The recent 2009 pandemic H1N1 virus infection in humans has resulted in nearly 5,000 deaths worldwide. Early epidemiological findings indicated a low level of infection in the older population (>65 years) with the pandemic virus, and a greater susceptibility in people younger than 35 years of age, a phenomenon correlated with the presence of cross-reactive immunity in the older population. It is unclear what virus(es) might be responsible for this apparent cross-protection against the 2009 pandemic H1N1 virus. We describe a mouse lethal challenge model for the 2009 pandemic H1N1 strain, used together with a panel of inactivated H1N1 virus vaccines and hemagglutinin (HA) monoclonal antibodies to dissect the possible humoral antigenic determinants of pre-existing immunity against this virus in the human population. By hemagglutinination inhibition (HI) assays and vaccination/challenge studies, we demonstrate that the 2009 pandemic H1N1 virus is antigenically similar to human H1N1 viruses that circulated from 1918–1943 and to classical swine H1N1 viruses. Antibodies elicited against 1918-like or classical swine H1N1 vaccines completely protect C57B/6 mice from lethal challenge with the influenza A/Netherlands/602/2009 virus isolate. In contrast, contemporary H1N1 vaccines afforded only partial protection. Passive immunization with cross-reactive monoclonal antibodies (mAbs) raised against either 1918 or A/California/04/2009 HA proteins offered full protection from death. Analysis of mAb antibody escape mutants, generated by selection of 2009 H1N1 virus with these mAbs, indicate that antigenic site Sa is one of the conserved cross-protective epitopes. Our findings in mice agree with serological data showing high prevalence of 2009 H1N1 cross-reactive antibodies only in the older population, indicating that prior infection with 1918-like viruses or vaccination against the 1976 swine H1N1 virus in the USA are likely to provide protection against the 2009 pandemic H1N1 virus. This data provides a mechanistic basis for the protection seen in the older population, and emphasizes a rationale for including vaccination of the younger, naïve population. Our results also support the notion that pigs can act as an animal reservoir where influenza virus HAs become antigenically frozen for long periods of time, facilitating the generation of human pandemic viruses. Influenza A viruses generally infect individuals of all ages and cause severe respiratory disease in very young children and elderly people (>65 years). However, the 2009 pandemic H1N1 virus infection is predominantly seen in children and adults (<35 years of age), but rarely in people older than 65 years of age. Recent serological studies indicate that older people carry antibodies that recognize the 2009 H1N1 virus. This suggests that they may have been exposed to or vaccinated with an influenza virus similar to 2009 H1N1 virus. In this study, we wanted to identify the older H1N1 virus(es) that may confer protection to the elderly population. Using 11 different inactivated influenza A viruses that have circulated between 1918 to 2007, we immunized mice and challenged them with a lethal dose of the 2009 novel H1N1 virus. We find that mice vaccinated with human H1N1 viruses that circulated in 1918 and in 1943 were protected from the 2009 H1N1 virus. Also, the 1976 swine origin H1N1 virus, against which nearly 40 million people were immunized in 1976 in the United States, protects mice from death by the 2009 H1N1 virus. This indicates that people carrying antibodies against H1N1 viruses that circulated between 1918–1943 and to the 1976 swine origin H1N1 virus are likely to be protected against the 2009 pandemic H1N1. Importantly, our data underscores the significance of vaccinating people under 35 year of age, since the majority of them do not have protective antibodies against the 2009 H1N1, and provide a possible mechanism by which pandemic viruses could arise from antigenically frozen influenza viruses harbored in the swine population. Orthomyxoviridae family, cause severe respiratory diseases in humans with an average mortality rate of 36,000/year in the United States alone th century, influenza virus caused three pandemics in humans: 1918 “Spanish influenza” (H1N1), 1957 “Asian influenza” (H2N2) and 1968 “Hong Kong influenza” (H3N2) Influenza A viruses (IAV), members of the Apart from humans, IAV can infect a variety of species including poultry, aquatic birds, horses, pigs, dogs and seals Although several infections with swine IAV have been reported in humans, such infections have been rare In this study, using a lethal mouse model system of the current 2009 H1N1 virus, we tested the efficacy of a panel of 11 different virus vaccines spanning from 1918 to 2009. We demonstrate that mice immunized with inactivated vaccines based on human H1N1 virus from 1918 to 1943 or based on classical swine H1N1 viruses confer complete protection from death against a lethal challenge with the 2009 H1N1 virus. In contrast, vaccination of mice with inactivated human H1N1 viruses isolated after 1950, including contemporary H1N1 viruses, offered only partial protection and resulted in greater morbidity due to 2009 H1N1 virus infection. In agreement with vaccination/challenge studies, pre-challenge sera from mice vaccinated with 1918, and classical swine antigens show neutralization of hemagglutination of 2009 H1N1 virus. In addition, we isolated anti-HA specific monoclonal antibodies (mAb) that have cross-reactivity against 1918 and 2009 H1N1 HA's. In challenge experiments, these mAbs offer complete protection against death by the 2009 H1N1 virus following passive immunization. Analysis of escape mutant viruses selected in the presence of these mAbs mapped to antigenic site Sa. Together, these data suggest that the novel 2009 pandemic H1N1 virus shares a great level of antigenic similarity to the 1918 virus and that site Sa is highly conserved in these two viruses. These results agree well with epidemiological data which indicate that the older population (age >65) are less susceptible to the 2009 H1N1 All animal procedures performed in this study are in accordance with Institutional Animal Care and Use Committee (IACUC) guidelines, and have been approved by the IACUC of Mount Sinai School of Medicine.Human embryonic kidney (293T) cells were maintained in DMEM supplemented with 10% FBS and 1000u/ml penicillin/streptomycin. Madin–Darby canine kidney (MDCK) cells were maintained in MEM supplemented with 10% FBS and penicillin/streptomycin. Reagents for cell culture were purchased from Gibco Life Technologies.in vitro work, in accordance with guidelines of the Centers for Disease Control and Prevention. Cal/09 and Neth/09 virus stocks used for mice experiments were all grown in MDCK cells. All other viruses were grown in 10d old eggs at 37°C for 2–3 days.H1N1 viruses used in this study are as follows: A/Swine/Iowa/30 (Sw/30), A/Puerto Rico/8/34 -MSSM (PR8), A/Weiss/43 (Wei/43), A/New Jersey/8/1976 (NJ/76), A/USSR/92/77 (USSR/77), A/Houston/20593/84 (Hou/84), A/Texas/36/1991 (Tx/91), A/Brisbane/59/2007 (Bris/59/07), A/California/04/2009  and A/Netherlands/602/2009 (Neth/09). A/Northern Territory/60/1968 (NT/68) and A/Brisbane/10/2007 (Bris/10/07) were used as H3N2 controls. All experiments involving 2009 H1N1 viruses were conducted under Biosafety level 3 (BSL3) conditions for animal work and Biosafety level 2 with BSL3 practices laboratory conditions for The recombinant influenza A virus carrying HA/NA from Cal/09 and the internal six genes from PR8, referred to as Cal/09 6:2 in the reminder of the study, was generated as described previously All viruses used to prepare inactivated vaccines were grown in 10d old eggs at 37°C for 2 days. After the clarification of debris in the allantoic fluid by low speed centrifugation, the viruses were pelleted on a 30% sucrose cushion by centrifugation at 25,000 rpm for 2hr. The viral pellet was resuspended in calcium borate buffer pH = 7.0  at 1mg/ml concentration calculated by the Bradford method  and the virus was inactivated by formaldehyde . For the Cal/09 vaccination group, in place of wild type virus, a Cal/09 6:2 was used (described above). For the 1918 virus-based vaccine, virus-like particles (VLP) were used for vaccination. VLPs were produced by co-transfecting HA (A/South Carolina/1/18) and NA (A/Brevig Mission/1/18) into 293T cells as previously described 4 pfu of influenza virus Cal/09. Four weeks later, the mice were given 2×107 pfu of Cal/09 6:2 intravenously to boost Cal/09 HA-specific B cells. Three days after the boost the spleen was harvested and B cells from the spleen were fused with SP2/0 myeloma cells by the addition of polyethylene glycol. The supernatants of the resulting hybridomas were tested by immunofluorescence staining on 293T cells transfected with pDZ-HA . Positive hybridomas were subcloned and re-tested. To generate 1918 virus HA-specific antibodies, mice were immunized by DNA vaccination with pCAGGS-HA of the influenza A/South Carolina/1/18 H1N1 virus and then boosted with whole inactivated virus as above. Hemagglutination inhibition (HI) assays were used to select cross-reactive antibodies against homologous HA using 1918 VLPs. The 1918 HA-specific mAbs 6B9 and 39E4 correspond to isotype IgG2a and the Cal04/09 mAb 29E3 correspond to an IgG2b isotype. The mAbs were isotyped using the IsoStrip kit  and purified using a Protein A sepharose column.Pandemic H1N1 2009 and 1918 HA-specific mouse mAbs were generated by the hybridoma shared research facility at Mount Sinai School of Medicine, New York, NY. For the generation of 2009 H1N1 HA-specific antibodies, 6-week-old BALB/C mice were infected intranasally with 5×10Mouse sera were inactivated using a trypsin-heat-periodate treatment as previously described mAb escape mutants were generated as previously described BALB/C or C57B/6 mice  were anesthetized with ketamine-xylaxine and intranasally infected with the indicated dose of virus diluted in 50 µl of PBS. Body weight and survival were measured every day for 14 days. All mouse experiments were carried in strict accordance with institutional protocols. Mice showing more than 25% of body weight loss were considered to have reached the experimental end point and were humanely euthanized.Lungs of infected mice were excised on indicated days post-infection and homogenized using a mechanical homogenizer . The viral titers in the homogenates were quantified by plaque assay on MDCK cells. Each data point presents the average titer from 2–3 mice.2, 5×103, 5×104, 5×105 or 5×106 pfu virus in 50 µl (n = 5/group). The mice were monitored daily for survival and body weight loss over a period of 14 day post infection (p.i.) Mice showing more than 25% of body weight loss were considered to have reached the experimental end point and were euthanized. LD50 were calculated by the Reed & Muench method Female C57B/6 mice (9 weeks old) were anesthetized with ketamine-xylaxine and intranasally infected with doses 5×1050 (7.9×105 pfu). Survival and body weight loss were monitored for 14 days. Mice were 9–10 week old at the time of lethal challenge.Five-week-old female C57B/6 were immunized with 15 µg of inactivated virus or 1918 VLP by intramuscular injection in the hind leg. Two-weeks later the mice were boosted with the same amount of inactivated virus. Four weeks after the initial immunization mice were challenged with Neth/09 at a dose equivalent to 50 LD50. Survival and body weight loss were monitored for 14 days post challenge (p.c.), and lung viral titers were obtained at days 3 to 6 p.c.Monoclonal antibodies 6B9 and 39E4 were generated against influenza A/South Carolina/1/18 (H1N1) virus (1918) HA, and monoclonal antibody 29E3 was raised against Cal/09 HA. Nine-week-old C57B/6 mice were passively immunized with a total 150 µg of indicated monoclonal antibody  or 200 µl of polyclonal sera from mice previously infected with Cal/09, 24h prior to lethal challenge. A mAb (isotype IgG2a) raised against an unrelated viral protein, Nipah W, was administered as control. The mice were challenged with Neth/09 at a dose equivalent to 50 LDAll of the vaccinated and passively immunized mice were challenged at the same time. A single group of control mice were used for comparison in both experiments and refer to either “No vaccination” or “No Ab” controls shown below.The structural models for HA of Cal//09 and Bris/59/07 were generated using structural automated protein structure homology-modeling prediction server (Swiss-Model) with best fitting templates  3 pfu  and monitored for weight loss and survival over a period of 14 days p.i.  assays and challenge experiments. Analysis of sera from mice infected with Cal/09 revealed similar levels of HI activity against the Cal/09 and Neth/09 isolates, suggesting that they are antigenically very similar, despite significant differences in pathogenicity in mice (data not shown).6 pfu of Neth/09 (>50 LD50). As expected, all mice previously infected with Cal/09 survived , and the vaccine efficacy was evaluated by assessment of weight loss and survival over a 14 day period p.c., and also by measuring the virus titer in the lower respiratory track  that might be responsible for the cross-protection.Next, to test whether the protection observed in 1918–1943 and NJ/76 vaccinated correlates with cross-reactive neutralizing antibodies in the sera, we tested the pre-challenge sera from vaccinated mice for HI activity against different H1N1 viruses . All thein vivo) against lethal Neth/09 challenge, mice were given 150 µg of mAb as a prophylactic treatment 24h prior to challenge (50 LD50). To assess the level of HI titers in the blood prior to challenge, the HI activity in passively immunized mice sera was measured (in vivo (Poly sera). Mice in this group were completely protected from lethal challenge and showed a decrease in lung virus titers on day 3 and 6 compared to untreated controls. Nonetheless, these mice showed a significant weight loss during the first 4 days  showed HI activity against Cal/09 and against 1918 VLPs . To evalmeasured . Sera frmeasured . As expemeasured . Polyclot 4 days . Treatmet 4 days . Taken tTo identify the shared cross-protective epitope(s) in HA, we generated mAb escape mutants of Cal/09 6:2 virus. Escape mutants generated by pre-incubation of Cal/09 virus (6:2) with excess of each of the cross-reactive mAbs  resulted in a loss of HI activity against the same mAb. Of interest, all the escape mutant viruses generated with either 1918- or 2009 HA-specific mAbs carried mutations in the antigenic site Sa ; Fig. 5.Seasonal influenza viruses predominantly cause severe disease in very young children and in the older population. However, infections with the 2009 pandemic H1N1 are considerably lower in people 65 years or older, likely due to immunity from prior exposure and/or vaccination to an antigenically similar influenza virus www.who.int).Usually human influenza viruses do not show substantial replication in mice without prior adaptation to this host Although at this moment we can not exclude the possibility that the NA protein contributed at least in part to the protection seen with the inactivated vaccines, our results from the passive immunization and vaccination studies suggest that human H1N1 viruses from or prior to 1943, including the 1918 virus, share common protective epitope(s) in the HA protein. The antigenic sites of influenza virus HA's has been extensively characterized by monoclonal antibody epitope mapping studies To characterize the antigenic similarity, we generated mAbs against the HA of 1918 and 2009 H1N1 viruses. Interestingly, two 1918 HA-specific mAbs, 6B9 and 39E4, and one Cal/09 HA specific mAb, 29E3, showed HI activity against both 1918 VLP and Cal/09 virus . PassiveIn 1976, an H1N1 virus closely related to classical swine H1N1 isolates from the 1930's caused an influenza outbreak at an American military facility, Fort Dix, in the state of New Jersey. After the 1976 swine H1N1 outbreak, nearly 40 million people in the United States were immunized with an NJ/76 vaccine. Our study in mice, in agreement with previous serology done with ferret sera Two recent human serology studies showed a high prevalence of neutralizing antibodies against 2009 pandemic H1N1 in people born before 1930 Our data are consistent with a picture in which domestic pigs have served as a reservoir for an “antigenically frozen” H1 hemagglutinin derived from the 1918 influenza virus. A significant drift in humans of that same 1918 H1N1 virus has resulted in a lack of significant pre-existing immunity against the 2009 pandemic H1N1 virus in humans born after the 1940–50s. Of concern, swine influenza viruses containing HA genes derived from more modern human H3 and H1 viruses have also established lineages in North American pigs during 1997–1998 and 2003–2005, respectively


The highly pathogenic H5N1 avian influenza emerged in the year 1996 in Asia, and has spread to Europe and Africa recently. At present, effective monitoring and data analysis of H5N1 are not sufficient in Chinese mainland.Anas platyrhynchos) had the highest prevalence (4.37%). A difference of prevalence was found among 14 provinces. Qinghai had a higher prevalence than the other 13 provinces combined (3.88% versus 0.43%). The prevalence in three species in Qinghai province (Pintail (Anas acuta), Mallard (Anas platyrhynchos) and Tufted Duck (Aythya fuligula)) were obviously higher than those in other 13 provinces. The results of sequence analysis indicated that the 17 strains isolated from wild birds were distributed in five clades , which suggested that genetic diversity existed among H5N1 viruses isolated from wild birds. The five isolates from Qinghai came from one clade (2.2) and had a short evolutionary distance with the isolates obtained from Qinghai in the year 2005.During the period from April of 2004 to August of 2007, we collected 14,472 wild bird samples covering 56 species of 10 orders in 14 provinces of China and monitored the prevalence of flu virus based on RT-PCR specific for H5N1 subtype. The 149 positive samples involved six orders. Anseriformes had the highest prevalence while Passeriformes had the lowest prevalence (2.70% versus 0.36%). Among the 24 positive species, mallard (We have measured the prevalence of H5N1 virus in 56 species of wild birds in 14 provinces of China. Continuous monitoring in the field should be carried out to know whether H5N1 virus can be maintained by wild birds. Influenza A virus contains eight segments of single-strand negative RNA. Segment 4 codes hemagglutinin (HA) gene and segment 6 codes neuraminidase (NA) gene. According to the antigenic characteristics of HA and NA, avian influenza A virus has 16 subtypes HA and nine subtypes NA Highly pathogenic H5N1 bird flu emerged in the year 1996 in Asia From 2003, there have been a series of infections of humans by H5N1 bird flu virus in Asia, and the reports of death have aroused the extensive attention among the general public China is in a critical position along the migration routes for the migratory birds across Eurasia. At present, effective monitoring and data analysis of H5N1 virus among wild birds are quite insufficient in Chinese mainland. During the period from April, 2004 to August, 2007, we collected the samples from wild birds in 14 provinces in Chinese mainland and monitored H5N1 virus based on reverse transcription-polymerase chain reaction (RT-PCR). We also discuss the prevalence of H5N1 virus in wild birds in Chinese mainland.We launched a sample collection from the wild birds living in the major lakes and wetlands covering 14 provinces of China from 2004 to 2007 . The sam2-test, ρ<0.001).149 samples were H5/N1 positive after the test based on RT-PCR . The posTadorna ferruginea) had the lowest prevalence (1.52%), while Mallard (Anas platyrhynchos) had the highest prevalence (4.37%). Four strains of H5N1 subtype were isolated from Anseriformes, which were named as: A/wDk/Shanghai/59/04 (H5N1), A/BarHeadedGoose/Qinghai/F/06 (H5N1), A/Swan/Qinghai/01/06 (H5N1) and A/BarHeadedGoose/Qinghai/369/07 (H5N1) respectively. In Qinghai province, two strains of virus were isolated from the samples of bar-headed goose from 2006 to 2007 and one strain of virus was isolated from the samples of swan in the year 2006.The positive samples of Anseriformes were collected from ducks, geese and swans. The prevalence among 9 species of Anseriformes varied from 1.52% to 4.37% and did not show obvious difference. Ruddy Shel Duck  was relatively high (2.39%). Three H5N1 viruses were isolated from Lariformes, which were named as: A/BrownHeadedGull/Qinghai/3/06 (H5N1), A/SlatyBackedGull/Shandong/59/04 (H5N1) and A/PallasGull/Qinghai/81/07 (H5N1). Among the samples of Anseriformes and Lariformes, five H5N1 viruses were isolated from Qinghai provinces from 2006 to 2007, which suggested that wild birds in Qinghai province might maintain H5N1 virus.The positive samples of Lariformes were only collected from gulls. The prevalence of Brown-headed Gull (Passer montanus) was relatively high among all. Eight H5N1 viruses were isolated from the 26 positive samples and they were named as: A/TreeSparrow/Henan/1/04 (H5N1), A/TreeSparrow/Henan/2/04 (H5N1), A/TreeSparrow/Henan/3/04 (H5N1), A/TreeSparrow/Henan/4/04 (H5N1), A/Babbler/Fujian/320/04(H5N1), A/BlackBird/Hunan/1/04 (H5N1), A/GoldenMountainThrush/Fujian/376/04 (H5N1), and A/BlackBulbul/Fujian/439/04 (H5N1).We collected 7,320 samples from the 33 species of Passeriformes . The preColumba livia). Six samples were H5N1 positive and the prevalence is 1.05%, which is similar to that of Tree Sparrow (Passer montanus). One H5N1 virus was isolated and named as: A/Pigeon/Zhejiang/17/05(H5N1). The samples of Charadriiformes were only colleted from Eurasian Curlew (Numenius arquata) and the prevalence is 0.40%. One H5N1 virus was isolated (A/Curlew/Shandong/61/04 (H5N1)). The prevalence of Pelecaniformes was 1.73%, but no virus was isolated. It was noted that H5N1 viruses  were isolated from the samples of migratory bird in Shanghai and Shandong province in the year 2004 and the sites for samples are located in coastal wetlands which belong to the Asia-Australia fly way.Samples of Columbiformes were only collected from Rock Dove (Passer montanus). The relative high prevalence in Henan might be related with the high prevalence of Tree Sparrow. As shown in A certain difference of prevalence was found among 14 provinces as shown in Qinghai province, located on the Qinghai-Tibet plateau of Northwest China, had special environments and various wildlife species. According to pervious study Anas platyrhynchos) had the highest prevalence (11.19%) while Ruddy Shelduck (Tadorna ferruginea) had the lowest prevalence (2.17%). The prevalence of three species in Qinghai province (Pintail (Anas acuta), Mallard (Anas platyrhynchos) and Tufted Duck (Aythya fuligula)) were obviously higher than those in other 13 provinces . Except Common Teal (Anas crecca) and Red-crested Pochard (Rhodonessa rufina), the prevalence of other six species were relatively higher than those in other 13 provinces.As shown in 17 strains of H5N1 virus were isolated from 149 positive samples covering 24 species. We sequenced the HA genes of these 17 strains. Based on the results of sequence analysis, it was known that all of the HA proteins had a series of basic amino acids at the HA cleavage site (RRRKKR/G), which is a characteristic of highly pathogenic influenza viruses. All amino acids relevant to receptor binding  did not have mutations.The phylogenetic tree of HA genes was constructed to determine the relationship with other H5N1 viruses . All the2-test, ρ<0.001). It was reported that H5N1 viruses were isolated from dead wild birds Anser indicus), Whooper Swan (Cygnus cygnus), Brown-headed Gull , Pallas's Gull (Larus ichthyaetus), Tufted Duck (Aythya fuligula), Rock Dove (Columba livia), Tree Sparrow (Passer montanus). However, the number of theses species with signs of disease was very small and the mortality was closed to zero.The positive samples involved 24 species of six orders, among which the prevalence of H5N1 virus varied. The samples of Anseriformes had the highest prevalence and those of Passeriformes had the lowest prevalence  As shown in Moreover, a certain difference of prevalence was found between 14 provinces as shown in In the paper, we found the samples of 24 species were positive for H5N1 virus and the prevalence for Anseriformes was relatively high, which suggested that H5N1 virus might be maintained by wild birds, especially by water fowls. However, it was not excluded that H5N1 viruses were transmitted repeatedly from ongoing outbreaks in poultries to wild birds. In China, water fowl could be closely contact with poultries  in the water environment. Excrements of poultries infected by H5N1 viruses could contaminate the living environment for water fowls. Moreover, the resident birds living around the farms could be infected by poultry H5N1 virus and carry virus from farm to the water environment around water fowls. H5N1 Viruses existed in the environment could infect water fowls, which could transmit H5N1 viruses when they are migrating. For example, Qinghai Lake is a major breeding site for migratory birds and did not have livestock breeding, but the range of migration for migratory birds included Xinjiang, Neimenggu, Gansu, Shannxi, Hunan, Xizang, Yunnan, where many poultry cases were reported from 2004 . During Passer montanus) had a relative high prevalence (1.09%), which is similar to Pigeons . These resident birds often appeared in the cultivation zones of poultries and the habitats of water fowls and could come in contact with infected birds or infected environment. The analyzed results suggested that these resident birds had a high risk of transmitting of bird flu virus between wild birds and poultries Outbreaks of bird flu among migratory birds were reported in Qinghai province in 2005, 2006, 2009 In the paper, the prevalence of H5N1 virus among wild birds in China were relatively higher than those in Europe or Africa As shown from the phylogenetic tree, the 17 strains isolated from wild birds distributed in 5 groups , which suggested that genetic diversity existed among H5N1 viruses of wild birds and that the genotypes in wild birds in Chinese mainland was complicated The outbreak of bird flu H5N1 subtype proved that our common knowledge on the flu in wild birds was still insufficient. It is very important to know if H5N1 virus existed in wild birds continuously and continuous monitoring in the field should be carried out.All animals were handled in strict accordance with good animal practice as defined by Hubei Animal Welfare Council, China, and all animal work was approved by State Forestry Administration, China.We launched a sample collection about the wild birds living in the major lakes and wetlands covering 14 provinces in China from 2004 to 2007 . The samFrom 2004 to 2007, we collected 14,472 wild bird samples in total, covering 56 wild bird species of 10 orders . All the5′-AGCAAAAGCAGG-3′. The primers for PCR was designed according to the WHO recommended manual for H5N1 identification based on conventional RT-PCR 5′- GCCATTCCACAACATMCACCC-3′ for forward primers 5′- CTCCCCTGCTCRTTGCTATG-3′ for reverse primers. For each run, the samples were prepared and processed in parallel with negative and positive controls. PCR products were routinely processed with 1% agarose gel electrophoresis. The expected length of the specific amplified fragment was about 0.2 kb.For cloacal swabs, RNA was extracted using QIAamp Viral RNA Mini kit  according to the standard protocol. 140 µL transport medium were used to extract RNA, and then RNA was eluted by 50 ul elution buffer. For tissues and droppings, it was homogenized and centrifuged firstly. 140 ul supernatant liquor was used to extract RNA as above. RNA was stored in −80°C. Conventional reverse transcription polymerase chain reaction (RT-PCR) was performed using a one-step RNA PCR kit  to identify the subtype according to the standard protocol. The primer for reverse transcription of viral RNA genome was H5N1 positive samples were subsequently processed for virus isolation. Cloacal samples were diluted with phosphate-buffered saline (PBS) and centrifugation. Tissues and droppings were homogenized and centrifuged, and supernatant were collected. All positive samples were mixed with equal volume of PBS containing antibiotics  overnight at 4°C, and then centrifuged and inoculated into the allantoic cavities of 10-day-old specific-pathogen-free embryonated eggs . After incubation at 37°C for 24 to 72 h, the allantoic fluid of the inoculated eggs was harvested. All the experiments were performed in a Bio-safety level 3 laboratory.According to the PCR protocol described previously 2-test were used for analysis of the dataset used in this study.The Pearson χTable S1Bird species involved in the study. Table S1 includes data on all species in which H5N1 virus was detected by RT-PCR, including geographical sampling location and sample size.(0.07 MB XLS)Click here for additional data file.


Envelope (E) glycoprotein E2 of the hepatitis C virus (HCV) mediates binding of the virus to target cell receptors. Nevertheless, the precise role of E1 in viral entry remains elusive.To understand the involvement of the fusion peptide-like domain positioned at residues 264 to 290 within envelope glycoprotein E1 in HCV infection, mutants with Ala and Asn substitutions for residues conserved between HCV and E proteins of flaviviruses or the fusion proteins of paramyxoviruses were constructed by site-directed mutagenesis and their effects on membrane fusion and viral infectivity were examined.None of these mutations affected the synthesis or cell surface expression of envelope proteins, nor did they alter the formation of a non-covalent E1-E2 heterodimer or E2 binding to the large extracellular loop of CD81. The Cys residues located at positions 272 and 281 were unlikely involved in intra- or intermolecular disulfide bond formation. With the exception of the G267A mutant, which showed increased cell fusion, other mutants displayed reduced or marginally inhibited cell fusion capacities compared to the wild-type (WT) E1E2. The G267A mutant was also an exception in human immunodeficiency virus type 1 (HIV-1)/HCV E1E2 pseudotyping analyses, in that it showed higher one-cycle infectivity; all other mutants exhibited greatly or partially reduced viral entry versus the WT pseudotype. All but the G278A and D279N mutants showed a WT-like profile of E1E2 incorporation into HIV-1 particles. Since C272A, C281A, G282A, and G288A pseudotypes bound to Huh7 cells as effectively as did the WT pseudotype, the reduced infectivity of these pseudotypes was due to their ability to inhibit cell fusion.Our results indicate that specific residues, but not the structure, of this fusion peptide-like domain are required for mediating cell fusion and viral entry. Hepacivirus genus of the family Flaviviridae with a genome of 9.6 kb. More than six distinct genotypes exist, with genotypes 1a and 1b being the most prevalent worldwide. The viral genome is translated in host cells into a ~3000-amino acid (aa)-long polyprotein, which is cleaved co-translationally and post-translationally by both host signal peptidases and viral proteases to generate several distinct polypeptides, including three structural proteins located at the N-terminal region of the polyprotein, i.e., the core protein and two envelope (E) glycoproteins , the p7 protein, and six nonstructural proteins . E1 and E2 form a non-covalent heterodimer . After centrifugation at 18,000 × escribed . Equal v35S]methionine  for 30 min and chased with excess cold methionine as described previously [g at 4°C for 10 min. Equal volumes of cell lysates were precipitated with 10 μl of anti-E2 MAb H53. The antibody was prebound to a mixture containing 30 μl of protein A-Sepharose 4B (Amersham Biosciences). After incubation at 4°C for 2 h, the immune complexes were washed six times with RIPA buffer  and then boiled in 30μl of Laemmli buffer. The immunoprecipitates were examined by nonreducing 10% SDS-PAGE.Two days after transfection, 293T cells expressing WT or mutant E1E2 proteins were metabolically labeled with [eviously  for 4 h.-E-Luc and HCV WT or mutant E1E2 expression plasmids at 24, 48, and 72 h after transfection, combined, filtered through 0.45-μm membrane discs, and concentrated by centrifugation at 100,000 × g for 2 h at 4°C in an SW28 rotor. Viral pellets were resuspended in DMEM containing 2.5% FBS to concentrate the viral particles by 100-fold. Pseudovirions were normalized by reverse transcriptase (RT) activity as previously described [4/well) were cultured for 24 h, and then infected with the WT or mutant pseudotypes containing 5 × 105 cpm of RT activity in the presence of 8 μg/ml polybrene at 37°C overnight. Each infection assay was performed in triplicate wells. After removing the viral suspensions, cells were incubated at 37°C for an additional 48 h before being lysed with 60 μl per well luciferase cell lysis buffer . Fifty microliters of lysates was added to 50 μl of luciferase substrate solution (Promega), and the luciferase activity, in terms of relative light units, was measured for 10 s in a Sirius luminometer .Culture supernatants were collected from 293T cells cotransfected with pNL4-3Rescribed . Huh7 ce5/well) and Huh7 (2 × 105/well) cells were seeded in 6-well plates 1 day prior to the assay. As effector cells, 293T cells were cotransfected with 2 μg of pCAG-T7pol [293T  seeded in 12-well plates were cultured for 24 h before HCVpp binding. Cells were incubated with WT or mutant HCV pseudotypes containing 107 cpm of RT activity in a total volume of 300 μl of DMEM containing 10% FBS and 0.1% of NaN3 at 37°C for 1 h. After three washes with 1 ml of PBS containing 2% FBS and 0.1% NaN3, cells were incubated with 4 μg of rabbit anti-E2 in a total volume of 300 μl on ice for 2 h. After washing, cells were incubated with a 1:100 concentration of FITC-conjugated anti-rabbit IgG on ice for 1 h. After washing, cells were fixed with 300 μl of 4% paraformaldehyde on ice for 30 min, and then analyzed by FACS. Alternatively, cells were fixed, permeabilized, and then stained with rabbit anti-E2 or anti-p24. After incubation with FITC-conjugated anti-rabbit IgG, the immunostained cells were analyzed by FACS.Binding of HCVpp to Huh7 cells was performed as previously reported . Huh7 ceTo understand the involvement of the E1 putative fusion domain, which spans residues 264~290, in viral infection, we characterized the effects of amino acid substitutions for conserved residues in this region on viral infection. This internal hydrophobic region of E1 bears similarities to the predicted fusion peptide from E proteins of flaviviruses . First, Accordingly, substitutions were introduced at these conserved residues, and their effects on the phenotypes of E1E2 proteins were examined. In the first group, mutants were constructed by Ala or Asn substitutions for the conserved residues located in the N-terminal half of this region, i.e., G267A, C272A, G278A, D279N, and C281A. The first single-letter amino acid code indicates that the residue located at that position was replaced by an Ala (A) or Asn (N) residue, as shown by the second code letter. Another group contained Ala substitutions for the conserved Gly residues located in the C-terminal segment of this region, i.e., G278A, G282A, and G288A mutants. All of these mutants were constructed by oligonucleotide-directed PCR overlap extension mutagenesis Fig.  from a pTo understand whether these E1 mutant proteins are normally expressed and processed, 293T cells were transfected with each of the WT and mutant plasmids, and cell lysates were subjected to SDS-PAGE followed by Western blotting using MAbs specific for E1 and E2. All N-terminal half mutants, i.e., G267A, C272A, G278A, D279N, and C281A, produced comparable levels of E1 and E2 proteins to those produced by the WT plasmid upon transfection  and the subsequent entry process . AlthougInterestingly, we also noted that the severity of reductions in cell fusion and viral entry of the C272A, C281A, G282A, and G288A mutants paralleled the conserved nature of these residues among HCV genotypes. For instance, the Cys-272 and Cys-281 residues are highly conserved among the HCV and E proteins of flaviviruses Fig.  and alsoIt was shown that the fusion sequences from alphaviruses and flaviviruses include Cys residues for specific disulfide bridges which are essential for the folding stability of fusion peptides ,82. The The observation that the G267A mutant exhibited higher viral entry ability compared to the WT proteins Fig  correlatOur study has implications in understanding the molecular basis of how the E1 fusion peptide-like domain acts during cell fusion and viral entry.The authors declare that they have no competing interests.HFL carried out all experiments, and participated in experimental design. CHH participated in the CD81-E1E2 protein pull-down assay and incorporation of E1 and E2 proteins into HIV-1 virons. LSA performed the control pseudotype binding and the flow cytometric analyses with the E2 MAb. CKC performed the sCD81 and E mutant construction. SSC conceived of the study, coordinated the work, and wrote the manuscript. All authors read and approved the final manuscript.


The cardioprotective potential of human recombinant erythropoietin  (Epo) against ischemia-reperfusion-induced injury is well known. But, the underlying mechanisms are not well elucidated. The aim of this study was to characterize the mechanism involved in the cardioprotective effect of Epo-induced preconditioning in isolated rat heart.The heart was mounted on a Langendorff apparatus. After 10 min of stabilization, four cycles of ischemic preconditioning (IPC) were given followed by 30 min of global ischemia and 120 min of reperfusion. Epo preconditioning was induced by four cycles of 5-min perfusion of K-H solution containing Epo (1.0 U/ml) followed by 5 min perfusion with K-H solution. Myocardial infarct size was estimated macroscopically using the triphenyltetrazolium chloride staining technique. The extent of myocardial injury was measured by release of lactate dehydrogenase and creatine kinase-MB in the coronary effluent.The present study demonstrates that Epo preconditioning was almost as effective as IPC. Administration of Wortmannin (100 nM), a PI-3K inhibitor, or Chelerythrine (1 µM), a protein kinase-C (PKC) inhibitor, or AG490 (5 µM), a JAK-2 inhibitor, significantly attenuated the cardioprotective effects of Epo-induced preconditioning.Our result suggest that the cardioprotective potential of Epo-induced preconditioning in isolated rat heart was due to an interplay of the JAK-2, PI-3K and PKC pathways. Inhibition of any one of the three pathways was sufficient to block the cardioprotective effect of Epo-induced preconditioning in isolated rat heart. Ischemic preconditioning (IPC) is an endogenous protective phenomenon in which brief episodes of sublethal ischemia followed by reperfusion increase the resistance of the myocardium to subsequent sustained ischemia of longer duration. It has b2et al.[It has been reported that both IPC and Epo produce cardioprotection through common signaling pathways: PI-3K, PKC and JAK/STAT.515–22 Gl524et al. reportedThe experimental protocol used in the present study was approved by the Institutional Animal Ethics Committee.Human recombinant Epo  , Wortmannin, chelerythrine and AG490  were dissolved in dimethyl sulfoxide (DMSO) and the final concentration of DMSO in K-H solution was 0.02%. All other reagents used in this study were of analytical grade and were always freshly prepared before use.2 2.5 mM; MgSO4.7H2O 1.2 mM; KH2PO4 1.2 mM; C6H12O6 11 mM), pH 7.4 and bubbled with 95% O2 and 5% CO2. The flow rate was maintained at 7–9 ml/min using a Hoffman’s screw. The heart was enclosed in a double-wall jacket, the temperature of which was maintained by circulating water heated at 37°C. IPC was produced by closing the inflow of K-H solution for 5 min followed by 5 min of reperfusion. Four such episodes were employed. Global ischemia was produced for 30 min followed by 120 min of reperfusion. The coronary effluent was collected before ischemia, immediately, 5 min and 30 min after reperfusion for estimation of lactate dehydrogenase (LDH) and creatine kinase-MB (CK-MB).Rats were administered heparin  20 min before their sacrifice by cervical dislocation. The heart was rapidly excised and immediately mounted on Langendorff’s apparatus and was Four cycles, each cycle consisting of 5-min perfusion of K-H solution containing Epo (1.0 U/ml) followed by 5 min perfusion with K-H solution (free of Epo), and followed by 30 min global ischemia and 120 min reperfusion were used.The myocardial infarct size was measured using the triphenyltetrazolium chloride (TTC) staining method while the level of LDH  and CK-MB  in coronary effluents was estimated using commercially available kits. Values of LDH and CK-MB were expressed in international units per liter (IU/L).The heart was removed from the Langendorff’s apparatus. Both the atria and the root of the aorta were excised and the ventricles were kept overnight at a temperature of -4°C. Frozen ventricles were sliced into uniform sections of about 1–2 mm thickness. The slices were incubated in 1% w/v TTC at 37°C in 0.2M tris-chloride buffer for 30 min. The normal myocardium was stained brick red while the infarcted portion remained unstained. Infarct size was measured by the volume method.A diagrammatic representation of the experimental protocol is shown in n = 6) After stabilization, the heart was perfused continuously with K-H buffer for 190 min without subjecting it to global ischemia. Group 2: (Vehicle control: n = 6) After stabilization, the heart was perfused for 5 min with K-H containing DMSO (0.02%) followed by 5 min perfusion with K-H solution free of DMSO; four such cycles were repeated. Then, the preparation was subjected to 150 min of perfusion. Group 3:  After stabilization, the heart was subjected to 30 min of global ischemia followed by 120 min of reperfusion. Group 4:  Four cycles of IPC were given just after stabilization. Each cycle comprised of 5 min ischemia and 5 min reperfusion with K-H solution and then followed by 30 min global ischemia and 120 min reperfusion. Group 5: (Epo preconditioning: n = 6) Just after stabilization, the heart was subjected to four cycles of Epo preconditioning, each cycle comprising of 5 min perfusion with K-H solution containing Epo (1.0 U/ml) followed by 5 min perfusion with K-H solution free of Epo and further followed by 30 min of global ischemia and 120 min of reperfusion. Group 6:  During 10 min of stabilization, the heart was perfused with K-H solution containing 100 nM of Wortmannin followed by four repeated episodes of 5 min perfusion of K-H solution containing Epo (1.0 U/ml) followed by 5 min perfusion with K-H solution containing (100 nM) Wortmannin. Then, the preparation was subjected to 30 min global ischemia followed by 120 min reperfusion. Group 7:  During stabilization, the heart was perfused with K-H solution containing 1 µM Chelerythrine followed by four repeated episodes of 5 min perfusion of K-H solution containing Epo (1.0 U/ml) followed by 5 min perfusion with K-H solution containing (1 µM) Chelerythrine followed by 30 min global ischemia and 120 min reperfusion. Group 8:  The heart was perfused with K-H solution containing 5 µM AG490 during the stabilization followed by four repeated episodes of 5 min perfusion of K-H solution containing Epo (1.0 U/ml) followed by 5 min perfusion with K-H solution containing (5 µM) AG490 further followed by 30 min of global ischemia and 120 min of reperfusion.In all groups, isolated rat heart was perfused with K-H solution and allowed to stabilize for 10 min. Group 1: . Statistical analysis was performed using Sigmastat Software. The values were statistically analysed using one-way analysis of variance (ANOVA) followed by Tukey’s multiple comparison test. Value of Effect of IPC, Epo preconditioning and pharmacological interventions on myocardial infarct size:P < 0.05) reversed the Epo preconditioning-induced decrease in myocardial infarct size [P > 0.05).Both IPC and Epo preconditioning significantly attenuated ischemia–reperfusion-induced increase in myocardial infarct size. Administration of Wortmannin or Chelerythrine or AG490 significantly (rct size . The difP < 0.05) reversed the Epo preconditioning-induced decrease in the release of LDH in the coronary effluent [P > 0.05).Both IPC and Epo preconditioning significantly attenuated ischemia-reperfusion-induced increase in release of LDH in the coronary effluent. Administration of Wortmannin or Chelerythrine or AG490 significantly (effluent . The difP < 0.05) reversed the Epo preconditioning-induced decrease in the release of CK-MB in the coronary effluent [P > 0.05).Both IPC and Epo preconditioning significantly attenuated ischemia-reperfusion-induced increase in release of CK-MB in the coronary effluent. Administration of Wortmannin or Chelerythrine or AG490 significantly  and EPO preconditioning-induced cardioprotection in the isolated rat heart is mediated through an interplay of the JAK-2, PI-3K and PKC pathways. The order of effectiveness for attenuation of erythropoietin preconditioning was Wort>Che>AG490 in the dosages used, but these inter-se differences in the efficacy were not statistically significant.Based on the results of the present study, it may be concluded that Epo-induced preconditioning was almost as effective as IPC (


A3 gene encodes a protein with one or two conserved zinc-coordinating motifs . The presence of one A3 gene in mice (Z2–Z3) and seven in humans, A3A-H , suggests extraordinary evolutionary flexibility. To gain insights into the mechanism and timing of A3 gene expansion and into the functional modularity of these genes, we analyzed the genomic sequences, expressed cDNAs and activities of the full A3 repertoire of three artiodactyl lineages: sheep, cattle and pigs.APOBEC3 (A3) proteins deaminate DNA cytosines and block the replication of retroviruses and retrotransposons. Each A3 genes, A3Z1, A3Z2 and A3Z3, whereas pigs only have two, A3Z2 and A3Z3. A comparison between domestic and wild pigs indicated that A3Z1 was deleted in the pig lineage. In all three species, read-through transcription and alternative splicing also produced a catalytically active double domain A3Z2-Z3 protein that had a distinct cytoplasmic localization. Thus, the three A3 genes of sheep and cattle encode four conserved and active proteins. These data, together with phylogenetic analyses, indicated that a similar, functionally modular A3 repertoire existed in the common ancestor of artiodactyls and primates . This mammalian ancestor therefore possessed the minimal A3 gene set, Z1-Z2-Z3, required to evolve through a remarkable series of eight recombination events into the present day eleven Z domain human repertoire.Sheep and cattle have three A3 genes is consistent with the modular nature of the locus and a model in which most of these events  were selected by ancient pathogenic retrovirus infections.The dynamic recombination-filled history of the mammalian  We have therefore established a critical foundation for understanding the function(s) and evolutionary history of the A3 repertoire of any other placental mammal.Prior to the present studies, it was clear that most  placental mammals had Z2- and Z3-type  and cow ,42,54,72s Figure . Second,A3 genes . Thus, unfortunately, these two interesting non-placental mammals are unlikely to provide significant insights into the earliest stages of A3 gene evolution . Perhaps data from the other two placental mammal super-orders, Afrotheria and Xenarthra , will help shed light on earlier stages of A3 gene evolution, when presumably an AID-like gene transposed between CBX6 and CBX7 and duplicated to give rise to the ancestral Z1-Z2-Z3 locus. Nevertheless, because all current data indicate that the A3 genes are specific to placental mammals, we hypothesize that a unique role of these genes may relate to the placenta itself, where the A3 proteins may function to help protect the developing fetus from potentially harmful retrotransposition events and/or retroviral infections.It is noteworthy that our pan-species Z1 PCR analyses failed to generate product from opossum genomic DNA and that the recently released opossum and platypus genomic sequences lack A3 genes of mammals is to provide an innate immune defense to retrovirus and retrotransposon mobilization. This is supported by the fact that the single A3 gene of mice is dispensable and that many of the mammalian A3 genes show evidence for a strong diversifying selection 6-Z1-Z3 repertoire? We propose that large-scale events such as gene expansions were selected by extremely pathogenic or lethal retroviral epidemics, because rare expansions would have been easily lost amongst a population of non-expanded alleles. A powerful selective pressure such as a lethal epidemic has the potential to produce a population bottleneck such that mostly (or only) pathogen-resistant individuals would survive . Such powerful selective pressures would have the potential to promote and perhaps even cause speciation events. We further predict that such events may be marked by changes in A3 Z domain copy number. It is therefore quite plausible that at least some of the eight recombination events required to transform the ancestral Z1-Z2-Z3 repertoire into the present day human Z1-Z2-Z1-(Z2)6-Z1-Z3 repertoire may have protected our human ancestors from ancient retroviral infections and thereby facilitated the evolution of primates (a process that we have termed primatification).Finally, a major question is what selective pressure(s) drove the A3 locus of sheep and cattle consists of three genes, A3Z1, A3Z2 and A3Z3, and the potential to encode four functional proteins, three directly and one (A3Z2-Z3) by read-through transcription and alternative splicing. The A3 locus of pigs experienced a deletion and therefore lacks A3Z1. The artiodactyl A3 repertoire demonstrates a unique modularity centered upon the conserved zinc-coordinating motifs. DNA deaminase activity data and subcellular localization studies suggest that this modularity may also correspond to a broader functionality. All of the data combined to indicate that the common ancestor of artiodactyls and humans possessed a sheep/cattle-like A3 gene set, with the organization and capacity to evolve into the present day repertoires. The remarkable A3 gene expansion in the primate lineage – from the three ancestral genes (A3Z1-A3Z2-A3Z3) to the present-day eleven Z-domain human repertoire – was predicted to require a minimum of eight recombination events, most of which may have been required to thwart an ancient retroviral infections.The A3-, CBX6- and CBX7-specific PCR was used to identify one A3-positive BACs for sheep  and two for pigs . Purified BAC DNA was sheared to an average of approximately 3000 bp . Fragment ends were blunted with T4 and Klenow DNA polymerases (NEB) and ligated into pBluescriptSK- (Stratagene) or pSMART-HC (Lucigen). Individual subclones were picked randomly and sequenced . Phrap  and Sequencher 4.8 (Gene Codes Corp.) were used to assemble DNA sequences and they were groomed manually. Sequence coverage for the sheep A3 locus averaged 4.5 sequences and the pig 27 sequences. The genomic sequences were compared using Jdotter software . E. coltware . RepetiA3 exons were identified by directly comparing the genomic DNA sequences with cDNA, EST and RACE sequences . Other CBX6 and CBX7, sequences, respectively, were NM_014292.3 and NM_175709.2 (human), NM_001103094 and XM_604126 (cow), NM_028763.3 and NM_144811 (mouse) and NM_001016617.2 &NM_001005071 (frog).A3Z1 genes: 5'-GCC ATG CRG AGC TSY RCT TCY TGG and 5'-GTC ATD ATK GWR AYT YKG GCC CCA GC-3'. Two PCR rounds were used to achieve the final number of cycles . Amplicons were analyzed by agarose gel electrophoresis, TOPO-cloned (Invitrogen) and subjected to DNA sequencing. In all instances, the expected A3Z1 fragments were recovered . 30 PCR cycles using identical conditions and degenerate primers for the ALDOA gene were used as a positive control (5'-CGC TGT GCC CAG TAY AAG AAG GAY GG-3' and 5'-CTG CTG GCA RAT RCT GGC YTA).Genomic DNA was isolated from the following tissues or cell lines: opossum kidney tissue, mouse NIH-3T3 cells, pig PK-15 cells, peccary brain tissue, cow MDBK cells, sheep FLK cells, horse blood cells (PBMC), African green monkey COS7 cells and human 293T cells . 10ng genomic DNA was used as template for PCR using primers designed to anneal to all known Sus scrofa Landrance/Yorkshire cross), sheep (Ovis aries Hampshire) and cattle (Bos taurus Hereford) PBMCs using the QIAamp RNA Blood mini kit (Qiagen). 5' and 3' RACE was performed using reagents from the FirstChoice RLM-RACE kit (Ambion). The protocol was modified slightly by using SAP (Roche) instead of CIP to remove 5'-phosphates. A3 cDNA 5' and 3' ends were amplified using Phusion high-fidelity polymerase (NEB), purified and TOPO-cloned (Invitrogen). All A3-specific primers used in conjunction with the 5' and 3' RACE primers are listed in Additional File RNA was extracted from fresh pig . It was amplified from PBMC cDNA (above) using primers 5'-NNN NGA GCT CAG GTA CCA C CA TGG ACG AAT ATA CCT TCA CT and 5'-NNN NGT CGA CGT TTT GCT GAG TCT TGA G and TOPO-cloned into pCR-BLUNT-II-TOPO (Invitrogen). As a control, human A3A was amplified using 5'-NNN NGA GCT CGG TAC CAC CAT GGA AGC CAG CCC AGC and 5'-NNN NGT CGA CCC CAT CCT TCA GTT TCC CTG ATT CTG GAG and TOPO-cloned. Catalytic mutant derivatives of the cow A3Z1 and human A3A plasmids were constructed by site-directed mutagenesis (Stratagene) using oligonucleotides 5'-CCT GCC ATG CAG CGC TCT ACT TCC TG & 5'-CAG GAA GTA GAG CGC TGC ATG GCA GG and 5'-GGC CGC CAT GCG GCG CTG CGT TCT TG & 5'-CAA GAA GCG CAG CGC CGC ATG GCG GCC, respectively.The artiodactyl A3 proteins were expressed in Hela cells as N-terminal fusions to eGFP . Cow and pig A3Z2-Z3-eGFP and the human A3A-, A3C-, A3F- and A3H-eGFP constructs were reported previously ,79. Cow A3Z1 in E. coli, an A3Z1::intron-eGFP plasmid was made by overlapping PCR to join 3 separate fragments: A3Z1 exons 1 and 2 (primers 5'-NNN NGA GCT CAG GTA CCA C CA TGG ACG AAT ATA CCT TCA CT and 5'-CCT GGA CTC ACC TTG TTG CGC), an L1-derived intron ([A3Z1 exons 3–5 (primers 5'-CAT CTC AAC AGG GTT TGG ATC A and 5'-NNN NGT CGA CGT TTT GCT GAG TCT TGA G). The resulting PCR amplicon was digested with EcoRI and SalI and then ligated into a similarly cut pEGFP-N3 (Clontech).Due to the toxicity of cow  intron . All. AllR muTo observe subcellular localization of A3 proteins, 5000 Hela cells were incubated for 24 h in Labtek chambered coverglasses (Nunc), transfected with 200 ng of the pEGFP-N3 based constructs and, after an additional 24 h visualized on a Zeiss Axiovert 200 microscope at 400× magnification. HsA3F, HsA3C, HsA3H, HsA3A, BtA3Z2-Z3 and SsA3Z2-Z3 fusion constructs were previously reported ,42,79.Z domain exons were used for all phylogenetic, positive selection and modelling studies. GARD showed no evidence for recombination breakpoints within the Z domain exons . T_coffeFree ratio model positive selection studies were based on a phylogenetic tree generated through Bayesian inference using MrBayes version 3.1 . Each trPositive selection was also evaluated in specific phylogenetic lineages using the NsSites model in the PAML codeml program Table . IndividA3 locus. Instead of considering each gene as an individual element, we subdivided it into its N-terminal and C-terminal Z-domains. Hence, the present-day human locus configuration was represented as follows: Z1-Z2-Z1-(Z2)6-Z1-Z3. The considered duplications are 'multiple tandem duplications' resulting from unequal crossing over [The aim was to infer the most likely histories of duplications and deletions that gave rise to the human ing over . In otheA3 locus and the classification of the Z domains into three distinct categories made it useful here  with identified interspersed repeats was downloaded from the RepeatMasker web site . A dot plot of this sequence with itself was obtained using Gepard [To infer the most recent evolutionary events  the sheep and pig and (B) the sheep and cow A3 genomic sequences.Click here for fileA3 and AID sequencesMammalian . A table summarizing all DNA sequences used in this study, including GenBank accession numbers.Click here for fileAPOBEC3promoter element conservation. Predicted interferon-stimulating response elements (ISRE) in the promoter regions of the indicated A3 genes and known interferon-inducing genes ISG54 and ISG15. The ISRE sequences are shown relative to the translation initiation codon ATG. Identities to human sequences are shaded gray.Click here for fileE. coli-based DNA cytosine deaminase activity data. DNA cytosine deaminase activity of the pig A3Z2-Z3 and A3Z2 proteins in E. coli. Conditions and labels are identical to those used in Figure Click here for fileEvidence for positive selection inAPOBEC3gene evolution. Phylogenetic trees showing relative relationships and ω values for the indicated (A) Z1 domains, (B) Z2 domains, (C) Z3 domains and (D) the Z domain of AID. The phylogenetic trees were determined using MrBayes, and the ω values were calculated using the PAML free ratio model. ω values are shown in red adjacent to  each phylogenetic branch. Asterisks denote branches where the ω value was infinity . The units for the scale bars are nucleotide changes per codon. The dotted line in panel (C) was used to provide more space to depict the human and non-human primate Z3 tree branches. See the main text and Methods for additional details.Click here for fileProposedAPOBEC3 gene diversification events during primatification. An alternative representation of the 8-event model for the duplication and deletion history of the human A3 repertoire. Z1, Z2 and Z3 domains are colored green, orange and blue, respectively. The Z domain(s) involved in each event are shaded gray. Dark black and red lines mark duplications , crosses designate deletions and light gray lines indicate no change. See the main text, Figure Click here for filePrimers used to identify expressedAPOBEC3transcripts from cow, sheep and pig PBMCs. A table summarizing the oligonucleotide primers used in this study.Click here for file


However, the anti-proliferative activity of capsaicin has not been studied in human SCLCs. The present manuscript fills this void of knowledge and explores the anti-proliferative effect of capsaicin in SCLC in vitro and in vivo.Small cell lung cancer (SCLC) is characterized by rapid progression and low survival rates. Therefore, novel therapeutic agents are urgently needed for this disease. Capsaicin, the active ingredient of chilli peppers, displays anti-proliferative activity in prostate and epidermoid cancer in vivo as ascertained by CAM assays and nude mice models. The second part of our study attempted to provide insight into molecular mechanisms underlying the anti-proliferative activity of capsaicin. We found that the anti-proliferative activity of capsaicin is correlated with a decrease in the expression of E2F-responsive proliferative genes like cyclin E, thymidylate synthase, cdc25A and cdc6, both at mRNA and protein levels. The transcription factor E2F4 mediated the anti-proliferative activity of capsaicin. Ablation of E2F4 levels by siRNA methodology suppressed capsaicin-induced G1 arrest. ChIP assays demonstrated that capsaicin caused the recruitment of E2F4 and p130 on E2F-responsive proliferative promoters, thereby inhibiting cell proliferation.BrdU assays and PCNA ELISAs showed that capsaicin displays robust anti-proliferative activity in four human SCLC cell lines. Furthermore, capsaicin potently suppressed the growth of H69 human SCLC tumors Our findings suggest that the anti-proliferative effects of capsaicin could be useful in the therapy of human SCLCs. Small cell lung cancer (SCLC) is an aggressive malignancy representing 13% of all lung cancer cases, with an overall 5-year survival rate of less than 5% Capsaicin, the major active ingredient of chili peppers, is used topically to treat pain and inflammation associated with a variety of diseases Apart from causing apoptosis, capsaicin has been found to induce cell cycle arrest in human cancer cells. Several convergent studies have shown that capsaicin-induced G1 arrest in CE 81T/VGH human epidermoid carcinoma cells and prostate cancer cells occur via induction of p53 and the cyclin-dependent kinase (cdk) inhibitor p21 The E2F family of transcription factors, consisting of eight member genes (E2F1-E2F8), plays a pivotal role in regulating cell cycle progression and cell proliferation Studies in recent years have shown that the activity of E2Fs is stringently regulated by the pocket protein family, namely Rb, p130 and p107. E2Fs 1–3 can bind to the Rb protein, but E2Fs 4 and 5 preferentially bind to p107 and p130 proteins in vivo models. Previous studies have shown that a majority of human SCLCs have mutations in Rb and p53, as well as a dysregulation in the E2F-Rb pathway The anti-proliferative activity of capsaicin has not been studied in human SCLCs. The present manuscript fills this gap of knowledge and shows for the first time that capsaicin can potently inhibit the proliferation of human SCLCs using multiple cell culture and in vitro. Furthermore, our results show that capsaicin suppresses the growth of human SCLC tumors in CAM and nude mice models. We have also explored the contribution of the E2F family of transcription factors in the growth-inhibitory effects of capsaicin in SCLC cells. We observed that capsaicin inhibits the proliferation of human SCLCs via a specific member of the E2F family, namely E2F4. Ablation of E2F4 levels by two independent siRNA was found to reverse the anti-proliferative effect of capsaicin. Furthermore, capsaicin-induced growth arrest was associated with a decrease in the expression of the E2F target genes cyclin E, TS, cdc25A and cdc6. Finally, chromatin IP (ChIP) analysis of human SCLC cells demonstrated that treatment of capsaicin decreased the recruitment of activator E2Fs, namely E2F2 and E2F3, to proliferative promoters like cyclin E, TS, cdc25A and cdc6. On the other hand, the recruitment of repressor E2F4 was enhanced by capsaicin treatment. Taken together, our data suggest that capsaicin displays potent anti-proliferative activity in human SCLC cells by differentially regulating the E2F family of transcription factors. Furthermore, our results raise the possibility that nutritional agents like capsaicin may be useful for the therapy of human SCLC.The present manuscript is an initial study aimed at investigating the anti-proliferative activity of capsaicin in human SCLC in both cell culture and animal models. Here, we demonstrate for the first time that capsaicin displays potent anti-proliferative activity in four human SCLC cell lines ad libitum access to food and water in HEPA-filtered racks and closely monitored by animal facility staff. All procedures involving nude mice were conducted according to the Animal Care and Use guidelines in a facility accredited by the Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC) International and were approved by the Institutional Animal Care and Use Committee (IACUC) of Joan C. Edwards School of Medicine, Marshall University (protocol#371).Nude mice were obtained from Charles River Laboratories and acclimatized for one week. They were housed in autoclaved cages with The human SCLC cell lines NCI-H69, NCI-H82 , DMS53 and DMS114 were obtained from American Type Culture Collection, Rockville, MD. These cell lines were chosen because they have been extensively studied, and their physical and molecular characteristics closely resemble SCLC in patients. Gazdar et al., (1985) originally isolated and characterized the H69 and H82 cell lines H69 and H82 were maintained in RPMI-1640 supplemented with 2 mM glutamine, 100 units/ml penicillin, 50 µg/ml streptomycin and 10% fetal bovine serum (FBS). DMS53 human SCLC cells were cultured in Waymouth's MB752/1 media containing 2 mM glutamine, 100 units/ml penicillin, 50 µg/ml streptomycin and 10% FBS. The culture media for DMS114 human SCLC cells was identical to DMS53, except that the media contained an additional 2% sodium bicarbonate.Primary normal human bronchial epithelial cells (NHBE) and small airway epithelial cells (SAEC) were obtained from Lonza Technologies, Switzerland. NHBEs were maintained in BEBM media containing growth factors. Similarly SAECs were maintained in SABM media supplemented with growth factors. Both BEMB and SABM media were prepared according to the manufacturer's instructions. All experiments using NHBEs and SAECs were performed between passages 3–8 MTT assays were performed as described by Heo et al., (1990). H69 and H82 cells were plated in 96-well plates at a density of 50,000 cells/well. DMS53 and DMS114 cells were plated in 96-well plates at a density of 5,000 cells/well. The plates were incubated for 24 hours to allow complete reattachment of the cells. Subsequently, cells were treated with 50 µM capsaicin for 24 hours, 48 hours or 72 hours. After the indicated time points, 50 µl of MTT solution (5 mg/ml) was added to each well, and the plates were incubated for 4 hours at 37°C Bromodeoxyuridine (BrdU) labeling enzyme linked immunosorbent assay (ELISA) kits were obtained from Roche Biochemicals and were used to examine the effects of capsaicin on the proliferation of four human SCLC cell lines, H69, H82, DMS53 and DMS114. BrdU is a thymidine nucleotide analog that is incorporated during S-phase (instead of thymidine) only in the DNA of proliferating cells H69 and H82 cells were plated in 96-well plates at a density of 50,000 cells/well. DMS53, DMS114, SAEC and NHBE cells were plated in 96-well plates at a density of 10,000 cells/well. DMS53 and DMS114 were incubated with serum-free media for 36 hours to remove the effect of endogenous growth factors. After 36 hours, these cells were then re-stimulated with 10% FBS in the presence or absence of indicated concentrations of capsaicin for 18 hours, which is the time required for S-phase entry Cell proliferation was also assessed by measuring the levels of proliferating cell nuclear antigen (PCNA) using a PCNA ELISA kit from Calbiochem. H69, H82, DMS53 and DMS114 were plated in 96-well plates and treated in an identical manner as the BrdU assay described above. Subsequently, the media was removed, and re-suspension buffer  was added to the cells. The level of PCNA in the cells was quantitated by measuring the absorbance at 405 nm, according to the manufacturer's protocol. Each sample was tested in duplicate, and the assay was repeated twice for each cell type.5 cells were used per sample. Each sample was incubated with serum-free media for 36 hours to remove the effect of endogenous growth factors. After 36 hours, the cells were then re-stimulated with 10% FBS in the presence or absence of 50 µM capsaicin for 18 hours, which is the time required for S-phase entry H69 SCLC cells were used for cell cycle analysis, using a modification of the propidium iodide technique Lysates for each cell line were made using the NP-40-based lysis protocol The relative expression of the indicated proteins was analyzed by western blotting. E2F1 monoclonal and polyclonal E2F2–6 antibodies were obtained from Santa Cruz Biotechnology. Monoclonal antibodies to TS, cdc25A and cdc6 were also obtained from Santa Cruz Biotechnology. Monoclonal antibody to cyclin E was obtained from BD Biosciences. Monoclonal β-actin antibody was obtained from Sigma Chemical Company, USA. Polyclonal GAPDH antibody was obtained from Trevigen, Inc. The secondary antibodies were obtained from Pierce Biotechnologies. The signal obtained in the western blot experiments was detected by the SuperSignal West Dura Extended Duration Substrate (Pierce Biotechnologies). The results of the western blotting assays were quantitated by densitometric analysis (BioRad Gel Documentation System) using the analysis software Quantity 4.5.2.6) were suspended in 100 µl cold serum-free medium, mixed with 100 µl cold BD Matrigel Matrix  and 50 µM capsaicin. These cells were applied to the CAM of each chicken embryo. Eggs were incubated at 37°C for 4 days before tumor implants were removed, photographed and weighed. A total of 12 eggs were assayed for each group Specific pathogen-free (SPF) fertile chicken eggs  were incubated at 37.5°C with 75% relative humidity, and continuously rotated slowly by an automatic egg turner . At Day 9, eggs were candled and windows opened on the shell to expose the CAM ad libitum access to food and water in HEPA-filtered racks and closely monitored by animal facility staff. All procedures were conducted according to the Animal Care and Use guidelines in a facility accredited by the Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC) International and were approved by the Institutional Animal Care and Use Committee (IACUC) of Joan C. Edwards School of Medicine, Marshall University (protocol#371).Eight 4-week-old male nude mice were obtained from Charles River Laboratories and acclimatized for one week. They were housed in autoclaved cages with 3, the mice were switched to control AIN-76A based diet containing 10% corn oil until the tumors reached 800 mm3. Subsequently, the mice were divided into two groups. The treatment group (N = 4) was changed to a diet containing 50 mg capsaicin/kg food (which is about 10 mg capsaicin/kg body weight of mouse per day). The control group (N = 4) was continued on the control diet. Mice were weighed once per week. Their food consumption was monitored by weighing the leftover food once per week. The administration of capsaicin caused no discomfort or weight loss in mice. Additionally, food intake was similar between control and capsaicin-treated mice.H69 cells were harvested and re-suspended in a 1∶1 (v/v) solution of serum-free media and Matrigel matrix (BD Biosciences). Two million cells in 100 µL were injected subcutaneously between the scapulae of each mouse 3. Tumor lengths (l), widths (w) and height (h) were measured daily (for 6 days out of a week) for each mouse. Tumor volumes were calculated as (l x w x h)/2 The drug treatment was continued until tumors of the control group reached 2000 mmCaspase cleavage assay was performed with tumor lysates prepared from control mice and capsaicin-treated mice using the caspase-3 cleavage kit . Tumor lysates were prepared using T-Per lysis buffer as described above. An aliquot of 60 µl lysate was used for each reaction. Additionally, 60 µl of cisplatin-treated H69 lysate (made out of treating H69 cells with 30 µM of cisplatin for 72 hours) was used as the positive control, according to manufacturer's protocol. Each sample was tested in duplicate, and the assay was repeated twice for each cell type.2O2 solution in methanol for 30 minutes. The sections were blocked using the Avidin-Biotin blocking kit . The sections were incubated with anti-PCNA (BioGenex Inc.) monoclonal primary antibody (1∶100 dilution) for one hour at room temperature. The sections were washed in PBS to remove excess antibody and developed using the M.O.M. and peroxidase DAB kit, obtained from Vector Laboratories . Sections were counterstained with hematoxylin, dehydrated, mounted in Permount Mounting Medium (Fisher Biotech) and photographed under Olympus BX41 bright field microscope. Hemotoxylin and eosin (H and E) pictures were photographed at 40X magnification. PCNA staining was photographed at 1000X magnification using oil. PCNA positive cells, which are the proliferating cells, were quantitated by counting 5 fields of 100 cells. Data is presented as the percentage of PCNA positive cells.Immunostaining was performed using the M.O.M. staining kit . Paraffin-embedded H69 xenograft mouse tissue sections (4 µm) were dewaxed in xylene and subsequently rehydrated in ethanol. Sections were subjected to antigen retrieval treatment using the Antigen Retrival kit (BioGenex Inc.), according to the manufacturer's protocol. The sections were then treated with Proteinase K treatment (20 µg/ml) for 15 minutes and quenched of endogenous peroxidases in 0.3% HChemically synthesized, double-stranded siRNA for E2F1, E2F2, E2F3, E2F4, E2F5 and E2F6 was purchased from Santa Cruz Biotechnology. The transfection experiments were performed in H69 and DMS114 cells The results of the E2F4 transfection experiments were verified using a second set of independent siRNA obtained from Ambion Biotechnologies 5 cells seeded in T-10 flasks (Midwest Scientific) in RPMI containing 10% FBS without antibiotics. In the case of the DMS114 cells, the transfection was performed in 6–well plates, using 5×105 cells/well. In both the H69 cells and DMS114 cells, each transfection was performed in duplicate, and the whole experiment was repeated twice. The transfection of the indicated siRNA was performed using Oligofectamine reagent (Invitrogen Corporation), according to the manufacturer's protocol. A non-targeting control-siRNA was used as the negative control. After 36 hours of transfection, lysates were made as described above and the expression of E2F family of proteins was determined by western blotting analysis.Western blotting experiments were performed to assess the expression of proteins after siRNA transfection H69 cells were subjected to serum starvation for 36 hours and subsequently stimulated with 10% FBS for 8 hours 5′TTCTTGAGCAACACCCTCTTCTGCAGCC3′.Cyclin E (forward primer): 5′TCGCCATATACCGGTCAAAGAAATCTTGTGCC3′.Cyclin E (reverse primer): 5′CTGCCAGCTGTACCAGAGAT3′.TS (forward primer): 5′ATGTGCATCTCCCAAAGTGT3′.TS (reverse primer): 5′CCCCATGATTGTGTTGGTAT3′.Cdc6 (forward primer): 5′TTCAACAGCTGTGGCTTACA3′.Cdc6 (reverse primer): 5′CTCAACACGGGAAACCTCAC3′.18S (forward primer): 5′AAATCGCTCCACCAACTAAGAA3′.18S (reverse primer): Real-time PCR was performed on a Bio-Rad iCycler.H69 were serum-starved for 36 hours by incubating them in serum-free RPMI-1640 5′CCCCGTCCCTGCGCCTCGCTG3′.Cyclin E (forward primer): 5′CGGCGGCGGCGACGGCAGTGG3′.Cyclin E promoter (reverse primer): 5′GGCCTCACAGCGACTCTAAGA3′.Cdc6 promoter (forward primer): 5′CTCGGACTCACCACAAGC3′.Cdc6 promoter (reverse primer): 5′TGGCGCACGCTCTCTAGAGC3′.TS promoter (forward primer): 5′GACGGAGGCAGGCCAAGTG3′.TS promoter (reverse primer): The cdc25A, c-fos primers and PCR conditions are described in All data was expressed as the mean ± SEM and represented using Graph Pad Prism 5. Differences between control and treated samples were analyzed by analysis of variance (ANOVA), followed by a Dunnett's multiple comparison test. The tumor growth rates in athymic mice were analyzed by ANOVA followed by Neuman-Keuls test. In the immunohistochemistry experiments, all PCNA positive nuclei were counted in five independent fields by two independent observers in a randomized double blind fashion. Chi-square analysis was done to determine differences in the percentage of PCNA positive nuclei. BrdU assays were performed in triplicate for each data point and the whole assay was repeated twice. PCNA ELISAs and caspase cleavage assays were performed in duplicate and the whole assay was repeated twice. All analyses were completed using a 95% confidence interval. Data was considered significant when the P value was less than 05.in vitroMTT assays were performed to determine whether capsaicin can suppress the growth of human SCLC cells The next question we asked was whether capsaicin could cause inhibition of cell proliferation in human SCLC cells. BrdU assays were used to examine the anti-proliferative effects of capsaicin in four human SCLC cell lines, namely H69, H82, DMS53 and DMS114 Capsaicin inhibited the proliferation of H69 human SCLC cells in a concentration dependent manner . The antThe results of the BrdU assays were further verified by measurement of the PCNA levels in human SCLC cells treated with capsaicin. Quiescent human SCLC cells were stimulated with serum for 18 hours (to induce S-phase entry) in the presence or absence of 50 µM capsaicin. After 18 hours, PCNA levels were measured by ELISA. PCNA ELISA assays showed that treatment of H69, H82, DMS53 and DMS114 human SCLC cells with 50 µM capsaicin resulted in about a 50% inhibition of S-phase entry of cells .in vitro.Finally, we performed cell cycle analysis by flow cytometry to confirm the results obtained from the BrdU and PCNA experiments Next, we wanted to examine whether capsaicin-induced growth arrest was correlated with any changes in the expression of E2F-target proliferative genes. We selected cyclin E, TS, cdc25A and cdc6. Since these are E2F target genes, we examined whether capsaicin could decrease the mRNA level by quantitative real-time-PCR using RNA extracted from serum-stimulated cells and cells treated with serum and 50 µM capsaicin. Our results showed that serum-stimulated expression of cyclin E, TS, cdc25A, and cdc6 was significantly down-regulated by 50 µM capsaicin P<01; . These rin vivo3) H69 tumors xenotransplanted in nude mice  constitute an established model to study tumor growth ude mice .in vivo.Tumors were harvested at the end of the treatment. Sections were stained with H and E and analyzed by immunohistochemical staining with monoclonal antibody to PCNA . There wIn the next series of experiments, the effect of E2F1–6 in the anti-proliferative activity of capsaicin was assessed by siRNA methodology. E2F1, E2F2, E2F3, E2F4, E2F5 and E2F6 siRNAs were individually transfected in H69 cells. Eighteen hours after transfection, H69 cells were serum-starved for 36 hours (by incubating them in serum-free RPMI) and subsequently re-stimulated with 10% FBS in the presence of 50 µM capsaicin for 18 hours. BrdU assays were performed to measure the percentage of cells undergoing S-phase entry We observed that E2F4-siRNA reversed the anti-proliferative effect of capsaicin in H69 and DMS114 cells, whereas E2F1–3-, E2F5- and E2F6-siRNA did not have any effect on the growth-inhibitory effects of capsaicin . The conSince the E2F-family of transcription factors have been shown to play a pivotal role in cell proliferation, we wanted to examine whether E2F-siRNAs would affect proliferation of human SCLC cells untreated with capsaicin. E2F1–6-siRNAs were individually transfected in H69 cells using Oligofectamine reagent. Eighteen hours post-transfection, H69 cells were serum-starved for 36 hours (by incubating them in serum-free RPMI) and subsequently re-stimulated with 10% FBS for 18 hours. BrdU assays were performed to measure the percentage of cells undergoing S-phase entry The results of the siRNA experiments were verified by using two independent sets of E2F-siRNAs. BrdU assays showed that both independent E2F4-siRNA ablated the effects of 50 µM capsaicin in H69 cells , whereasOur data seemed to indicate that E2F4 plays a vital role in the anti-proliferative effects of capsaicin. Therefore, ChIP assays were performed to examine the differential occupancy of E2F family of proteins on E2F-responsive proliferative promoters  upon capsaicin-treatment of H69 human SCLC cells High amounts of the proliferative E2Fs, namely E2F1, E2F2 and E2F3, were bound to all E2F-responsive promoters in the control serum-stimulated H69 cells (The dysfunction of the E2F/Rb pathway is a hallmark of greater than 90% of lung cancers in vivo models. Our results showed that capsaicin suppresses the growth of established H69 human SCLC tumors in both CAM and nude mice models. We observed that the administration of capsaicin did not cause any gross discomfort, toxicity or weight loss in these animals. Taken together, our data suggest that capsaicin has the potential for therapy and management of human SCLCs.Emerging evidence shows that capsaicin is a promising anti-cancer agent that causes potent apoptosis in prostate cancer, non small cell lung cancer, gliomas and gastric cancers The majority of studies exploring the anti-cancer activity of capsaicin have focused on the mechanisms underlying capsaicin-induced apoptosis One of the interesting findings of our study was that E2F1-, E2F2- and E2F3-siRNA by themselves had no effect on the serum-induced proliferation of H69 cells. This observation can be explained by the fact that there is considerable redundancy in the proliferative functions of the E2F family of proteins Our study demonstrated for the first time that E2F4 primarily mediates the anti-proliferative activity of capsaicin. Our data is consistent with previous studies that have shown that E2F4 is a repressor of transcription and inhibits cell proliferation. In addition, the E2F4/p130 pathway has been implicated in the growth and progression of lung cancer. E2F4−/− mice were found to have defects in small airway epithelial cells, suggesting a role for this protein in lung development ChIP assays were performed to examine the relative distribution of E2Fs on E2F-responsive promoters in human SCLC cells. Our studies revealed that the treatment of human SCLC cells with capsaicin leads to differential recruitment of E2Fs on E2F-responsive promoters like cyclin E, TS, cdc25A and cdc6. Control H69 SCLC cells contained E2F1, E2F2 and E2F3 bound to proliferative promoters. Our results are consistent with previous studies in that proliferative responses are primarily mediated by E2F1, E2F2 and E2F3, whereas the recruitment of E2F4 on the promoters suppresses cell proliferation In summary, the data presented in this paper show that capsaicin displays potent anti-proliferative activity against human SCLC, and this effect is mediated by the E2F4 pathway. Aberrancies in the E2F pathway are one of the hallmarks of human SCLC


The hemagglutinin (HA) of influenza A viruses has been classified into sixteen distinct subtypes (H1–H16) to date. The HA subtypes of influenza A viruses are principally defined as serotypes determined by neutralization or hemagglutination inhibition tests using polyclonal antisera to the respective HA subtypes, which have little cross-reactivity to the other HA subtypes. Thus, it is generally believed that the neutralizing antibodies are not broadly cross-reactive among HA subtypes. In this study, we generated a novel monoclonal antibody (MAb) specific to HA, designated MAb S139/1, which showed heterosubtypic cross-reactive neutralization and hemagglutination inhibition of influenza A viruses. This MAb was found to have broad reactivity to many other viruses  in enzyme-linked immunosorbent assays. We further found that MAb S139/1 showed neutralization and hemagglutination-inhibition activities against particular strains of H1, H2, H3, and H13 subtypes of influenza A viruses. Mutant viruses that escaped neutralization by MAb S139/1 were selected from the A/Aichi/2/68 (H3N2), A/Adachi/2/57 (H2N2), and A/WSN/33 (H1N1) strains, and sequence analysis of the HA genes of these escape mutants revealed amino acid substitutions at positions 156, 158, and 193 (H3 numbering). A molecular modeling study showed that these amino acids were located on the globular head of the HA and formed a novel conformational epitope adjacent to the receptor-binding domain of HA. Furthermore, passive immunization of mice with MAb S139/1 provided heterosubtypic protection. These results demonstrate that MAb S139/1 binds to a common antigenic site shared among a variety of HA subtypes and neutralizes viral infectivity in vitro and in vivo by affecting viral attachment to cells. The present study supports the notion that cross-reactive antibodies play some roles in heterosubtypic immunity against influenza A virus infection, and underscores the potential therapeutic utility of cross-reactive antibodies against influenza. Neutralizing antibodies play a critical role in protection from influenza A virus infection. Most neutralizing antibodies recognize hemagglutinin (HA), which is the major surface glycoprotein of influenza viruses. The HA has been classified into sixteen antigenically distinct subtypes. Since HA subtypes of influenza A viruses are principally defined as serotypes determined by neutralization or hemagglutination inhibition tests using polyclonal antisera to the respective HA subtypes, which have little cross-reactivity to the other HA subtypes, it is generally believed that the neutralizing antibodies are not broadly cross-reactive among HA subtypes. Herein we present a novel cross-neutralizing monoclonal antibody that reacts with a variety of HA subtypes in vitro and provides heterosubtypic protection against influenza A virus infections in mice. We demonstrate that this antibody recognizes a common epitope adjacent to the receptor binding region of HA and inhibits virus binding to the cells. The present study supports the notion that cross-reactive antibodies, as well as cytotoxic T lymphocytes, play some roles in heterosubtypic immunity against influenza A virus infection, and underscores the potential therapeutic utility of cross-reactive monoclonal antibodies for multivalent prophylaxis and treatment against infection with influenza A viruses, including the hypothetical new pandemic influenza viruses. Neutralizing antibodies play a critical role in protection from influenza virus infection. Most neutralizing antibodies recognize hemagglutinin (HA), which is the major surface glycoprotein of influenza viruses. The HA of influenza A viruses has been classified into sixteen antigenically distinct subtypes (H1–H16) that are maintained in avian and mammalian species in nature HA is responsible for virus entry into target cells, virus binding to the host receptor, internalization of the virus, and subsequent membrane-fusion events. It is initially synthesized as a precursor polypeptide, HA0, that requires proteolytic cleavage into disulfide-linked HA1 and HA2 before it is functional and virus particles are infectious. The major part of HA1 forms the “globular head” region, which contains the necessary structure for binding to the sialic acid receptors. The “stem” region is mostly formed by HA2, which contains the fusion peptide and membrane anchor domain. It has been recognized that there is considerable amino acid variability (antigenic difference) in the globular head region among HA subtypes, whereas the structure of the stem region is relatively conserved.The HA antigenic structure of the H3 subtype has been well characterized by using the sequence information on naturally occurring and laboratory-selected antigenic variants In general, HA subtypes of influenza A viruses are principally defined as serotypes determined by neutralization or hemagglutination inhibition (HI) tests using polyclonal antisera to the respective HA subtypes, which have little cross-reactivity to the other HA subtypes. Furthermore, since the structures of HA antigenic sites vary among not only different subtypes of viruses but also the same subtype, it is generally believed that the neutralizing antibodies are not broadly cross-reactive among HA subtypes. Therefore, studies on cross-reactive HA-specific antibodies to multiple HA subtypes have been limited Recently, it has been shown that intranasal immunization with inactivated viruses provided heterosubtypic protection in a mouse model, suggesting a role for cross-reactive antibodies in the heterosubtypic immunity against influenza viruses MAb S139/1 (IgG2a) was originally produced as an H3 HA-specific antibody. The cross-reactivity of MAb S139/1 to multiple subtypes of influenza A virus HAs was then tested by enzyme-linked immunosorbent assay (ELISA) using several H1, H2, H3, H5, H9, and H13 subtypes . We founWe next tested HI activity of MAb S139/1 to various influenza virus strains . MAb S13p<0.05 and p<0.01, respectively)  or WSN (H1). Control groups were given an irrelevant MAb (ZGP12/1.1) ctively) . ImportaTo determine the epitope for MAb S139/1, escape mutants of Aichi (H3), WSN (H1), and Adachi (H2) were selected in the presence of this antibody as described in We compared deduced amino acid sequences of the region including the MAb S139/1 epitope among H1, H2, H3, H5, H9, and H13 subtypes of HA . Among tIn three-dimensional structural analysis of the Aichi (H3) HA molecule, positions of the amino acid substitutions found in the escape mutants were mapped in the globular head of the HA . We founTo confirm the importance of the amino acid residues at positions 156, 158, and 193 for binding of MAb S139/1, we generated recombinant PR8 (H1) mutant viruses with modified epitopes whose amino acid sequences at these positions were replaced with those of Aichi (H3), and tested neutralizing activities of MAb S139/1 to the mutant viruses . Of the It has been generally known that hetrosubtypic immunity can be provided by subtype cross-reactive cytotoxic T lymphocytes that recognize conserved epitopes of viral internal proteins of influenza A viruses such as nucleoprotein and matrix protein For example, MAb IVA1B10 In the present study, we found that MAb S139/1 bound to all the strains of H1, H2, H3, H5, H9, and H13 subtypes tested by ELISA, whereas it showed neutralization and HI activities to some particular strains tested. Since we assume that MAb S139/1 recognizes a single epitope on the HA molecules of all the subtypes, it is likely that the different binding affinity of MAb S139/1 to each HA subtype influences the neutralization and HI activities. Indeed, our data demonstrated that there was an appreciable correlation between its binding affinities tested by ELISA and by HI or the neutralization test. Furthermore, our sequence analyses and reverse genetics approaches revealed the major contribution of the HA amino acid residues at positions 156, 158, and 193 to the binding capacity of MAb139/1.We found three independent substitutions at positions 156, 158, or 193 in the Aichi mutant viruses that escaped from neutralization by MAb S139/1. Three-dimensional structural analyses revealed a conformational epitope consisting of these amino acid residues. Of these, the amino acid residue at position 193 of H3 HA was shown to interact with the host receptor molecule  Three-dimensional structural analyses based on HA molecules cocrystallized with a sialylated glycan receptor analogue (pentasaccharide) suggest that molecular contacts between HA and the sialylated glycan receptor are divided into base and extension regions which include contacts with the terminal sialylgalactose moiety and the subsequent sugar rings, respectively It is well-known that HAs of avian and human influenza viruses bind preferentially to α2-3 and α2-6 sialylated (SA) glycan receptors, respectively. Although it has been generally believed that amino acid substitutions at positions 226 and 228 are primarily responsible for the differences in the receptor specificity between avian and human H3 viruses, several studies have reported that the mutation at position 193 of H3 HA might also alter the receptor binding properties Recently, passive transfer of MAbs specific to viral proteins has been tested in clinical studies, providing models for the use of MAbs for prophylaxis or treatment of infectious diseases. In fact, a humanized MAb specific to RSV F protein is already approved by the US Food and Drug Administration and used in clinical cases. It has been experimentally shown that this approach is effective for influenza virus infection in mice Together with previous studies by others Influenza virus strains, A/PR/8/34 (PR8) (H1N1), A/WSN RG/33 (WSN) (H1N1), A/Adachi/2/57 (Adachi) (H2N2), A/Singapore/1/57 (H2N2), A/duck/Hong Kong/836/80 (H3N1), A/Aichi/2/68 (Aichi) (H3N2), A/Memphis/1/96 (Memphis) (H3N2), A/duck/Hokkaido/5/77 (H3N2), A/chicken/Hong Kong/37/78 (H3N2), A/duck/Hokkaido/8/80 (H3N8), A/Hong Kong/483/97 (H5N1), A/rgViet Nam/1194ΔHA/2004 (rgVNΔHA) (H5N1), A/swan/Hokkaido/67/96 (Hok67) (H5N3), A/swine/Hong Kong/10/98 (HK10) (H9N2), A/duck/Hong Kong/W213/97 (W213) (H9N2), A/duck/Hokkaido/49/98 (H9N2), A/gull/Maryland/704/77 (Maryland) (H13N6), and B/Lee/40 were used . rgVNΔHASix-week-old female BALB/c mice, were intranasally immunized twice at 2-week intervals with 100 µg of formalin (0.2%)-inactivated purified Aichi together with cholera toxin B (Sigma). Two weeks after the second immunization, the mice were intranasally boosted with inactivated virus alone. Three days later, the spleen cells from the mice and mouse myeloma Sp2/0 cells were fused and maintained according to a standard procedure o-phenylendiamine dihydrochloride (Sigma). HI activity of the purified MAb was tested by the standard method using 0.5% chicken erythrocytes. Neutralizing activity of the MAb was measured by a plaque reduction assay using MDCK cells. Ten-fold dilutions of MAb were mixed with 100–200 plaque forming unit (PFU) of viruses and incubated for 1 h at room temperature. The confluent monolayers of MDCK cells on 12-well plates were inoculated with the mixture. After 1 h adsorption, the virus inoculums were removed and the cells were overlaid with MEM containing 1% Bact-agar and trypsin (5 µg/ml). The plaques were enumerated after incubation at 35°C, 5% CO2 for 2 days. Western blotting was performed as follows. Virus proteins were separated by 10% SDS-polyacrylamide gel electrophoresis under reducing or non-reducing conditions, and transferred to PVDF membranes (Millipore). The membranes were blocked with 3% skim-milk (SM) in PBS containing 0.05% Tween-20 (PBST) and exposed to MAb S139/1 (1 µg/ml) in 1% SM-PBST, and then probed with horseradish peroxidase-conjugated goat anti-mouse IgG (H+L) (Jackson), and the reacted bands were visualized by Immunostaining HRP-1000 (Konica Minolta).ELISA was performed essentially as previously described The experimental protocols were reviewed and approved by the Hokkaido University Animal Care and Use Committee (08-0234). Six-week-old female BALB/c mice were passively immunized by intraperitoneal injection with 200 µg of purified MAb S139/1 or ZGP12/1.1 (IgG2a) 5′-AGCAAAAGCAGG), HA genes were amplified by PCR using gene-specific primer sets Viral RNA was extracted using a QIAamp Viral RNA Mini Kit (Qiagen). After reverse transcription with M-MLV reverse transcriptase (Invitrogen) using Uni12 primer (Escape mutants were selected by culturing WSN (H1), Adachi (H2), and Aichi (H3) strains in MDCK cells in the presence of MAb S139/1. Viruses were incubated with purified MAb S139/1  for 1 h, and then the mixtures were inoculated into confluent MDCK cells in 6-well tissue culture plates. After 1 h adsorption, the cells were overlaid with MEM containing 1% Bacto-Agar (Difco) and MAb S139/1 ascites  and trypsin (5 µg/ml), and then incubated for 2 days at 35°C. Escape mutants were purified from single isolated plaques, and propagated in MDCK cells with serum-free MEM containing trypsin. The nucleotide sequences of the HA genes of the parent strains and the escape mutants were determined and deduced amino acid sequences were compared among these viruses.The plasmid pWH194-HA The HA structures of WSN (H1) and Adachi (H2) were constructed using Modeller 8v2