scholarly journals Identification of Virulence Associated Region during Highly Pathogenic Porcine Reproductive and Respiratory Syndrome Virus during Attenuation In Vitro: Complex Question with Different Strain Backgrounds

Viruses ◽  
2021 ◽  
Vol 14 (1) ◽  
pp. 40
Author(s):  
Yifeng Jiang ◽  
Wu Tong ◽  
Lingxue Yu ◽  
Liwei Li ◽  
Fei Gao ◽  
...  
Keyword(s):  
Virus Replication ◽  
Cytokine Response ◽  
Lung Damage ◽  
Respiratory Syndrome Virus ◽  
Cdna Clones ◽  
Highly Pathogenic ◽  
Prrsv Strain ◽  
Chimeric Viruses

Highly pathogenic porcine reproductive and respiratory syndrome virus PRRSV (HP-PRRSV) was one of the most devastating diseases of the pig industry, among various strategies, vaccination was one of the most useful tools for PRRS control. Attenuated live vaccine was used worldwide, however, the genetic basis of HP-PRRSV virulence change during attenuation remain to be determined. Here, to identify virulence associated regions of HP-PRRSV during attenuation in vitro, six full-length infectious cDNA clones with interchanges of 5′UTR + ORF1a, ORF1b, and ORF2-7 + 3′UTR regions between HP-PRRSV strain HuN4-F5 and its attenuated vaccine strain HuN4-F112 were generated, and chimeric viruses were rescued. Piglets were inoculated with chimeric viruses and their parental viruses, and rectal temperature were recorded daily, and serum were collected for future experiments. Our results showed that ORF1a played an important role on virus replication, cytokine response and lung damage, the exchange of ORF1b and ORF2-7 in different backbone led to different exhibition on virus replication in vivo/vitro and cytokine response. Among 9 PRRSV attenuated series, consistent amino acid changes during PRRSV attenuation were found in NSP4, NSP9, GP2, E, GP3 and GP4. Our study provides a fundamental data for the investigation of PRRSV attenuation, the different results of the virulence change among different studies indicated that different mechanisms might be used during PRRSV virulence enhancement in vivo and attenuation in vitro.

scholarly journals A Synthetic Porcine Reproductive and Respiratory Syndrome Virus Strain Confers Unprecedented Levels of Heterologous Protection

2015 ◽  
Vol 89 (23) ◽  
pp. 12070-12083 ◽  
Author(s):  
Hiep L. X. Vu ◽  
Fangrui Ma ◽  
William W. Laegreid ◽  
Asit K. Pattnaik ◽  
David Steffen ◽  
...  
Keyword(s):  
Reverse Genetics ◽  
Vaccine Coverage ◽  
Rna Virus ◽  
Respiratory Syndrome Virus ◽  
Full Genome ◽  
Genome Sequences ◽  
Prrsv Strain ◽  
Heterologous Protection

ABSTRACTCurrent vaccines do not provide sufficient levels of protection against divergent porcine reproductive and respiratory syndrome virus (PRRSV) strains circulating in the field, mainly due to the substantial variation of the viral genome. We describe here a novel approach to generate a PRRSV vaccine candidate that could confer unprecedented levels of heterologous protection against divergent PRRSV isolates. By using a set of 59 nonredundant, full-genome sequences of type 2 PRRSVs, a consensus genome (designated PRRSV-CON) was generated by aligning these 59 PRRSV full-genome sequences, followed by selecting the most common nucleotide found at each position of the alignment. Next, the synthetic PRRSV-CON strain was generated through the use of reverse genetics. PRRSV-CON replicates as efficiently as our prototype PRRSV strain FL12, bothin vitroandin vivo. Importantly, when inoculated into pigs, PRRSV-CON confers significantly broader levels of heterologous protection than does wild-type PRRSV. Collectively, our data demonstrate that PRRSV-CON can serve as an excellent candidate for the development of a broadly protective PRRSV vaccine.IMPORTANCEThe extraordinary genetic variation of RNA viruses poses a monumental challenge for the development of broadly protective vaccines against these viruses. To minimize the genetic dissimilarity between vaccine immunogens and contemporary circulating viruses, computational strategies have been developed for the generation of artificial immunogen sequences (so-called “centralized” sequences) that have equal genetic distances to the circulating viruses. Thus far, the generation of centralized vaccine immunogens has been carried out at the level of individual viral proteins. We expand this concept to PRRSV, a highly variable RNA virus, by creating a synthetic PRRSV strain based on a centralized PRRSV genome sequence. This study provides the first example of centralizing the whole genome of an RNA virus to improve vaccine coverage. This concept may be significant for the development of vaccines against genetically variable viruses that require active viral replication in order to achieve complete immune protection.

scholarly journals The 30-Amino-Acid Deletion in the Nsp2 of Highly Pathogenic Porcine Reproductive and Respiratory Syndrome Virus Emerging in China Is Not Related to Its Virulence

2009 ◽  
Vol 83 (10) ◽  
pp. 5156-5167 ◽  
Author(s):  
Lei Zhou ◽  
Jialong Zhang ◽  
Jingwen Zeng ◽  
Shuoyan Yin ◽  
Yanhua Li ◽  
...  
Keyword(s):  
Amino Acid ◽  
Parental Virus ◽  
Respiratory Syndrome Virus ◽  
Cdna Clones ◽  
Coding Region ◽  
Amino Acid Deletion ◽  
Highly Pathogenic ◽  
Specific Pathogen ◽  
Prrsv Strain ◽  
Highly Pathogenic Prrsv

ABSTRACT During the past 2 years, an atypical clinical outbreak, caused by a highly pathogenic porcine reproductive and respiratory syndrome virus (PRRSV) with a unique 30-amino-acid deletion in its Nsp2-coding region, was pandemic in China. In this study, we generated four full-length infectious cDNA clones: a clone of the highly virulent PRRSV strain JXwn06 (pWSK-JXwn), a clone of the low-virulence PRRSV strain HB-1/3.9 (pWSK-HB-1/3.9), a chimeric clone in which the Nsp2 region containing the 30-amino-acid deletion was replaced by the corresponding region of the low-virulence PRRSV strain HB-1/3.9 (pWSK-JXwn-HB1nsp2), and a mutated HB-1/3.9 clone with the same deletion in Nsp2 as JXwn06 (pWSK-HB1-ND30). We also investigated the pathogenicities of the rescued viruses (designated RvJXwn, RvJXwn-HB1nsp2, RvHB-1/3.9, and RvHB1-ND30, respectively) in specific-pathogen-free piglets in order to determine the role of the 30-amino-acid deletion in the virulence of the highly pathogenic PRRSV. All the rescued viruses could replicate stably in MARC-145 cells. Our findings indicated that RvJXwn-HB1nsp2 retained high virulence for piglets, like RvJXwn and the parental virus JXwn06, although the survival time of piglets infected with RvJXwn-HB1nsp2 was obviously prolonged. RvHB1-ND30 exhibited low virulence for piglets, like RvHB-1/3.9 and the parental virus HB-1/3.9. Therefore, we conclude that the 30-amino-acid deletion is not related to the virulence of the highly pathogenic PRRSV emerging in China.

scholarly journals Two Residues in NSP9 Contribute to the Enhanced Replication and Pathogenicity of Highly Pathogenic Porcine Reproductive and Respiratory Syndrome Virus

2018 ◽  
Vol 92 (7) ◽  
Author(s):  
Kuan Zhao ◽  
Jia-Cong Gao ◽  
Jun-Yao Xiong ◽  
Jin-Chao Guo ◽  
Yong-Bo Yang ◽  
...  
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Molecular Mechanisms ◽  
Nonstructural Protein ◽  
Respiratory Syndrome Virus ◽  
Replication Capacity ◽  
Cdna Clones ◽  
Highly Pathogenic ◽  
Replication Efficiency ◽  
Prrsv Strain

ABSTRACT Highly pathogenic porcine reproductive and respiratory syndrome virus (HP-PRRSV) possesses greater replicative capacity and pathogenicity than classical PRRSV. However, the factors that lead to enhanced replication and pathogenicity remain unclear. In our study, an alignment of all available full-length sequences of North American-type PRRSVs ( n = 204) revealed two consistent amino acid mutations that differed between HP-PRRSV and classical PRRSV and were located at positions 519 and 544 in nonstructural protein 9. Next, a series of mutant viruses with either single or double amino acid replacements were generated from HP-PRRSV HuN4 and classical PRRSV CH-1a infectious cDNA clones. Deletion of either of the amino acids led to a complete loss of virus viability. In both Marc-145 and porcine alveolar macrophages, the replicative efficiencies of mutant viruses based on HuN4 were reduced compared to the parent, whereas the replication level of CH-1a-derived mutant viruses was increased. Plaque growth assays showed clear differences between mutant and parental viruses. In infected piglets, the pathogenicity of HuN4-derived mutant viruses, assessed through clinical symptoms, viral load in sera, histopathology examination, and thymus atrophy, was reduced. Our results indicate that the amino acids at positions 519 and 544 in NSP9 are involved in the replication efficiency of HP-PRRSV and contribute to enhanced pathogenicity. This study is the first to identify specific amino acids involved in PRRSV replication or pathogenicity. These findings will contribute to understanding the molecular mechanisms of PRRSV replication and pathogenicity, leading to better therapeutic and prognostic options to combat the virus. IMPORTANCE Porcine reproductive and respiratory syndrome (PRRS), caused by porcine reproductive and respiratory syndrome virus (PRRSV), is a significant threat to the global pig industry. Highly pathogenic PRRSV (HP-PRRSV) first emerged in China in 2006 and has subsequently spread across Asia, causing considerable damage to local economies. HP-PRRSV strains possess a greater replication capacity and higher pathogenicity than classical PRRSV strains, although the mechanisms that underlie these characteristics are unclear. In the present study, we identified two mutations in HP-PRRSV strains that distinguish them from classical PRRSV strains. Further experiments that swapped the two mutations in an HP-PRRSV strain and a classical PRRSV strain demonstrated that they are involved in the replication efficiency of the virus and its virulence. Our findings have important implications for understanding the molecular mechanisms of PRRSV replication and pathogenicity and also provide new avenues of research for the study of other viruses.

scholarly journals Insertion of a Multibasic Cleavage Motif into the Hemagglutinin of a Low-Pathogenic Avian Influenza H6N1 Virus Induces a Highly Pathogenic Phenotype

2010 ◽  
Vol 84 (16) ◽  
pp. 7953-7960 ◽  
Author(s):  
Vincent J. Munster ◽  
Eefje J. A. Schrauwen ◽  
Emmie de Wit ◽  
Judith M. A. van den Brand ◽  
Theo M. Bestebroer ◽  
...  
Keyword(s):  
Avian Influenza ◽  
Virus Replication ◽  
Influenza A ◽  
Virus Genotype ◽  
Specific Nature ◽  
Highly Pathogenic ◽  
Pathogenic Avian Influenza ◽  
Low Pathogenic Avian Influenza

ABSTRACT The highly pathogenic avian influenza (HPAI) virus phenotype is restricted to influenza A viruses of the H5 and H7 hemagglutinin (HA) subtypes. To obtain more information on the apparent subtype-specific nature of the HPAI virus phenotype, a low-pathogenic avian influenza (LPAI) H6N1 virus was generated, containing an HPAI H5 RRRKKR↓G multibasic cleavage site (MBCS) motif in HA (the downward arrow indicates the site of cleavage). This insertion converted the LPAI virus phenotype into an HPAI virus phenotype in vitro and in vivo. The H6N1 virus with an MBCS displayed in vitro characteristics similar to those of HPAI H5 viruses, such as cleavage of HA0 (the HA protein of influenza A virus initially synthesized as a single polypeptide precursor) and virus replication in the absence of exogenous trypsin. Studies of chickens confirmed the HPAI phenotype of the H6N1 virus with an MBCS, with an intravenous pathogenicity index of 1.4 and systemic virus replication upon intranasal inoculation, the hallmarks of HPAI viruses. This study provides evidence that the subtype-specific nature of the emergence of HPAI viruses is not at the molecular, structural, or functional level, since the introduction of an MBCS resulted in a fully functional virus with an HPAI virus genotype and phenotype.

scholarly journals Evaluation of the Efficacy of an Attenuated Live Vaccine against Highly Pathogenic Porcine Reproductive and Respiratory Syndrome Virus in Young Pigs

2012 ◽  
Vol 19 (8) ◽  
pp. 1199-1206 ◽  
Author(s):  
Xue Leng ◽  
Zhenguang Li ◽  
Mingqi Xia ◽  
Yanliang He ◽  
Hua Wu
Keyword(s):  
Respiratory Syndrome Virus ◽  
Effective Protection ◽  
Lethal Challenge ◽  
Highly Pathogenic ◽  
Live Virus ◽  
Pork Industry ◽  
Live Virus Vaccine ◽  
Young Pigs ◽  
Prrsv Strain

ABSTRACTHighly pathogenic porcine reproductive and respiratory syndrome virus (HP-PRRSV) is characterized by high fever and high mortality in pigs of all ages and has severely affected the pork industry of China in the last few years. An attenuated HP-PRRSV strain, TJM, was obtained by passaging HP-PRRSV strain TJ on MARC-145 cells for 92 passages. Porcine reproductive and respiratory syndrome virus (PRRSV)- and antibody-free pigs were inoculated intramuscularly with TJM (105.050% tissue culture infective doses [TCID50]) and challenged at 28, 60, 120, and 180 days postimmunization (dpi). The results showed that 5/5, 5/5, 5/5, and 4/5 immunized pigs were protected from the lethal challenge and did not develop fever and clinical diseases at each challenge, respectively. Compared to control pigs, vaccinated pigs showed much milder pathological lesions and gained significantly more weight (P< 0.01). Sequence analysis of different passages of strain TJ showed that the attenuation resulted in a deletion of a continuous 120 amino acids (aa), in addition to the discontinuous 30-aa deletion in the nsp2 region. The analysis also demonstrated that the 120-aa deletion was genetically stablein vivo. These results suggested that HP-PRRSV TJM was efficacious against a lethal challenge with a virulent HP-PRRSV strain, and effective protection could last at least 4 months. Therefore, strain TJM is a good candidate for an efficacious modified live virus vaccine as well as a useful molecular marker vaccine against HP-PRRSV.

scholarly journals Genomic Sequencing Reveals Mutations Potentially Related to the Overattenuation of a Highly Pathogenic Porcine Reproductive and Respiratory Syndrome Virus

2013 ◽  
Vol 20 (4) ◽  
pp. 613-619 ◽  
Author(s):  
Xiuling Yu ◽  
Nanhua Chen ◽  
Xiaoyu Deng ◽  
Zhen Cao ◽  
Wei Han ◽  
...  
Keyword(s):  
Structural Proteins ◽  
Respiratory Syndrome Virus ◽  
Genomic Sequencing ◽  
Nucleotide Substitutions ◽  
Highly Pathogenic ◽  
Genomic Variants ◽  
High Virulence ◽  
Prrsv Strain ◽  
Amino Acid Mutations

ABSTRACTHighly pathogenic porcine reproductive and respiratory syndrome virus (HP-PRRSV) continues to evolve when serially passaged in Marc-145 cells. In this study, we analyzed the genomic and antigenic variants of HP-PRRSV strain JXA1 duringin vitropassage. Protective efficacies of JXA1 from passages 100, 110, 120, 140, and 170 against the high-virulence parental virus were evaluated by inoculating pigs with each of these viruses and then challenging with JXA1 from passage 5 at 28 days postimmunization. We found that the antigenicities of JXA1 from passages after 110 were significantly reduced. Inoculation with JXA1 from passages after 110 provided only insufficient protection against the parental strain challenge, indicating that the immunogenicity of JXA1 is significantly decreased when it isin vitropassaged for 110 times and more. To identify the genomic variants that emerged during the overattenuation, eight complete genomes of highly passaged JXA1 were sequenced. One guanine deletion in the 5′ untranslated region (UTR), two nucleotide substitutions in the 3′ UTR, and 65 amino acid mutations in nonstructural and structural proteins that accompanied with the attenuation and overattenuation were determined. Genomic sequencing ofin vitroserially passaged HP-PRRSV first identified the mutations potentially correlated with the overattenuation of a HP-PRRSV strain. These results facilitate the research aimed at elucidating the mechanisms for PRRSV genomic and antigenic changes and may also contribute to developing a safe and effective PRRSV vaccine.

scholarly journals Engineered Small-Molecule Control of Influenza A Virus Replication

2018 ◽  
Vol 93 (1) ◽  
Author(s):  
Elizabeth J. Fay ◽  
Stephanie L. Aron ◽  
Ian A. Stone ◽  
Barbara M. Waring ◽  
Richard K. Plemper ◽  
...  
Keyword(s):  
Influenza A Virus ◽  
Virus Replication ◽  
Small Molecule ◽  
Human Population ◽  
Influenza A ◽  
Health Concern ◽  
Highly Pathogenic ◽  
Significant Safety

ABSTRACT Influenza A virus (IAV) remains a global health concern despite the availability of a seasonal vaccine. It is difficult to predict which strains will circulate during influenza season, and therefore, it is extremely challenging to test novel vaccines in the human population. To overcome this obstacle, new vaccines must be tested in challenge studies. This approach poses significant safety problems, since current pharmacological interventions for IAV are poorly efficacious. New methods are needed to enhance the safety of these challenge studies. In this study, we have generated a virus expressing a small-molecule-assisted shutoff (SMASh) tag as a safety switch for IAV replication. The addition of the SMASh tag to an essential IAV protein allows for small-molecule-mediated inhibition of replication. Treatment with this drug controls the replication of a SMASh-tagged virus in vitro and in vivo. This model for restriction of viral replication has potential for broad applications in vaccine studies, virotherapy, and basic virus research. IMPORTANCE Influenza A virus (IAV) causes significant morbidity and mortality annually worldwide, despite the availability of new formulations of the vaccine each season. There is a critical need to develop more-efficacious vaccines. However, testing novel vaccines in the human population in controlled studies is difficult due to the limited availability and efficacy of intervention strategies should the vaccine fail. There are also significant safety concerns for work with highly pathogenic IAV strains in the laboratory. Therefore, novel strategies are needed to improve the safety of vaccine studies and of research on highly pathogenic IAV. In this study, we developed an IAV strain engineered to contain a small-molecule-mediated safety switch. This tag, when attached to an essential viral protein, allows for the regulation of IAV replication in vitro and in vivo. This strategy provides a platform for the regulation of virus replication without targeting viral proteins directly.

scholarly journals An infectious cDNA clone of a highly pathogenic porcine reproductive and respiratory syndrome virus variant associated with porcine high fever syndrome

2008 ◽  
Vol 89 (9) ◽  
pp. 2075-2079 ◽  
Author(s):  
Jian Lv ◽  
Jianwu Zhang ◽  
Zhi Sun ◽  
Weiquan Liu ◽  
Shishan Yuan
Keyword(s):  
Clinical Symptoms ◽  
Animal Experiments ◽  
High Fever ◽  
Respiratory Syndrome Virus ◽  
Cdna Clones ◽  
Highly Pathogenic ◽  
Virus Variant ◽  
Recombinant Viruses ◽  
Prrsv Strain ◽  
Highly Pathogenic Prrsv

Since May 2006, a so-called ‘porcine high fever syndrome’ (PHFS) has spread all over China. The arterivirus porcine reproductive and respiratory syndrome virus (PRRSV) was believed to be the main causative agent, although the involvement of other pathogens was not formally excluded. The genome of a representative Chinese PRRSV strain, named JX143, was sequenced and used to develop infectious cDNA clones, pJX143 and pJX143M, with the latter containing an engineered MluI site that served as a genetic marker. In various virological assays, the rescued viruses, vJX143 and vJX143M, were indistinguishable from their parental virus. Animal experiments showed that these recombinant viruses retained the high pathogenicity and induced the typical clinical symptoms observed during PHFS outbreaks. This is the first report describing infectious cDNA clones of this highly pathogenic PRRSV. Our results unambiguously fulfil Koch's postulates and define highly pathogenic PRRSV as the aetiological agent of PHFS in China.

scholarly journals Targeting highly pathogenic coronavirus-induced apoptosis reduces viral pathogenesis and disease severity

2021 ◽  
Vol 7 (25) ◽  
pp. eabf8577
Author(s):  
Hin Chu ◽  
Huiping Shuai ◽  
Yuxin Hou ◽  
Xi Zhang ◽  
Lei Wen ◽  
...  
Keyword(s):  
Disease Severity ◽  
Lung Damage ◽  
Intrinsic Apoptosis ◽  
Highly Pathogenic ◽  
In Vivo Models ◽  
Apoptosis Pathway ◽  
Intrinsic Apoptosis Pathway ◽  
Induced Apoptosis

Infection by highly pathogenic coronaviruses results in substantial apoptosis. However, the physiological relevance of apoptosis in the pathogenesis of coronavirus infections is unknown. Here, with a combination of in vitro, ex vivo, and in vivo models, we demonstrated that protein kinase R–like endoplasmic reticulum kinase (PERK) signaling mediated the proapoptotic signals in Middle East respiratory syndrome coronavirus (MERS-CoV) infection, which converged in the intrinsic apoptosis pathway. Inhibiting PERK signaling or intrinsic apoptosis both alleviated MERS pathogenesis in vivo. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and SARS-CoV induced apoptosis through distinct mechanisms but inhibition of intrinsic apoptosis similarly limited SARS-CoV-2– and SARS-CoV–induced apoptosis in vitro and markedly ameliorated the lung damage of SARS-CoV-2–inoculated human angiotensin-converting enzyme 2 (hACE2) mice. Collectively, our study provides the first evidence that virus-induced apoptosis is an important disease determinant of highly pathogenic coronaviruses and demonstrates that this process can be targeted to attenuate disease severity.

scholarly journals In vivo and in vitro studies on the antiviral activities of viperin against influenza H1N1 virus infection

2012 ◽  
Vol 93 (6) ◽  
pp. 1269-1277 ◽  
Author(s):  
Kai Sen Tan ◽  
Farzad Olfat ◽  
Meng Chee Phoon ◽  
Jung Pu Hsu ◽  
Josephine L. C. Howe ◽  
...  
Keyword(s):  
Virus Replication ◽  
Influenza A ◽  
H1n1 Virus ◽  
Lung Damage ◽  
Structural Protein ◽  
Significant Difference ◽  
Antiviral Activities ◽  
Influenza H1n1

Influenza A virus has caused a number of pandemics in past decades, including the recent H1N1-2009 pandemic. Viperin is an interferon (IFN)-inducible protein of innate immunity, and acts as a broad-spectrum antiviral protein. We explored the antiviral activities and mechanisms of viperin during influenza virus (IFV) infection in vitro and in vivo. Wild-type (WT) HeLa and viperin-expressing HeLa cells were infected with influenza A/WSN/33/H1N1 (WSN33) virus, and subjected to virological, light and electron microscopic analyses. Viperin expression reduced virus replication and titres, and restricted viral budding. Young and old viperin-knockout (KO) mice and WT control animals were challenged with influenza WSN33 at lethal doses of 103 and 104 p.f.u. via the intratracheal route. Lungs were subjected to histopathological, virological and molecular studies. Upon lethal IFV challenge, both WT and KO mice revealed similar trends of infection and recovery with similar mortality rates. Viral quantification assay and histopathological evaluation of lungs from different time points showed no significant difference in viral loads and lung damage scores between the two groups of mice. Although the in vitro studies demonstrated the ability of viperin to restrict influenza H1N1 virus replication, the viperin-deficient mouse model indicated that absence of viperin enhanced neither the viral load nor pulmonary damage in the lungs of infected mice. This may be due to the compensation of IFN-stimulated genes in the lungs and/or the influenza non-structural protein 1-mediated IFN antagonism dampening the IFN response, thereby rendering the loss of viperin insignificant. Nevertheless, further investigations that exploit the antiviral mechanisms of viperin as prophylaxis are still warranted.

Sign in / Sign up

Export Citation Format

Share Document