Current Trends in Viral Gene Therapy for Human Orthopaedic Regenerative Medicine

BACKGROUND: Viral vector-based therapeutic gene therapy is a potent strategy to enhance the intrinsic reparative abilities of human orthopaedic tissues. However, clinical application of viral gene transfer remains hindered by detrimental responses in the host against such vectors (immunogenic responses, vector dissemination to nontarget locations). Combining viral gene therapy techniques with tissue engineering procedures may offer strong tools to improve the current systems for applications in vivo. METHODS: The goal of this work is to provide an overview of the most recent systems exploiting biomaterial technologies and therapeutic viral gene transfer in human orthopaedic regenerative medicine. RESULTS: Integration of tissue engineering platforms with viral gene vectors is an active area of research in orthopaedics as a means to overcome the obstacles precluding effective viral gene therapy. CONCLUSION: In light of promising preclinical data that may rapidly expand in a close future, biomaterial-guided viral gene therapy has a strong potential for translation in the field of human orthopaedic regenerative medicine.

Novel variants of human herpesvirus 2 from Brazilian HIV-1 coinfected subjects

BACKGROUND Human herpesvirus 2 (HHV-2) have DNA genome with a limited genetic variability and have been classified into two clades. OBJECTIVES To identify and characterise six HHV-2 isolates derived from Brazilian women. METHODS HHV-2 isolates were performed polymerase chain reaction (PCR) and sequencing of 2250 pb of the glycoprotein B (gB) coding regions. FINDINGS Four HHV-2 isolates were classified into clade B, while the remaining two, derived from HIV-1 co-infected women, showed a notable genetic divergence (> 1%). MAIN CONCLUSION The results reveal novel HHV-2 variants. The impact of these novel variants on HHV-2 pathogenesis and HIV/HHV-2 coinfection need to be investigated.

Nucleocapsid protein from porcine epidemic diarrhea virus isolates can antagonize interferon-λ production by blocking the nuclear factor-κB nuclear translocation / 浙江大学学报（英文版）（B辑：生物医学和生物技术）

Porcine epidemic diarrhea virus (PEDV) is a highly infectious pathogen that can cause severe diseases in pigs and result in enormous economic losses in the worldwide swine industry. Previous studies revealed that PEDV exhibits an obvious capacity for modulating interferon (IFN) signaling or expression. The newly discovered type III IFN, which plays a crucial role in antiviral immunity, has strong antiviral activity against PEDV proliferation in IPEC-J2 cells. In this study, we aimed to investigate the effect of PEDV nucleocapsid (N) protein on type III IFN-λ. We found that the N proteins of ten PEDV strains isolated between 2013 and 2017 from different local farms shared high nucleotide identities, while the N protein of the CV777 vaccine strain formed a monophyletic branch in the phylogenetic tree. The N protein of the epidemic strain could antagonize type III IFN, but not type I or type II IFN expression induced by polyinosinic-polycytidylic acid (poly(I:C)) in IPEC-J2 cells. Subsequently, we demonstrated that the inhibition of poly(I:C)-induced IFN-λ3 production by PEDV N protein was dependent on the blocking of nuclear factor-κB (NF-κB) nuclear translocation. These findings might help increase understanding of the pathogenesis of PEDV and its mechanisms for evading the host immune response.

Possible Transmission of Irkut Virus from Dogs to Humans / 生物医学与环境科学（英文）

Lyssaviruses, including Rabies virus, Duvenhage virus, European bat lyssavirus 1, European bat lyssavirus 2, Australian bat lyssavirus, and Irkut virus (IRKV), have caused human fatalities, but infection of IRKV in dogs has not been previously reported. In China, a dead dog that previously bit a human was determined to be infected with IRKV. Pathogenicity tests revealed that IRKVs can cause rabies-like disease in dogs and cats after laboratory infection. The close relationship between humans and pets, such as dogs and cats, may generate a new spillover-spreading route for IRKV infection. Therefore, additional attention should be paid to trans-species infection of IRKV between bats and dogs or dogs and humans through investigation of the prevalence and circulation patterns of IRKV in China.

Detection and genotyping of human adenovirus and sapovirus in children with acute gastroenteritis in Belém, Pará, between 1990 and 1992: first detection of GI. 7 and GV. 2 sapoviruses in Brazil

Abstract INTRODUCTION: Acute gastroenteritis (AGE) is one of the most common causes of morbidity and mortality, especially among children from developing countries. Human adenovirus (HAdV) and sapovirus (SaV) are among the agents that cause AGE. The present study aimed to detect and genotype HAdV and SaV in 172 fecal samples from children with AGE, collected during a surveillance study carried out in a low-income community in Belém, Pará, between 1990 and 1992. METHODS: HAdV was detected by nested PCR, using primers Hex1deg/Hex2deg and NeHex3deg/NeHex4deg. SaV was assayed by reverse transcription PCR (RT-PCR), nested PCR, and quantitative PCR. The nucleotide sequence was determined by direct cycle sequencing. RESULTS: Overall, 43% (74/172) of samples were positive for HAdV, of which 70.3% (52/74) were sequenced and classified as belonging to five different species, mostly A and F. For SaV, positivity was 5.2% (9/172) and genotypes GI.1, GI.7, GII.1, and GV.2 were detected. CONCLUSIONS: The present results reinforce the need for further studies to obtain epidemiological data about the circulation of these viruses in Brazil, especially in the Amazon Region, where data from the early 1990's are scarce. Furthermore, the study describes for the first time the detection of SaV genotypes GI.7 and GV.2 in Brazil, showing that these types circulated in the region more than 25 years ago.

A highly sensitive method for the detection of Chrysanthemum virus B

Background: Chrysanthemum plants are subject to serious viral diseases. The viruses cause severe losses of the quantity and quality of chrysanthemum. The most problematic pathogen of chrysanthemum is typically considered Chrysanthemum virus B (CVB). Thus, a method for the simultaneous detection of CVB is needed. Results: We used gene-specific primers, which were derived from the coat protein gene region of the virus, for reverse transcription to obtain cDNA. Nested amplification polymerase chain reaction (PCR) was employed to detect the viral gene. This method was sensitive enough to detect the virus at up to 10-9 dilution of the cDNA. Conclusion: A highly specific and sensitive nested PCR-based assay has been described for detecting CVB. This new method is highly specific and sensitive for the detection of CVB, which is known to infect chrysanthemum plants in the fields. Further, this protocol has an advantage over traditional methods as it is more cost-effective. This assay is ideal for an early stage diagnosis of the disease.

Experimental infection of dogs with highly pathogenic avian influenza virus (H5N8)

During the highly pathogenic avian influenza (HPAI) H5N8 virus outbreak in Korea, a dog in layer farm contaminated by H5N8 was reported seropositive for HPAI H5N8. To investigate the possibility of adaptation and transmission of HPAI H5N8 to dogs, we experimentally inoculated dogs with H5N8. Viral genes were weakly detected in nasal swabs and seroconversions in inoculated and contact dogs. Although the H5N8 virus did not induced severe clinical signs to dogs, the results suggest that surveillance of farm dogs should continue as a species in which the avian influenza virus may acquire infectivity to mammals through frequent contact with the virus.

Diversity of group A rotavirus genes detected in the Triângulo Mineiro region, Minas Gerais, Brazil

ABSTRACT Group A rotaviruses are the main causative agent of infantile gastroenteritis. The segmented nature of the viral genome allows reassortment of genome segments, which can generate genetic variants. In this study, we characterized the diversity of the VP7, VP4 (VP8*), VP6, NSP4, and NSP5 genes of the rotaviruses that circulated from 2005 to 2011 in the Triângulo Mineiro (TM) region of Brazil. Samples with genotypes G2 (sublineages IVa-1 and IVa-3), G1 (sublineage I-A), G9 (lineage III), G12 (lineages II and III), G8 (lineage II), G3 (lineage III), P[4] (sublineages IVa and IVb), P[8] (sublineages P[8]-3.6, P[8]-3.3, and P[8]-3.1), I2 (lineage VII), E2 (lineages VI, XII, and X), and H2 (lineage III) were identified. The associations found in the samples were G1, G9, or G12 with P[8]-I1-E1-H1; G2 or G8 with P[4]-I2-E2-H2; G12 with I3-E3-H6; and G3 with P[4]-I2-E3-H3 (previously unreported combination). Reassortment events in G2P[4] strains and an apparent pattern of temporal segregation within the lineages were observed. Five TM samples contained genes that exhibited high nucleotide and amino acid identities with strains of animal origin. The present study includes a period of pre- and post-introduction of rotavirus vaccination in all Brazilian territories, thereby serving as a basis for monitoring changes in the genetic constitution of rotaviruses. The results also contribute to the understanding of the diversity and evolution of rotaviruses in a global context.

Molecular characterization and phylogenetic analysis of pseudorabies virus variants isolated from Guangdong province of southern China during 2013–2014

Outbreaks of pseudorabies (PR) have occurred in southern China since late 2011, resulting in significant economic impacts on the swine industry. To identify the cause of PR outbreaks, especially among vaccinated pigs, 11 pseudorabies virus (PRV) field strains were isolated from Guangdong province during 2013–2014. Their major viral genes (gE, TK, gI, PK, gD, 11K, and 28K) were analyzed in this study. Insertions or deletions were observed in gD, gE, gI and PK genes compared with other PRV isolates from all over the world. Furthermore, sequence alignment showed that insertions in gD and gE were unique molecular characteristics of the new prevalent PRV strains in China. Phylogenetic analysis showed that our isolates were clustered in an independent branch together with other strains isolated from China in recent years, and that they showed a closer genetic relationship with earlier isolates from Asia. Our results suggest that these isolates are novel PRV variants with unique molecular signatures.

Percepción parental de la salud psicofísica, estado nutricional y salud bucal, en relación con características sociodemográficas en niños de Bariloche, Argentina: estudio epidemiológico / Parental perception of psychophysical health, nutritional status and oral health in relation to sociodemographic characteristics in children in Bariloche, Argentina: an epidemiological study

Introducción. Existen evidencias de la asociación de determinantes sociales con la salud infantil. Objetivo. Identificar características sociodemográficas asociadas a desigualdades en la salud infantil y evaluar el efecto acumulado sobre la salud de factores de riesgo basados en estas características. Población y métodos. Evaluamos niños de 4-13 años, de Bariloche, entre junio de 2008 y mayo de 2009. Características sociodemográficas consideradas: nivel socioeconómico, educación materna, embarazo adolescente, cobertura médica, inseguridad y hábitos familiares. Valoramos la percepción parental de la salud física y socioemocional, el estado nutricional y la salud bucal en relación con dichas características y con la acumulación de factores de riesgo. Utilizamos encuesta, antropometría y examen bucal. Resultados. Participaron 180 escolares. El nivel educativo materno se asoció con la salud física, socioemocional y bucal del niño. El porcentaje de niños con piezas faltantes o caries fue 77% entre aquellos cuyas madres, como máximo, habían completado el primario, comparado con 13% entre aquellos cuyas madres habían completado estudios terciarios/universitarios. La posibilidad de percepción de salud física y socioemocional no óptima aumentó con cada factor de riesgo 1,8 y 1,4 veces, respectivamente, y la posibilidad de caries o piezas faltantes se duplicó con cada factor de riesgo adicional. El 27,3% de los escolares presentó sobrepeso y el 8,7%, obesidad, y no se encontró asociación con características sociodemográficas. Conclusiones. El bajo nivel socioeconómico familiar y educativo materno se asoció con una mayor prevalencia de resultados de salud desfavorables. Múltiples factores de riesgo tienen un efecto acumulado sobre la percepción parental de la salud física y socioemocional y la salud bucal.

Introduction. There is evidence of an association between social determinants and child health. Objective. To identify sociodemographic characteristics related to child health inequalities and to analize the cumulative effect on health of risk factors based on these characteristics. Population and Methods. We evaluated 4-13 year-old children in Bariloche between June 2008 and May 2009. The following sociodemographic characteristics were taken into account: socioeconomic level, maternal education, adolescent pregnancy, medical coverage, unsafeness, and family habits. We assessed parental perception of physical, and social and emotional health, nutritional status and oral health in relation to these characteristics and the accumulation of risk factors. We used survey, anthropometry and oral examination. Results. One hundred and eighty students participated. The level of maternal education was associated with the child's physical, social and emotional, and oral health. The percentage of children with missing teeth or cavities reached 77% among those whose mothers had, at most, completed primary school, compared to 13% among those whose mothers had completed tertiary school or university. The possibility of perceiving a non-optimal physical, and social and emotional health increased 1.8 and 1.4 times with each risk factor, respectively, and the possibility of having missing teeth or cavities was twice as much with each additional risk factor. Overweight and obesity was observed in 27.3% and 8.7% of students, respectively, and no relationship was found with sociodemographic characteristics. Conclusions. A low family socioeconomic level and a low maternal education level were associated with a higher prevalence of unfavorable health outcomes. Multiple risk factors have an cumulative effect on parental perception of physical, social and emotional, and oral health.

Monitoring and molecular characterization of group Drotavirus in brazilian poultry farms / Monitoramento e caracterização molecular de rotavírus do grupo D em criações comerciais brasileiras

Rotaviruses are etiological agents of diarrhea both in humans and in several animal species. Data on avian Group D rotaviruses (RVD) are scarce, especially in Brazil. We detected RVD in 4 pools of intestinal contents of broilers, layer and broiler breeders out of a total of 111 pools from 8 Brazilian states, representing an occurrence of 3.6%, by a specific RVD RT-PCR targeting the VP6 gene. Phylogenetic tree confirmed that the Brazilian strains belong to group D and 3 of the sequences were identical in terms of amino acid whereas one showed 99.5% identity with the others. The sequences described in this study are similar to other sequences previously detected in Brazil, confirming the conserved nature of the VP6 protein.

Rotavírus são agentes etiológicos de diarreia tanto em humanos como em várias espécies animais. Dados sobre rotavírus do grupo D (RVD) em aves são escassos, especialmente no Brasil. Nós detectamos RVD em 4 pools de conteúdo intestinal de frango de corte, poedeiras e matrizes de um total de 111 pools originários de 8 estados brasileiros, representando uma ocorrência de 3,6% a partir de uma RT-PCR específica para RVD, tendo como alvo o gene VP6. A árvore filogenética confirmou que as amostras brasileiras pertencem ao grupo D e três das sequências obtidas foram idênticas em termos de aminoácidos enquanto uma apresentou 99,5% de identidade com as demais. As sequências aqui definidas são semelhantes a outras sequências previamente definidas no Brasil, confirmando a natureza conservada da proteína VP6.

Characterization and evaluation of apoptotic potential of double gene construct pVIVO.VP3.NS1.

Viral gene oncotherapy, targeted killing of cancer cells by viral genes, is an emerging non-infectious therapeutic cancer treatment modality. Chemo and radiotherapy in cancer treatment is limited due to their genotoxic side effects on healthy cells and need of functional p53, which is mutated in most of the cancers. VP3 (apoptin) of chicken infectious anaemia (CIA) and NS1 (Non structural protein 1) of Canine Parvovirus-2 (CPV-2) have been proven to have oncolytic potential in our laboratory. To evaluate oncolytic potential of VP3 and NS1 together these genes needed to be cloned in a bicistronic vector. In this study, both these genes were cloned and characterized for expression of their gene products and its apoptotic potential. The expression of VP3 and NS1 was studied by confocal microscopy and flowcytometry. Expression of VP3 and NS1 in pVIVO.VP3.NS1 transfected HeLa cells in comparison to mock transfected cells indicated that the double gene construct expresses both the products. This was further confirmed by flowcytometry where there was increase in cells expressing VP3 and NS1 in pVIVO.VP3.NS1 transfected group in comparison with the mock control group. The apoptotic inducing potential of this characterized pVIVO.VP3.NS1 was evaluated in human cervical cancer cell line (HeLa) by DNA fragmentation assay, TUNEL assay and Hoechst staning. This double construct was observed to induce apoptosis in HeLa cells.

Microevolutionary trends of dengue virus-3 in Kerala, India.

Envelope gene is of great evolutionary significance and had been targeted as the vaccine candidate for dengue virus. We analyzed partial sequences of this gene to understand its genetic variability among viral isolates from Kerala state, India, if any. The current study focused on the evolutionary trends of this phylogenetically important gene among DENV-3 isolates through 2008 to 2010 outbreaks. The results gave an insight into the microevolutionary trends of the dengue viral genome. A unique mutation was recorded in the Domain II of the Envelope gene (EDII) of the viral genome at the amino acid position 219 (A219T). The evolutionary implication of this non-synonymous mutation near the EDI/EDII hinge remains to be explored. The study also provided knowledge on the genetic ancestral history of the viral isolates. Two variants of different phylogenetic origin were recorded in Kerala State. The findings in the study have significant implications on the development of dengue vaccines based on the Envelope gene of the virus.

Epstein-Barr virus latent genes

Latent Epstein-Barr virus (EBV) infection has a substantial role in causing many human disorders. The persistence of these viral genomes in all malignant cells, yet with the expression of limited latent genes, is consistent with the notion that EBV latent genes are important for malignant cell growth. While the EBV-encoded nuclear antigen-1 (EBNA-1) and latent membrane protein-2A (LMP-2A) are critical, the EBNA-leader proteins, EBNA-2, EBNA-3A, EBNA-3C and LMP-1, are individually essential for in vitro transformation of primary B cells to lymphoblastoid cell lines. EBV-encoded RNAs and EBNA-3Bs are dispensable. In this review, the roles of EBV latent genes are summarized.

Discovery and whole genome sequences analysis of a dengue virus type 1 strain isolated in Henan province / 中华预防医学杂志

<p><b>OBJECTIVE</b>To diagnose imported dengue fever case from Henan province, and to sequence and analyze the characteristics of whole genome sequence, and to explore the possible viral origin source.</p><p><b>METHODS</b>A suspected dengue fever case was reported in Yuzhou city, Henan province. The patient returned from foshan, Guangdong province on September 19, 2014, after the epidemiological investigation and serum specimen collected, which dengue fever case was diagnosed in the laboratory, then it was inoculated on Vero cells. Whole genome sequence was amplified by several pairs primers and characterized using biologic software.</p><p><b>RESULTS</b>The imported case was diagnosed as dengue virus 1 serotype infection. Dengue 1 strain was isolated using Vero cells successfully. Whole genome was 10,670 nt, which belonged to dengue virus 1 serotype V genotype and didn't found any recombination event. The phylogenetic analysis demonstrated that the strain was closed to Indian starins isolated in 2008-2011, and the homology of nucleotide sequence was between 98.2%-99.4%.</p><p><b>CONCLUSION</b>It was the first time to discover imported dengue 1 serotype case in Henan province. However, according to the patient has been to Guangdong province before onset, it inferred that the Indian strain had been imported to Guangdong province before this case in Henan province.</p>

Differential Regulation of NF-kappaB Signaling during Human Cytomegalovirus Infection

NF-kappaB transcription factors are key regulators of immune and stress responses, apoptosis, and differentiation. Human cytomegalovirus (HCMV) activates or represses NF-kappaB signaling at different times during infection. An initial increase in NF-kappaB activity occurs within a few hours of infection. The virus appears to adapt to this change since initial viral gene expression is promoted by the elevated NF-kappaB activity. Because NF-kappaB upregulates innate immune responses and inflammation, it has also been suggested that HCMV needs to downregulate NF-kappaB signaling. Recent studies have shown that HCMV has various mechanisms that inhibit NF-kappaB signaling. HCMV reduces cell surface expression of tumor necrosis factor receptor 1 (TNFR1) and blocks the DNA binding activity of NF-kappaB. Furthermore, some HCMV tegument proteins antagonize NF-kappaB activation by targeting the key components of NF-kappaB signaling at late stages of infection. In this review, we summarize the recent findings on the relationship between HCMV and NF-kappaB signaling, focusing, in particular, on the viral mechanisms by which the NF-kappaB signaling pathway is inhibited.

Severe diarrhea outbreak in beef calves (Bos indicus) caused by G6P[11], an emergent genotype of bovine rotavirus group A / Surto de diarreia neonatal em bezerros de corte (Bos indicus) ocasionado por um genotipo emergente G6P[11] de rotavírus bovino grupo A

The episodes of diarrhea caused by neonatal bovine rotavirus group A (BoRVA) constitute one of the major health problems in the calf rearing worldwide. The main G (VP7) and P (VP4) genotypes of BoRVA strains involved in the etiology of diarrhea in calves are G6P[1], G10P[11], G6P[5], and G8P[1]. However, less frequently, other G and P genotypes have been described in BoRVA strains identified in diarrheic fecal samples of calves. This study describes the identification and molecular characterization of an emerging genotype (G6P[11]) in BoRVA strains involved in the etiology of a diarrhea outbreak in beef calves in a cattle herd of high production in extensive management system. The diarrhea outbreak, which showed high morbidity (60%) and lethality (7%) rates, occurred in calves (n= 384) Nelore (Bos indicus) up to 30-day-old from the State of Mato Grosso do Sul, Brazil. BoRVA was identified in 80% (16/20) of the fecal samples analyzed by polyacrylamide gel electrophoresis (PAGE) technique. In all PAGE-positive fecal samples were amplified products with 1,062-bp and 876-bp in the RT-PCR assays for VP7 (G type) and VP4 (VP8*) (P type) of BoRVA, respectively. The nucleotide sequence analysis of VP7 and VP4 genes of four wild-type BoRVA strains showed G6-III P[11]-III genotype/lineage. The G6P[11] genotype has been described in RVA strains of human and animal hosts, however, in calves this genotype was only identified in some cross-sectional studies and not as a single cause of diarrhea outbreaks in calves with high morbidity and lethality rates as described in this study...

Os episódios de diarreia neonatal ocasionados pelo rotavírus bovino grupo A (BoRVA) constituem-se em um dos principais problemas sanitários na criação de bezerros em todo o mundo. Os principais genotipos G (VP7) e P (VP4) de cepas de BoRVA envolvidos na etiologia da diarreia em bezerros são G6P[1], G10P[11], G6P[5] e G8P[1]. No entanto, com menor frequência, outros genotipos G e P têm sido descritos em cepas de BoRVA identificadas em amostras de fezes diarreicas de bezerros. Este estudo descreve a identificação e caracterização molecular de um genotipo emergente (G6P[11]) em cepas de BoRVA envolvidas na etiologia de um surto de diarreia em bezerros de um rebanho bovino de corte de alta produção em sistema de manejo extensivo. O surto, que apresentou altas taxas de morbidade (60%) e de letalidade (7%), ocorreu em bezerros (n=384) da raça Nelore (Bos indicus) com até 30 dias de idade, provenientes do estado do Mato Grosso do Sul, Brasil. O BoRVA foi identificado em 80% (16/20) das amostras fecais analisadas pela técnica de eletroforese em gel de poliacrilamida (PAGE). Em todas as amostras fecais PAGE-positivas foi possível a amplificação por RT-PCR de produtos com 1.062 pb e 876 pb referentes aos genes VP7 (G tipo) e VP4 (VP8*) (P tipo), respectivamente, de BoRVA. A análise da sequência de nucleotídeos dos genes VP7 e VP4 de quatro cepas de BoRVA demonstrou a presença do genotipo/linhagem G6-III P[11]-III. O genotipo G6P[11] tem sido descrito em cepas de RVA de hospedeiros humanos e animais. Contudo, em bezerros, este genotipo foi apenas identificado em alguns estudos transversais e não como a única causa de surtos de diarreia em bezerros com altas taxas de morbidade e...

Drug susceptibility and UL97 gene mutation analysis of cytomegalovirus in recipients of hematopoietic stem cell transplantation / 南方医科大学学报

<p><b>OBJECTIVE</b>To monitor human cytomegalovirus (HCMV) drug resistance in recipients of hematopoietic stem cell transplantation by phenotypic and genotypic methods.</p><p><b>METHODS</b>HCMV clinical isolates was isolated from the urine of hematopoietic stem cell transplantation recipients treated with GCV. Tissue cell infection median dose (TCID50) of the isolates was calculated using Reed-Muench method, and their drug susceptibility was determined by plaque reduction assay. We amplified the UL97 DNA fragment of the virus by nested PCR followed by automated DNA sequencing.</p><p><b>RESULTS</b>HCMV clinical strain isolated from the urine samples of the recipients using a human fibroblast cell line showed a TCID50 value of 10(-4.618)/0.1 ml and a 50% inhibitory concentration (IC50) to GCV of 5.847 µmol/L, suggesting its sensitivity to GCV. Alignment with the AD169 DNA reference sequence identified 4 point mutations of the virus at 1509 (T-C), 1575 (C-T), 1794 (T-C), and 1815 (C-G), and only the last mutation resulted in one amino acid mutation to D605E. No gene mutation was found in relation to GCV resistance.</p><p><b>CONCLUSIONS</b>Phenotypic and genotypic assays were established to examine antiviral drug resistance of HCMV in recipients of hematopoietic stem cell transplantation. We did not find any drug resistance of the clinical HCMV isolate.</p>

The construction of a novel recombinant virus Δ67R-RGV and preliminary analyses the function of the 67R gene / 病毒学报

The Rana grylio virus (RGV) is a member of the genus Ranavirus. It belongs to the family Iridoviridae, and contains the gene 67R encoding dUTPase. In order to investigate the function of 67R in the replication and infection of RGV, we constructed Δ67R-RGV, a recombinant virus with deletion of 67R. First, we constructed the plasmid pGL3-67RL-p50-EGFP-67RR which carried an enhanced green fluorescence gene (EGFP) as a selectable marker. After homologous recombination between pGL3-67RL-p50-EG- FP-67RR and the RGV genome, Epithelioma papulosum cyprini (EPC) cells were infected with the resulting mixture. Through ten successive rounds of plaque isolation via EGFP selection, all plaques emitted green fluorescence, and finally Δ67R-RGV was generated. Total DNA of Δ67R-RGV infected cells was extracted for PCR analyses. Simulateously, mock infected and wild-type RGV (wt-RGV) infected cells were used as a comparison. Results showed that 67R could be detected in wt-RGV infected cells, but that only the EGFP gene was detected in Δ67R-RGV infected cells. Furthermore, one-step growth curves of wt-RGV and Δ67R-RGV were similar. Therefore, 67R and its encoding product dUTPase might not be essential for the growth of RGV. These results suggest that, homologous recombination and recombinant rana- virus could be used to study the gene function of viruses in aquatic animals.

Analysis of B/C recombinant structure of C gene in a newly found HBV subtype / 病毒学报

Hepatitis B is one of the most serious global threats to human health. Phylogenetic analysis of hepatitis B virus (HBV) can reveal the evolutionary relationship between HBV sequences and thus provide a basis for the prediction and treatment of hepatitis B and other aspects. In this study, we performed sequence analyses on the HBV sequences of five clinical HBV samples and the HBV sequences retrieved from the GenBank, EMBL, and DDBJ to construct a phylogenetic tree and analyze sequence structures. The experimental results revealed that the C gene of one cloned sequence had a recombinant structure of HBV B/ C subtype. Moreover, the phylogenetic results proved the existence of a newly found subtype HBV/B6 in Xishuangbanna of Yunnan Province, China. The experimental conclusion represents certain value for phylogenetic studies of HBV in Yunnan ethnic minority groups.