Structural and Functional Changes in Pulmonary Macrophages and Lungs of Mice Infected with Influenza Virus A/H5N1 A/goose/Krasnoozerskoye/627/05

C57Bl/6 mice were intranasally infected with influenza virus A/H5N1 A/goose/Krasnoozerskoye/627/05. The mortality rate of animals reached 70% on day 14 of the disease. The lungs of animals were characterized by necroses, destruction of vessels, hemorrhagic and thrombotic complications, edematous syndrome, and early fibrosis of the interstitium. On days 6-10 after infection, fibrosis was found in the zones of postnecrotic inflammatory infiltration. The expression of lysozyme and myeloperoxidase by pulmonary macrophages was initially increased, but decreased on day 10 of the study. The number of cathepsin D-expressing macrophages was elevated up to the 10th day of examination.     

Structural Changes in the Brain of Mice Infected with Influenza A/H5N1 Virus

Structural changes in the brain of outbred mice were studied after infection with infuenza A/H5N1 strain isolated in the Novosibirsk region. High mortality was observed after intranasal infection. Examination of brain specimens revealed vasculopathies with thrombosis of the microcirculatory vessels, pericellular and perivascular edema with multifocal ischemic necrosis, hyperplasia of glial cells, caspase-dependent apoptosis of neurons caused by the cytopathic effect of the virus, and hypercytokinemia.     

Changes in the Structure of Mouse Kidney in the Acute Period after Infection with Influenza Viruses A/H5N1 and A/H1N1

Bulletin of Experimental Biology and Medicine - Changes in the kidney structure in outbred and inbred male BALB/c mice were analyzed in the acute period after infection with influenza viruses...     

Influenza A/H5N1 virus infection in humans in Cambodia.

BACKGROUND: Between January 2005 and April 2006, six patients of influenza A/H5N1 virus infection were reported in Cambodia, all with fatal outcome. OBJECTIVES: We describe the virological findings of these six H5N1 patients in association with clinical and epidemiologic findings. STUDY DESIGN: Broncho-alveolar lavage, nasopharyngeal, throat and rectal swabs and sera were cultured for virus isolation and viral load quantified in clinical specimens by real-time RT-PCR. We compared sequences obtained from different body sites within the same patient to detect viral quasi-species. RESULTS: H5N1 virus strains isolated in Cambodia belong to genotype Z, clade 1 viruses. H5N1 viruses were isolated from serum and rectal swab specimens in two patients. The haemagglutinin gene sequences of the virus in different body sites did not differ. Amino acid substitutions known to be associated with a change in virus binding were not observed. CONCLUSION: The high frequency of virus isolation from serum and faecal swabs highlights that H5N1 is likely to be a disseminated infection in humans and this has implications for antiviral treatment, biosafety in clinical laboratories and on risks for nosocomial and human-to-human transmission. There were no tissue-specific adaptive mutations in the HA gene from viruses isolated from different organs.     

Pathogenesis of Infectious Disease of Mice Caused by H5N1 Avian Influenza Virus

The pathogenesis of a disease caused by Qinghai-like H5N1 influenza virus in BALB/c mice was studied. Clinical, morphological, and immunological characteristics of the experimental infection caused by highly pathogenic A/duck/Tuva/01/06/ (H5N1) virus are described. Translated from Byulleten’ Eksperimental’noi Biologii i Meditsiny, Suppl. 1, pp. 52–55, 2008     

Influenza A/H5N1 virus outbreaks and prepardness to avert flu pandemic

This review emphasizes the need to improve the knowledge of the biology of H5N1 virus, a candidate for causing the next influenza pandemic. In-depth knowledge of mode of infection, mechanisms of pathogenesis and immune response will help in devising an efficient and practical control strategy against this flu virus. We have discussed limitations of currently available vaccines and proposed novel approaches for making better vaccines against H5N1 influenza virus. They include cell-culture system, reverse genetics, adjuvant development. Our review has also underscored the concept of therapeutic vaccine (anti-disease vaccine), which is aimed at diminishing 'cytokine storm' seen in acute respiratory distress syndrome and/or hemophagocytosis.     

Anti-Inflammatory Effect of Thalidomide on H1N1 Influenza Virus-Induced Pulmonary Injury in Mice

The purpose of this study is to investigate the anti-inflammatory effect of thalidomide (Thd) on H1N1-induced acute lung injury in mice. BALB/C mice were infected intranasally with influenza A virus (H1N1) and then treated with Thd at a dose of 100 or 200 mg/kg/day for 7 days. Weight loss and survival of mice were monitored for 14 days after virus challenge, and the serum and lung tissues were collected at 4 days for histological and biochemical analysis. The results showed that Thd significantly improved the survival rate, reduced the infiltration of inflammatory cells and cytokine (e.g., IL-6, TNF-α) and chemokine (e.g., RANTES, IP-10) levels, and inhibited activated p-NFκB p65 in infected mice. These findings suggested that Thd may attenuate H1N1-induced pulmonary injury and thus may find use in the treatment of viral diseases.     

Antiviral Activity of Anaferon (Pediatric Formulation) in Mice Infected with Pandemic Influenza Virus A(H1N1/09)

Anaferon (pediatric formulation) administered in the therapeutic-and-prophylactic regimen to mice receiving intranasally 100% infecting dose of A/California/07/2009(H1N1)v influenza virus exhibited an antiviral effect and 10-fold reduced the production of influenza virus in the lungs of infected mice on days 4, 6, and 8 after infection compared to the control (distilled water). The efficiency of Anaferon (pediatric formulation) administered before and after infection with A/California/07/2009(H1N1)v influenza virus was not inferior to the use of Tamiflu after infection.     

Morphological Changes in Bird Viscera in Experimental Infection by Highly Pathogenic H5N1 Avian Influenza Virus

Intravenous infection of chicken with H5N1 avian influenza virus (strain A/Gs/Krasnoozerskoye/627/05) causes rapid lethal outcome. Pathomorphological study of bird viscera showed manifestations of disseminated intravascular coagulation syndrome, generalized inflammatory reaction, and wide-scale necrobiotic changes in tissues. Translated from Byulleten’ Eksperimental’noi Biologii i Meditsiny, Suppl. 1, pp. 56–59, 2008     

Intranasal Delivery of an IgA Monoclonal Antibody Effective against Sublethal H5N1 Influenza Virus Infection in Mice

Highly pathogenic avian H5N1 influenza viruses are endemic in poultry in Asia and pose a pandemic threat to humans. Since the deployment of vaccines against a pandemic strain may take several months, adequate antiviral alternatives are needed to minimize the effects and the spread of the disease. Passive immunotherapy is regarded as a viable alternative. Here, we show the development of an IgA monoclonal antibody (DPJY01 MAb) specific to H5 hemagglutinin. The DPJY01 MAb showed a broad hemagglutination inhibition (HI) profile against Asian H5N1 viruses of clades 0, 1.0, 2.1, 2.2, and 2.3 and also against H5 wild bird influenza viruses of the North American and Eurasian lineages. DPJY01 MAb displayed also high neutralization activity in vitro and in vivo. In mice, DPJY01 MAb provided protection via a single dose administered intranasally before or after inoculation with a sublethal dose of H5N1 viruses of clades 1.0 and 2.2. Pretreatment with 50 mg of DPJY01 MAb kg of body weight at either 24, 48, or 72 h before highly pathogenic H5N1 virus (A/Vietnam/1203/2004 [H5N1]) inoculation resulted in complete protection. Treatment with 50 mg/kg at either at 24, 48, or 72 h after H5N1 inoculation provided 100%, 80%, and 60% protection, respectively. These studies highlight the potential use of DPJY01 MAb as an intranasal antiviral treatment for H5N1 influenza virus infections.Influenza type A viruses are negative-sense segmented RNA viruses that belong to the family Orthomyxoviridae (11). They are further subdivided into subtypes based on the antigenic properties of the two surface glycoproteins, hemagglutinin (HA) and neuraminidase (NA). Among these subtypes, the highly pathogenic avian H5N1 influenza viruses have been intensively studied since the first report of lethal human infections in 1997 (36). H5N1 viruses continue to circulate in poultry in Asia and occasionally are transmitted from birds to humans, posing a potential pandemic threat (1). As of 6 April 2010, the World Health Organization (WHO) had reported 493 human infections with 292 deaths, a fatality rate exceeding 60%. These strains have shown significant evolutionary changes and are currently divided into 10 HA clades (36). Among these clades, clade 2 is further classified into five subclades (2.1 to 2.5), and within each subclade there are several lineages (35). Clade 2.1 is predominant in Indonesia, the country in which H5N1 has become endemic and in which the highest number of human infections and associated fatalities have been reported. In Indonesia, of the 163 cases confirmed to date by the WHO, 135 have been fatal. The latest human infections with H5N1 viruses have been reported in Egypt, where viruses from clade 2.2.1 are endemic. In Egypt since 2006, H5N1 viruses have been identified as the causative agent in 109 human infections with 34 deaths according to the WHO. More importantly, some of these strains have developed resistance to available antiviral drugs (17, 21). For example, most clade 1 H5N1 viruses are resistant to adamantanes (10), and oseltamivir-resistant H5N1 viruses with neuraminidase mutations (H274Y and N294S) have been also identified in infected patients during or after treatment (7, 12). These limitations and others, such as the poor immunogenicity of H5N1 vaccines (3, 16, 26, 31), call for the development of alternative intervention strategies.Several groups have reported the development of monoclonal antibodies (MAbs) against the HA of influenza viruses, particularly against the H1, H3, and H5 subtypes (9, 14, 38). Some of these MAbs have broad subtype cross-reactions (38). Human and mouse monoclonal antibodies against H5 HA have been shown to provide protection against lethal infection in a mouse model (4, 20, 24). These anti-H5 MAbs are usually of the IgG1 or IgG2a subtypes and are administered by parenteral routes. Retrospective studies have suggested that those patients with influenza pneumonia during the 1918 Spanish influenza pandemic who received influenza convalescent-phase human blood products may have experienced a reduction in the risk of death (15), and H5N1-infected patients treated with convalescent H5N1 plasma recovered from the infection (39). Therefore, passive antibody immunotherapy is an attractive and potentially efficient alternative for the treatment of H5N1 infections. To our knowledge, intranasal administration of antibodies against H5N1 has not been reported. Although intranasal administration of drugs depends largely on the health status of the patient, it does represent an alternative intervention strategy. Intranasal administration of antibodies would allow the antibodies to directly reach their target in the respiratory track, which is the major site for influenza virus replication in humans and other mammals (29, 33). IgA-mediated neutralization monoclonal antibody therapy against H5N1 has not been reported, and only a few IgA MAbs against A/Puerto Rico/8/34 (H1N1) have been reported to show antiviral activity when given intravenously (2). In this study, we generated an IgA monoclonal antibody (DPJY01) with a broad HI profile and high neutralization activity against the H5N1 virus in vitro and in vivo. Remarkably, DPJY01 provided protection against sublethal H5N1 infection after a single dose through intranasal administration.     

A/鹅/广东/2/96(H5N1)流感毒株7和8 RNA节段核苷酸序列测定

目的　弄清A鹅广东296(H5N1)毒株RNA7和8核苷酸全序列及它们与AHK15697(H5N1)毒株RNA7和8之间的内在关系,并为今后流感病毒M和NS基因研究打下基础。方法　病毒粒RNA经逆转录合成cDNA,经聚合酶链反应(PCR)扩增,产物纯化,采用双脱氧链末端终止法进行核苷酸序列测定。结果　A鹅广东296(H5N1)毒株RNA7长度为1027个核苷酸,编码M1(含252个氨基酸)和M2(含97个氨基酸)的蛋白。其RNA8长度为890个核苷酸,编码NS1(含230个氨基酸)和NS2(含121个氨基酸)非结构蛋白。其M1,M2,NS1和NS2蛋白分子上氨基酸序列与AHK15697(H5N1)毒株间同源性分别为976%,928%,657%和769%。结论　A鹅广东296(H5N1)毒株RNA7和8长度分别为1027和890个核苷酸,此两节段RNA均属禽类毒株。AHK15697(H5N1)毒株的RNA7和8不是来自A鹅广东296(H5N1)毒株。     

Microneedle Delivery of H5N1 Influenza Virus-Like Particles to the Skin Induces Long-Lasting B- and T-Cell Responses in Mice

A simple method suitable for self-administration of vaccine would improve mass immunization, particularly during a pandemic outbreak. Influenza virus-like particles (VLPs) have been suggested as promising vaccine candidates against potentially pandemic influenza viruses, as they confer long-lasting immunity but are not infectious. We investigated the immunogenicity and protective efficacy of influenza H5 VLPs containing the hemagglutinin (HA) of A/Vietnam/1203/04 (H5N1) virus delivered into the skin of mice using metal microneedle patches and also studied the response of Langerhans cells in a human skin model. Prime-boost microneedle vaccinations with H5 VLPs elicited higher levels of virus-specific IgG1 and IgG2a antibodies, virus-specific antibody-secreting cells, and cytokine-producing cells up to 8 months after vaccination compared to the same antigen delivered intramuscularly. Both prime-boost microneedle and intramuscular vaccinations with H5 VLPs induced similar hemagglutination inhibition titers and conferred 100% protection against lethal challenge with the wild-type A/Vietnam/1203/04 virus 16 weeks after vaccination. Microneedle delivery of influenza VLPs to viable human skin using microneedles induced the movement of CD207⁺ Langerhans cells toward the basement membrane. Microneedle vaccination in the skin with H5 VLPs represents a promising approach for a self-administered vaccine against viruses with pandemic potential.Influenza viruses typically cause seasonal epidemics resulting in over 200,000 hospitalizations and approximately 36,000 annual deaths in the United States (45). In addition to seasonal outbreaks, a new pandemic influenza virus strain may emerge at any time. For example, the novel 2009 H1N1 virus has spread rapidly throughout the world, resulting in the first influenza pandemic in the 21st century (6). Indeed, the recent experience with the 2009 H1N1 virus demonstrates the need to develop improved methods of immunization, as conventional vaccination programs showed a significant delay in controlling the new pandemic spread despite a half-century of experience with influenza vaccines.In 1997, the first human cases of infection by highly pathogenic H5N1 avian influenza viruses were reported, with 6 fatalities out of 18 confirmed cases (7, 41). Since 2003, more than 400 human infections with H5N1 viruses have occurred from recurring H5N1 outbreaks. Accumulating data indicated that the fatality rates among H5N1-infected individuals are about 60% (http://www.who.int/csr/disease/avian_influenza). Although H5N1 viruses isolated from humans retain characteristic features of avian influenza viruses, direct transmission of this virus among family members has been observed in Vietnam, Thailand, and Indonesia (31, 46, 50). If these H5N1 viruses were to acquire the properties for efficient transmission among humans, like the 2009 H1N1 pandemic virus, and if the fatality rate remains high, this virus would pose a significant health threat.It is highly desirable to develop pandemic influenza vaccines that can be rapidly produced on a large scale and at low cost, as well as vaccine delivery methods that can achieve mass vaccination within weeks rather than months. Virus-like particles (VLPs) have been suggested as a promising candidate vaccine against influenza viruses. Such influenza VLPs have been demonstrated to provide protective immunity in experimental animal models (12, 15, 19, 37, 44), and VLP vaccines against other diseases are in widespread clinical use (18).The skin is considered an attractive site for vaccination due to the abundance of Langerhans and dermal dendritic cells (11, 17, 27, 30). Intradermal (i.d.) immunization, i.e., delivering antigens to the dermal layer in the skin, has been investigated in many clinical trials (1, 3, 20). In particular, i.d. delivery of influenza vaccines was reported to induce greater protective immunity in the high-risk elderly population (16), possibly by stimulating effective cellular immune responses (51). While the use of syringes and needles to deliver a liquid formulation of vaccines is the most common method for delivering i.d. vaccines, the injection is painful and difficult to perform in a reproducible manner and requires highly trained medical personnel.Microneedles have been developed to facilitate simple and effective vaccination without using hypodermic needles (13, 34). Because they can be prepared in a patch format, microneedles are envisioned to be administered easily and quickly by minimally trained personnel, or possibly by patients themselves. Metal microneedles coated with whole inactivated influenza viruses were demonstrated to deliver the antigen cargo through the restrictive stratum corneum skin barrier, eliciting protective immunity (23, 36, 53). In this study, we tested the feasibility of microneedle vaccination using influenza H5 (A/Vietnam/1203/04) VLPs. Vaccination by microneedles coated with H5 VLPs in the skin induced protective immunity in mice equivalent to or higher than that from conventional intramuscular immunization. Importantly, we provide evidence that these findings could be relevant for human vaccines because human Langerhans cells (LCs) responded to influenza VLP vaccines delivered by coated microneedles.     

鹅流感病毒2／96—H5N1亚型毒株RNA1—3及RNA5节段核？…

目的 明确广东鹅流感病毒２／９６－Ｈ５Ｎ１亚型毒株ＲＮＡ１－３和ＲＮＡ５节段核芏酸全序列及其所编码蛋白的氨基酸序列,以及这些基因节段与香港禽流感病毒１５６／９７－Ｈ５Ｎ１亚型毒株相应节段间的关系。方法 病毒粒ＲＮＡ经逆转录合成ｃＤＮＡ,经聚合酶链反应（ＰＣＲ）扩增,产物纯化,采用双脱氧链末端终止法进行核苷酸序列测定。结果 广东鹅流感病毒２／９６－Ｈ５Ｎ１亚型毒株ＲＮＡ１－３和ＲＮＡ５节段长度分别为     

New Strategies for the Development of H5N1 Subtype Influenza Vaccines

The emergence and spread of highly pathogenic avian influenza (H5N1) viruses among poultry in Asia, the Middle East, and Africa have fueled concerns of a possible human pandemic, and spurred efforts towards developing vaccines against H5N1 influenza viruses, as well as improving vaccine production methods. In recent years, promising experimental reverse genetics-derived H5N1 live attenuated vaccines have been generated and characterized, including vaccines that are attenuated through temperature-sensitive mutation, modulation of the interferon antagonist protein, or disruption of the M2 protein. Live attenuated influenza virus vaccines based on each of these modalities have conferred protection against homologous and heterologous challenge in animal models of influenza virus infection. Alternative vaccine strategies that do not require the use of live virus, such as virus-like particle (VLP) and DNA-based vaccines, have also been vigorously pursued in recent years. Studies have demonstrated that influenza VLP vaccination can confer homologous and heterologous protection from lethal challenge in a mouse model of infection. There have also been improvements in the formulation and production of vaccines following concerns over the threat of H5N1 influenza viruses. The use of novel substrates for the growth of vaccine virus stocks has been intensively researched in recent years, and several candidate cell culture-based systems for vaccine amplification have emerged, including production systems based on Madin-Darby canine kidney, Vero, and PerC6 cell lines. Such systems promise increased scalability of product, and reduced reliance on embryonated chicken eggs as a growth substrate. Studies into the use of adjuvants have shown that oil-in-water-based adjuvants can improve the immunogenicity of inactivated influenza vaccines and conserve antigen in such formulations. Finally, efforts to develop more broadly cross-protective immunization strategies through the inclusion of conserved influenza virus antigens in vaccines have led to experimental vaccines based on the influenza hemagglutinin (HA) stem domain. Such vaccines have been shown to confer protection from lethal challenge in mouse models of influenza virus infection. Through further development, vaccines based on the HA stem have the potential to protect vaccinated individuals against unanticipated pandemic and epidemic influenza virus strains. Overall, recent advances in experimental vaccines and in vaccine production processes provide the potential to lower mortality and morbidity resulting from influenza infection.