Tissue factor pathway inhibitors disrupt structures of rhabdovirus/ranairidovirus and inhibit viral infection in Chinese perch, Siniperca chuatsi.

Viral diseases have caused great economic losses to the aquaculture industry. However, there are currently no specific drugs to treat these diseases. Herein, we utilized Siniperca chuatsi as an experimental model, and successfully extracted two tissue factor pathway inhibitors (TFPIs) that were highly distributed in different tissues. We then designed four novel peptides based on the TFPIs, named TS20, TS25, TS16, and TS30. Among them, TS25 and TS30 showed good biosafety and high antiviral activity. Further studies showed that TS25 and TS30 exerted their antiviral functions by preventing viruses from invading Chinese perch brain (CPB) cells and disrupting Siniperca chuatsi rhabdovirus (SCRV)/Siniperca chuatsi ranairidovirus (SCRIV) viral structures. Additionally, compared with the control group, TS25 and TS30 could significantly reduce the mortality of Siniperca chuatsi, the relative protection rates of TS25 against SCRV and SCRIV were 71.25 % and 53.85 % respectively, and the relative protection rate of TS30 against SCRIV was 69.23 %, indicating that they also had significant antiviral activity in vivo. This study provided an approach for designing peptides with biosafety and antiviral activity based on host proteins, which had potential applications in the prevention and treatment of viral diseases.

Water-in-oil adjuvant challenges in fish vaccination: An experimental inactivated adjuvanted vaccine against betanodavirus infection in Senegalese sole.

The extensive growth of intensive fish farming has led to a massive spread of infectious diseases. Nervous necrosis virus (NNV) is the causative agent of the viral encephalo- and retinopathy disease which has become a major threat for fish farming all over the globe. The devastating mortality rates recorded in disease outbreaks, especially when infected specimens are at early stages of development, have a high economic impact on the sector. Currently, vaccines are the most cost-effective preventing tool in the fight against viruses. Inactivated vaccines have the advantage of simplicity in their development at the same time as present the antigen in a similar manner than the natural infection in the host. Nevertheless, they usually trigger weaker immune responses needing adjuvants to boost their effectiveness. In this work, we have intraperitoneally vaccinated Senegalese sole juveniles (Solea senegalensis) with a previously designed inactivated vaccine against NNV based on binary ethylenimine (BEI), mixed or not with an oil-adjuvant. Our results demonstrated the potential activation of different immune pathways when the vaccine was administered alone compared to the oil-adjuvanted vaccine, both resulting in an equivalent partial improvement in survival following a NNV challenge. However, whilst the vaccine alone led to a significant increase in specific antibodies, in the adjuvanted version those antibodies were kept basal although with a slight improvement in their neutralization capacity. At transcriptional level, neither vaccine (adjuvanted or not) triggered the immune system activation during the vaccination period. However, after NNV infection, the BEI-inactivated vaccines alone and oil-adjuvanted both elicited the stimulation of antiviral responsive genes (rtp3, herc4), antigen presentation molecules (mhcii) and T-cell markers (cd8a) in the head-kidney. Additionally, the oil-adjuvanted vaccine appears to stimulate mediator cytokines (il6) and B-cell markers (ight and ighm). Surprisingly, when the adjuvant was administered alone, fish showed the highest survival rates concomitantly with a lack of NNV-IgM production, pointing to the possible induction of different immune pathways than the B-cell responses via antibodies by the adjuvant. Since this combined vaccine did not succeed in the full extension of protection against the pathogen, further studies should be performed focusing on unravelling the molecular mechanisms through which adjuvants trigger the immune response, both independently and when added to a vaccine antigen.

IFN-λ1 Displays Various Levels of Antiviral Activity In Vitro in a Select Panel of RNA Viruses.

Type III interferons (lambda IFNs) are a quite new, small family of three closely related cytokines with interferon-like activity. Attention to IFN-λ is mainly focused on direct antiviral activity in which, as with IFN-α, viral genome replication is inhibited without the participation of immune system cells. The heterodimeric receptor for lambda interferons is exposed mainly on epithelial cells, which limits its possible action on other cells, thus reducing the likelihood of developing undesirable side effects compared to type I IFN. In this study, we examined the antiviral potential of exogenous human IFN-λ1 in cellular models of viral infection. To study the protective effects of IFN-λ1, three administration schemes were used: 'preventive' (pretreatment); 'preventive/therapeutic' (pre/post); and 'therapeutic' (post). Three IFN-λ1 concentrations (from 10 to 500 ng/mL) were used. We have shown that human IFN-λ1 restricts SARS-CoV-2 replication in Vero cells with all three treatment schemes. In addition, we have shown a decrease in the viral loads of CHIKV and IVA with the 'preventive' and 'preventive/therapeutic' regimes. No significant antiviral effect of IFN-λ1 against AdV was detected. Our study highlights the potential for using IFN-λ as a broad-spectrum therapeutic agent against respiratory RNA viruses.

NK-lysin peptides ameliorate viral encephalopathy and retinopathy disease signs and provide partial protection against nodavirus infection in European sea bass.

Antimicrobial peptides (AMP) comprise a wide range of small molecules with direct antibacterial activity and immunostimulatory role and are proposed as promising substitutes of the antibiotics. Additionally, they also exert a role against other pathogens such as viruses and fungi less evaluated. NK-lysin, a human granulysin orthologue, possess a double function, taking part in the innate immunity as AMP and also as direct effector in the cell-mediated cytotoxic (CMC) response. This molecule is suggested as a pivotal molecule involved in the defence upon nervous necrosis virus (NNV), an epizootic virus provoking serious problems in welfare and health status in Asian and Mediterranean fish destined to human consumption. Having proved that NK-lysin derived peptides (NKLPs) have a direct antiviral activity against NNV in vitro, we aimed to evaluate their potential use as a prophylactic treatment for European sea bass (Dicentrarchus labrax), one of the most susceptible cultured-fish species. Thus, intramuscular injection of synthetic NKLPs resulted in a very low transcriptional response of some innate and adaptive immune markers. However, the injection of NKLPs ameliorated disease signs and increased fish survival upon challenge with pathogenic NNV. Although NKLPs showed promising results in treatments against NNV, more efforts are needed to understand their mechanisms of action and their applicability to the aquaculture industry.

Oral immunization with recombinant protein antigen expressed in tobacco against fish nervous necrosis virus.

Nervous necrosis virus (NNV), also known as betanodavirus, has been recently implicated in mass mortalities of cultured marine fish. An effective vaccine is urgently needed to protect fish against this virus. However, parenteral immunization methods are very stressful. Individual immunization for thousands of fish is very labor intensive and expensive. Therefore, we expressed NNV coat protein in tobacco chloroplasts and used it as an oral vaccine to induce immunities in fish followed by challenges with NNV. Our results revealed that mice (IgG and IgA) and fish (IgM) immunized with the oral vaccine developed significantly higher antibody titers against the NNV coat protein. Fish were partially protected against viral challenge. Taken together, our results demonstrated that a plant-based vaccine could effectively induce immune response and protect groupers against NNV. The present method could be used to develop oral fish vaccine in the future.

Structural analysis and insertion study reveal the ideal sites for surface displaying foreign peptides on a betanodavirus-like particle.

Betanodavirus infection causes fatal disease of viral nervous necrosis in many cultured marine and freshwater fish worldwide and the virus-like particles (VLP) are effective vaccines against betanodavirus. But vaccine and viral vector designs of betanodavirus VLP based on their structures remain lacking. Here, the three-dimensional structure of orange-spotted grouper nervous necrosis virus (OGNNV) VLP (RBS) at 3.9 Å reveals the organization of capsid proteins (CP). Based on the structural results, seven putative important sites were selected to genetically insert a 6× histidine (His)-tag for VLP formation screen, resulting in four His-tagged VLP (HV) at positions N-terminus, Ala220, Pro292 and C-terminus. The His-tags of N-terminal HV (NHV) were concealed inside virions while those of 220HV and C-terminal HV (CHV) were displayed at the outer surface. NHV, 220HV and CHV maintained the same cell entry ability as RBS in the Asian sea bass (SB) cell line, indicating that their similar surface structures can be recognized by the cellular entry receptor(s). For application of vaccine design, chromatography-purified CHV could provoke NNV-specific antibody responses as strong as those of RBS in a sea bass immunization assay. Furthermore, in carrying capacity assays, N-terminus and Ala220 can only carry short peptides and C-terminus can even accommodate large protein such as GFP to generate fluorescent VLP (CGV). For application of a viral vector, CGV could be real-time visualized to enter SB cells in invasion study. All the results confirmed that the C-terminus of CP is a suitable site to accommodate foreign peptides for vaccine design and viral vector development.

Emerging and changing viral diseases in the new millennium.

Most viral infections encountered in resource-rich countries are relatively trivial and transient with perhaps fever, malaise, myalgia, rash (exanthema) and sometimes mucosal manifestations (enanthema), including oral in some. However, the apparent benignity may be illusory as some viral infections have unexpected consequences - such as the oncogenicity of some herpesviruses and human papillomaviruses. Infections are transmitted from various human or animal vectors, especially by close proximity, and the increasing movements of peoples across the globe, mean that infections hitherto confined largely to the tropics now appear worldwide. Global warming also increases the range of movement of vectors such as mosquitoes. Thus recent decades have seen a most dramatic change with the emergence globally also of new viral infections - notably human immunodeficiency viruses (HIV) - and the appearance of some other dangerous and sometimes lethal infections formerly seen mainly in, and reported from, resource-poor areas especially in parts of Asia, Latin America and Africa. This study offers a brief update of the most salient new aspects of the important viral infections, especially those with known orofacial manifestations or other implications for oral health care.

Coxsackievirus cloverleaf RNA containing a 5' triphosphate triggers an antiviral response via RIG-I activation.

Upon viral infections, pattern recognition receptors (PRRs) recognize pathogen-associated molecular patterns (PAMPs) and stimulate an antiviral state associated with the production of type I interferons (IFNs) and inflammatory markers. Type I IFNs play crucial roles in innate antiviral responses by inducing expression of interferon-stimulated genes and by activating components of the adaptive immune system. Although pegylated IFNs have been used to treat hepatitis B and C virus infections for decades, they exert substantial side effects that limit their use. Current efforts are directed toward the use of PRR agonists as an alternative approach to elicit host antiviral responses in a manner similar to that achieved in a natural infection. RIG-I is a cytosolic PRR that recognizes 5' triphosphate (5'ppp)-containing RNA ligands. Due to its ubiquitous expression profile, induction of the RIG-I pathway provides a promising platform for the development of novel antiviral agents and vaccine adjuvants. In this study, we investigated whether structured RNA elements in the genome of coxsackievirus B3 (CVB3), a picornavirus that is recognized by MDA5 during infection, could activate RIG-I when supplied with 5'ppp. We show here that a 5'ppp-containing cloverleaf (CL) RNA structure is a potent RIG-I inducer that elicits an extensive antiviral response that includes induction of classical interferon-stimulated genes, as well as type III IFNs and proinflammatory cytokines and chemokines. In addition, we show that prophylactic treatment with CVB3 CL provides protection against various viral infections including dengue virus, vesicular stomatitis virus and enterovirus 71, demonstrating the antiviral efficacy of this RNA ligand.

Niclosamide is a proton carrier and targets acidic endosomes with broad antiviral effects.

Viruses use a limited set of host pathways for infection. These pathways represent bona fide antiviral targets with low likelihood of viral resistance. We identified the salicylanilide niclosamide as a broad range antiviral agent targeting acidified endosomes. Niclosamide is approved for human use against helminthic infections, and has anti-neoplastic and antiviral effects. Its mode of action is unknown. Here, we show that niclosamide, which is a weak lipophilic acid inhibited infection with pH-dependent human rhinoviruses (HRV) and influenza virus. Structure-activity studies showed that antiviral efficacy and endolysosomal pH neutralization co-tracked, and acidification of the extracellular medium bypassed the virus entry block. Niclosamide did not affect the vacuolar H(+)-ATPase, but neutralized coated vesicles or synthetic liposomes, indicating a proton carrier mode-of-action independent of any protein target. This report demonstrates that physico-chemical interference with host pathways has broad range antiviral effects, and provides a proof of concept for the development of host-directed antivirals.

Update on childhood and adolescent immunizations: selected review of US recommendations and literature: part 2.

PURPOSE OF REVIEW: To provide a clinically relevant synopsis of research findings regarding childhood and adolescent vaccines. RECENT FINDINGS: Vaccine coverage is relatively static or improving for the vaccines included in the 2010 annual harmonized immunization schedules. Providers should be reviewing patients' immunization records at each visit to take advantage of any opportunity to administer indicated, age-appropriate vaccines. There have been infectious disease outbreaks among highly immunized populations, although unvaccinated or undervaccinated individuals continue to play large roles in the spread of disease. Infants, many of whom are too young to be vaccinated, continue to bear a large disease burden, which underscores the importance of cocooning and, in some cases, vaccination of pregnant women. Influenza, measles, mumps, and rubella, varicella, hepatitis A, meningococcal conjugate, human papillomavirus, diphtheria and tetanus toxoids and acellular pertussis, and tetanus and diphtheria toxoids and acellular pertussis vaccines are reviewed in this second of two articles. SUMMARY: New research on childhood and adolescent vaccines is anticipated to shape the practice of pediatric providers. Research will continue to provide the science to optimize protection and to promote the health and well being of all children and adolescents.

Epidemiology of influenza-like illness during Pandemic (H1N1) 2009, New South Wales, Australia.

To rapidly describe the epidemiology of influenza-like illness (ILI) during the 2009 winter epidemic of pandemic (H1N1) 2009 virus in New South Wales, Australia, we used results of a continuous population health survey. During July-September 2009, ILI was experienced by 23% of the population. Among these persons, 51% were unable to undertake normal duties for <3 days, 55% sought care at a general practice, and 5% went to a hospital. Factors independently associated with ILI were younger age, daily smoking, and obesity. Effectiveness of prepandemic seasonal vaccine was ?20%. The high prevalence of risk factors associated with a substantially increased risk for ILI deserves greater recognition.

Treatment of viral conjunctivitis with antiviral drugs.

Viral conjunctivitis is one of the most common disorders observed in ophthalmic emergency departments, yet no established treatment exists. Lately, antiviral medications have been introduced into clinical practice; however, a systematic review focusing on their use and effectiveness in the treatment of viral conjunctivitis has not been previously reported. We systemically reviewed the literature to identify studies where antiviral drugs were used to treat viral conjunctivitis. Currently, aciclovir, trifluridine and valaciclovir are commonly used as antiviral agents to treat herpesvirus infections. Cidofovir has been used successfully to treat some cases of adenoviral conjunctivitis, although toxicity has also been reported. The use of other medications, such as idoxuridine, has been minimized in clinical practice due to their high toxicity. Interestingly, most of the antiviral drugs developed are used to treat herpesvirus infections, while less progress has been made in the field of adenoviral infections. For other viral causes of conjunctivitis, no effective remedy is currently available, and treatment focuses on the relief of symptoms. Caution should be exercised when coadministering other pharmacological agents, such as corticosteroids, because of emerging adverse effects.

RNA recognition and signal transduction by RIG-I-like receptors.

Viral infection is detected by cellular sensor molecules as foreign nucleic acids and initiates innate antiviral responses, including the activation of proinflammatory cytokines and type I interferon (IFN). Recent identification of cytoplasmic viral sensors, such as retinoic acid-inducible gene-I-like receptors (RLRs), highlights their significance in the induction of antiviral innate immunity. Moreover, it is intriguing to understand how they can discriminate endogenous RNA from foreign viral RNA and initiate signaling cascades leading to the induction of type I IFNs. This review focuses on the current understanding of the molecular machinery underlying RNA recognition and subsequent signal transduction by RLRs.

Functional evolution of the TICAM-1 pathway for extrinsic RNA sensing.

The type I interferon (IFN) is a host defense factor against microbial pathogens in vertebrates. In mammals, retinoic acid-inducible gene I (RIG-I) and melanoma differentiation-associated gene 5 (MDA5) in the cytoplasm are regarded as sensors for double-stranded RNA (dsRNA) and trigger IFN regulatory factor-3 (IRF-3) activation followed by type I IFN induction through the mitochondrial antiviral signaling (MAVS) adapter. This intrinsic pathway appears to link the main protective responses against RNA virus infection in mammals. On the other hand, human Toll-like receptor 3 (TLR3) is localized in the endosomal membrane or cell surface and signals the presence of extrinsic dsRNA. In response to RNA stimulation, TLR3 recruits the Toll-interleukin 1 receptor domain (TIR)-containing adapter molecule 1 (TICAM-1) adapter and induces IRF-3 activation followed by IFN-beta promoter activation. Human TLR3 is localized limitedly extent in myeloid dendritic cells, fibroblasts, and epithelial cells. The TICAM-1 and cytoplasmic MAVS pathways converge at the IRF-3-activating kinase in human cells. The reason for the involvement of this extrinsic mode of IFN-inducing pathways in the dsRNA response remains unknown. In fish, two TLRs, i.e. endoplasmic TLR3 and cell surface TLR22, participate in teleost IFN production without the activation of IRF-3. TLR22 is distinct from mammalian TLR3 in terms of cellular localization, ligand selection, and tissue distribution. TLR22 may be a functional substitute for human cell surface TLR3 and may serve as a surveillance molecule for detecting dsRNA virus infection and alerting the immune system for antiviral protection in fish. In this review, we discuss the fundamentals of the extrinsic dsRNA recognition system, which has evolved to induce cellular effectors to cope with dsRNA virus infection across different vertebrate species.

Approaching the RNA ligand for RIG-I?

Innate and antigen-specific antiviral immunity are triggered by immunorecognition of viral nucleic acids. The helicase retinoic acid-inducible gene I (RIG-I) (also known as DDX58) is the key sensor of negative strand RNA viruses in the cytosol of cells. RNA containing a triphosphate at the 5'-end was shown to activate RIG-I, but the exact structure of RNA supporting 5'-triphosphate recognition, the requirement of a 5'-triphosphate group, as well as the existence of RNA structures detected by RIG-I in the absence of 5'-triphosphate remain controversial. Here, we revisit the literature on RIG-I and RIG-I ligands. The literature proposes at least six different RIG-I ligands: (i) single strand with a 5'-triphosphate, (ii) double-stranded RNA with a 5'-triphosphate, (iii) 5'-triphosphate single-stranded RNA with A- and U-rich 3'-sequences, (iv) double-stranded RNA of intermediate length (>300 and <2000 bp) without 5'-triphosphate, (v) blunt-end short double-stranded RNA (23-30 bp) without 5'-triphosphate, and (vi) short double-stranded RNA (23-30 bp) with 5'-monophosphate. RIG-I thus seems promiscuous for a variety of different RNA molecules, very similar to the Toll-like receptors, of which 10 family members are sufficient for the safe detection of the microbial cosmos. In the light of these outstanding publications, it seems an unlikely possibility that there is a fundamental shortcoming in the design of all studies. Looking closely, the only issue that comes to mind is the in vitro transcription technique used by all investigators without confirming the identity of RNA products. This technique, together with the different biological systems used, the lack of dose responses and of proper comparison of different published ligands and controls leave us with more questions than answers as to what the exact RIG-I ligand is, if in fact it exists.

Innate immunity to virus infection.

The innate immune system is essential for the initial detection of invading viruses and subsequent activation of adaptive immunity. Three classes of receptors, designated retinoic acid-inducible gene I (RIG-I)-like receptors (RLRs), Toll-like receptors (TLRs), and nucleotide oligomerization domain (NOD)-like receptors (NLRs), sense viral components, such as double-stranded RNA (dsRNA), single-stranded RNA, and DNA. RLRs and TLRs play essential roles in the production of type I interferons (IFNs) and proinflammatory cytokines in cell type-specific manners. While the RLRs play essential roles in the recognition of RNA viruses in various cells, plasmacytoid dendritic cells utilize TLRs for detecting virus invasion. NLRs play a role in the production of mature interleukin-1 beta to dsRNA stimulation. Activation of innate immune cells is critical for mounting adaptive immune responses. In this review, we discuss recent advances in our understanding of the mechanisms of viral RNA recognition by these different types of receptors and its relation to acquired immune responses.

Immunodominant epitopes mapped by synthetic peptides on the capsid protein of avian hepatitis E virus are non-protective.

Avian hepatitis E virus (avian HEV) was recently discovered in chickens with hepatitis-splenomegaly syndrome in the United States. The open reading frame 2 (ORF2) protein of avian HEV has been shown to cross-react with human and swine HEV ORF2 proteins, and immunodominant antigenic epitopes on avian HEV ORF2 protein were identified in the predicted antigenic domains by synthetic peptides. However, whether these epitopes are protective against avian HEV infection has not been investigated. In this study, groups of chickens were immunized with keyhole limpet hemocyanin (KLH)-conjugated peptides and recombinant avian HEV ORF2 antigen followed by challenge with avian HEV virus to assess the protective capacity of these peptides containing the epitopes. While avian HEV ORF2 protein showed complete protection against infection, viremia and fecal virus shedding were found in all peptide-immunized chickens. Using purified IgY from normal, anti-peptide, and anti-avian HEV ORF2 chicken sera, an in-vitro neutralization and in-vivo monitoring assay was performed to further evaluate the neutralizing ability of anti-peptide IgY. Results showed that none of the anti-peptide IgY can neutralize avian HEV in vitro, as viremia, fecal virus shedding, and seroconversion appeared similarly in chickens inoculated with avian HEV mixed with anti-peptide IgY and chickens inoculated with avian HEV mixed with normal IgY. As expected, chickens inoculated with the avian HEV and anti-avian HEV ORF2 IgY mixture did not show detectable avian HEV infection. Taken together, the results of this study demonstrated that immunodominant epitopes on avian HEV ORF2 protein identified by synthetic peptides are non-protective, suggesting protective neutralizing epitope on avian HEV ORF2 may not be linear as is human HEV.

Potential of antiviral therapy and prophylaxis for controlling RNA viral infections of livestock.

With intensification of trade, livestock are increasingly exposed to severe animal diseases caused by a range of RNA viruses. Recent prime examples include outbreaks of foot-and-mouth disease (FMD), peste des petits ruminants, Rift Valley fever and bluetongue. To minimise their impact, controlling the spread of virus is of utmost importance. Good quality, reliable vaccines exist for some, although not all, of these diseases, but suffer from a set of drawbacks, not the least of which being the time needed to trigger the immune response (i.e. "immunity-gap"). Effective, rapid control tools are, therefore, urgently needed and antiviral compounds could serve this purpose. Although limited in vitro and in vivo research has been performed, encouraging results for FMD suggest that livestock could be protected against infection within 24h following antiviral treatment and up to 12h post-infection. Such prophylactic/therapeutic antiviral drugs could complement emergency vaccination in a previously disease-free setting or be applied to treat valuable zoological collections and breeding stocks in endemic and previously disease-free regions alike. This paper will primarily focus on the effects of FMD on livestock and other sectors, and on appropriate control tools. The outlined principles can be extrapolated to other RNA viral diseases.

Immunity against infectious diseases: predictive value of self-reported history of vaccination and disease.

OBJECTIVE: To determine whether self-reported history of disease and/or vaccination is predictive of immunity against hepatitis B, varicella, rubella, mumps, and measles. DESIGN: The seroprevalence of viral antibodies and the predictive value of a self-report questionnaire were determined for 616 paramedical students who matriculated into Padua Medical School (Padua, Italy) during 2003-2005. RESULTS: The majority of subjects (86.9%) remembered being vaccinated against hepatitis B but had no recollection of disease. Among vaccinees, 1.5% showed markers of previous infection, 6.7% tested negative for anti-hepatitis B virus surface antigen (anti-HBsAg) antibodies, and 91.8% tested positive for anti-HBsAg. Self-reported vaccination history had a positive predictive value of 93.2% for test results positive for immunity against hepatitis B. Immunity against varicella (93.7% of subjects) and rubella (95.5%) was high, compared with immunity against mumps (79.9%) and measles (83.1%). In addition, results of tests for detection of immunity against mumps and measles were equivocal for more than 7% of subjects, probably because their vaccination regimen was not completed. Self-reported histories of varicella disease and rubella disease and vaccination had high positive predictive values (greater than 98% each) for testing positive for antiviral antibodies, compared with self-reported histories of mumps disease and vaccination and measles disease and vaccination; however, high positive predictive values were observed for self-reported histories of mumps only (92.0%) and measles only (94.7%). CONCLUSIONS: The self-report questionnaire used in this study did not accurately predict immunity against 5 transmittable but vaccine-preventable diseases. A complete serological evaluation of healthcare workers, followed by vaccination of those with negative or equivocal results of serological tests, is an appropriate measure to decrease the risk of infection in this population.

Effect of cDNA fragments in different length derived from potato virus Y coat protein gene on the induction of RNA-mediated virus resistance.

We have reported that cDNA derived from entire coat protein (CP) gene of potato virus Y (PVY) could induce resistance to PVY infection in transgenic tobacco plants, and the resistance was further demonstrated to be RNA-mediated rather than coat protein-mediated. In this study, we cloned cDNA fragments of 202 bp, 417 bp, and 603 bp in length derived from the 3' end of the PVY CP gene, and the cDNA fragments were introduced into tobacco (var. NC89) plants via Agrobacterium-mediated transformation system. The results of resistance assay showed that the CP cDNA fragments of 417 bp and 603 bp could confer resistance of the transgenic plants to PVY infection, but the fragment of 202 bp in length could not. Molecular analysis revealed that the resistance was RNA-mediated, which is believed to be a result of post-transcriptional gene silencing. The results indicate that the length of cDNA fragments needed for resistance induction was located somewhere between 202 bp and 417 bp from the 3' end of PVY CP gene.