in a NOD/SCID mouse model of acute myeloid leukemia (AML) and the best strategy for targeted therapy for this disease. METHODS: CD34(+)CD38(-) leukemia stem cells (LSCs) were separated and transplanted into NOD/SCID mice to establish a mouse model of acute monocytic leukemia (AMoL). Anti-human CD47 antibody, alone or combined with cytosine arabinoside (Ara-C), was used to treat the mice with AMoL for 7-14 days, and therapeutic efficacy was assessed. LSCs were cultured together with mouse macrophages in culture medium containing anti-CD47 or anti-CD45 monoclonal antibody for 2 hours, to observe the phagocytic ability of macrophages to LSCs. RESULTS: CD34(+)CD38(-) LSCs existed among THP-1 cells, with a content of about (0.12 ± 0.06)%, and a mouse model of AML was successfully established after the purified CD34(+)CD38(-) LSCs (97.0% ± 1.7%) were transplanted into NOD/SCID mice. The in vivo experiment showed that mice with AMoL had the most significant decrease in CD33(+)CD45(+) leukemia cells in peripheral blood and bone marrow and survived the longest after being treated with Ara-C (7 days) plus anti-CD47 monoclonal antibody (14 days) (P < 0.01). After 2 hours of in vitro culture, the phagocytic index in the culture medium containing anti-CD47 monoclonal antibody was significantly higher than in the culture medium containing anti-CD45 monoclonal antibody (76.9% ± 12.2% vs 7.60% ± 2.4%; P < 0.05). CONCLUSIONS: High expression of CD47 is an adverse prognostic factor in AML. Combination therapy with anti-CD47 monoclonal antibody and Ara-C can effectively eliminate leukemia cells and LSCs, demonstrating great clinical significance in curing AML.

Sorting, identification and enrichment of side population cells in THP-1 acute monocytic leukemia cells.

The objective of the present study was to examine and determine whether the human acute monocytic leukemia cell line THP-1 contains side population (SP) cells, and, if so, to increase the proportion of SP cells using arabinosylcytosine (Ara-C). Fluorescent microscopy and flow cytometry were employed to detect the percentage of SP cells in THP-1 cells. Then, SP and non-SP (NSP) cell subpopulations were collected and identified. THP-1 cells were incubated with different concentrations of Ara-C for 24 h and the proportion of SP cells was detected. Our results demonstrated that the percentage of SP cells was 1.81 ± 0.99% in THP-1 cells. A majority of the SP cells remained in the G0/G1 phase, and the expression of CD34⁺ and CD34⁺CD38⁻ and the proliferation ability of the SP cells were higher compared to NSP cells (P<0.05). The mRNA expression of multidrug resistance genes (ABCG2 and ABCB1), apoptosis regulation genes (Bcl-2) and the Bcl-2/Bax value of SP cells were higher than those of NSP cells. SP cells have been shown to be more tumorigenic than NSP cells. Following co-culture with Ara-C, the proportion of SP cells increased significantly and subsequently the Ara-C concentration increased. These findings suggest that the THP-1 cell line contains SP cells and that SP cells possess certain intrinsic stem cell properties and may contain a larger proportion of leukemia stem cells (LSCs). The concentrations of SP cells can be increased with Ara-C by co-culture, and this technique is a useful and important application for the study of LSCs.

Localization of a region in the fusion protein of avian metapneumovirus that modulates cell-cell fusion.

The genus Metapneumovirus within the subfamily Pneumovirinae of the family Paramyxoviridae includes two members, human metapneumovirus (hMPV) and avian metapneumovirus (aMPV), causing respiratory tract infections in humans and birds, respectively. Paramyxoviruses enter host cells by fusing the viral envelope with a host cell membrane. Membrane fusion of hMPV appears to be unique, in that fusion of some hMPV strains requires low pH. Here, we show that the fusion (F) proteins of aMPV promote fusion in the absence of the attachment protein and low pH is not required. Furthermore, there are notable differences in cell-cell fusion among aMPV subtypes. Trypsin was required for cell-cell fusion induced by subtype B but not subtypes A and C. The F protein of aMPV subtype A was highly fusogenic, whereas those from subtypes B and C were not. By construction and evaluation of chimeric F proteins composed of domains from the F proteins of subtypes A and B, we localized a region composed of amino acid residues 170 to 338 in the F protein that is responsible for the hyperfusogenic phenotype of the F from subtype A. Further mutagenesis analysis revealed that residues R295, G297, and K323 in this region collectively contributed to the hyperfusogenicity. Taken together, we have identified a region in the aMPV F protein that modulates the extent of membrane fusion. A model for fusion consistent with these data is presented.

Development and optimization of a direct plaque assay for human and avian metapneumoviruses.

The genus Metapneumovirus within the subfamily Pneumovirinae and family Paramyxoviridae includes only two viruses, human metapneumovirus (hMPV) and avian metapneumovirus (aMPV), which cause respiratory disease in humans and birds, respectively. These two viruses grow poorly in cell culture and other quantitation methods, such as indirect immuno-staining and immuno-fluorescent assays, are expensive, time consuming, and do not allow for plaque purification of the virus. In order to enhance research efforts for studying these two viruses, a direct plaque assay for both hMPV and aMPV has been developed. By optimizing the chemical components of the agarose overlay, it was found that both hMPV with a trypsin-independent F cleavage site and aMPV formed clear and countable plaques in a number of mammalian cell lines (such as Vero-E6 and LLC-MK2 cells) after 5 days of incubation. The plaque forming assay has similar sensitivity and reliability as the currently used immunological methods for viral quantitation. The plaque assay is also a more simple, rapid, and economical method compared to immunological assays, and in addition allows for plaque purification of the viruses. The direct plaque assay will be a valuable method for the quantitation and evaluation of the biological properties of some metapneumoviruses.

[Efficacy of immunosuppressive therapy for children with aplastic anemia].

OBJECTIVE: To study the effectiveness and safety of immunosuppressive therapy (IST) in the treatment of childhood aplastic anemia (AA) and to study the main factors influencing the effectiveness. METHODS: The clinical data of 55 children with severe aplastic anemia (SAA) and 51 children with chronic aplastic anemia (CAA) were retrospectively analyzed. All patients received IST from January 2007 to December 2010. RESULTS: In children with CAA, the effective rate of antithymocyte globulin (ATG) plus cyclosporine A(CsA) combination therapy was significantly higher than that of CsA alone (80% vs 44%; P<0.05); in children with SAA, the effective rate of ATG plus CsA combination therapy was also significantly higher than that of CsA alone (75% vs 40%; P<0.05). No patients developed clonal disease such as myelodysplastic syndrome, paroxysmal nocturn hemoglobinuria or acute myelocytic leukemia. In patients treated with the ATG plus CsA combination therapy, the response rate was relatively high for children whose disease course was less than six months, bone marrow hematopoietic area was more than 40%, had no severe infections, and experienced granulocyte colony stimulating factor (G-CSF) reaction during the early treatment; however, it was not related to AA subtypes and age. CONCLUSIONS: ATG plus CsA combination therapy is effective and safe in the treatment of childhood AA. The disease course, bone marrow hematopoietic area, severe infections and G-CSF reaction to early treatment are the main factors influencing the therapeutic effects.

Identification of aromatic amino acid residues in conserved region VI of the large polymerase of vesicular stomatitis virus is essential for both guanine-N-7 and ribose 2'-O methyltransferases.

Non-segmented negative-sense RNA viruses possess a unique mechanism for mRNA cap methylation. For vesicular stomatitis virus, conserved region VI in the large (L) polymerase protein catalyzes both guanine-N-7 (G-N-7) and ribose 2'-O (2'-O) methyltransferases, and the two methylases share a binding site for the methyl donor S-adenosyl-l-methionine. Unlike conventional mRNA cap methylation, the 2'-O methylation of VSV precedes subsequent G-N-7 methylation. In this study, we found that individual alanine substitutions in two conserved aromatic residues (Y1650 and F1691) in region VI of L protein abolished both G-N-7 and 2'-O methylation. However, replacement of one aromatic residue with another aromatic residue did not significantly affect the methyltransferase activities. Our studies provide genetic and biochemical evidence that conserved aromatic residues in region VI of L protein essential for both G-N-7 and 2'-O methylations. In combination with the structural prediction, our results suggest that these aromatic residues may participate in RNA recognition.

Novel parvovirus sublineage in the family of Parvoviridae.

Parvoviridae, which are classified into two subfamilies Parvovirinae and Densovirinae, can infect both vertebrate and insects and are related to a wide range of diseases in insects, animals, and humans. In this report, several new parvoviruses were identified in swine sera collected in southeastern China. The sequence analyses showed that the parvoviruses detected in southeastern China formed a distinct sublineage within the subfamily Parvovirinae. Based on these results, we propose a novel parvovirus sublineage, Cnvirus, to describe these parvoviruses.

Reassortant infectious bursal disease virus isolated in China.

Infectious bursal disease virus (IBDV) is a bi-segmented, double-stranded RNA virus which belongs to the genus Avibirnavirus of the family Birnavirideae. In this study, we determined the complete nucleotide sequences of a reassortment IBDV strain TL2004 with segments A and B derived from attenuated and very virulent strains of IBDV. This strain is pathogenic to SPF-embryonated eggs and chickens, although it is not as virulent as very virulent strain. Genomic sequence in GenBank analysis showed that both types of natural genetic reassortment of infectious bursal disease virus emerged in China. Our findings, which strongly suggest genetic exchange between attenuated and very virulent strains of IBDV, emphasizes the risk of generating uncontrolled chimeric viruses by using live attenuated vaccines.